.. . 1...: a» asuthh...c.«.u.§la::z I: $1.315." .9 JIAEI. v1.13»: ‘v. £n’.‘ 1|:- :31. .c: 3.1.»... than , : . my acute .. 30.35 If Jr . 3:... . r13}... I... ‘mavmwrmmfivm. ”(6% . a .... a.» 3 king... t t as ‘4. I ‘- mil: .3 $25.: . 1 and. 30 A iguir. ,wm. A, . . Sawflyv u . l 9.“, 31" ‘65 Sfiii 9 F I'I x32“): 3-fs§:3~1‘€: “infiit “ 1“ Q ‘I ~ 4» 1 '. - ,J 23. .2- f .5. c.» 53.8.... . an? :1 x lg. 5. .w & 4‘ . 3' ' 3r ' I ’;§5"13‘:;EEF ' I V vi ..\A‘0U:Y in b.» '- ikb!..l. i A .I~V- v»: 11:31.. A (I 3);- tr! \h!‘~ .! 2...!!! .53 1‘ bl (It... i n . .2 nva‘ 0|.)qu- avvx. \ .1. .u I x 1!}! 6.1:} 5. [Alta ’r.‘..\. A )Vlwt .. I. . .91.: £22... I 3. 7i Exfihfifixvauolaiu J.......H!.... 9.0.35. s. Ms ; .. ,“ llllllllllllllllllllllllllllllllllllllll 3 1293 01410 K J This is to certify that the dissertation entitled Characterization of the regulatory regions of cell- interaction-dependent genes in Myxococcus xanthus presented by Makda Fisseha has been accepted towards fulfillment of the requirements for P ‘h' D degreein MKmB‘CIOS’J 01321 ’km Major professor Date “('6 [(177 7 l MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE ll RETURN BOX to romovo this ohookout from your rooord. TO AVO|D FINES Mum on or boron doto duo. DATE DUE DATE DUE DATE DUE l l l MSU lo An Affirm-rm ActloNEqal Opportunity Inflation WA CHARACTERIZATION OF THE REGULATORY REGIONS OF CELL INTERACTION-DEPENDENT GENES IN M YXOCOC C US XANT H US By Makda Fisseha A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1995 ABSTRACT CHARACTERIZATION OF THE REGULATORY REGIONS OF CELL INTERACTION-DEPENDENT GENES IN MYXOCOC C US XANT HUS By Makda Fisseha Myxococcus xanthus is a gram-negative, rod-shaped, soil bacterium that undergoes multicellular development when starved for nutrients. Development involves the coordinate movement of cells into aggregation centers, formation of fruiting bodies, and sporulation of cells within fruiting bodies. The coordination of this multicellular fruiting body formation and sporulation process requires at least five different cell-cell signaling interactions. These intercellular signaling interactions are also required for the proper expression of a temporally regulated set of genes. Extracellular C-signaling is required for development starting at six hours after the initiation of the developmental process. Cells that are defective in C-signaling initiate development normally but fail to form fruiting bodies and spores. Expression of genes that normally begin after six hours of development is either reduced or abolished in C-signaling defective cells indicating that C-si gnalin g is required for gene expression after six hours of development. To understand the mechanism by which extracellular C-signalin g regulates gene expression during development, the upstream DNA regulatory elements of two fusions between developmentally regulated M. xanthus promoters and a promoterless lacZ gene were characterized. The Tn5 lac Q4403 fusion, whose expression has an absolute requirement for C- signaling, is expressed from a promoter bearing some resemblance to E. coli promoters transcribed by 670-RNA polymerase. DNA between 80 and 72 nucleotides upstream of the transcriptional initiation site is required for the expression of this fusion in a developmentally regulated manner and DNA downstream of -80 is sufficient for C-signal- dependent activation of the promoter, indicating that proreins in addition to RNA polymerase may be required for C-signal-dependent expression of Q4403. Tn5 lac 94499 is a developmentally regulated fusion whose expression only partially requires extracellular C-signalin g. C-signal dependent expression of this fusion requires 3.2 kb of DNA upstream of the fusion point. Tn5 lac Q4499 appears to be inserted into an operon that encodes enzymes involved in secondary metabolism. TO Almaz Zewde for her love and for teaching me that hard work and at principles should not be compromised. Haeran and Yonas Fisseha for their love, friendship, their high standards which have been a source of inspiration, and Yonas for coming to my rescue in my times of computer crises. iv ACKNOWLEDGEMENTS Many people helped me in my endeavors in graduate school and my gratitude and appreciation goes out to all of them. I have to especially thank Lee Kroos with whom I have been honored to work. I thank him for teaching my the art of science and for all his patience, kindness and understanding. I would also like to thank Mary, Mark, David, and Heidi Kroos for their friendship throughout the years. I would like to thank my father, Fisseha Haile-Meskal, whose work and interest in science have always been an inspiration. Richard Halberg was both a colleague and friend whose humor, advice, and camaraderie I will always remember and value. Sijie Lu shared with me not only his cloning expertise but also his enthusiasm for science, which made the late hours in lab fun. I would like to thank both him and Mimi Lu for their friendship and kindness. I also thank Jamie White, the pioneer Myxo worker at Michigan State University, for his sincerity and for his offer of a (risky) canoe excursion. I have shared the joys and travails of working with Myxo with Janine Brandner who has given me many invaluable advice in my experiments and in my writing, and for which I am grateful. I thank Tong Hao for her friendship, for her interest in my project and for continuing with the mobility shift experiment. Dvora Biran has shown me that trying new experiments can be not only challenging but also exciting. I thank her for this and for all her encouragement; I also thanks Jennifer Loconto who has made the lab livlier, Hiroshi Ichikawa for being a kind and conscientious lab mate, which made working in the lab very pleasant, Bin Zhang for his candor and for allowing me to laugh at his absent mindedness, and Michelle Anderson for all her helpful suggestions. Among my family and friends I would like to especially thank Eleni Gabre-Medhin for showing me that where there is a will there is a way. I also thank Raw], Saleela and Nisha Hollingsworth for their warm friendship and for welcoming me into their family, Rena, Ron and Arden Azarbarzin for all the good times I have had with them. Thanks also to Lilu Tesfa, Roya Mavaddat and Ghislaine Lydon for their special friendship. vi TABLE OF CONTENTS Page List of tables ....................................................... . .................... ix List of figures ......................................................................... x List of abbreviations and symbols ............................................. xii Introduction ............................................................................ 1 Chapter 1. Literature Review Social growth and motility ............................................ 4 Social development ..................................................... 5 A-signaling .............................................................. 10 B-signaling .............................................................. 13 D-signaling .............................................................. 14 E-signaling .............................................................. 15 C-signaling .............................................................. 16 Chapter 2. Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus Abstract ...................................................................... 29 Introduction ................................................................ 30 Materials and Methods .................................................. 33 Results Cloning DNA upstream of Q4403 and testing it for promoter activity ................................................................. 45 Accumulation of the Tn5 lac Q4403 fusion mRNA during development ........................................................... 53 DNA sequence of the Q4403 upstream region .................... 56 Precise mapping of the Q4403 mRNA 5’ end .................... 61 Further deletion analysis of the Q4403 promoter ................. 61 Possible explanations for the low level of expression from fusions integrated at Mx8 at: ........................................ 64 Dependence of expression on C-signaling ......................... 72 Discussion .................................................................... 76 Chapter 3. Characterization of the DNA upstream of a partially C-signal-dependent operon A bstra ct ...................................................................... 86 Introduction ................................................................ _ 87 Materials and Methods ................................................. 88 Results Analysis of the Q4499 upstream region for promoter activity. 96 C-signal-dependent expression of Q4499 ........................ 105 Analysis of the Q4499 transcript .................................. 108 DNA sequence analysis of the Q4499 upstream region ........ 111 Discussion .................................................................. 118 Summary and Perspectives ..................................................... 122 Appendices ............................................................................. 126 Bibliography ............................................................................. 137 viii LIST OF TABLES Table 2.1 Bacterial strains and plasmids .................................................. 3.1 Bacterial strains and plasmids .................................................. Appendix 3 Table l Myxococcus xanthus sporulation efficiency ............................. ix Page 34 90 135 LIST OF FIGURES Figure l . 1 Dependence of developmental markers on A, B, C, and D, factors ....... 2 . 1 Physical map of the Q4403 insertion region and summary of deletions tested for promoter activity ................................................... 2.2 Map of the plasmid used to test for promoter activity ..................... 2 . 3 Expression of developmental lacZ under the control of the Q4403 promoter ........................................................................ 2 . 4 Levels of fusion mRNA and B—galactosidase specific activity from Tn5 lac Q4403 during development ......................................... 2 . 5 Nucleotide sequence of 616 bp directly upstream of of Tn5 lac Q4403. 2 . 6 Alignment of the amino acid sequence of the Q4403 partial ORF and the N-terminal region of aqualysin I .......................................... 2.7 Primer extension analysis of the Q4403 promoter ........................... 2.8 Deletion analysis of the Q4403 promoter .................................... 2 . 9 Developmental lacZ expression from fusions at the native Q4403 position in the chromosome ................................................... 2 . l 0 Extracellular complementation of developmental lacZ expression from the Q4403 promoter in csgA cells ............................................ 3.1A Physical map of the Q4499 insertion region ................................ 3. IB Developmental lacZ expression from the Q4499 promoter ................ 3.2 Deletion analysis of the Q4499 promoter region ............................ 3.3 Developmental lacZ expression in csgA mutant cells ....................... 3.4 Northern blot analysis of the Q4499 transcript .............................. 3.5 Nucleotide sequence of 2.1 kb upstream of Tn5 lac Q4499 ................ Page 47 49 52 55 58 63 66 70 74 99 101 104 107 110 113 3 . 6 Analysis of the Q4499 upstream sequence for ORFs likely to be translated ......................................................................... Appendices 1 Northern blot analysis of Q4403 ............................................. 2A DNA structure of pREGl666-derived plasmids integrated at Mx8 att .. 23 DNA analysis of developing MMF31-7 cells ............................... xi 116 128 131 133 bp CaC12 dCTP DEPC dGTP DNA EDTA kb LIST OF ABBREVIATIONS xii ampicillin base pair cytidine calcium chloride cytosine-5’-triphosphate deoxycytosine—S’-triphosphate diethyl pyrocarbonate deoxyguanosine-S’-triphosphate deoxyribonucleic acid dithiothreitol (ethylenedinitriol)tetraacetic acid guanosine kilobase pairs kilodalton kanamycin Luria-Bertani microgram microliter micromolar millirnolar molar PCR SDS Tc xiii nanomolar polymerase chain reaction ribonucleic acid sodium dodecylsulfate tetracycline terminal deoxynucleotide transferase volume INTRODUCTION Myxococcus xanthus is a gram-negative, rod—shaped, soil bacterium that has a multicellular form of existence. Upon nutrient depletion, cells undergo a complex developmental process which involves the movement of cells into aggregation centers and the formation of fruiting bodies. Within fruiting bodies, some of the cells lyse, whereas others mature into ovoid spores known as myxospores. Both the regulation of morphogenesis and the transcriptional regulation of gene expression during development require intercellular signaling. To begin to understand how one extracellular signaling event, known as C—signalin g, controls gene expression, the DNA regulatory regions of two developmental genes whose expression is regulated by C- signaling was characterized. In chapter one, a review of cell-cell interactions in M. xanthus is presented and is compared with cell-cell interactions in other systems. Chapter two describes the characterization of the upstream DNA regulatory region of Q4403, a fusion of Tn5 lac with a developmentally regulated gene whose expression has an absolute requirement for C-si gnalin g. This fusion appears to be with a gene encoding a protease, and its expression requires DNA more than 72 base pairs upstream of the transcriptional initiation site. This suggests that activation proteins may be involved in the expression of Q4403. Studies showing that the position in the chromosome affects expression of this fusion are also presented in this chapter. The contents of this chapter 2 will be submitted to the Journal of Bacteriology with Monica Gloudemans (a former graduate student in our lab) and Ronald E. Gill (University of Colorado) as second and third authors, respectively. Monica Gloudemans was responsible for cloning the 8.5 kb of DNA upstream of Q4403 into pREGl666 and pREGl 175 and testing it for promoter activity. Monica also generated the 4.0 and 2.0 kb deletions and tested them for promoter activity, and performed the Sl-nuclease analyses. Ron Gill constructed pREGl666 and pREGl727, which I used extensively, and which have not been described adequately in the literature. Chapter three describes the characterization of the DNA regulatory region of a second Tn5 lac fusion, Q4499, which has only a partial requirement for extracellular C- signaling. This fusion appears to be with a gene encoding a cytochrome P-450 enzyme, based on deduced amino acid sequence similarities. This gene appears to be part of an operon whose transcription requires DNA more than 2.1 kb upstream of the Q4499 insertion site. The operon appears to encode at least one other enzyme, an NADH-dependent reductase. The final chapter contains the conclusions and significance of these results. The regulatory region of the absolutely C-signalin g dependent gene requires sequences upstream of what is typically recognized by RNA polymerase, and the same may hold true for the regulatory region of the operon whose expression is partially C-signal dependent operon. These studies provide the basis for identifying regulatory proteins that mediate the C-signaI-dependent response. Chapter 1 Literature Review 4 Differentiation and development are important processes that have been widely studied in a variety of multicellular organisms, ran gein g from the simple eukaryote Dictyostelium discoideum to C aenorhabditis elegans, Drosophila melanogaster, and embryos. Myxococcus xanthus is a gram-negative, rod-shaped, soil bacterium that has a multicellular form of existence. The highly cooperative processes of multicellular feeding, swarming, and fruiting in M. xanthus resemble the developmental processes of more complex multicellular organisms in their requirement for cell migration and intercellular communication. Because M. xanthus is readily amenable to genetic and biochemical analyses, and because its developmental process is relatively short and simple, it provides a good model system for studying how cell-cell interactions control multicellular deveIOpmental and cellular differentiation. Social growth and motility. During vegetative growth on a solid surface, M. xanthus cells glide as swarms toward nutrient sources and secrete bacteriolytic, cellulolytic, and other digestive enzymes to hydrolyze macromolecules (Kaiser & Kroos, 1993). Growth of M. xanthus is cell is density dependent, as indicated by experiments in which growth of cells on casein as the sole nutrient source increased proportionally as the cell density was raised in liquid culture (Rosenberg et al., 1977). This cell density dependent growth is attributed to the increased concentration of nutrients available for growth that results from the increased concentration of extracellular hydrolytic enzymes at high cell concentrations. The multicellular nature of grthh and development of M. xanthus is also evident in its motility. M. xanthus cells move in large groups known as swarms. Within the swarm, movement of cells in small groups is known as social (8) motility. Occasionally, individual cells briefly move outside of a swarm before returning, and this is known as adventurous (A) motility. These two types of motility are controlled by different systems that function largely independently. The S-motility system involves at least ten genes 5 (Shimkets, 1990). Cells defective in any of the S-motility genes fail to move in clusters but are able to move as individual cells (Shimkets, 1990). The A-motility system involves at least 23 gene products (Fink & Zissler, 1989; Hodgkin & Kaiser, 1977; Sodergren & Kaiser, 1983). Defects in any of the A-motility genes results in the failure of cells to move individually; however, these cells can still move in groups. A third locus that controls motility is the mgl locus. Mutations in the mgl locus render cells completely nonmotile. Thus, cells that are nonmotile both as individual cells and in swarms have mutations either in mgl or in both an A- and a S-motility gene (A—S-) (Hodgkin & Kaiser, 1977; Hodgkin & Kaiser, 1979b). The mgl locus consists of two genes, mglA and mng (Stephens et al., 1988). The predicted amino acid sequence of mglA shows significant similarity to a class of GTP-binding proteins, and the region of similarity spans the putative GTP binding and hydrolysis sites (Hartzell & Kaiser, 1991). The product of the mng gene appears to be important for the synthesis and/or stability of the MglA protein since mutations in mng that do not affect mglA transcription have lower levels of the MglA protein. Since lacZ expression of transcriptional fusions of A- or S-motility genes with Tn5 lac was not affected in mgl strains, it is unlikely that the mgl gene products are involved in regulating the transcription of A- or S- motility genes (MacNeil et al., 1994). Motility of M. xanthus cells is dependent upon cell-cell interactions since increasing cell density also increases motility (Kaiser & Crosby, 1983). Further evidence that cell-cell interactions are essential for motility is that cells with defects in some motility genes can be transiently induced to be motile by placing them in contact with wild-type cells (Kaiser & Crosby, 1983). Social development. When Myxococcus cells are starved at a sufficiently high density on a solid surface they initiate a developmental process. Early in development cells move synchronously, arranging themselves in a series of equidistant ridges separated by regions devoid of cells. At this stage, cells move in a pulsating manner and are said to be 6 rippling since the ridges resemble ripples on the surface of water (Shimkets, 1990). Sager and Kaiser (Sager & Kaiser, 1993a) have proposed that there is no net movement of cells during rippling. They suggest that the appearance of propagating waves is due to the back-and-forth movement of cells as they reverse the direction of their gliding upon end-to-end contact with cells in adjacent ridges. The second morphological feature of development is the movement of cells into aggregation centers where they form structures known as fruiting bodies. Fruiting bodies consist of about 105 cells and within a fruiting body some of the cells lyse, whereas a small proportion of cells forms spherical spores, known as myxospores, which are resistant to harsh environmental conditions such as freezing, heat, dessication and UV irradiation (Shimkets, 1990). The morphological changes that occur during development are also accompanied by the coordinated expression of many developmentally regulated genes (Kuspa et al., 1986) and the synthesis of new proteins. At least 15 new proteins that appear as bands on protein gels were observed in extracts prepared from developing cells (Inouye et al., 1979b). Among the most abundant proteins shown to be expressed during development are the spore coat proteins S (found on the outer surface of spores) (Inouye et al., 1979a) and S-l (found inside spores) (Teintze et al., 1985), which are the products of the lps and ops genes respectively. These proteins are 90% identical at the DNA sequence level (Inouye et al., 1983a; Inouye et al., 1983b), but are expressed at different times during development. The tps gene is expressed starting at about 5 hours into development, whereas the ops gene is expressed at about 24 hours into development (Inouye et al., 1983a; Inouye et al., 1983b; Downard & Zusman, 1985; Downard et al., 1984). Other proteins that are expressed during development include myxobacterial hemagglutanin (encoded by mbhA ), which is expressed 10 hours into development (Romeo & Zusman, 1991), protein C, a spore coat protein that is expressed early in development (McCleary et al., 1991), and phosphatases (Weinberg & Zusman, 1990). 7 Multicellular development in M. xanthus is coordinated by at least five extracellular signals known as the A-, B-, C-, D—, and E- signals. These extracellular signaling systems were identified by isolating conditional nonsporulatin g mutants that fail to undergo development (Hagen et al., 1978). When allowed to co-develop with wild-type cells, these mutants develop normally, indicating that they are defective in producing but not perceiving extracellular developmental signals. In a similar extracellular complementation experiment, development of these mutants was rescued when mixed in certain pair-wise combinations. These complementation experiments identified four classes of cell-cell signaling interactions (the A-, B-, C- and D-signaling interactions), and more recently a fifth signaling class, E-signaling, has been identified (Downard et al., 1993). Kroos et. a1. (Kroos & Kaiser, 1987; Kroos et al., 1986) demonstrated that extracellular signaling interactions regulate the expression of developmental genes using a Tn5 lac promoter probe to generate transcriptional fusions of lacZ to developmentally regulated genes and assaying for the production of developmental ligaIactosidase in cells that were defective in an extracellular signaling interaction. These and later studies showed that genes expressed during the early hours of development require the A- and B- signaling interactions but do not require the C- and D- signaling interactions (Figure 1.1). Genes that are expressed starting at about four hours into deve10pment require the D-signaling interaction in addition to the earlier signal, and genes that are expressed after six hours require have an additional requirement for the C-signaling interaction. The requirement for the E-signaling interaction also appears to be early in development since the expression of rps (which is expressed at about 5 hours) is reduced in E-signaling deficient cells (Downard et al., 1993). These analyses suggested that the extracellular signaling interactions lie on the same developmental pathway, but also showed that genes within a given temporal class can exhibit different levels of dependence on a signaling event. 8 Figure 1.1 Dependence of developmental markers on A, B, C, and D, factors. Each marker is placed above an arrow positioned along the x axis according to the time at which its expression normally begins during development. Position along the y axis indicates the marker’s dependence on cell-cell interactions, as determined by measuring its expression in asg, bsg, csa, and dsg mutants. For example, a marker whose expression is reduced in an asg mutant exhibits partial dependence on A factor (abbreviated “partial A”). Similarly, a marker whose expression whose expression is abolished in a bsg mutant exhibits absolute dependence on B factor (abbreviated “absolute” B). Dependences are cumulative, so a higher position along the y axis indicates greater dependence on cell-cell interactions. Superecript a, d, c, or (1 means that the marker has not been tested in asg, bsg, csg, or dsg mutant cells, respectively. Tn5 Iac insertions are indicated by Q followed by a four digit number, and brackets connect insertions that probably lie in the same transcription unit. Reprinted with permission from authors. Kaiser, D. and Kroos, L. (1993) In Intercellular signalling. (Dworkin, M. and Kaiser, D., eds), 257-283, American Society for Microbiology. oesoe ouee‘ were start. an Aesowre o '” ouor . . . Eouer ' ' nus: Canoe :°'°‘ ouee "‘ ouoe ‘ one: 0415:! AISOLUTE C fi' 0453“" oeeee "‘ Conn" auoo ‘ on r e ouu‘ J AISOLU'I'E e and Aesow'r: o . ‘ 0.4 ellteloe . 04427 oesta one: "neonate“ pAanAL c " ‘1 PARTIAL O on“. oeaer 0442;; case ‘°‘°‘ nus our 1 ”mm 04521 , ems ‘ mm“... Assault! A . Pm o “r eAln'IAt. A emu. c ' auee PARTIAL o eegA' 0445. once um I protein c ABSOLUTE A PARTIAL o are . paeepueteee PAn'I'IAL A «A4 I I r r r r r 1 “-4—; e z 4 e e to r: 14 re 1e 28 17 H00” 0' WENT 10 A-signaling. A-signaling is required early in development. A-signaling mutants fail to form compact aggregates, are defective in sporulation and fail to express genes that are expressed early during development (Hagen et al., 1978; LaRossa et al., 1983; Kuspa et al., 1986). Mutations affecting A-signaling map to three genetic loci, asgA, asgB, and asgC (Kuspa & Kaiser, 1989). These loci have been cloned and sequenced (Plamann et al., 1995; Plamann et al., 1994; Davis et al., 1995). The deduced amino acid sequence of asgA shows a high degree of similarity to proteins of the two-component signal transduction systems. These systems are widely used in prokaryotes to regulate cellular responses to environmental changes (Parkinson, 1993). They regulate a variety of responses including nitrogen regulation, nitrogen fixation, chemotaxis, sporulation, transformation, competence, pathogenicity, gliding and flagellar motility, membrane transport and intermediary metabolism (Stock et al., 1990). A two-component regulatory system typically consists of a sensor protein (a histidine kinase) and response regulator protein. The sensor has a C-terminal transmitter domain coupled to an N-terminal input domain, whereas the response regulator typically consists of an N- terminal receiver module coupled to one or more C—terminal output domains (Parkinson, 1993). In most cases, the sensor protein is localized in the cytoplasmic membrane, and environmental stimuli detected by the input domain cause autophosphorylation at a histidine residue in the transmitter domain. The activated sensor protein then phosphorylates the response regulator at its receiver domain (typically an aspartate residue). The activated response regulator mediates the response (e. g., controls expression of one or more genes via its DNA-binding activity). The AsgA protein contains two domains that have a high degree of amino acid identity to the transmitter domain of the sensor protein and the receiver domain of the response regulator (Plamann et al., 1995). This configuration is characteristic of a group of the two-component signal transduction proteins such as VirA and FrzE, which contain 11 both the sensor and response regulator in a single protein (Stock et al., 1990). Unlike these proteins, however, neither the sensor nor the response regulator of AsgA contain the input or output domains. Thus, it has been proposed (Plamann et al., 1995) that AsgA may function in a phosphorelay-type signal transduction mechanism similar to that used in the early stages of development in Bacillus subtilis. The deduced protein product of the asgB gene also appears to be a regulatory protein based on the presence of a helix-turn-helix motif near the C-terminus, and its high degree of amino acid similarity to region 4.2 of major sigma factors (Plamann et al., 1994) which is involved in recognizing the -35 sequence of promoters. It has, therefore, been hypothesized that AsgB may recognize sequences similar to the -35 hexamer, TTGACA, and that it may also be a component of a phosphorelay system that regulates gene(s) involved in A-signal production. The asgC mutation localizes to the gene encoding the putative major vegetative sigma factor of M. xanthus, rpoD (Davis et al., 1995), which may also be involved in the expression of genes required for A-factor production. Upstream of moD, other genes encoding proteins involved in the initiation of DNA synthesis and protein synthesis were identified (Davis et al., 1995). These genes, which include dnaG (encoding DNA primase) and rpsU (encoding ribosomal protein S21), have been proposed to make up the macromolecular synthesis operon that is conserved among many different groups of bacteria . The A-factor itself has been purified from media conditioned by developing cells by monitoring the expression of an early expressed Tn5 lac fusion marker, Q4521 , as an assay to identify fractions containing A-factor activity (Kaplan et al., 1991). Since Q4521 is the earliest expressed Tn5 lac fusion that is dependent on A-signaling, it provides a useful assay for the earliest appearance of A—factor. The A—factor isolated from conditioned media consisted of amino acids, peptides, and proteases (Plamann et al., 1992; Kuspa et 12 al., 1992b). Since single amino acids could rescue normal aggregation and sperulation of A-signaling mutant cells to wild-type levels, it is thought that the A-signal consists primarily of amino acids, peptides and proteases that are connected by a pathway in which proteases degrade proteins into peptides and finally into amino acids (Kuspa et al., 1992b). Based on the observation of Kuspa et al. (1992a) that a threshold level of amino acids was required to rescue deVelopment, it was suggested that the concentration of extracellular amino acids (which increases proportionally to cell density) may serve as a mechanism for monitoring the density of starving cells. This model is supported by the finding that wild-type cells fail to develop when their density falls below the predicted density required for the production of the threshold level of A factor. Development of wild-type cells at this low density is rescued by the addition of amino acids or peptides (Kuspa et al., 1992b). Other components of the A-si gnalin g pathway were identified by isolating second- site suppressors of asgB that restore expression of an A-signal dependent gene (Kaplan et al., 1991). The sasA (suppressor of mg) gene product appears to be a member of the ATP-binding cassette (ABC) family of transport proteins (Guo et al., 1995), and the sasB gene product is similar to the histidine kinase family of proteins (Yang & Kaplan, 1995). The emerging model for the A-signaling pathway is that starvation is sensed by Asg proteins (such as AsgA, AsgB and AsgC) which induce the expression of genes involved in A factor production and secretion. When cells reach the appropriate cell density, monitored by the concentration of A-factor, cells proceed to the next stage of development. Similar observations have been made in B. subtilis where the initiation of starvation is at least partially dependent on an extracellular peptide or peptides (known as the extracellular differentiation factor, EDF-A) (Kroos & Cutting, 1994). EDF-A is sensed by an ATP-binding cassette (ABC) transport protein and is used to measure cell density that functions in the decision whether to initiate endosporulation (Grossman & Losick, 1988; 13 Rudner et al., 1991). Perhaps M. xanthus SasA senses A-factor and functions in a similar manner. In Streptomyces coelicolor an extracellular peptide, SapB, was shown to serve as an extracellular signal during development (Willey et al., 1993). The mating behavior of Enterococcusfacealis (Clewell & Weaver, 1989; Dunny, 1990) and of Saccharomyces cerevisiae (Herskowitz, 1989) depend on extracellular peptide signals. In addition to the high cell density requirement for the initiation of the developmental process, cells must also be starved. The initial nutrient depletion sensed by the Asg proteins has been proposed to affect intracellular levels of (p)ppGpp. M. Singer and D. Kaiser (Singer & Kaiser, 1995) suggest that M. xanthus responds to starvation by integrating two starvation signals, intracellular (p)ppGpp concentration and extracellular A- signal concentration. These two signals may initiate and coordinately control entry into the developmental process. According to this model, nutritional limitations induce (intracellular) (p)ppGpp production. Theintracellular (p)ppGpp regulates a number of cellular functions, including the expression of starvation dependent genes such as genes involved in the production of A-signal. The accumulation of the A-factor serves as a population starvation signal. Thus, when the threshold level of A-signal is attained, cells initiate entry into development. This model integrates the sensing of starvation in single cells with the coordination of the response to starvation within the population of cells. B-signaling. Another signaling system required for development is B-signaling, which is required within two hours after the onset of development. B-signaling defective cells fail to form fruiting bodies and spores and do not express early developmental genes. Some B-signaling mutations map to the bsgA gene (Gill et al., 1988a). The bsgA gene product, BsgA, is an ATP-dependent cytoplasmic protease with a high degree of amino acid similarity to the Lon protease of E. coli and Bacillus brevis (Gill et al., 1993). Thus the role of BsgA in development may be to modulate the half-life of protein(s) involved in producing the B-signal. The activity of some regulatory proteins is modulated by 14 proteolysis. The CII protein of bacteriophage it is a well known example. Proteolytic cleavage inactivates the LexA repressor, causing induction of the SOS response in E. coli (Ptashne, 1987). In B. subtilis, activation of the development-specific sigma factors OE and 0K requires the proteolytic cleavage of precursor proteins. The proteolytic activity of BsgA may affect a regulatory protein and/or the proteins of the B—factor. However, the nature of the B-signal is not yet known. D-signaling. D-signaling mutant (dsg) cells arrest development after about four hours. These mutants fail to complete aggregation, are defective in sporulation, and fail to express Tn5 lac fusions whose expression normally begins about 5 to 6 hours into development. dsg cells are also defective in their vegetative colony morphology due to defects in motility but their vegetative growth is unaffected (Cheng & Kaiser, 1989b; Kalman et al., 1994). The two dsg point mutations identified map to the same locus, dsgA (Cheng & Kaiser, 1989a). Sequence analysis showed that the dsgA gene product is very similar to translation initiation factor IF3 of E. coli and Bacillus stearothermophilus (Chen g & Kaiser, 1989b), suggesting that dsgA mutant cells are defective in the translation of a gene or genes required for D-factor production. Although it is not known what the D-factor itself is, it has been proposed to be a mixture of fatty acids since fatty acids rescue the development of dsgA mutant cells (Rosenbluh & Rosenberg, 1989). An alternative explanation for this rescue is that fatty acids may be increasing membrane permeability and facilitating the transfer of other molecule(s) that are the D-signal. In addition to the cellular requirement for DsgA during development, dsgA is also essential for cell viability (Cheng & Kaiser, 1989b). IF3 is normally required for the initiation of translation, and since there is only one copy of dsgA in M. xanthus, a null mutant cannot be obtained. The role of IF3 during translation initiation is in the selection of the AUG, GUG, or UUG translation initiation codons. The translation initiation codon 15 for the gene encoding IF3 is AUU, and it has been suggested to be involved in autoregulation (Butler et al., 1987; Gold et al., 1984). A low level of IF3 in the cell causes less stringent selection of the translation initiation codon by the 308 ribosomal subunit, and translation initiation can occur at rare codons, such as AUU. This allows translation of the IF3 gene when IF3 is in low abundance; and when the level of 1133 is high, selection of the initiation codon is stringent and translation is prevented from the rare AUU initiation codon. It has been hypothesized that in M. xanthus, the dsgA mutant gene may encode an IF3 that is more stringent in its selection of a translation initiation codon. Thus, the mutant IF3 does not recognize rare translation initiation codons, and would fail to translate some vegetative as well as developmental genes. E-signaling. Cells defective in E-signaling also fail to form fruiting bodies and spores. Expression of an early developmental gene (tps) is reduced in these cells, indicating that E—signaling affects early events. The mutation affecting E-signaling maps to the esg locus (Downard et al., 1993). The esg locus consists of two ORFs with similarities to the a and b subunits of the E1 component of the branched-chain keto acid dehydrogenase (BCKAD), a multienzyme complex (T oal et al., 1995). BCKAD is involved in the synthesis of branched-chain fatty acids (BCFAs) from branched chain amino acids (BCAAs). During vegetative growth, BCFAs are synthesized and incorporated into membrane phospholipids. However, since the esg mutation reduces but does not abolish synthesis of these fatty acids, alternative routes must exist for their synthesis. Furthermore, esg cells grown vegetatively in the presence of short-chain fatty acids are rescued for development, indicating that BCFAs synthesized during vegetative growth are used later in cell-cell signaling . Thus, one or more of the fatty acids may be the E-signal. Alternatively, these fatty acids may function less directly in E-signaling, for example, they may modify the activity of the protein that is transmitted between cells. 16 C-signaling. C-signaling is one of the best characterized signaling systems in M. xanthus. All of the mutations affecting C-si gnalin g map to a single gene known as csg/1 (Shimkets & Asher, 1988) (Shimkets et al., 1983). C-signaling mutant cells initiate . aggregation but fail to form tight mounds and do not sporulate. Unlike wild-type cells which form compact translucent mounds by 12 hours, csgA cells are still in larger, less compact mounds at 18 hours into development (Kim & Kaiser, 1990a). In addition to defecrs in fruiting body formation and sporulation, csg/1 cells fail to form ripples and they have reduced or abolished expression of genes that are normally expressed after six hours into development (Kroos & Kaiser, 1987; Shimkets & Kaiser, 1982b). Defects in C-signaling can be rescued by co-development of csg/1 cells with wild- type cells or with mutant cells in a different signaling group. Additionally, many compounds, including several carbohydrates and amino acids that are biochemical precursors to peptidoglycan such as N-acetylglucosamine, N ~acetylmuramic acid, D- alanine and diaminopimelate can induce csgA cells to sporulate (Shimkets & Kaiser, 1982b; Shimkets, 1990). However, the actual nature of the C-signal has yet to be established. Extensive effort has been devoted to identifying and characterizing the C-signal. Kim and Kaiser showed that a 17 kDa protein tightly associated with cells rescues fruiting body formation and sporulation of csgA cells (Kim & Kaiser, 1990b; Kim & Kaiser, 1990a). In addition to this result, the following observations indicate that C-signaling involves a short-range signal whose transmission is restricted to contiguous cells. First, when csg/1 responder cells were separated from wild-type C—factor donor cells by a 0.45 pm nucleOpore membrane, the csgA cells could not be rescued for development, indicating that the C-factor is not diffusible (Kim & Kaiser, 1990a). Second, extracts made by extensive washing of wild-type cells at various developmental stages also failed to rescue development of csgA cells, whereas extracts made from lysed developing wild-type cells restored both fruiting body formation and sporulation to csg cells (Kim & Kaiser, 1990b). l7 Cloning of the csgA gene and initial sequence analysis suggested that it encodes a 17.7 kDa protein (Shimkets et al., 1983; Hagen & Shimkets, 1990), consistent with the results that a 17 kDa protein purified from developing cells rescues development of csg/1 mutant cells. The N-terminal amino acid sequence of the purified 17 kDa protein was the same as the deduced N-tenninal amino acid sequence of csgA. Moreover, polyclonal antibodies raised against a lacZ-csgA fusion protein reacted with the purified 17 kDa C-factor protein. Thus, it appears that the purified 17 kDa protein is encoded by the csgA gene. Recent studies have shown that the csgA gene producr, ngA, may actually be larger than previously reported. Extending the csgA ORF 5' to another possible translation initiation codon increases the size of the predicted protein to 24 kDa (Lee & Shimkets, 1994). The amino acid sequence of this protein is 19-39 % similar to the sequences of a family of short-chain alcohol dehydrogenases. These alcohol dehydrogenases have a wide variety of substrates and are involved in the production of many different signaling molecules including steroids, prostaglandins, and nodulation factors in plants (Baker, 1994; Persson et al., 1991). In addition to amino acid sequence similarity, several results are consistent with csgA encoding a protein in the alcohol dehydrogenase group. First, one class of csgA suppressors, socA (suppressor of csg), identified an operon that has a gene, socAI, whose deduced amino acid sequence is also similar to the family of short-chain alcohol dehydrogenases. Second, site-directed mutagenesis of the predicted active site of the ngA alcohol dehydrogenase causes a ng- phenotype (Lee et al., 1995a). Third, NAD+, a coenzyme predicted to bind short-chain alcohol dehydrogenases, stimulates a MalE-ngA fusion protein to rescue development of csg/1 cells (Lee et al., 1995a). Thus, ngA may be an enzyme and it may be involved in the production of C-factor rather than being the C-factor itself. Several genes that circumvent the need for the C-signal transduction have been 18 isolated as second-site suppressors of a csgA mutant. The 3001 cistron encodes three proteins (Lee & Shimkets, 1994). In addition to socAI (whose deduced amino acid sequence is similar to alcohol dehydrogenases), the predicted protein product of socA2 has a 48% amino acid sequence identity with the Proteus vulgarisfrd gene product, whose function is to anchor fumarate reductase to the membrane. The third gene, socA3, contains the Tn5 supressor mutation. However, the deduced amino acid sequence of socA3 does not show any similarity with known proteins. It has been proposed that the wild-type socA3 gene product negatively regulates the socA operon (Lee & Shimkets, 1995). Thus, cells containing the socA suppressor mutation are unable to produce SocA3, resulting in overproduction of SocAl. Since the socAI gene product belongs to the same group of alcohol dehydrogenases as ngA, increased production of SocAl is thought to overcome the csg/1 defect (Lee & Shimkets, 1995). However, even though a transposon insertion in socA3 restores sporulation to csgA mutant cells, the spores produced by these double mutants germinate prematurely and are unstable upon prolonged plating on starvation medium. This indicates that suppression is incomplete and may explain why csgA has been retained through evolution. socD500 is a second mutation that suppresses the sporulation defect of csgA cells under normal developmental conditions (low nutrient levels, high cell density and presence of a solid surface at 32°C). socDSOO also allows sporulation of csg+ or csgA cells at 15'C in the presence of nutrients (Rhie & Shimkets, 1991). In addition to these phenotypic changes, at 15'C socDSOO activates expression of tps, whose expression precedes that of csgA, and of ops, whose expression is after that of csg/1. This indicates that both early and late gene expression are affected by socDSOO. Furthermore, socD500 restores sporulation to csgA cells in the absence of nutrients without restoring aggregation, suggesting that the socDSOO mutation may be in a component of the nutrition sensory mechanism that has a regulatory function at several points in the developmental pathway. 19 The socDSOO gene encodes a 61.6 kDa histidine protein kinase (Lee et al., 1995b), and the mutations in socD that lead to the suppression of csgA map to the N-terminal sensor domain of the protein. It has thus been hypothesized that an input signal binds 800D and perpetuates growth, and mutations in this regulatory protein trigger sporulation. Consistent with this model, null mutations in socDSOO could not be obtained (Lee et al., 1995b) suggesting that SocD is required for growth. Different concentrations of purified ngA trigger distinct morphogenetic and transcriptional responses (Kim & Kaiser, 1991). When purified 17 kDa polypeptide was added to starving csg/1 cells at a concentration of 0.8 nM, aggregation and the expression of a lacZ fusion that is normally expressed earlier in development and is only partially dependent on C-signaling was rescued. At a concentration of 1 nM or more, the purified 17 kDa polypeptide rescued aggregation, sporulation and the expression of a lacZ fusion that is normally expressed late in development and has an absolute requirement for C- signaling (Kim & Kaiser, 1990a). This indicates that M. xanthus cells can discern and respond to small changes in C-factor concentrations. Similarly, Li et al. showed that increased expression of csgA in vivo over the course of development entrains the natural sequence of morphogenetic changes during development (Li et al., 1992). The regulation of the csg/l gene itself appears to be complex. During growth, lacZ expression in csg cells containing a csgA-lacZ transcriptional fusion is low, suggesting that csgA is expressed at very low levels in vegetatively growing cells (Kim & Kaiser, 1991). As cells enter the developmental stage, lacZ expression increases by about two-fold. When starving csgA cells (containing the csgA-lacZ fusion) are mixed with either an equal number of wild-type cells or with 1 nM of purified C-factor, lacZ expression increases by about four-fold. This suggests that the initial increase in C-factor production triggers the positive regulation of ngA synthesis and the aggregation of cells. As cells progress through development, aggregation leads to an increase in local C-factor concentration due 20 to the increase in cell density and increased efficiency of C-factor transmission (see below). At about 18-20 hours spore formation is initiated and the level of ngA decreases due to lysis of cells. Based on the autoregulation of csgA and the requirement for a threshold level of C- factor for the induction of the different developmental stages, Kim and Kaiser (Kim & Kaiser, 1991) proposed that the C-factor may be functioning as both a morphogenetic trigger and as a developmental timer whose increasing concentration during development triggers different morphogenetic and transcriptional changes. According to this model, low levels of C-factor, present early in development, are sufficient for the initiation of aggregation and the expression of early developmental genes. As cells aggregate, the local concentration of C-factor increases, inducing the production of a higher level of C-signal and, subsequently, inducing sporulation and late gene expression. As a morphogenetic signal, C-signal appears to allow cells to sense when they have moved into a fruiting body and then triggers their differentiation into spores. An interesting aspect of M. xanthus development is its requirement for motility. Nonmotile mutant cells (AS- or mgI-) have similar defects in development as csgA null mutants; they fail to complete aggregation, they do not sporulate and do not fully express developmental markers that are normally turned on between five and nine hours into development (Kroos et al., 1988). This suggests that nonmotile cells may be defective in the same signaling interactions as csg/1 mutant cells (Kim & Kaiser, 1990a). Unlike csg/1 mutants, however, nonmotile mutants are proficient in the production of C-signal. Development of csgA cells was restored by adding cell-free extracts of developing mgl/1 cells, demonstrating that mglA cells produce the C-signal. Addition of purified ngA to non-motile cells restores sporulation and expression of developmentally regulated lacZ fusions, demonstrating that mglA cells can perceive the C-signal (Kim & Kaiser, 1990a). On the other hand, aggregation was not restored, indicating that the motility defect was not 21 overcome by the addition of purified ngA. Mixing mglA cells(which do produce ngA) either with wild-type cells or with csg/1 cells does not restore sporulation or expression of developmental lacZ fusions in mglA cells to wild-type levels (Kim & Kaiser, 1990a), supporting a model in which both donor and responder cell motility is required for C-signal transmission. The role of motility in development appears to be to allow cells to aggregate and increase the local cell density since increasing the initial cell density of starving mglA cells increases sporulation efficiency (Kroos et al., 1988). However, even a ten-fold increase in the initial density of the mglA cells over the concentration of wild-type cells that is normally required for development increased the sporulation efficiency to only 1 % of the wild-type levels (and the expression of developmental lacZ fusions did not increase above background levels). These results indicate that a mere increase in cell density is not sufficient for restoring development in mglA cells. Kim and Kaiser therefore suggested that C-factor transmission may require a critical spatial orientation of cells which is acheived after cells move into the dense, highly aligned organization (of cells) in a nascent fruiting body. Indeed, scanning electron microscopic observations show that rod-shaped cells within a fruiting body are densely aligned in parallel rows (Kim & Kaiser, 1990a; O'Connor & Zusman, 1989). The movement of cells is thought, therefore, to align cells in a pattern that greatly increases the local cell density and efficiency of C-factor transmission. In support of this model, manipulating the orientation of nonmotile cells on the surface of starvation agar so that the cells are aligned in parallel rescues the developmental defect, and the expression of late developmental Tn5 lac fusions (Kim & Kaiser, 1990a). Similar alignment of csgA cells rescues neither development nor the expression of late developmental markers, supporting the idea that motility is required to mediate C-signaling. C-signaling in M. xanthus has characteristics that are similar to signaling processes 22 in eukaryotes. The most striking similarity is with CAMP signaling during the starvation- induced development of Dictyostelium discoideum. D. discoideum is a common soil protozoan that grows unicellularly in the presence of nutrients, but undergoes multicellular development when nutrients are depleted (Kaiser, 1986). Development in D. discoideum entails aggregation of cells into a mass of about 105 cells, forming a nascent structure known as a slug. The cells that form the slug are motile, and later in development the slug migrates and forms a fruiting body consisting of two types of differentiated cells. The stalk consists of cells which secrete cellulose and eventually mature into nonviable stalk cells containing vacuoles and thick cellulose walls. Atop the stalk, the majority of cells differentiate into dormant spores which initiate vegetative growth when nutrients are available. Initiation of development in Dictyostelium requires signaling by CAMP. Upon starvation adenylate cyclase activity is induced, increasing levels of both intracellular and extracellular CAMP. Production of CAMP is autoregulatory, with the binding of extracellular CAMP to the cAMP receptor further inducing the cellular adenylate cyclase activity. The CAMP positive feedback loop is, however, tempered by an adaptive chemosensory system that is responsive to the level of receptor-bound CAMP. In addition to adaptation, the CAMP level is also reduced by phosphodiesterase activity. Together, the adenylate cyclase feedback loop, adaptation and phosphodiesterase activity cause oscillations in the levels of CAMP. These oscillations coordinate movement of cells in a way that resembles the C-signal-dependent rippling observed in the early stages of M. xar'tthus deve10pment. Clearly, despite their evolutionary distance, interesting parallels can be drawn between the developmental processes of Myxococcus and Dictyostelium. In both cases starvation induces intercellular signaling (C-signaling in M. xanthus and CAMP signaling in D. discoidewn), directed cell movement into aggregates, and differentiation of vegetative 23 cells into environmentally resistant spores. Directed cell movement and the positive feedback loop of cAMP production in Dictyostelium are analogous to the C-signal mediated movement of cells into aggregates and the autoregulation of of C-factor production (Kim, 1991). This suggests that elucidation of the mechanisms of multicellular development that occurs in Myxococcus may contribute to our understanding of developmental processes in other multicellular organisms. The organization of M. xanthus cells within a fuiting body also offers a simple model system for studying pattern formation and spatial regulation of gene expression. The nascent fruiting body of M. xanthus consists of an outer hemispherical domain of highly organized, densely packed, motile, rod-shaped cells that are aligned in parallel rows (Sager & Kaiser, 1993b; O'Connor & Zusman, 1989; Kaiser et al., 1985). Cells within this outer domain move in a bidirectional stream orbiting the inner domain which consists of relatively less ordered, loosely packed nonsporulatin g cells (Sager & Kaiser, 1993b). Because spores are nonmotile, they are passively transported towards the center of the fruiting body by the driving force of the rod-shaped cell that move in circular orbits within the outer domain (Sager & Kaiser, 1993a). Since cells in the outer domain of fruiting bodies are at high cell density, ordered and motile, C-signaling is most efficient in this domain. Differentiation and expression of C-signal-dependent lacZ fusions, therefore, initiates in the outer domain (Sager & Kaiser, 1993a). As the undifferentiated rod-shaped cells continue to circle the inner domain, and proceed through development, the differentiating spores lose their motility and accumulate in the inner domain. Accordingly, expression of developmentally regulated lacZ fusions is higher in the inner domain as these cells have been expressing the fusion for a longer time. Thus, the short-range, spatially restricted, C-signaling system that regulates both the spatial and temporal expression of developmental genes in M. xanthus provides a good model system for studying the mechanism by which short-range signals are used in 24 differentiation of a homogeneous population of cells. Perhaps the best understood casein which the importance of short-range signaling in regulating differentiation has been demonstrated is vulval development of C aenorhabditis elegans. In addition to long-range diffusible signals, vulval precursor cells respond to short-range signals that determine their fates. These signals are responsible for the formation of precise spatial patterns of three cell types that develop into the vulva (Stemberg & Horvitz, 1989). Thus, an understanding of the mechanims by which C-signaling regulates differentiation in a fruiting body may shed new light on the mechanisms involved in pattern formation in eukaryotes. Additionally, short-range signaling is also important in the interaction between plant and animal hosts and microbes. These species-specific interactions establish the outcome of the interactions as beneficial or detrimental (Clark et al., 1992). The long-term goal of our studies is to unravel the molecular mechanism(s) by which extracellular C-signalin g regulates gene expression during starvation-induced development in M. xanthus. M. xanthus has a simple genetic system which is amenable to manipulation, and many genetic tools are available that facilitate these studies. Biochemical studies are possible as large quantities of cells can be grown and developed synchronously for isolation of proteins. Thus, M. xanthus provides a simple and tractable multicellular system for studying the mechanism by which intercellular signaling regulates gene expression. Regulation of gene expression in response to environmental cues is mediated by a variety of mechanisms in prokaryotes. Typically, regulation occurs at the transcriptional level. Many bacteria possess multiple sigma subunits of RNA polymerase and use alternative forms of RNA polymerase holoenzyme to express different sets of genes in response to a wide variety of environmental signals, including heat and cold shock, osmotic pressure, nitrogen limitation and starvation. A well studied system in which alternative 0 factors have been shown to regulate gene expression is the sporulation process 25 of B. subtilis (Haldenwang, 1995). Starvation-induced sporulation in B. subtilis involves transcription of developmentally regulated sets of genes. A cascade of o—factors, that are activated at different stages during development at the transcriptional as well as post-transcriptional levels, transcribe genes in a spatially and temporally regulated manner. At least two post-transcriptional activation mechanisms activate sigma factors in B. subtilis; proteolytic removal ofenzy inhibitory sequences at the N-terminus (the pro-region) and competition for binding of inhibitory proteins (anti-sigma factors). Thus, expression of sporulation genes is fine-tuned at different levels by regulating the expression as well as the activation of regulatory proteins. Multiple sigma factors have also been identified in M. xanthus (Inouye, 1990; Apelian & Inouye, 1990; Apelian & Inouye, 1993). The sigA gene product, 0", is thought to be the major sigma factor in vegetatively growing cells and has amino acid sequence similarity to 07° of E. coli and 0'43 of B. subtilis (Inouye, 1990). At least three sigma factors are produced during development, O'C’ the sigC gene product, is expressed early in development (Apelian & Inouye, 1993), and 03, the sigB gene product, is expressed late in development (Apelian & Inouye, 1990). Furthermore, there is evidence for a third developmental sigma factor, which has similarity to the 054 family, and appears to be involved in early developmental gene expression (Keseler & Kaiser, 1995). Thus gene expression during development in M. xanthus also entails the use of alternative sigma factors for differential gene expression. The regulation of many bacterial genes is mediated by transcriptional activator proteins. In genes transcribed by 054 RNA polymerase, activator proteins bind upstream of the promoter and activate transcription by catalyzing the formation of a tertiary complex. In some cases, these activation sequences are far removed from the promoter, and DNA 26 looping mediates their interaction with the promoter. Several M. xanthus genes have been identified that appear to be transcribed by 654-type RNA polymerase and which require upstream activation sequences. The mbhA gene, which is expressed early in development and encodes myxobacterial hemagglutinin, is transcribed from a 054-type promoter and requires cisacting sequences between 86 and 276 bp upstream of the promoter (Romeo & Zusman, 1991). Expression of the developmentally regulated Tn5 lac Q4521 fusion also appears to be from a 054-type promoter, and requires sequences upstream for transcription activation. Other genes are also present in M. xanthus that require upstream activation sequences for transcription. A DNA segment located between 131 and 311 bp upstream of the ops transcriptional start site activate not only ops transcription but also transcription of tps promoter which is located about 2 kb downstream of ops (Kil et al., 1990). The activation of tps by this DNA was orientation independent, and thus appears to be mediated by sequences that function much like eukaryotic enhancers. The ops UAS segment has also been shown to have a DNA-binding activity (Downard & Kroos, 1993), suggesting that proteins that activate transcription may bind in this region. Expression of tps is also activated by two UASs regions between 82 and 110 and 173 to 375 bp upstream of the its transcriptional start site (Downard & Kroos, 1993). The promoter region of csg/1 also requires a fairly large upstream sequence to modulate its expression in response to nutrient levels, peptidoglycan components and B- signaling (Li et al., 1992). At least 400 bp upstream from the start of transcription initiation were required for maximum expression of csgA. In the presence of low levels of nutrients, sequences extending to about 930 bp were required for the developmentally regulated expression of lacZ. Another widely used mechanism of gene regulation in prokaryotes is the two- 27 component signal transduction system. Chemotaxis in E. coli employs this system with several sensor proteins regulating the rate of swimming and tumbling in the presence of attractants and repellants, and M. xanthus proteins similar to these Chemotaxis proteins have been shown to regulate reversal of gliding movement. Mutations in these regulatory prOteins affect fruiting body formation and sporulation. Similarly, mechanisms that employ transcriptional or post-transcriptional activation of sigma factors or two-component systems may be involved in C-signal-mediated gene expression in M. xanthus. On the other hand, novel mechanisms may be involved in coordinating extracellular signaling with gene expression. Interestingly, eukaryotic-type protein serine/threonine kinases and tyrosine kinase(s) have been identified in M. xanthus (Munoz-Dorado et al., 1991; Frasch & Dworkin, 1995). Deletion of at least three different protein kinases, PKl, PK5, and PK6, (Udo et al., 1995; Zhang et al., 1995) has been demonstrated to affect development. It, therefore, seems possible that signal transduction systems similar to those in eukar'yotes may be utilized by the C-signaling system. Chapter 2 Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus (The work described in this chapter will be submitted for publication with Monica Gloudemans, Ron Gill, and Lee Kroos as second, third, and fourth authors, respectively). 28 29 Abstract Q4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-si gnalin g, are required for expression of Tn5 lac Q4403. We cloned DNA upstream of the Q4403 insertion site, localized the promoter, and identified a potential open reading frame. Based on the deduced amino acid sequence, the gene disrupted by Tn5 lac Q4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or B-galactosidase accumulation in cells containing Tn5 lac Q4403. The putative transcriptional start site was mapped and sequences centered near -10 and -35 show some similarity to these regions of promoters transcribed by E. coli 070 RNA polymerase. However, deletions showed that an essential promoter element lies between ~80 and -72, suggesting the possible involvement of an upstream activator prorein. DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two capies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression. 30 Introduction Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development (Dworkin & Kaiser, 1993). When starved at high cell density on a solid surface, cells move into aggregation centers where they construct a fruiting body comprised of about 105 cells. Within the fruiting body cells differentiate into dormant, ovoid spores. In addition to morphological changes, M. xanthus development involves a highly ordered program of gene expression controlled by cell-cell interactions (Kaiser & Kroos, 1993). At least five cell-cell signals, known as the A-, B-, C-, D- and E- signals, are required for normal development. Cells with mutations in genes needed for any of these cell interactions arrest development at a specific stage, but are rescued when they are co-developed with wild-type cells or with mutant cells from a different signaling group (Downard et al., 1984; Hagen et al., 1978; LaRossa et al., 1983). To study the role of cell-cell interactions in controlling M. xanthus gene expression, Tn5 lac has been used to identify developmentally regulated genes. Tn5 lac is a transposable promoter-probe containing a promoterless lacZ gene inserted near one end (Kroos & Kaiser, 1984). Transposition of Tn5 lac into the M. xanthus chromosome can generate a transcriptional fusion between lacZ and an M. xanthus promoter. Among 2,374 Tn5 lac insertions, Kroos et al. (Kroos et al., 1986) found 29 in distinct transcriptional units whose activity increases during development. Dependence of developmental gene expression on cell-cell interactions was examined by monitoring B-galactosidase expression of the lac fusions in cell-interaction mutants. A- and B-signaling were required for normal gene expression beginning at one to two hours into development (Gill & Cull, 1986; KI'OOS & Kaiser, 1987; Kuspa et al., 1986). D-signaling was first required at about four hours (Cheng & Kaiser, 1989a). C-signaling was required for normal expression of nearly all genes that begin to be expressed after six hours into deve10pment (Kroos & Kaiser, 1987; 31 Li & Shimkets, 1993). It has recently been shown that E-signaling is required early in development, as demonstrated by the lack of expression of an early developmental gene (tps) in an E-signaling mutant (Downard & Kroos, 1993). We are focusing on the regulation of deve10pmental gene expression by C-signaling for several reasons. First, the gene expression studies mentioned above show that the C-signal-mediated cell-cell interaction occurs later in development than the other signaling interactions. Since the the A-, B-, D- and E-signals are all required early in development, it may be difficult to decipher the role of each of these signals in controlling gene expression. In addition, nutritional downshift signals may also control early developmental gene expression. Another reason for focusing on C-signal-dependent gene expression is that the csg group of mutants, which are defective in C—signalin g, are genetically simple and well-characterized. All of the csg mutations map to a single gene, csgA, whose product appears to mediate the cell-cell interaction (Hagen & Shimkets, 1990; Kim & Kaiser, 1990a; Shimkets & Asher, 1988; Shimkets et al., 1983). ngA is associated with the cell surface membrane and/or extracellular matrix (Kim & Kaiser, 1990b; Shimkets & Rafiee, 1990). Upon solubilization and partial purification, ngA at approximately 1 to 2 nM restores normal development to csgA mutant cells (Kim & Kaiser, 1990a). Recent results suggest that ngA may function as an enzyme to generate an unknown signal molecule that is perceived by other cells (Lee et al., 1995b; Lee & Shimkets, 1995). C-signaling is also interesting because it requires cell movement for transmission (Kim & Kaiser, 1990b; Kim & Kaiser, 1990a)(Kroos et al., 1988). During development, cells move into alignment and become densely packed in the outer domain of a nascent fruiting body (Sager & Kaiser, I993b). Sager and Kaiser (Sager & Kaiser, 1993a) have proposed that end-to—end and/or side-to-side contacts between cells in the outer domain permit efficient C-signaling, triggering sporulation. Consistent with this model, patches of spore precursors and expression of several C-signal-dependent lacZ 32 fusions is first observed in the outer domain (Sager & Kaiser, 1993a). Hence, expression of C-signal-dependent genes is regulated spatially, as well as temporally. C-signaling appears to couple morphogenesis of the fruiting body with expression of late genes and differentiation of cells into spores. In addition to coordinating the aggregation and sporulation behavior of M. xanthus cells, C-signaling is required for rippling (Sager & Kaiser, 1994; Shimkets & Kaiser, 1982a). Ripples form early in development when cells organize themselves into ridges that move as traveling waves. A rising level of ngA has been proposed to entrain the developmental sequence of rippling, then aggregation, and finally sporulation (Li & Shimkets, 1988). A higher level of ngA has been shown to be required to activate a later-expressed C-signal-dependent gene than an earlier-expressed gene (Kim & Kaiser, 1991). To begin to understand how C-signaling regulates gene expression during M. xanthus development, we are characterizing the DNA regulatory regions of C-signal-dependent genes. Here, we report the characterization of the regulatory region of Q4403, a gene identified by Tn5 lac insertion (Kroos et al., 1986). Expression from Tn5 lac Q4403 starts at about 11-15 hours into development and has an absolute requirement for C-signaling 0(roos & Kaiser, 1987). We constructed deletions to localize the sequences required for promoter activity, determined the nucleotide sequence of the promoter region, and mapped the transcriptional start site. Promoter activity required sequences farther upstream of the transcriptional start site than is usually required for RNA polymerase binding, suggesting the involvement of an additional regulatory protein(s). Analysis of the minimum promoter region in csgA mutant cells showed that it requires C-signaling for activity. These studies lay the foundation to start identifying regulatory proteins required for C-signal-dependent expression of genes. 33 Materials and Methods Bacterial Strains and plasmids. Strains and plasmids used in this work are listed in Table 2.1. Growth and Development. Escherichia coli cells were grown at 37’C in LB medium containing 50 ug ampicillin or 25 pg kanamycin per ml when necessary. M. xanthus was grown at 32-34’C in C'I'l‘ medium (Hodgkin & Kaiser, 1977) in liquid cultures or on agar plates as described previously (Kroos et al., 1986). Forty ug of kanamycin or 12.5 pg of oxytetracycline per ml were used when required for selective growth. Fruiting body development was performed on TPM (10 mM Tris-HCl, pH 8.0, 1 mM KH2P04, 8 mM MgSO4, final pH 7.6) agar (1.5%) plates as described previously (Kroos et al., 1986). Molecular cloning and construction of plasmids. Recombinant DNA ' work was performed using standard techniques (Sambrook et al., 1989). Plasmid DNA was prepared from E. coli DH5a or J M83. M. xanthus chromosomal DNA used for cloning was prepared as described previously (Laue & Gill, 1994). M. xanthus DNA upstream of Tn5 lac Q4403 was cloned by digesting chromosomal DNA from strain DK4368 with XhoI and ligating into XhoI-digested pGEM-7Zf, resulting in pME8003 (Table 1). Restriction fragments of M. xanthus DNA from pME8003 were gel-purified and ligated into vectors as indicated in Table l. Vectors were digested with the same restriction enzymes used to produce the fragments, except as indicated below. To test M. xanthus DNA fragments for promoter activity, we constructed pREGl666 and its derivative pREG 1727. pREGl666 was derived from pREG1175 (Gill et al., 1988a) as follows. A 3 kbp Smal fragment from phage Mx8 (Stellwag et al., 1985) was ligated into Smal-digested pUCl 8 (Yanisch-Perron et al., 1985), then excised 34 Table 2.] Bacterial Strains and Plasmids Strain or plasmid Relevant characteristics Source or reference E.coli DH5a (p80 AlacZ AM15 AlacUI69 recAI end/11 {Iggghan’ Du hstI7 supE44 thi-I gyrA rel/11 ”“33 ara Alac-pro strA thi ¢d1acz AMIS ll‘ggsgsing’ 1)., M. xanthus DK1622 Wild-type Kaiser, D., 1979 DK4368 Tn5 (ac (Km!) Q4403 Kroos et al., 1986 DK4499 Tn5 lac (Kml’) Q4499 Kroos et al., 1986 JW103,104, 107 attB::pREGl666 Jamie White MMF1727-7, -9, attB::pREGl727 This work -1 1 NLESOOS, 008, atthpMES004 This work and 012 MIWF71-6, - 15, attB::pMF71-l This work and -22 IVIESOB4, 036, atthszESll6 This work 047 NIESOSB, O64, attB::pME8115 This work 077 MIMI-73 1 -3, -7, attB::pMF31 This work and —9 MNIFSl-l, -8, atthszF81 This work and -32 ML/IFlOO-6, -17, attB::pMF100 This work and -24 MNIF72-2, - 10, attB::pMF72 This work and -32 MMF62-4, -34 attB::pMF62 This work Table 2.1 (cont’d) 35 19552-35, -44, attB::pMF52 This work MMF301-3, -4, attB::pMF301 This work I/SIMFZOO-S, -6, atthszFZOO This work rings-314, 016, Q4403::pMESlO8 This work and 017. MSHl Q4403 ::pSH1 Stacie Hill JPB07 Tn5 lac (Tc!) Q4403 Janine Brandner $12-3, 4. Q4403zszK1Tn5 lac (T c1) Q4403 This work DK5203 csgA::Tn5-132 (T or) Q205 Erhcilmkt’vts. L's Asher, 1988 MESl l9 csgA::Tn5-I32 (rcr) (2205 attB::pREGl666 This work #2140006. csgA::Tn5 -132 (Tel) Q205 atthszFIOO This work WOOC-‘l, csgA::Tn5-132 (Tor) Q205 attB::pMP200 This work pUCl 9 Apr lac a 'cr'taarlrlrsclhg-ggrron pom-72f Apr laca “omega 81:1732671666IPRE Apl'KmrPl-inc art)” This work pREG 1 175 Apr M 1:14,“; Gill, R. and Bomemann, M., 1988 pREG429 Apr Kmr pum- Gill et a1. 1988 pME8003 Apr (pGEM-7Zf), M. xanthus Q4403, Kmr This work - (aphll), 16.7 kb XhoI fragment from DK4368 pMESOO4 Apr Kmr Pl-inc attP (pREGl666), 8.5 kb This work X hoI-BamHI fragment from pMES003 Table 2.1 (cont’d) pMESllO pMESllZ pMESl 15 pMES] 16 pMF01 pMF31 PMF3.4 pMF81 pMF072 pMF72 pMF062 pIVfl=62 pMF052 pIVIFS 2 pMF30 1 pMFo3or 36 Apr (pGEM-7ZF) 4.0 kb ClaI-BamHI fragment from pMESOO3 Apr (pUC19), 2.0 kb PstI—BamHI fragment from pMESOO3 Apr Kmr Pl-inc attP (pREGl666), 2.0 kb HindIII—BamHI fragment from pMESl 12 Apr Kmr Pl-inc attP (pREGl666), 4.0 kb XhoI-BamHI fragment from pMES 1 10 Apr (pUC19), 1.0 kb SalI-BamHI fragment from pMESl 12 Apr Kml' Pl-inc attP (pREGl666), 1.0 kb HindIII-BamHI fragment from pMF01 Apr (pUC19), 674 bp Sphl-BamHI from pMF01 Apr Kmr Pl-inc attP (pREGl727), 680 bp HindIII-BamHI from pMF3.4 Apr (pGEM-7ZF), M. xanthus 513 bp XhoI- BamHI generated by PCR Apr Kml‘ Pl-inc attP (pREGl727), XhoI- BamHI fragment from pMF7 2 Apr (pGEM-7ZF), M. xanthus 503 bp XhoI- BamHI generated by PCR Apr Kmr Pl-inc attP (pREGl727), Xhol- BamI-II fragment from pMF0062 Apr (pGEM-7ZF), M. xanthus 493 bp Xhol- BamI-II generated by PCR Apr Kmr Pl—inc attP (pREGl727), XhoI- BamHI fragment from pMF0052 ‘ Apr Kmr Pl-inc attP (pREGl727), M. xanthus 483 bp XhoI-BamHI generated by PCR Apr (pGEM-7ZF), M. xanthus 483 bp X hol- BamHI from pMF301 This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work Table 2.1 (cont’d) pMFZOO pMFlOO pMFOIOO pMES 108 pSHl pLKl pMF71-1 37 Apr Kmr Pl-inc attP (pREGl727), 452 bp This work RsaI-BamI-II fragment from pMF01 Apr Kmr Pl-inc attP (pREGl727), 521 bp This work I-IaeII-BamHI fragment from pMF01 Apr (pGEM-7ZF), 521 bp XhoI-BamHI This work fragment from pMF 100 Apr Kmr Pl-inc (pREG1175), 8.5 kb XhoI- This work BamHI fragment from pMESOO3 Apr Kmr Pl-inc (pREG429), 4 kb ClaI-BamHI Stacie Hill fragment from pMES l 10 Apr Kmr Pl-inc (pREG429), M. xamhus 17 kb This work EcoRI-BamI-II fragment from MSHl Apr Kmr Pl-inc attP (pREGl666), 17 kb This work BamHI-EcoRI from pLKl 38 as an EcoRI-BamHl fragment and ligated into EcoRI-Baml-II-digested pREGl 175. The resulting plasmid was digested with BamHI and a 71 1 bp Sau3AI fragment from pR8577 (Sirnons etal., 1987) containing 4 copies of the T1 terminator of the E. coli rrnB operon was inserted. The Sau3Al end upstream of the terminators does not generate a BamHI site, but the Sau3AI end downstream does, upon insertion into BamHI-digested vector. A plasmid in which the unique remaining BamHI site was proximal to lacZ was digested with BamHI and a 49 bp BglIl-BamHI fragment containing multiple restriction sites was inserted. pREGl666 has the polylinker in the orientation that leaves the unique remaining BamHI site proximal to lacZ. The polylinker fragment was generated by deleting the Bng site in the polylinker of pSP7 2 (Promega), digesting the resulting plasmid with XhoI and filling-in the ends with the Klenow fragment of DNA polymerase I, ligating on Bng linkers (5’-GAAGATCTI‘C—3’), and digesting with Bglll and Baml-Il. pREGl666 was used initially to test several M. xanthus fragments for promoter activity upon integration at the Mx8 phage attachment site (Mx8 art). To identify M. xanthus suains containing a single copy of the plasmid integrated at Mx8 art, the 3 kbp EcoRI-BamHI fragment from pREGl666 containing Mx8 attP was labeled and used to probe Southern blots of EcoRI-XhoI-digested chromosomal DNA as described below. The desired strains show fragments of 2 and 10 kbp in length that hybridize the probe and fail to show a 4 kbp fragment corresponding to unrecombined attP. Occasionally, a fragment slightly less than 2 kbp was detected, rather than the expected 2 kbp fragment. We examined the structure of pREGl666 and found that the terminator-containin g fragment was slightly larger than expected. We do not know the source of additional DNA. pREG 1727 is identical to pREGl666, except the terminator-containing fragment is the expected size. pREGl727 was used subsequently to test M. xanthus fragments for promoter activity, but this did not solve the problem of occasional integrants (about 15%) showing a fragment slightly smaller than 2 kbp upon Southern blotting. We suspect that recombination within the 39 tenninator-containing fragment accounts for this observation. We avoided integrants showing a fragment smaller than 2 kbp. In addition, we compared lacZ expression from pREGl666or pREGl727 with no insert of M. xanthus DNA or with the 1.0 kbp Q4403 upstream region inserted, and detected no significant difference between the two vectors. pMF3.4 was constructed by digesting pMF01 with Sphl, which cuts once in the 1.0 kb M. xanthus insert and once in the multiple cloning site of pUC19 near the end of the insert more distal from the Q4403 insertion site, releasing a fragment of about 400 bp. The 3.4 kb vector-containing fragment was gel-purified and ligated, generating pMF3.4. To construct pMF 100, the insert fragment was generated by digesting pMF01 with HaeII and making these ends blunt by digesting with mun g-bean nuclease (Sambrook et al., 1989). After phenol-chloroform extraction and ethanol precipitation the DNA was digested with BamHI and fractionated on an agarose gel. The supposed 523 bp fragment was purified and directionally cloned into pREGl727 that had been digested with HindIII, subjected to end-filling using the Klenow fragment of DNA polymerase I (Sambrook et al., 1989), digested with BamHI and gel-purified. The HindIII-HaeII junction in pMF100 was sequenced by first subcloning the insert as a X hoI-BamI-II fragment into XhoI-BamHI-digested pGEM-7Zf, generating pMFOIOO. The M13 pUC forward sequencing primer (United States Biochemical) was used to sequence this junction as descibed below. The sequence showed that mung-bean nuclease had over-digested the HaeII end by two base pairs. Thus, pMFlOO contains a 521 bp insert rather than the 523 bp insert predicted by the location of the HaeII restriction site. pMF200 was constructed by directional cloning of the 452 bp RsaI-BamI-II fragment from pMF01 into pREGl727 that had a filled-in HindIII blunt-end and a BamHI cohesive end (prepared as described above for pMFlOO). prIFO72, 062, and 052 contain PCR-generated fragments of Q4403 upstream DNA. The regions between -72, -62, and -52 (relative to the putative Q4403 4O transcriptional start site) and the BamHI site at the left end of Tn5 lac were amplified using pMF01 as template. For this purpose, the downstream primer was the M13 pUC forward sequencing primer (United States Biochemical) that binds in pUCl9 downstream of the BamHI site, and the upstream primers were 34-mers that bind between 454 and 429, 444 and 419, 434 and 409 bp upstream of the Q4403 insertion site. The 5' ends of the upstream primers had a 9 b sequence containing a X hoI site that does not bind to the template. The amplified fragments were digested with BamHI and XhoI, gel-purified, and ligated into XhoI-BamHI digested pGEM-7Zf, resulting in pMF72, 062, and 052. The inserts in these plasmids were sequenced. pMF301 also contains a PCR-generated fragment of Q4403 upstream DNA. In this case, the upstream primer binds between 424 and 405 bp upstream of the Q4403 insertion site and the downstream primer was the same as above. The amplified fragment was digested with X hoI and BamHI, gel-purified, and ligated into XhoI-BamHI—digested pREG 1727, generating pMF301. To sequence the insert fragment, the X hoI-BamHI fragment from pMF301was first cloned into XhoI-BamHI digested pGEM-7Zf, generating pMF0301, and sequencing was performed on this plasmid. To clone the 17 kb EcoRl-BamHI fragment upstream of Q4403, pSHl (Table 1) was transduced into DK1622 with selection for kanamycin-resistance (Kmr). One of the Km transductants, MSHI, had pSHl integrated at the native site (designated Q4403 in Table l) by homologous recombination, as verified by Southern blot analysis (data not shown). Chromosomal DNA from MSHI was digested with EcoRI, which cuts M. xanthus DNA 17 kb upstream of the Q4403 insertion site and cuts downstream at the distal end of the integrated vector. Religation at low DNA concentration to favor recircularization and transformation into E. coli DHSa with selection for Kmr and ampicillin resistance (Apr) resulted in the cloning of pLKl. To test the 17 kb Q4403 upstream segment in pLKl for promoter activity, the plasmid was digested with EcoRI and the ends were filled-in 41 using the Klenow fragment of DNA polymerase I, followed by digestion with BamHI. The 17 kb fragment was gel-purified and ligated to pREGl727 that had a filled-in HindIII blunt end and a BamHI cohesive end (prepared as described above for pMFlOO), resulting in pMF71-1. DNA sequencing. DNA fragments to be sequenced were cloned into either pUC19 or pGEM-7Zf and sequenced on both strands using synthetic oligonucleotide primers. Double-stranded sequencing was performed by the Sanger method (Sanger et aL, 1977) using the Sequenase kit (United States Biochemical). Ambiguities arising from premature termination were resolved using the protocol of Fawcett and Bartlett (Fawcett & Bartlett, 1990). Briefly, 1 pl of a reaction mixture containing terminal deoxynucleotide transferase (1 uM of each dNTP, pH 7.0, 2 units/u] terminal deoxynucleotide transferase, 1 X sequenase reaction buffer) was added to each of the termination reactions (7 ul total) and incubated at 37'C for 30 min. The reaction was terminated using 4 [.11 of stop buffer (United States Biochemical). 7-deaza dGTP reaction mixes (United States Biochemical) were used to resolve regions of compression. DNA and protein sequence analyses were performed using the University of Wisconsin Genetics Computer Group software package. Construction of M. xanthus strains. Strains containing pREGl666 or pREGl727, or derivatives of these plasmids, integrated at the Mx8 phage attachment site (designated attB in Table 1) were constructed by P1 specialized transduction from the rec+ E. coli strain JM83 into wild-type M. xanthus strain DK1622 or the csgA mutant strain DK5208 as described previously (Gill et al., 1988a). For each plasmid, three transductants (listed in Table 1), each containing a single copy of the plasmid integrated at Mx8 art, were identified using Southern blot analysis (data not shown). Similarly, pMES 108 was transduced into DK1622 and three transductants containing a single copy of the plasmid integrated at the native site (designated Q4403 in Table l) by homologous 42 recombination were identified by Southern blouin g (data not shown). Strain JPBO7 was constructed by transducin g bacteriophage P1::Tn5 lac (T or) into DK4368 with selection for oxytetracyclin resistance (T ct). Screening for kanamycin-sensitive transductants identified JPBO7 in which the Kmr gene was replaced by the Tcr gene, as verified by Southern blot analysis (Brandner & Kroos). The maximum level of developmental lacZ expression from JPBO7 (and similar, independent isolates) is consistently about two-fold lower than from DK4368 (Brandner & Kroos), and we do not know the reason for this difference. MLKl-3, -4, and —5 are Kmr, Tcr strains resulting from P1 specialized transduction (Gill et al., 1988a) of pLKl from E. coli JM83 into M. xanthus JPBO7. Southern blot analysis. M. xanthus chromosomal DNA was isolated using the method of Avery and Kaiser (Avery & Kaiser, 1983). Three to 5 ug of DNA was digested with the appropriate restriction enzyme(s) and fractionated on a 0.5% agarose gel. DNA was transferred as described previously (Davis et al., 1980), except nylon membranes were used. Probe synthesis and hybridization of the membrane were canied out using the Genius kit (Boeringer Mannheim) according to the manufacturer's specifications. RNA isolation. Total RNA was purified from DK4368 (which contains the Tn5 lac Q4403 fusion transcript) or DK4499 (which contains the wild-type Q4403 transcript) using the protocol of lgo and Losick (lgo & Losick, 1986), but with the following modifications. Cells were harvested from exponentially growing cultures (50 ml of 5 x 103 cells/ml) or from an equivalent number of cells at 24 h of development on TPM agar plates, washed with ice-cold TPM, and resuspended in 2 ml LETS buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris pH 7.8 with LiCl, 1 % SDS). The nucleic acid was resuspended in 200 [.11 of diethylpyrocarbonate (DEPC)-treated water (0.1% v/v) and treated with RNase-free DNase (Boeringer Mannheim) to eliminate DNA. DNase was removed by 43 extracting with phenol-chloroform and precipitating with ethanol. RNA was resuspended in DEPC-treated water and used for further analysis. Sl-nuclease protection experiments. Low resolution mapping of the 5' end of the Q4403-associated transcript was performed as described by Burton et al. (Burton et al., 1983). Hybridization buffer, 81 nuclease mapping buffer, stop solution and fonnamide loading buffer were prepared as described previously (Sambrook et al., 1989). The mapping strategy was designed to determine whether the start site for transcription was located between the BamHI andSaIl sites (0 to 1 kb) or the SalI andPstII sites (1 to 2 kb) upstream of the Q4403 insertion. Plasmid pMES] 12 was digested with BamHI or SalI, phosphatase treated, 5' end-labelled with 32P-‘yATP and T4 polynucleotide kinase, and used as a probe. Developmental or vegetative RNA (20 to 50 ug) was precipitated with probe (0.5 ug) and the pellet was resuspended in 10 ll] of hybridization buffer. After denaturing the nucleic acids at 85'C for 10 min, the samples were incubated at 53'C for 16 h. Unhybridized, single-stranded DNA and RNA was digested for 1 h at 37'C with $1 nuclease (Boehringer Manneheim , 25 to 250 units) in a final volume of 200 [.1] containing 1X 81 nuclease mapping buffer (Sambrook et al., 1989). The reaction was stopped by the addition of 40 ill of stop solution and was extracted with phenol-chloroform (150 ill). The samples were precipitated with ethanol, resuspended in forrnamide loading buffer, and the loaded onto a 5% polyacrylamide-8M urea gel. The protected products were separated by electrophoresis at 25 mA and visualized by autoradiography. For quantitative Sl nuclease mapping, experiments, the procedure of Gilman and Chamberlin (Gilman & Chamberlin, 1983) was used. Yeast tRNA was added to some samples to maintain a constant total amount of input RNA (50 pg). BamHI-digested pMES l 12 was 5' end-labelled and used as probe (0.5 ug). Hybridization, Sl nuclease digestion, and polyacrylamide gel electrophoresis conditions were identical to those described above. Signals were quantified using a Visage Digital Imager and fell within the 44 linear response range of the assay as determined with different amounts (0-100 pg) of RNA from DK4368 cells harvested at 18 h of development. Primer extension analysis. Primer extension reactions were performed as described previously (Sambrook et al., 1989) using 20 pg of RNA. The oligonucleotide (5'-CGCCCCCATCAGCAACATCATGCCCACGGA-3') used for the primer extension analysis corresponds to a sequence approximately 100 bp downstream of the putative transcriptional start site mapped by 81 nuclease protection. The primer was end-labeled using T4 polynucleotide kinase and 32P-yATP, and purified as described previously (Ausubel et al., 1989). Primer extension products were electrophoresed at 60 W on a sequencing gel (6 % polyacrylamide, 5 M urea) in parallel with sequencing reactions performed with the same end-labelled primer. Results Cloning DNA upstream of Q4403 and testing it for promoter activity. To clone the putative promoter region upstream of the developmentally regulated Tn5 lac insertion Q4403, we took advantage of a XhoI restriction site approximately 8 kb upstream of the site of insertion Q4403 (Kroos et al., 1986) and a X hoI site in Tn5 lac about 9 kb from the left end (Figure 2.1). Since the X hol site in Tn5 lac is downstream of the aphll gene (encoding aminoglycoside phosphotransferase, which confers kanamycin resistance), X hoI digestion of chromosomal DNA from M. xanthus containing Tn5 lac Q4403 should yield a 17 kb fragment able to confer kanamycin resistance when cloned in E. coli. We " isolated DNA from M. xanthus strain DK4368 containing Tn5 lac Q4403, digested it with Xhol, ligated the fragments with X hoI-di gested pGEM-7Zf, and transformed the mixture into E. coli strain DHSa, selecting for both ampicillin resistance of the vector and 45 kanamycin resistance of the desired insert. One transforrnant with a plasmid bearing an insert of the expected size was characterized further. Restriction mapping of the plasmid, pMESOO3, showed the expected patterns based on restriction sites in DNA upstream of Q4403 that had been mapped by Southern blotting (Kroos et al., 1986) and on known restriction sites in Tn5 lac and the vector (data not shown). Figure 2.1 shows a restriction map of DNA upstream of Q4403 based on these results. To test the DNA upstream of Q4403 for promoter activity, the XhoI-BamI-II restriction fragment from pMESOO3 that includes 8.5 kb of M. xanthus DNA and about 50 bp of the left end of Tn5 lac (Figure 2.1) was subcloned into X hoI-BamHI-digested pREGl666 (Figure 2.2) to construct pMESOO4. Because the multiple cloning site in pREGl666 is upstream of the same lacZ-containing segment found in Tn5 lac, pME8004 contains Q4403 upstream DNA fused to lacZ in exactly the same way as in the chromosome of M. xanthus strain DK4368, which has Tn5 lac inserted at site Q4403 46 Figure 2.1. Physical map of the Q4403 insertion region and summary of deletions tested for promoter activity. The top part shows restriction sites in Tn5 lac and the adjacent M. xanthus chromosome that were used in cloning experiments. The distance of restriction sites from the Tn5 lac Q4403 insertion is give in kb. B, BamHI; C, ClaI; E, EcoRI; S, SalI; Sp, Sphl. The bottom part shows different segments of Q4403 upstream DNA that were fused to lacZ to permit testing for promoter activity as described in the Materials and Methods. The maximum B-galactosidase specific activity during a 48 h developmental time course of wild-type M. xanthus strain DK1622 derivatives containing single copies of the plasmids at Mx8 art are given as a percentage of the maximum activity observed for strain DK4368 containing Tn5 lac Q4403. In each case, the maximum activity of three independent transductants was measured and the average is given. 47 1'3 lacZ aphII )f ——> E X C P S Sp l——-\\ i i i i i 17.0 8.5 4.0 2.0 1.0 0.6 . B-galactosrdase Activity pMF71-1 I__\\ J 32 % pMESOO4 I I 26 % pMES] 16 I I 28 % pMESl 15 I..___l 27 % pMF31 L_l 26 % pMF81 r_| 26 % 48 Figure 2.2. Map of the plasmid used to test for promorer activity. Unique cloning sites in this vector include B, BamHI; H, HindIII; Xb, X baI; X, X hol. Additional restriction sites indicated are E, EcoRI and M, Smal. This plasmid contains the Mx8 phage attachment site, Mx8 art, E.coli transcriptional terminators, TT, the promoterless lacZ gene, the aphll gene (conferring Kmr) with its own promoter, part of pBR322 including the origin of replication and the bla gene (conferring Apr), and a P1 incompatibility fragment, P1 inc. 49 0/20,785 bp E M 50 (Kroos et al., 1986). When pMESOO4 was transduced from E. coli strain JM83 into the wild-type M. xanrhus strain DK1622 using bacteriophage P1 specialized transduction, it integrated into the chromosome efficiently at the Mx8 prophage attachment site (Mx8 art). Transductants containing a single copy of the pMESOO4 integrated at Mx8 art were identified by Southern blot hybridization (data not shown, see the Materials and Methods). Several of these transductants were assayed for B-galactosidase activity during development. In parallel, developmental lacZ expression of M. xanrhus strain DK4368 containing Tn5 lac Q4403 was measured. Also, as a negative control, expression of transductants containing a single copy of pREGl666 (with no M. xanrhus DNA insert) integrated at Mx8 art, was examined. Figure 2.3 shows that transductants containing pMESOO4 integrated at Mx8 arr expressed lacZ with similar timing as the strain containing Tn5 lac Q4403, but reached only about 25% of the maximum level (after subtracting the background expression observed for the negative control). These results indicate that the 8.5 kb Q4403 upstream DNA has a promoter that is able to drive development-specific expression of a lacZ reporter gene. To determine whether a higher level of promoter activity could be observed by cloning additional upstream DNA, we cloned 17 kb of DNA upstream of Q4403 (see the Materials and Methods) and tested it for promoter activity as described above. Like the 8.5 kb segment, the 17 kb segment directed only about 25% of the maximum level of developmental lacZ expression seen with M. xanrhus strain DK4368 containing Tn5 lac Q4403 (Figure 2.1). This result suggests that the lower activity of fusions integrated at Mx8 art as compared to the fusion created by insertion of Tn5 lac at site Q4403 is unlikely to be due to insufficient upstream DNA. To determine the approximate location of the promoter in the 8.5 kb Q4403 upstream DNA, smaller fragments were tested for promoter activity after fusion to lacZ and integration at Mx8 art as described above. Figure 2.1 shows that promoter activity 51 Figure 2.3. Expression of developmental lacZ under the control of the Q4403 promoter. Developmental lacZ expression was determined as described previously (Kroos, et al., 1986) for M. xamthus strain DK4368 containing Tn5 lac Q4403 (I) and for for three independently isolated transductants containing a single copy of the 8.5 kb Q4403 upstream DNA fused to lacZ and integrated at Mx8 art (strains MESOOS, 008, and 012 in Table l) (D) or the vector with no insert DNA at Mx8 arr (JW103, 104, and 107) (O). The average B-galactosidase activity from three determinations for DK4368 or from one determination for each of three independent transductants is plotted. The units of B- galactosidase specific activity are nanomoles of GNP (ortho nitrophenyl phosphate) minutes-1 milligram of protein-1. B-galactosldase specific activity 100‘ a O 1 or O r uh O r 20‘ 52 —I— 94403 —D'— 8.5kb —0— No Insert ll 24 36 48 so ‘ 72 Hours of Development 53 localized to 0.6 kb of DNA immediately upstream of Q4403. No significant difference was observed in the tinting or level of developmental lacZ expression for any of the fragments that showed promoter activity. Accumulation of the Tn5 lac Q4403 fusion mRNA during development. Localization of promoter activity within 0.6 kb upstream of Q4403 indicated that an RNA 5’ end should map within this region. A probe labeled at the BamHI site near the left end of Tn5 lac (Figure 2.1) and including about 2 kb of M. xanrhus DNA upstream of Q4403, was hybridized to RNA from M. xanrhus strain DK4368, which contains Tn5 lac Q4403 (Kroos et al., 1986), and subjected to SI nuclease protection analysis. RNA from 24 h developing cells, but not from growing cells, protected a fragment of about 430 b in length. We used this assay to quantitate the relative amount of fusion mRNA from Tn5 lac Q4403 in strain DK4368 during development. Figure 2.4 shows that the fusion mRNA was first detected at 12 h of development and reached a maximum level at 24 h. B-galactosidase activity was measured in samples collected in the same experiment. Figure 2.4 shows that accumulation of B-galactosidase activity lagged slightly behind accumulation of fusion mRNA. We conclude that Tn5 lac Q4403 identifies a transcriptional unit with a putative start site approximately 430 b upstream of the BamHI site in Tn5 lac. The increase in the level of fusion mRNA during deve10pment appears to account for the observed deve10pmentally regulated B—galactosidase activity of strain DK4368. The increase in fusion mRNA probably results from activation of a developmentally regulated promoter, although we cannot exclude the possibility that the fusion mRNA is stabilized in a developmentally regulated fashion. We were unable to detect any discrete species of fusion mRNA by Northern blotting of RNA from developing DK4368 cells (Appendix 1). Apparently, the large fusion mRNA is susceptible to degradation. The ability to map the 5’ end of the fusion mRNA suggests that the SI nuclease assay is more sensitive and/or may involve 54 Figure 2.4. Levels of fusion mRNA and B-galactosidase Specific activity from Tn5 lac Q4403 during development. The top part shows quantitative S l -nuclease protection analysis of developmental RNA from M. xanrhus strain DK4368, performed as described in the Materials and Methods with 50 pg of RNA harvested from cells at 0, 6, 12, 18, 24, and 36 h of development in lanes 1-6, respectively. The numbers on the left indicate the length of markers in bases. The lower panel shows quantitation of the Sl-nuclease protected products in the upper panel (0), as well as B-galactosidase specific activity (0) of samples harvested in the same experiment. B-galactosldase Specific Activity . ' (100 140' 120' '80 100' ' '60 80' 60‘ '40, 40‘ ' '20 20‘ at t 0. 55 0 6 12182430364248 Hours of Development Relative mRNA level 0 56 hybridization of probe to partial breakdown products of the fusion mRNA. Using M. xanrhus cells that do not contain Tn5 lac Q4403, and DNA immediately upstream of Q4403 as the labeled probe, we detected a 1.5 kb transcript by Northern blotting of RNA isolated from 24 h developing cells, but not from growing cells (Appendix 1). Since the native Q4403 mRNA has the same 5’ end as the Tn5 lac Q4403 fusion mRNA (see below), the 1.5 kb size of the native transcript suggests that transcription terminates about 1.1 kb downstream of the Q4403 insertion site. DNA sequence of the Q4403 upstream region. Figure 2.5 shows the nucleotide sequence of 616 bp of M. xanrhus DNA immediately upstream of the Q4403 insertion site. An open reading frame (ORF) beginning with ATG at position 376 is preceded 6 nucleotides upstream by the sequence 5’-AGCAGG-3’, which is complementary (except for 1 mismatch) to a sequence near the 3’ end of M. xanthus l6S rRNA, suggesting it could serve as a ribosome binding site. The ORF exhibits codon preference typical of M. xanrhus genes (Hagen & Shimkets, 1990), as well as a strong bias towards usage of guanine or cytosine at the third position of codons, which is also typical of GC-rich organisms like M. xanthus (Shimkets, 1990). The ORF remains uninterrupted for at least 80 amino acids, extending to the Q4403 insertion site at the end of the sequenced region. The deduced amino acid sequence of the ORF shows 38% amino acid identity and 73% amino acid similarity over a 63 amino acid stretch to aqualysin I (Figure 2.6), a secreted protease of Thermus aquaricus (T erada et al., 1990). Both the ORF and aqualysin I exhibit a sequence highly similar to the sequence YIVGFK, which is conserved in subtilisin-type proteases and influences processing of precursor protein to the active protease (Jacobs et al., 1985; Lee et al., 1991). Based on these observations, we speculate that Tn5 lac Q4403 disrupts the first (and perhaps only) gene in a developmentally regulated transcriptional unit, and that this gene encodes a subtilisin-type serine protease. The function of the putative protease remains unclear because M. xanrhus 57 Figure 2.5. Nucleotide sequence of 616 bp directly upstream of Tn5 lac Q4403. The transcriptional start site is indicated by an arrow and the primer used for primer extension analysis is underlined. The hexanucleotide sequences in the -10 and -35 regions that resemble sequences recognized by E. coli 070 RNA polymerase are indicated by bold lettering. The putative start codon and a potential ribosome binding site are boxed in the deduced amino acid sequence of the Q4403 partial open reading frame is shown below the nucleotide sequence. Restriction sites used to generate some deletions in the Q4403 promoter region are shown. The numbers above the nucleotide sequence indicate the 5' end of deletions that were tested for promoter activity. 58 SphI GCATGCCCACCTGGTGAACGCAGCACGAGAGATGGGCATCAACGACATCCTGTCACGCCC CGGGAAACGTCCACTGGAAGCAGTCTTGTACGAATGGGGAACCACCATTCGTCCTTGGTA HaeII-8O -72 -62 GGTCATGGCCAGACATCGCGCCTTGAAGCGCCATGGCATGTTCAATCACGGACCGCCGTC —52 -42 -11 +1 TCATCCCTCCGGGTTGATTCATGAATAAGCCGTTTTTGATGTACACCCGTTTTACCCATC ACAACCATTTTCCTCTGGGTAAAACATGAATCGACACACACTGAAGCTGTTCGTCIQQGI EGGCAIGAIGIIGQIGAIGGGGGQGTGCGCATCGCGCAGGTGCCCTGCAAGGACCGCACG GCCAGCAGQCCCACGAIGGGCCAGAAGCAGACCGGCAAGTTCATCACTGTCCGAAAGAAG M G Q K Q T G K F I T V R K K ATTCCGGGTGAATACATTGTCGTCCTGAAGTCGCCCGCACAAAGCCTGGAACAGGTCGAG I P G E Y I V V L K S P A Q S L E Q V E GTCCAGCAAGCCACGACGAGCCTCATCACGGCTTACGGTGGCACCGCATTCGCGATGTAT V Q Q A T T S L I T A Y G G T A F A M Y GAGAATGCGTTGCGTGGTTTCGCGGCCAAGATGACGGAAGCCCAGGCGCGGGCCATGGCG E N A L R G F A A K M T E A Q A R A M A AACGACCCCGCAGCTG N D P A A 59 Figure 2.6. Alignment of the amino acid sequence of the Q4403 partial ORF and the N-terrninal region of aqualysin I. Identical amino acids are indicated by a vertical line and similar amino acids are indicated by two dots. The conserved amino acid sequence in subtilisin-type proteases (Jacobs, et al., 1985) is shown in bold lettering above the corresponding regions. 60 <.<0_u_n Stow ‘ ZODXOHQXEEExEVGmfiér—A- me>0mrm0jmc .2 :2. _n .____ "n ....— >ncm__u<=umm<_<<_uxXOXQDmEtOOD_ ...—r02»; atom a §zrmon>>x§m>o>n>z>zon; .u. x u.. : :2... I .. ... 355.... m. no.0<<<3> fio>roon>>mz>eo>rm>30meo85% of the Kmr transductants contain a single copy of the plasmid integrated 78 at Mx8 arr, which is easily determined by Southern blotting. Hence, the plasmid makes it simple to test a DNA segment for promoter activity under well-defined conditions of copy number and chromosomal position. The plasmid just described allowed us to test a series of deletions and localize Q4403 promoter activity to within 0.6 kb upstream of the insertion site (Figure 2.1). 81 nuclease analysis of RNA from developing cells identified a single 5’ end in this region, providing evidence for a single promoter with a start site located about 380 bp upstream of Tn5 lac Q4403 (Figure 2.4). The promoter appeared to be inactive in growing cells and 6 h developing cells since no signal was observed upon S1 nuclease analysis of RNA from these cells and lacZ expression from Tn5 lac Q4403 remained at a low background level. The promoter was clearly active by 12 h into deve10pment, as evidenced by accumulation of fusion mRNA (detected by the S lnuclease assay) and B-galactosidase activity fi'om Tn5 lac Q4403. The simplest explanation of these results is that the Q4403 promoter is developmentally regulated at the level of transcription initiation. We cannot exclude the possibility that the Q4403 promoter is active in growing cells and/or early developing cells, and that the fusion mRNA is rapidly degraded. According to this model, the rise in B-galactosidase activity from Tn5 lac Q4403 in 12 h developing cells would result from stabilization of the fusion mRNA. It seems unlikely a priori that a short (382 b) segment of M. xanrhus RNA at the 5’ end could stabilize the long (at least several kb) fusion mRNA in a developmentally regulated fashion. Indeed, we were unable to detect any discrete species of fusion mRNA in developing cells by Northern blotting, suggesting that it is unstable. The native Q4403 mRNA may be more stable, since a 1.5 kb species was detected by Northern blotting of RNA from developing cells lacking Tn5 lac Q4403. Several M. xanthus genes that appear to be developmentally regulated at the level of transcription initiation produce transcripts with unusually long half-lives (Downard et al., 1984; Nelson & Zusman, 1983; Romeo & Zusman, 1992). 79 The DNA sequence of the Q4403 upstream region revealed an ORF that has striking similarity to aqualysin I, a secreted protease of T hermus aquaticus (T erada et al., 1990). The ORF could extend farther upstream, beyond the predicted translational start at position 376 (Figure 2.5). Potential (GTG) start codons are located at positions 325 and 340, but neither of these exhibits a satisfactory ribosome binding site and the longer ORFs do not exhibit the codon preference and third codon position GC bias typical of M. xanthus genes. If translation starts at the ATG at position 376, the ORF would encode 80 amino acids and be interrupted downstream by the Tn5 lac Q4403 insertion. A 63 amino acid stretch within the ORF shows high similarity to aqualysin I (Figure 2.6), and our sequence ends a few amino acids after this stretch. It will be interesting to clone DNA downstream of Tn5 lac Q4403 and determine whether the similarity to aqualysin I can be extended. The 1.5 kb Q4403 mRNA could encode a polypeptide similar in size to the aqualysin I precursor (51 kDa) (T erada et al., 1990), if it were monocistronic. Activation of the aqualysin I precursor involves self-processing of N - and C-terminal pro-sequences (T erada et al., 1990). The Q4403 ORF is similar to the N~terminal pro-sequence of aqualysin I, and both contain a sequence highly similar to the sequence YIVGFK; which is conserved in subtilisin-type proteases (Jacobs et al., 1985). Amino acid substitutions in the YIVVFK sequence of aqualysin I enhanced processing of the precursor protein to the active protease, but deletion of this six amino acid sequence abolished accumulation of precursor and active enzyme (Lee et al., 1991). Unlike aqualysin I, the Q4403 ORF does not appear to encode an N-terminal signal sequence, which is found in many proteins that are translocated across the inner membrane of gram-negative bacteria (Pugsley, 1993). Perhaps the Q4403 gene product is not secreted. Alternatively, the protein might exit the cytoplasm by a mechanism that does not require a signal peptide, as appears to be the case for some extracellular and periplasmic developmental proteins of M. xanrhus (Guespin-Michel et al., 1993). If the Q4403 genes does encode a developmentally regulated subtilisin-type 80 protease as we have speculated, this protease does not seem to be essential for aggregation or sporulation. The insertion of Tn5 lac at codon 81 of the putative protease gene would probably abolish function, yet the M. xanrhus strain containing Tn5 lac Q4403 aggregates normally and produces a normal number of heat- and sonication-resistant spores (measured after 3 days of development). Of course, the Q4403 gene product may have subtle effects on aggregation and/or spore viability that escaped detection. It is also possible that the Q4403 gene product is functionally redundant with another M. xanrhus protein(s). Our principal interest in the Q4403 gene is its mechanism of regulation. Inspection of the DNA sequence immediately upstream of the putative Q4403 transcriptional start site revealed hexanucleotide sequences in the ~35 and ~10 regions with some similarity to sequences found in these regions of E. coli (Lisser & Margalit, 1993) and B. subrilis (Helmann, 1995) promoters transcribed by the major vegetative RNA polymerase containing 070 and 043, respectively. However, the 20 bp spacing between the two hexanucleotide sequences in the Q4403 case is more than the optimal spacing. B. subtilis 043 RNA polymerase tolerates spacings of 22 and 21 bp between the ~35 and ~10 regions of the spoIlG and spoIlE promoters, respectively, but transcription requires an activator protein, SpoOA-phosphate (Baldus et al., 1994; York et al., 1992). A similar mechanism might regulate the Q4403 promoter, since M. xanrhus contains a gene, sigA, predicted to encode the major sigma factor, and SigA is very similar to B. subtilis 0’43 (and E. coli 070), including the domains that interact with the ~35 and ~10 regions of promoters (Inouye, 1990). Alternatively, the Q4403 promoter might be recognized by a development-specific form of RNA polymerase. Two developmentally regulafed sigma factors have been described in M. xanrhus, sigB and sigC, and more are likely to exist (Apelian & Inouye, 1990; Apelian & Inouye, 1993). Neither Si gB nor SigC appears to be required for Q4403 transcription because null mutations in these sig genes do not prevent developmental 81 expression of Tn5 lac Q4403-Tcr (Brandner & Kroos, ). Deletion analysis showed that DNA between ~80 and ~72 is critical for Q4403 promoter activity. Although RNA polymerase does interact with DNA upstream of the ~35 region of some promoters (Busby & Ebright, 1994), more typically a required sequence element upstream of the ~35 region is indicative of a binding site for a transcriptional activator protein (Collado—Vides et al., 1991). Transcriptional activators often bind to palindromic DNA sequences (Pabo & Sauer, 1992). The region critical for Q4403 promoter activity has two cepies of the palindromic sequence 5’~CATG~3’ separated by 1 bp (Figure 2.5); however, the ~80 deletion that retains promoter activity (Figure 2.8) replaces the CA of the upstream palindrome with GC from the vector. Nucleotide substitutions will be required to establish the importance of the palindromic sequence, or the ~35 and ~10 regions, for Q4403 promoter activity. Mutations that reduce promoter activity could provide a means to identify regulatory proreins by isolating suppressor mutants with restored promoter activity. We are currently pursuing biochemical approaches to identify proteins that bind to the Q4403 regulatory region. The Q4403 promoter is not unusual among deve10pmentally regulated M. xanrhus genes in requiring an upstream element. All such genes examined so far exhibit this requirement (Downard & Kroos, 1993). In some cases multiple elements appear to act over distances of several kb from the transcriptional start site (Kil et al., 1990). In the case of Q4403, however, our results show that 80 bp of DNA upstream of the transcriptional start site (Figure2.8), or 17 kb of upstream DNA, direct the same level of developmentally regulated gene expression, providing no evidence for additional upstream promoter elements. It seems unlikely that a promoter element lies more than 17 kb upstream of Q4403, or can still function even when located downstream of the 12 kb Tn5 lac element, yet cells containinng5 lac Q4403 express 3- to 4-fold more B—galactosidase activity during 82 development than any strains we tested with fusions integrated at Mx8 atr. We ruled out several possible explanations for this difference. The fusion-containing plasmid integrated at the phage attachment site did not appear to be unstable. Niether was it a hindrance to the normal developmental process. Tn5 lac Q4403 did not disrupt a gene whose product negatively autoregulates, as was observed for Tn5 lac Q4414 (Thony-Meyer & Kaiser, 1993). Cells with one copy of the Q4403 regulatory region fused to lacZ and another copy in tandem fused to the intact gene at the native site expressed lacZ at about 70% the maximum level observed for cells containing Tn5 lac Q4403 (Figure 2.9). This suggests that titration of a limiting positive-acting factor by a second copy of the Q4403 regulatory region in cells with fusions integrated at Mx8 art can, in part, account for their reduced expression as compared to cells containing Tn5 lac Q4403 (with only one copy of the regulatory region). An effect of chromosomal position may also hinder expression from some fusions located at Mx8 arr. Our results show that a putative promoter in the vicinity of the phage attachment site is down-regulated by C~signaling (Figure 2.10). Perhaps this effect is not confined to the putative promoter, but is also observed for some promoters integrated at Mx8 art. In particular, it seems to be difficult to observe full expression at Mx8 art of late developmental genes that depend absolutely on C~signaling. Li and Shimkets (Li & Shimkets, 1988) observed about 50% as much developmental B-galactosidase activity in cells with 1.3 or 11 kb of Q4435 upstream DNA fused to lacZ and integrated at Mx8 art as compared with cells containing Tn5 lac Q4435. Similarly, 1.3 or 10 kb of Q4459 upstream DNA inserted in the plasmid we described here and integrated at Mx8 arr drives only about 10~20% as much developmental lacZ expression as seen with cells containing, Tn5 lac Q4459 (Brandner et al.). Also, Thony-Meyer and Kaiser(Thony-Meyer & Kaiser, 1993) reported poor expression of devRS at Mx8 art as compared to the native site. These authors invoked differential condensation of the chromosome during development as a 83 possible explanation of the positional effects. Given that Tn5 lac Q4403 is within 525 kb of Mx8 arr (Shimkets, ), the differential condensation would have to be highly localized. Perhaps this involves a silencing mechanism similar to that used in yeast mating type switching (Dillin & Rine, 1995). Genes that are expressed earlier during development and/or that depend less strongly on C~signaling for expression seem to escape inhibition. Cells with Q4499 (Fisseha & Kroos) or Q4514 (Hao & Kroos) upstream DNA inserted in the plasmid shown in Figure 2 and integrated at Mx8 art show a comparable level of developmental lacZ expression as cells containing the corresponding Tn5 lac insertions. Taking all these observations together, we speculate that C~signaling leads to an altered chromosomal state of DNA integrated at the Mx8 phage attachment site and DNA nearby, partially inhibiting expression of genes that depend strongly on C~signaling. The Q4403 promoter was sufficiently active upon integration at Mx8 art to permit deletion analysis (Figures 2.1 and 2.8). For promoters less active at Mx8 arr, integration of plasmids by homologous recombination to produce structures like that depicted in Figure 2.9B might be the preferred approach. Unexpectedly, our plasmid vector alone, with no insert of M. xanrhus DNA, expressed lacZ upon integration at Mx8 arr in csg/1 mutant cells (Figure 2.10A). A promoter that is active in developing csgA mutant cells must lie upstream of lacZ in the vector or in adjoining M. xanthus DNA after integration at Mx8 arr. We did not map this promoter; however, it is likely to be located upstream of the multiple cloning site present in the vector. The same sequences downstream of the multiple cloning site in the plasmid are also present in Tn5 lac (Kroos & Kaiser, 1984), and many Tn5 lac insertions express only a low background level of B-galactosidase when present in csg/1 mutant cells (Kroos & Kaiser, 1987). The multiple cloning site itself cannot be the origin of the transcription because it is almost completely deleted in some of the plasmids that contain Q4403 upstream DNA inserts and show a similar pattern of lacZ expression as the vector alone. 84 Neither the transcriptional terminators engineered into the vector nor inserts of Q4403 upstream DNA up to 8.5 kb in size seemed to impede the transcription, suggesting that the RNA polymerase is in an antitennination mode. Interestingly, this transcription is negatively regulated by C~signaling. csgA+ cells express only a low background level of B-galactosidase when the vector is present at Mx8 arr (Figure 2.3). Moreover, mixing csg/1+ cells with csg/1 mutant cells containing the vector at Mx8 arr inhibits lacZ expression from the csgA cells (Figure 2.108). The ability of csg/1+ cells to donate C~signal and substantially reduce lacZ expression from csg/1 mutant cells containing the vector alone integrated at Mx8 arr allowed us to demonstrate that the minimal Q4403 promoter, as defined by deletions, is responsive to C~signaling. We conclude that elements needed for C~signal~dependent expression of Q4403 lie between ~80 and +382. Our deletion analysis showed that an element critical for Q4403 promoter activity lies between ~80 and ~72. A simple model is that this DNA element interacts with a transcriptional activator protein whose synthesis or activity is controlled directly or indirectly by C~signalling. The challenge now is to identify the regulatory protein(s). Chapter 3 Characterization of the DNA upstream of a C~signal~dependent operon 85 86 Abstract Tn5 lac Q4499 is a fusion of a promorerless lacZ gene to a deve10pmentally regulated promoter in the Myxococcus xanrhus chromosome. Expression of Tn5 lac Q4499 in wild-type cells starts about six hours after the initiation of development and peaks at about 24 hours of development. Normal expression from the Q4499 promoter requires exuacellular C~signaling, which is mediated by the product of the csg/1 gene. A lesion in csgA reduces lacZ expression from Tn5 lac Q4499 to approximately 25 % of the expression in wild-type cells (Kroos & Kaiser, 1987). Based on in vivo deletion analyses of the Q4499 upstream region, regulatory elements required for expression of lacZ in a C~signal~dependent manner were localized to between 2.1 and 3.2 kb upstream of the insertion. DNA sequence analysis showed that there are two open reading frames (ORFl and ORF2) in the 2.1 kb of upstream DNA. ORFl is similar to NADH-putidaredoxin reductase from Pseudomonas putida and ORF2 is similar to cytochrome P-450 from Streptomyces griseolus. The DNA sequence between ORFl and ORF2 shows no similarity to transcriptional termination sequences or to known promoter sequences from M. xanrhus or other bacteria. Together with RNA analyses, these results suggest that Q4499 is inserted into a developmentally regulated operon whose transcription initiates more than 2.1 kb upstream of the Q4499 fusion point; however, the multicistronic RNA appears to be very susceptible to degradation. 87 Introduction Myxococcus xanrhus is a gram-negative, soil bacterium that undergoes multicellular development when nutrient sources are depleted. The developmental process is complex and involves the movement of cells into mounds known as fruiting bodies and the differentiation of some cells into ovoid myxospores. This developmental process is coordinated by cell-cell interactions, five of which have been identified so far. These cell- cell interaction systems are known as the A-, B-, C-, D~, and E- signaling interactions(Hagen et al., 1978; Downard et al., 1993). Our studies are focused on understanding how extracellular C~si gnalin g regulates the expression of developmental genes. The relative genetic simplicity of the C~signalin g interaction make it an attractive model system for studying cell-cell interaction-regulated gene expression. All of the mutations that affect C~signaling map to a single genetic locus known as csgA(Shimkets & Asher, 1988; Shimkets et al., 1983). The csgA gene product is well-characterized and encodes an alcohol dehydrogenase (Lee & Shimkets, 1994). Extracellular addition of a purified csg/1 gene product rescues the developmental defect of C~signaling defective cells (Shimkets & Rafiee, 1990; Kim & Kaiser, 1990a). The C~signalin g interaction is required starting at about six hours after the onset of development, well separated from the time at which the other signaling systems are that required. Genes whose expression begins after six hours following the initiation of development require C~signalin g at least partially. To investigate how extracellular C- signaling regulates gene expression during development, the upstream regulatory region of Tn5 lac Q4499, a transcriptional fusion of a promoterless lacZ gene to a developmentally regulated transcriptional unit, whose expression has only a partial requirement for C~ signaling, was examined. This fusion was one of 29 fusions to developmentally regulated transcriptional units that were identified by Kroos et al. (Kroos et al., 1986). These 88 fusions were generated by transposition of a promoterless Tn5 lac promoter probe into the M. xanthus chromosome and identification of transductants that increased lacZ expression dming development but not during vegetative growth. The promoter region of one other gene, csgA, whose expression is stimulated by C~si gnalin g, has been characterized (Li et al., 1992). csg/1 requires a fairly large upstream sequence to modulate its expression in response to nutrient levels, peptidoglycan components and B-signaling. At least 400 bp upstream from the transcription initiation site was required for the maximum expression of csg/1 in the absence of nutrients. In the presence of low levels of nutrients, sequences farther upstream (~930 bp) were required for proper developmental expression. Deletion analyses were used to show that promoter elements required for C~signal- dependent expression are found between 3.2 and 2.1 kb upstream of the Q4499 insertion point. Transcription from the Q4499 promoter generates a transcript of at least 3.5 kb which appears to be unstable. Analysis of the 2.1 kb DNA sequence directly upstream of the fusion suggests that the Q4499 insertion is in an operon that encodes enzymes involved in metabolizing alternative carbon sources. In addition to localizing the promoter region, these studies provide information on the type of genes that are expressed during development. Materials and Methods Bacterial Strains and Plasmids. Strains and plasmids used in this Work are listed in Table 3.1. Growth and Development. Escherichia coli cells were grown at 37°C in LB medium containing 50 pg of ampicillin or 25 pg of kanamycin per ml when necessary. M. xanrhus was grown in CIT (1 % casitone, 10 mM Tris.HCl, 1 mM KHzPO4-K2HPO4, 8 89 mM MgSO4, pH 8.0) medium in liquid cultures or on agar plates as described previously (Kroos et al., 1986). 40 pg of kanamycin or 12.5 pg oxytetracycline per ml were used when required for selective growth. Fruiting body development was performed on TPM (10 mM Tris, pH 8.0, 1 mM KHzPO4, 8 mM MgSO4, final pH 7.6) agar plates (1.5 % Bacto-agar) as described previously (Kroos et al., 1986). Molecular cloning and construction of plasmids. Recombinant DNA work was performed using standard techniques (Sambrook et al., 1989). Plasmid DNA was prepared from E. coli strains J M83 or DHSOL. M. xanrhus DNA used for cloning was prepared as described previously (Laue & Gill, 1994). To test the Q4499 upstream DNA for promoter activity, fragments were subcloned into the promoter-testing plasmid, pREGl666 or pREGl727 (Chapter 2). To construct pMFl, pREG4499 was digested with X hol, and the 22.3 kb fragment was purified from a low melting point agarose gel and religated at a low DNA concentration. pMF500 was constructed by digesting pMF0051 with Ncol and making the ends blunt by digesting with mung-bean nuclease (Sambrook et al., 1989). After phenol~ 90 Table 3.1 Bacterial strains and plasmids Strain or plasmid Relevant characteristics or genotype Source or reference E.coli . DHSOI oso AlacZ AM15 AlacUI69 recAI end/11 $981513“, D» hstI7 supE44 rhi-I gyrA relAI JM83 ara Alac-pro strA thi fdlacZ AM15 III/aqsgsing, D" M. xanrhus DK1622 Wild-type Kaiser, D., 1979 DK4368 T1151“ (Km!) Q4403 K9roos et al., 1 86 DK4499 Tn5 lac (Kml') Q4499 K5008 Ct al., 1 86 JW 103,104, 107 artB::pREGl666 Jamie White MMFl727-7, ~9, atthszEGl727 This work ~1 l MMF21~1, ~2, arthszFZI This work ~4, ~10 MMF51~60, arthszFSI This work ~70,~80, MMF41~25, 40, attB::pMF4l This work 42 MMF521~6, ~16, arthszF521 This work ~20 MMF500~3, ~24, atthszFSOO This work ~38, ~39 DK5203 csg/1:: Tn5-132 (Tcr) Q205 Shimkets, L. and Asher, 1988 M58119 csg/1:: 1115-132 (rel) Q205artB::pREG1666 This work MMFZIC-S. csgA:: Tn5-I32 (Tel) Q205atthszF21 This work ~15, ~16, ~50 MMFSIC-7. csg/1:: Tn5-132 (Tel) Q205atrB::pMF51 This work ~23, ~36, ~40 Table 3.1 (cont’d) pGEM-7Zf pUCl9 pREGl666/pRE (31727 pIP110 pREG4499 pMF002 pMF21 pMFOOS 1 pMF5 1 pMF4l pMF2.3 pMF521 pMF400 pMF060- 1 pMF030-26 91 Apr laca Apr lacor Apr Kmr Pl-inc arrP Apr (pGEM-7Zf), M xanthus Q4499, Kmr (aphll), 20.4 kb HindIII fragment from DK4499 Apr Kml' Pl~inc arrP (pREGl666), 9.4 kb HindIII-BamI-II fragment from pIPl 10 Apr (GEM-72f) 4.5 kb SmaI-BamHI from pIPl 10 in Smal-BamHI Apr Kmr Pl-inc arrP (pREGl666), 4.5 kb XbaI-BamI-II fragment from pMF002 , Apr (pGEM-7Zt), 3.2 kb Per-BamHI from pMF002 Apr Kmr Pl-inc arrP (pREGl666), 3.2 kb HindIII-BamI-II fragment from pMF0051 Apr Kml' P1~inc arrP (pREGl727), 3.0 kb Smal-Xhol fragment from pMF002 Apr (pUC19), 2.1 kb NcoI-BamI-II fragment from pMF0051 in pUCl9 Apt Kmr Pl-inc attP (pREGl727), 2.1 kb HindIII-BamI-II fragment from pMF2.3 Apr Kml' Pl~inc attP (pREGl666) 1.5.kb X hoI-BamHI fragment from pREG4499 Apl' (pGEM-7Zf), 1.5 kb Xhol-BamHI fragment from pMFl Apr (pGEM-7Zf), M. xanthus 1.3 kb SpliI- SphI from pMF400 Apr (pGEM-7Zf), M. xanthus 300 bp NcoI- XhoI from pMF2.3 Yanisch-Perron etaL,1985 Promega chapter 2 Il Sun Park Beta Borer This work This work This work This work This work This work This work This work This work This work This work 92 Table 3.1 (cont’d) pMF040~110 Apr (pGEM-7Zf), M. xanthus 350 bp NcoI- This work NcoI from pMF 2.3 93 chloroform extraction and ethanol precipitation, the DNA was digested with BamHI and fractionated on a low melting point agarose gel. The 1.8 kb fragment was purified and directionally cloned into pREGl727 that had been digested with HindIII, subjected to end- filling using the Klenow fragment of DNA polymerase I (Sambrook et al., 1989), digested with BamHI and gel-purified. pMF2.3 was constructed by digesting pMF0051 to completion with Psrl followed by a partial digest with Ncol. The 4.8 kb fragment which contains pUC19 and the 2.1 kb NcoI-BamHI fragment was purified from a low melting point agarose gel, digested with mung-bean nuclease (Sambrook et al., 1989) and religated. Plasmids pMF030—26, 040—10, and 060-1 were constructed in pGEM-7Zf to serve as templates for the synthesis of riboprobes. In each case, the insert fragment was ligated into the multiple cloning site downstream of the SP6 promoter in the proper orientation so that transcription by SP6 RNA polymerase would produce a complement of the putative Q4499 message. To make pMF040-10, the 350 bp HindiIl-Ncol fragment from pMF2.3 (containing M. xanrhus DNA between 2.1 and 1.8 kb upstream of Q4499) was gel-purified and ligated into pGEM-7Zf that had been digested with Hindlll and dephosphorylated as described previously (Sambrook et al., 1989). Following ligation, the incompatible ends were made blunt by digesting with mun g-bean nuclease. The DNA was extracted with phenol and chloroform and reli gated. Similarly, pMF030-26 was made by gel-purifying the 300 bp NcoI-Xhol fragment from pMF2.3 (containing M. xanrhus DNA between 1.9 and 1.6 kb upstream of Q4499) and ligating it into Xhol-digested and dephosphorylated pGEM-7Zf. Following the ligation, the incompatible ends were made blunt by digesting with mung-bean nuclease and the DNA was extracted with phenol and chloroform, ethanol precipitated and reli gated. 94 Construction of M. xanrhus strains. Strains containing pREGl666 or pREGl727, or derivatives of these plasmids, integrated at the Mx8 phage attachment site (designated arrB in Table 3.1) were constructed by P1 specialized transduction from the rec+ E. coli strain J M83 into wild-type M. xanrhus strain DK1622 or the csgA mutant strain DK5208 as described previously (Gill et al., 1988a). For each plasmid, three or four transductants (listed in Table 3.1), each containing a single copy of the plasmid integrated at Mx8 art, were identified using Southern blot analysis (data not shown). Southern blot analysis. M. xanrhus chromosomal DNA was isolated using the method of Avery and Kaiser (Avery & Kaiser, 1983). Three to 5 pg of DNA was digested with the appropriate restriction enzyme(s) and fractionated on a 0.5% agarose gel. DNA was transferred onto a nylon membrane (Fisher Scientific) using the alkaline transfer method described by Sambrook et.al.(Sambrook et al., 1989). Probe synthesis and hybridization of the membrane was carried out using the Genius kit (Boeringer Mannheim) according to the manufacturer's specifications. Expression of B-galactosidase in M. xanthus. Strains were grown to the logarithmic stage (5 x 108 cells/ml), resuspended to 5 x 109 cells/ml in TPM, and plated for development as described previously (Kroos et al., 1986). Assays for B-galactosidase expression were performed as described by Kroos etal. (Kroos et al., 1986). RNA isolation. Total RNA was purified from DK4368 (which contains the wild-type Q4499 transcript) using the protocol of lgo and Losick (lgo & Losick, 1986), but with the following modifications. Cells were harvested from exponentially growing cultures (50 ml of 5 x 108 cells/ml) or from an equivalent number of cells at 24 h of development on TPM agar plates, washed with ice-cold TPM, and resuspended in 2 ml LETS buffer (100 mM LiCl, 10 mM EDTA, 10 mM Tris pH 7.8 with LiCl, l % SDS). The nucleic acid was resuspended in 200 pl of diethylpyrocarbonate (DEPC)-treated water 95 (0.1% v/v) and treated with RNase-free DNase (Boeringer Mannheim) to eliminate DNA. DNase was removed by extracting with phenol-chloroform and precipitating with ethanol. RNA was resuspended in DEPC-treated water and used for further analysis. Northern Blot Analysis. Northern b10t analysis was carried out as described previously (Ausubel et al., 1989) except with the following modifications. RNA was fractionated on a 0.6% agarose gel containing 1.1% (v/v) formaldehyde and transferred onto a nitrocellulose membrane (Schleicher & Schull). The membrane was baked for 2 h at 80°C. Riboprobes were synthesized from pMF400, pMF030-26, pMF040-10, and pMF060-1 linearized with restriction enzymes Sphl, Xbal, EcoRI and AarII, respectively. These restriction enzymes cut in the multiple cloning site downstream (relative to the SP6 promoter) of the insert fragment. Riboprobes were synthesized using the SP6 riboprobe kit from Promega and [0‘32P]CT P. All of the run-off transcripts except the one derived from Sphl- digested pMF400 were expected to be of the same length as the insert DNA. SphI cuts pMF400 about 230 bp upstream of BamHI near the junction of M. xanrhus DNA and Tn5 lac Q4499. Therefore, the expected size of the run-off transcript was about 230 bp. Samples of the riboprobes were electrophoresed on 5% polacrylamide, 7 M urea gels to verify the sizes. Prehybridization was in 50% formamide, 6 X SSPE, 0.5% SDS, 5 X Denhardt's solution, 100 pg/ml sheared salmon sperm DNA for 2-3 h at 42°C, and hybridization was overnight at 56°C. The hybridization solution was the same as the prehybridization solution except that it contained 1-2 x 107 CPM of the apprOpriate riboprobe. Following hybridization, the membranes were washed at 65°C for 30 minutes each time once in 2 X SSC 0.1% SDS, 1 X SSC 0.1% SDS and two times in 0.1 X SSC, 0.1% SDS. Membranes were exposed to X-ray films at 70°C with intensifying screens. DNA sequencing. Plasmids pMF0051 and pMF060-1 were used for double- stranded sequencing of the 2.1 kb of DNA upstream of Q4499. Sequencing was carried out on both strands using synthetic oligonucleotide primers. Double-stranded sequencing 96 was performed by the Sanger method (Sanger et al., 1977) using the Sequenase kit (United States Biochemical). Ambiguities arising from premature termination were resolved using the protocol of Fawcett and Bartlett (Fawcett & Bartlett, 1990). Briefly, 1 p1 of a reaction mixture containing terminal deoxynucleotide transferase (1 pM of each dNTP, pH 7.0, 2 units/pl terminal deoxynucleotide transferase, 1 X sequenase reaction buffer) was added to each of the termination reactions (7 pl total) and incubated at 37°C for 30 min. The reaction was terminated using 4 pl of stop buffer (United States Biochemical). 7-deaza dGTP reaction mixes (United States Biochemical) were used to resolve regions of compression. DNA and protein sequence analyses were done using the University of Wisconsin Genetics Computer Group software package. Results Analysis of the Q4499 upstream region for promoter activity. To identify the promoter region upstream of the partially C-signal-dependent Tn5 lac fusion Q4499, the upstream DNA had previously been cloned (Il-Seon Park, unpublished results) i using HindIII restriction sites located approximately 9.4 kb upstream of the Q4499 insertion (Kroos et al., 1986) and downstream of the aphll gene (which encodes aminoglycoside phosphotransferase, and confers resistance to kanamycin) in Tn5 lac (Figure 3.1A shows a partial restriction map). Thus, HindIII digestion of chromosomal DNA from M. xanrhus containing Tn5 lac Q4499 with Hindlll generates a fragment of about 20.4 kb containing the gene for kanamycin resistance. Chromosomal DNA isolated- from DK4499 cells was digested with Hindlll, ligated into HindIII-digested pGEM-7Zf and transformed into Escherichia coli strain DHSor. Transfonnants were selected for resistance to ampicillin (conferred by the vector) and kanamycin (conferred by the cloned 97 M. xanrhus DNA). One transforrnant carrying the plasmid with Q4499 upstream DNA, pIPl 10, was identified and analyzed by restriction digestion using sites that had previously been mapped in M. xanthus and in Tn5 lac (Kroos et al., 1986) (Figure 3.1A). pIPl 10 contains approximately 9.4 kb of Q4499 upstream DNA and 11 kb of DNA from Tn5 lac. To test the DNA upstream of Q4499 for promoter activity, a Smal-BamHI fragment that includes about 50 bp from the left end of Tn5 and 4.5 kb of DNA directly upstream of Q4499 (Figure 3.1A) was subcloned into the multiple cloning site of pREGl666. This creates a fusion of the upstream DNA with a promoterless lacZ gene in exactly the same configuration found in DK4499. This plasmid, pMF21, was introduced into wild-type M. xanrhus strain DK1622, by P1 specialized transduction. Kanamycin- resistant M. xanthus transductants contain pMF21 integrated into the chromosome. Since pREGl666 contains an Mx8 phage attachment site (Mx8 arr) the plasmid integrates either 98 Figure 3.1 A. Physical map of the Q4499 insertion region. The restriction sites in the Tn5 lac and the adjacent M. xanrhus DNA that were used in cloning experiments are shown. The distance between restriction sites andthe point of Tn5 lac Q4499 insertion is given in kb. B, BamHI; H, Hindlll; M, Smal; N, Ncol; P, Psrl; S, SphI; X, Xhol 99 lacZ aphII JH ———> --> B l 0.23 2.1 1.8 1.5 3.2 4.5 9.4 100 Figure 3.1 B. Developmental lacZ expression from the Q4499 promoter. 13- galactosidase specific activity during development was determined as described previously (Kroos, et al., 1986). The B-galactosidase activity from a single determination of M. xanrhus strain DK4499, containing Tn5 lac Q4499, is shown (I). The average lacZ expression from three independently isolated transductants containing 4.5 kb of Q4499 upstream DNA fused to lacZ and integrated at the Mx8 arr site in single c0py is plotted (Cl). The units of B-galactosidase specific activity are nanomoles ONP (ortho nitrophenyl phosphate) minutes-1 milligram of protein-1. w B-galactosldase Specific Actlvity 101 12 18 24 30 36 42 48 Hours of Development 102 by site-specific recombination at the arrB site in the chromosome or by homologous recombination through the cloned upstream region. Transducrants containing a single copy of pMFZl integrated at Mx8 art were identified by Southern blot analysis and several of these were analyzed for the production of B-galactosidase during development. Figure 3.1B shows that 4.5 kb of Q4499 upstream DNA directs lacZ expression with similar timing and level as observed for DK4499. This indicates that 4.5 kb of DNA upstream of Q4499 contains all of the regulatory elements required for proper expression of lacZ in a developmentally regulated fashion. To further localize the region upstream of Q4499 that is required for prOper expression of developmental lacZ, deletions of the upstream DNA were generated and tested for promoter activity in the same way as described above. Figure 3.2 shows that 3.2 kb of Q4499 upstream DNA directs the expression of lacZ with the same timing as in DK4499, indicating that the 3.2 kb region contains a developmentally regulated promoter. The maximum level of expression from this region was about 65% of the level in DK4499 (after subtracting background expression in the strains containing the vector alone with no insert). These results indicate that the DNA between 4.5 and 3.2 kb upstream of Q4499 contains a promoter(s) and/or promoter element(s) that elevates expression to the level observed for DK4499 cells. Further deletion of the upstream DNA to 2.1 kb or less abolished developmental B-galactosidase production, indicating that essential promoter elements lie more than 2.1 kb upstream of the Q4499 insertion point. The 3.0 kb region between 4.5 and 1.5 kb upstream of Q4499 directed expression of about 30% as much B- galactosidase activity as seen with DK4499 cells, and the timing of expression from this DNA segment was delayed by about 12 hours. This result is consistent with a developmentally regulated promoter being located between 2.1 and 3.2 kb upstream of Q4499 but shows that DNA within 1.5 kb immediately upstream of Q4499 is also 103 Figure 3.2. Deletion analysis of the Q4499 promoter region. Developmental lacZ expression was determined as described previously (Kroos, et al., 1986) for transductants containing a single copy of either different segments of Q4499 upstream DNA fused to lacZ, or the vector alone with no insert DNA (D), at the Mx8 arr site. The DNA segments tested for promoter activity included 3.2 (O) or 2.1 kb (A) directly upstream of Q4499 and 3.0 kb (0) between Smal and X hol (Figure 3.1 A). lacZ expression from DK4499 cells containing Tn5 lac Q4499 is also shown (I). The units of enzyme activity are given in the legend of Figure 1B. Points represent the average for three or more independently isolated transductants, except the DK4499 data was a single detennination made in the same experiment. B-galactosldase- 'specltlc actlvlty 104 . —I— 94499 140 ‘ —D— No Insert —0— 3.2 120 - - —O— 3.0 —¢— 2.1 100 - 80 - 60 q 40 - ° ° 20 1 . o ' I r I O 6 12 18 24 30 '36 42 48 Hours of Development 105 required for the normal tinting and levels of expression. C-signal dependent expression of Q4499. The Tn5 lac Q4499 fusion is one of many developmentally regulated fusions whose expression requires extracellular C- signaling (Kroos & Kaiser, 1987). C~signaling is mediated by the product of the csgA gene (Shimkets & Rafiee, 1990; Kim & Kaiser, 1990a). lacZ expression in csgA mutant cells containing the Tn5 lac Q4499 fusion is reduced to about 25% the level observed in wild-type cells, indicating that the expression of Q4499 is partially dependent on extracellular C—signaling (Kroos & Kaiser, 1987). To determine whether lacZ expression from the cloned regions that have promoter aetivity exhibit C-signal dependency, fusions of the 4.5 and 3.2 kb upstream regions with lacZ were integrated at the Mx8 art site of csg/1 cells and tested for deve10pmentally regulated promorer activity. Figure 3.3 shows that lacZ expression from these fusions is only 10-20% the level observed for wild-type cells containing Tn5 lac Q4499 measured in parallel, indicating that the cloned promoter region is partially dependent on C~signaling for proper expression. The negative control strain, which contains pREGl666 alone with no M. xanrhus insert DNA integrated at Mx8 arr, reached about 50 units of B-galactosidase activity at 48 hours of development. This activity is attributed to read-through transcription from a fortuitous promoter in the vector or in the adjacent M. xanrhus DNA. Since lacZ expression did not rise above above 15 units in wild-type cells containing pREGl666 at Mx8 art, this read-through transcription must normally repressed by C-signaling (Chapter 2). The expression of lacZ in transductants containing the 4.5 and 3.2 kb fusions was greater than in transductants containing the vector alone indicating that there is some expression from these regions in csgA cells, just as was observed for csg/1 mutant cells containing Tn5 lac Q4499. 106 Figure 3.3. Developmental lacZ expression in csgA mutant cells. B-galactosidase activity was measured as described previously for cells containing fusions of 4.5 (O) or 3.2 kb (A) of Q4499 upstream DNA to lacZ integrated in single copy at the Mx8 arr site. Points represent the average for three or more independently isolated transductants. A single determination of expression from csgA mutant cells containing a single copy of the vector alone (with no insert DNA) integrated at Mx8 alt (+) and from DK4499 cells containing Tn5 lac Q4499 (I) was made in the same experiment. B-galactosldase Specific Activity 300 ' 250i 200 " 150i 100‘ 107 l (24499 No Insert/csgA 4.5 kb/csgA 3.2 kb/csgA i i ll 12 18 24 30 36 42 48 Hours of Development 108 Analyses of the Q4499 transcript. To determine the size of developmental transcripts produced from the Q4499 region and to start localizing their 5' ends Northern blot analyses were performed. RNA was isolated from cells (containing the wild-type Q4499 gene) that were either grown vegetatively or that were developed for 24 hours on starvation agar. A 3.5 kb transcript was detected by riboprobes synthesized from pMF060-l (linearized by digesting with AarII) and from pMF400 (linearized by digesting with SphI) (Figure 3.4). These riboprobes are complementary to the 0.23 kb region directly upstream of Q4499 (detected by the pMF400 riboprobe), and to the region between 0.23 and 1.5 kb upstream of Q4499 (detected by the pMF060-1 riboprobe). Two additional transcripts of about 1.9 and 1.0 kb were also detected using these riboprobes. These may be degradation products of the larger transcript. The intensity of all three transcripts was greater in RNA isolated from 24 hour deveIOping cells compared to RNA isolated from vegetatively growing cells. This suggests that the Q4499 transcript accumulates more in developing cells, as might be expected from the lacZ fusion results. The greater accumulation of transcripts in developing cells may be due to increased synthesis and/or stability of the mRNA. Riboprobes synthesized from DNA more than 1.5 kb upstream of the Q4499 insertion point (using pMF030-26 and pMF040-10, data not shown) did not detect any of these transcripts. These results suggest that the 5’ end(s) of the Q4499 transcripts may lie within 1.5 kb upstream of Q4499. Alternatively, it is possible that the native Q4499 transcript is unstable at its 5’ end. 109 Figure 3.4. Northern blot analysis of the Q4499 transcript. RNA was isolated from vegetatively growing cells (lanes 1) and from 24 hour developing cells (lanes 2). Riboprobes were synthesized as described in the Materials and Methods, and used to detect right ward transcripts from the regions indicated above each autoradiogram. The sizes of the transcripts are indicated on the left. 110 PstI NcoI Xhol SphI BamHI I 3.2 2.1 1.5 0.23 XhoI-SphI SphI-BamHI V D V D 3.51:!) 1.9 kb 1.0 kb 3i 111 DNA sequence analysis of the Q4499 upstream region. Since the size and organization of open reading frames in DNA upstream of Q4499 might help distinguish between alternative models about the location of the transcriptional start site(s), 2.1 kb of DNA upstream of Q4499 was sequenced. Figure 3.5 shows the DNA sequence of this region. Two ORFs were identified based on the strong bias for guanine and cytosine nucleotides at the third position of codons (expected for G+C rich organisms like M. xarrthus), and the usage of codons preferred by M. xanrhus. Figure 3.6 shows the third position GC bias and codon preference within the 2.4 kb of Q4499 upstream DNA. The sequence between 2.1 and 2.4 kb was determined in one strand and contains some ambiguities. ORF2 begins 939 bp upstream of the Q4499 insertion point (shown in Figure 3.6 as the open bar in the first reading frame) and remains open downstream to the point of fusion with Tn5 lac Q4499. Starting at about nucleotide 1200, the third position GC bias and the codon preference are higher than would be expected for random occurrences (indicated in Figure 3.6 by the upper and lower graphs, respectively), indicating that there may be an ORF (designated ORF] in Figure 3.5) in this region. The absence of a translational initiation codon for ORFl suggests that translation may start beyond 2.1 kb upstream of the Q4499 insertion point. A translation stop codon is present at nucleotide 1066 (102 bp upstream of ORF2), and delimits the N-tenninal end of ORFI. However, the sequence between 1066 and 1200 (Figure 3.5) does not show a bias for G and C in the third position of codons (around nucleoride 1200 to 1400 in Figure 3.6). The 300 bp region between 2.4 and 2.1 kb upstream of Q4499 (Figure 3.6) exhibits poor third position GC bias, usage of many rare codons, and several stop codons in the first reading fiame. However, the same region in the third reading frame has a relatively high frequency of GC in the third position of codons and a high frequency of using codons preferred by M. xanthus, and remains open until it meets ORF] This suggests that a sequencing error may have shifted the reading frame in the sequence analysis. Hence, the translation initiation 112 Figure 3.5. Nucleotide sequence of 2.1 kb directly upstream of Tn5 lac Q4499. The two partial open reading frames and their deduced amino acid sequences are indicated. The translation termination codon of ORFl and the putative translation initiation codon of ORF2 are shown in bold letters. A putative ribosome binding site is shown by the overline. 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 113 ORFI CCGGCCArGCAGGTCCTCCTTGCTGACGGGAACAGGGTCCAGTTCGACCGGTTGCTGATT P A X Q V L L A D G N R V Q F D R L L I GCGACGGGGGCGCGTGCCCGGCCGTGGCCCAACAACGCGGAGGCGTCGCTGGAGGGCGTA A T G A R A R P W P N N A E A S L E G V GAGGTGGTGCGCACCCGCGACGATGCGGCCCAGTTGCAGCGCCGTCTGGCCGCCAGACCC E V V R T R D D A A Q L Q R R L A A R P GGCCGGGTTCTGGTCATCGGCGGCGGGTTCACCGGCTCGGAGGTCGCCTCCGCGTGCCGC G R V L V I G G G F T G S 'E V A S A C R GAGCTGGGTCTGCCGGTGACCGTCACGGAGCGGGGGGCCACTCCACTGGTGGGGGCGCTC E L G L P V T V T E R G A T P L V G A L GGCGGAGTTGTCGGGGCGGTCGCAGCCGCCTTGCAACGCGACCATGGCGTCGACCTGCGC G G V V G A V A A A L Q R D H G V D L R TGCGGCGTCACCGTCACGCGGCTGGAGGGGGATGCAAACGGGAGGCTGCGGCGGGCCCAC C G V T V T R L E G D A N G R L R R A H TTCTCCGACGGCTCGACACTGAACGTCGACGTGGCCGTGGTCGCGCTAGGGGCGATCCGC F S D G S T L N V D V A V V A L G A I R 'AACGTGGAGTGCGTGCGCGACTCGGGGCTGGCGGTGACTCGATGGGGAGTGGCCTGTGAT N V E C V R D S G L A V T R W G V A C D GCGGCCTGCCACGCGTTCGACATCAACGGCCTGGTGACCAGGAACATCTTCGTCGCCGGG A A C H A F D I N G L V T R N I F V A G GACGTGGCCCGCGTCCCTCACCCGATCTACGACTACCAGTTCCTGTCCCTCGAGCACTGG D V A R V P R P I Y D Y Q F L S L E H W GGCAACGCGGTCACCCAGGCCAAGATTGCGGCCCACAACATGCTCAGCCCGGAAGCGGAC G N A V T Q A K I A A H N M L S P E A D CGCTGGCCACACCTGACCATCCCCACCTTCTGGTCCACGCAATTCGGCGTCAACATCAAA R W P H L T I P T F W S T Q F G V N I K TCGGTGGGTGTGCCGACCTTCTCGGACGCGCTCGTCATCACCCAGGGCTCACTCGCCGGA S V G V P T F S D A L V I T Q G S L A G AGCGCCGATTCGTCGCCGTCTATGGCTACAAGGGGCGTGTCACCGCCGCGGTCGCTTTCG S A D S S P S M A T R G V S P P R S L S ACAGCCCAAATGGCTGGAGTTCTACCAGGGCTTGATCGATGCAGCCGCGCCGTTCCCGCC T A O M A G V L P G L D R C S R A V P A CGCCTTCCGCACCGTCCATCAGCCCGCCGAGATGCGGCCCGTCCCTCCCGAATTTCCGAA R L P H R P S A R R D A A R P S R I S E ACGGGCCGTGTCCGAGCACGAGGCCACCGTCATGGTCACCGGCCGTGAACCCTACGAGCG T G R V R A R G H R H G H R P * T L R A 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 114 GCGGGTTCCGGTGGGTGTACCGGCACCTTTGAGCCGAATGTCGGCAGCCGCGTCACCCCA A G S G G C T G T F E P N V G S R V T P ORF2 CCCCTCCAACGGACTCAAGGAGACCGCTTCATGGCACAGGCCAGCATCTTCGAGCAGATC P L Q R T Q G D R F M A Q A S I F E Q I CTCGACCCGGCCAACCGGGCCAACCCCTACCCGCTCTACGCCGAGTTGCGGAAGACTCCC L D P A N R A N P Y P L Y A E L R K T P GTGGCGCGCGAGGCCGACGGCACGTACATCGTCAGCACCTACGATGAGATTGTCGCGTTG V A R E A D G T Y I V S T Y D E I V A L CTCCACGACCCACGGGTCAGCTCCGACATACGCAACCTCGTCCGGCAGGCCGGTGCCACG L H D P R V S S D I R N L V R Q A G A T CCCTCCCCCCAGGAGGGCCCTCCAGGGCTGCCAGAGCCCTTCATCCGGCGCGACCCTCCC P S P Q E G P P G L P E P F- I R R D P P GACCACGACCGGCTCCGGAAGCTGGCGATGAGGCCCTTCGGGCGCACACCCCCGGACGCA D H D R L R K L A M R P F G R T P P D A TTGATGCCCTTCGTCCCTGGCTGGTCGAGACCACGACGGGCCTGCTTGACGCGCTCGCGG L M P F V P G W S R P R R A C L T R S R GCAAGAACCAAGTCGACATCGTCGGACGACGTGGCCTACCCATTCCCTGTGACTGTCATC A R T K S T S S D D V A Y P F _P V T V I TGCAAGCTGCTGGGAGTCCCTCGCGAGGACGAAGCGCGCTTTCACGAGTTGGCCGACGCG C K L L G V P R E D E A R F H E L A D A GGCGTCGAGACCCTTGACCCCACCACAGGGACGATCGAGCAGCGAAAGGCCAAGCGCGAC G V E T L D- P T T G T I E Q R K A K R D CGGACGAAAGCGGAGCTGGGGCAGTACCTCGCTGCGCTCGCCGATGCCCACCTGCGCCAG R T K A E L G Q Y L A A L A D A H L R Q CCGGGCGGCGACCTGCTCTCCGGGTTCCTGACGGATAACGGCCCCGACGGGCGGATGTCG P G G D L L S G F L T D N G P D G R M S CGGGAAGAGGTGTTGAGCACCGCCGCGTTGCTGCTCGTCGCTGGCCATGAGACCACCGTC R E E V L S T A A L L L V A G K E T T V AACCTCATCGCCAATGGCATGCTCACCCTGCTGCGCCACCCCGGCGTGTTCGAGCGGCTG N L I A N G M L T L L R H P G V F E R L CGCCGCGAGCCCGAGCTGAGCATCCCGCTGGTCGAGGAGCTCGTGCGCTATGAACCACCG R R E P E L S I P L V E E L V R Y- E P P GTCGAGTTCCTGCCTGACCGAGTCACTCTGGCCGACATCGACATTGCCGGCACCACCATT V E F L P D R V T L A D I D I A G T T I CCCCAGGGC PQG 115 Figure 3.6. Analysis of the 04499 upstream sequence for ORFs likely to be translated. The 2.1 kb DNA sequence presented in Figure 3.5, plus an additional about 300 bp of sequence between 2.4 and 2.1 kb upstream of 94499 (based on sequence obtained for only one DNA strand), was analyzed using the codonpreference program of the University of Wisconsin Genetics Computer Group software package, with codon usage data for M. xanthus (Hagen and Shimkets, 1990). The third position GC bias and the codon preference for all three reading frames is plotted. Relative to these plots, the direction of transcription is left to right and Tn5 lac Q4499 would be located at the right hand end of the plots. Open reading frames identified by the presence of an AUG translation initiation codon and a termination codon are shown as Open bars below the plots. The usage of rare codons is indicated by ticks at the bottom of each panel._ The numbers along the horizontal axis indicate the number of base pairs from the 5’ end of the sequence and the numbers along the vertical axes indicate the third codon-position GC bias (right) and the frequency of using codons preferred by M. xanthus (left). The upper and lower plots in each panel reflect the third position GC bias and the codon preference, respectively. Values above the horizontal line associated with each plot indicate favorable sequence for translation in M. xanthus. 116 Olin Profmo In. E gonna.“ arpnug in“. pap-«a g «tn—cu {*2- Svogitu a Pal g diet-fl 95 31. air—I... a 6!“. we... a... . ‘ a ..1 3.. ..1 L P F P P I: I: uh~n~l-1~ilmnl :5... II :I=II~.I:~1--~H.|=~ Ell. I...~.-I~ HE. nu: III-I -I~. .- 51.5.- 1 I: hr .1}... W 1. r. r21kl‘rfltr11 P ”I 11.1 1.1 11.11 JG. 1! EEEEEEEECEEC ThH P-Ytlc't C Ii- 117 codon of ORF] may be more than 2.4 kb upstream of the Q4499 insertion. Data bank searches using TFASTA and BLASTP showed that there is 32% identity between the deduced 355 amino acid sequence of ORF] shown in Figure 3.5 and NADH- putidaredoxin reductase, over a 279 amino acid overlap. NADH-putidaredoxin reductase is an enzyme in Pseudomonas putida, encoded by the camA gene (Koga et al., 1989). camA is a component of an operon that consists of three other genes, camB, which encodes putidaredoxin, (Koga et al., 1989), camC, which encodes a cytochrome P-450 (Koga et al., 1985), and camD which encodes S-exo-hydroxycamphor dehydrogenase (Koga etal., 1986). All four genes in this operon encode enzymes that are involved in the metabolism of camphor. ORF2 consists of 313 amino acids. The putative translation initiation codon of ORF2 (at 1171 bp) is preceded by a GGAGA sequence seven nucleotides upstream (Figure 3.5), which may serve as a ribosome binding site. The deduced amino acid sequence of ORF2 has 32% identity with two cytochrome P-450 proteins from Streptomyces griseolus, P-4505u1 and P-4505u2. The regions of identity consist of sequences that had previously been shown to be conserved between both P-450 proteins in S. griseolus and P-450 in P. putida (Omer et al., 1990). These include one of two regions (at positions 1855 to 1957, Figure 3.5) that form tat-helices involved in attaching and aligning the heme, and a proline residue (at position 1960) that terminates this helix. Additionally, two of the three conserved amino acids between P. putida and S. griseolus that were previously shown to be hydrogen bond donors to the proprionate groups of heme in the Pseudomonas P—450 are also present at the same positions in ORF2 (the two arginines at positions 1333 and 2059). Finally, the residues proposed to form part of the oxygen-binding site in P-450 are identical between the two P-450 proteins of S. griseolus and in the corresponding region of ORF2 (amino acids alanine, glycine, glutamic acid and threonine at positions 1900, 1903, 1909, and 1912, respectively). 118 The genes that encode P-4505u1 and 114505.12 in S. griseolus are components of operons that also encode ferredoxin and an additional less well-characterized ORF (Omer et al., 1990). Both P-450 and ferredoxin are involved in the metabolism of xenobiotics and it has been proposed that the third ORF may also encode an enzyme involved in the same metabolic pathway. By analogy, l speculate that 524499 is an insertion into an operon whose products include a reductase and a cytochrome P-450 enzyme involved in the metabolism of alternative carbon source. Sequences that resemble rho-independent transcriptional terminators or known bacterial promoters were not evident between ORFl and ORF2, supporting the idea that these genes are likely to be present in an operon whose transcriptional initiation site lies more than of 2.4 kb upstream of 94499. Discussion M. xanthus DNA extending 4.5 kb upstream of Tn5 lac Q4499 was sufficient to direct proper developmental lacZ expression upon integration at Mx8 art (Figure 3.1B). Unlike 94403 and other fusions that have an absolute requirement for C-signaling (chapter 2), expression of lacZ from fusions with 94499 upstream DNA at Mx8 att was not impaired. Perhaps expression of Q4499 upstream DNA at an earlier time during development and/or its partial (rather than absolute) C-signal dependency explain the ability of 94499 to escape inhibition of expression at Mx8 art. In chapter 2, it was proposed that an altered chromosomal state of DNA integrated at Mx8 at: might affect gene expression. If this model is correct, perhaps activation of Q4499 transcription occurs early in development and independent of C—signalin g prevents the altered chromosomal state from forming. Deletion analyses showed that a promoter element(s) required for developmentally regulated expression of 94499 is present more than 2.1 kb upstream 119 (Figure 3.2). This promoter element or elements could be an upstream activation sequence (UAS) recognized by a DNA-binding activator protein and/or a promoter recognized by RNA polymerase. The latter possibility is supported by the finding that the 3.0 kb region between 1.5 and 4.5 kb upstream of 94499 also directed developmental lacZ expression, albeit at a later time and at a lower level compared to the Tn5 lac Q4499 insertion—containing strain, DK4499 (Figure 3.2). Evidence for a UAS located between 4.5 and 3.2 kb upstream of (24499 comes from the finding that deleting this region reduced developmental lacZ expression by 35% (Figure 3.2). However, 3.2 kb of upstream DNA is sufficient for C-signal dependent expression of (24499, since the 3.2 kb segment in wild-type cells directs 65% as much expression as observed for DK4499 (Figure 3.2), but in csgA mutant cells it directs only 10-20% as much expression (Figure 3.3). Northern blot analyses with probes proximal to the Q4499 insertion site detected a transcript of 3.5 kb that was more abundant in deve10ping cells than in growing cells. If this RNA is the Q4499 transcript, its presence in growing cells is surprising since expression from Tn5 lac 94499 is very low (about the background level) in growing cells. Perhaps the fusion mRNA from the Tn5 lac insertion is less stable than the native Q4499 mRNA. Probes corresponding to DNA between 2.1 and 1.5 kb upstream of 94499 failed to detect the 3.5 kb transcript. This could mean that transcription starts within 1.5 kb of the insertion point; however, two results suggest otherwise. First, DNA between 4.5 and 1.5 kb upstream of Q4499 did exhibit promoter activity (Figure 3.2). Second, analysis of the upstream DNA sequence identified two ORFs that are likely to be cotranscribed based on analogy with other organisms, and the upstream ORF extends beyond 2.1 kb upstream of 04499. If transcription does start more than 2.1 kb upstream of Q4499, perhaps the transcript is unstable. The 3.5 kb transcript could be a breakdown product of a larger transcript that has lost sequences at its 5’ end. The two ORFs upstream of 94499 are separated by 102 bp (Figure 3.5). This 120 separation is rather large for genes in an operon, but separation of cotranscribed genes by as many as 93 bp has previously been reported (Adams et al., 1988). Transcriptional termination sequences or sequences that resemble known bacterial promoters are not evident within the 102 bp intergenic region, though, of course such sequences could go unrecognized. Perhaps the strongest evidence that (24499 is inserted into an operon is the similarity of the deduced amino acid sequences of the two ORFs to proteins that are encoded by genes found in operons in other organisms. ORF] remains open up to the 5' end of the region for which both DNA strands have been sequenced and this partial ORF would encode a 355 amino acid peptide that is similar to NADH-putidaredoxin reductase from Pseudomonas putida. This enzyme is encoded by the camA gene, which is present in an operon that also encodes three other enzymes (Koga et al., 1989), putidaredoxin, cytochrome P-450, and 5-exo-hydroxycamphor-dehydrogenase. All four enzymes are involved in the metabolism of D—camphor, which can serve as the sole carbon source for growth of P. putida. ORF2 would encode 313 amino acids and remains open downstream to the point of fusion with Q4499. The deduced amino acid sequence of ORF2 is similar to two cytochrome P-4505 from Streptomyces griseolus, P-4505u1 and P-4505u2. The genes that encode both cytochrome P-4505, suaC and subC, are components of two different operons. These operons also encode ferredoxin (Patel & Omer, 1992). Both cytochrome P-450 and ferredoxin are involved in the metabolism of sulfonylurea herbicide (Patel & Omer, 1992; Omer et al., 1990). A third ORF that is less well-characterized has also been identified in these operons (Patel & Omer, 1992), and is thought to encode enzymes that may be involved in the same metabolic pathway. Interestingly, both cytochrome P-4508 from S. griseolus have high similarity to the cytochrome P-450 from P. putida encoded by camC, which is also similar to ORF2. The initial oxidation of both D-camphor by P. putida and sulfonyl urea compounds by S. griseolus involves a monooxygenase system that is catalyzed by NADH- 121 putidaredoxin reductase, putidaredoxin, and cytochrome P-450 in P. putida and by ferredoxin and cytochrome P-450 in S. griseolus. Monooxygenases catalyze oxygen- dependent reactions that oxidize many organic compounds that are used in growth. This system requires an oxidizable cosubstrate which, in many bacteria, is provided by the reduced form of cytochrome P-450. Typically, enzymes catalyze this reaction according to the equation: monooxygenase substrate—H + 02 + All; --------------- > substrate-OH + A + H20 in which AHz is an oxidizable cosubstrate (Gottschalk, 1986). This system is used in a many different metabolic pathways such as those involved in metabolizing long chain hydrocarbons and ammonia (Gottschalk, 1986), and is present in a diverse group of microorganisms including fiavobacteria, mycobacteria, yeast and fungi (Gottschalk, 1986). By analogy with the Operons in P. putida and S. griseolus, it seems highly likely that 94499 is inserted into an operon encoding enzymes that catalyze a monooxygenase reaction(s). The induction of this operon during development suggests that M. xanthus may induce alternative metabolic pathways to scavange scarce nutrient sources. For example, compounds released by lysin g cells within fruiting bodies may serve as carbon source for the completion of the developmental process in sporulatin g cells. Since cells containing the 94499 insertion showed no obvious developmental defects (Kroos et al., 1986), the putative monooxygenase reaction catalyzed by enzymes encoded by the 94499 operon does not appear to be essential for normal development. However, it is possible that other enzymes can substitute for these deficiencies. In this respect, it will be interesting to determine whether the M. xanthus genome has other genes that cross- hybridize with genes in the 94499 Operon. Mutations in the 524499 operon could have subtle effects on sporulation efficiency, or effects on spore resistance or germination properties that are not evident upon visual inspection. Summary and Perspectives 122 123 The aim of the studies presented in this thesis was to characterize the DNA elements involved in regulating the expression of two developmentally regulated transcription units whose expression is modulated by extracellular C—signaling. Q4403 appears to be inserted into a gene that encodes a serine protease. Sequences between 72 and 80 bp upstream of the transcriptional initiation site were required for the transcription of this gene in a developmentally regulated manner. DNA extending to 80 bp upstream of the transcriptional start site was sufficient for expression in a C-signal-dependent fashion. The requirement for DNA more than about 50 bp upstream of the Q4403 transcriptional start site suggests that a protein(s) Other than RNA polymerase may activate transcription from the 04403 promoter. C~signaling may regulate expression of the Q4403 gene by modulating (directly or indirectly) either the activity of the putative activator protein or the RNA polymerase that transcribes this gene. The presence of an activator protein that associates with the -80 to -72 region can be examined by looking for proteins that bind to this region in vitro. Analysis of protein extracts from wild-type and csgA deve10pin g cells can be used to determine whether C- signaling affects the expression of this putative DNA-binding protein. In vitro reconstitution experiments would need to be done to demonstrate the role of this protein in activating transcription. An alternative approach is to mutagenize the Q4403 promoter, then look for suppressor mutations that restore expression. Expression of the 94403 gene is sensitive to its position in the chromosome. At the Mx8 phage attachment (Mx8 att) site, expression from the cloned 17 kb upstream DNA is decreased by about 45% compared to expression from the same region at the native site. We hypothesize that the chromosomal structure in the vicinity of the Mx8 at site may change as cells progress through development. The chromosome clearly condenses during B. subtilis sporulation (Setlow et al., 1991). The effect of chromosomal position on expression of the (24403 promoter could be tested further by constructing plasmids 124 containing Q4403 upstream DNA fused to lacZ that are capable of integrating at different positions in the M. xanrhus chromosome. UnderStanding the mechanism of C~signal- dependent inhibition of gene expression is potentially interesting and might be amenable to a genetic approach involving higher expression from the 94403 promoter at Mx8 att after mutagenesis. The analysis of DNA upstream of Tn5 lac Q4499 suggests that it is inserted into a gene that is a component of a developmentally regulated Operon. Two ORFs were identified in the 2.1 kb DNA upstream of Q4499. Since sequence analysis of the region between these ORFs failed to identify transcription termination or initiation signals, it appears that these genes are cotranscribed. Based solely on the sequence analysis, it is not possible to rule out models in which each ORF is transcribed from its own promoter, or the idea that the operon has an internal promoter. This question may be difficult to resolve since the Northern blot data suggest that the 94499 transcript may be unstable. An immediate goal is to extend the sequence farther upstream. This will provide information about the location of the likely ORF] translational start and the presence or absence of additional ORFs that may be part of the operon, and will guide additional deletion analysis of a putative promoter currently thought to be located between 3.2 and 2.1 kb upstream of 94499. It is interesting that both of the developmentally regulated fusions were with genes that may be involved in catabolism. The serine protease encoded by the 94403 gene, may be involved in catabolizing proteins and peptides during developmentgmd cytochrome P- 450 and NADH-dependent reductase could be involved in metabolizing organic compounds that could serve as carbon sources. The expression of these enzymes during starvation of- M. xanthus cells may indicate mechanisms used by starving cells to complete spore formation by utilizing carbon from a variety of sources. The absence of a phenotypic defect in cells harboring insertions in these genes may 125 indicate that there are other systems that are turned on during development that compensate for these defects. APPENDICES Appendix 1 126 127 Figure 1. Detection of the (24403 native message using Northern blot analysis. RNA was isolated from 24 hour developing cells and from vegetatively growing cells as described in Chapter 2. Northern blotting was performed as described previously (Ausubel et al., 1989). RNA was fractionated on a 0.6% agarose gel containing formaldehyde (1.1% v/v), transferred onto a nitrocellulose membrane (Schleicher and Schuell), and baked for two hours at 80°C. Prehybridization was performed at 42°C for 2-3 hours in a solution containing 50% formamide, 6X SSPE, 0.5% SDS, 5X Denhardt’s reagent, and 100 ug/ml sheared salmon sperm DNA. Hybridization was performed at 52'C in a fresh solution of prehybridization buffer containing 1-2 x 107 CPM of [a32P]dCFP labeled probes synthesized from the HaeIl-Haell fragment at ~900 to 520 bp upstream of BamHI and from the RsaI-BamHI fragment ( 451 bp upstream of BamHI) isolated from pMF01. Probes were synthesized using a random priming kit from Stratagene according to the manufacturers specifications. The numbers on the left indicate the molecular weight of . single stranded size markers (Bethesda Research Laboratories, Inc.). The RsaI-BamI-II fragment detected a transcript of about 1.5 kb in RNA isolated from DK4499 cell that were developed for 24 hours but not in the vegetative RNA. More than two faint transcripts were detected in the RNA isolated from 24 hour developing DK4368 cells. No transcripts were detected with the HaeII-HaeII fragment in RNA isolated from either strain (data not shown). 128 Hall HaeII Rsal BamHI I I l I Tn5 lac Q4403 Wild-type 024024 2.4 1.4 Appendix 2 129 130 Figure 2A. Chromosomal structure of M. xanthus containing a pREGl666-derived plasmid at Mx8 art. Strains containing a single copy of the integrated plasmid are identified by the 2.0 and 10.0 kb flanking fragments upon digestion with EcoRI and X hoI, and these fragments are detected by a probe corresponding to the attP fragment. Unintegrated plasmid or strains containing multiple copies of integrated plasmid are identified by a 4.0 kb band. 131 attB attP E°°R' Xh°' attP mm m 9.38,,” 12$— 1 l 2 kb 10 kb 132 Figure 28. DNA was isolated as described previously (Avery & Kaiser, 1983) from vegetatively growing MMF31-7 cells and from MMF31-7 cells that had developed for 24 hours on MC7 agarose plates (10 mM morpholine-propanesulfonic acid; pH7.0, 1 mM CaClg, 1% agarose). 4-20 pg of DNA was digested with EcoRI and Xhol, fractionated on a 0.5% agarose gel. Transfer of the gel onto a Nylon membrane, pehybridization, and hybridization were carried out as described previously (Avery & Kaiser, 1983). A 3.0 kb attP fragment (isolated as described in Chapter 2) was radioactively labeled with [a32P]dC1‘ P using a random printing kit from Stratagene, and 1-2 x107 CPM was used for hybridization. 133 Vegetative Developmental m: cm mam mac m: cm mum wzv 3 2 .7 9.4 6.6 4.4 2.0 Appendix 3 134 135 Table l. Sporulation efficiency of Myxococcus xamhus strains Myxococcus xanthus strains Sporulation efficiency a DK1622 0.3 DK4368 0.1 MMF31-7 0.1 8 Spore titers were deterrrrined three days after development using the procedure of Kroos and Kaiser (Kroos & Kaiser, 1987), except that cells were spotted onto TPM (10 mM Tris, pH 8.0, 1 mM KH2P04, 8 mM M gSO4, final pH 7.6) agar plates (1.5 % Bacto-agar). Sporulation efficiency is presented as a percentage of the number of cells initially plated. BIBLIOGRAPHY 136 137 Bibliography Adams, C. W., Fornwald, J. A., Schmidt, J. F., Rosenberg, M. & Brawner, M. E. (1988). Gene organization and structure of Streptomyces lividans gal operon. J. Bacteriol., 170, 203-212. Apelian, D. & Inouye, S. (1990). Development-specific o-factor essential for late-stage differentiation of Myxococcus xanthus. Genes Dev., 4, 1396-1403. Apelian, D. & Inouye, S. (1993). A new putative sigma factor of Myxococcus xanthus. J. Bacteriol., 175, 3335-3342. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. & Struhl, K. (1989). In Current Protocols in Molecular Biology. eds), Pages, John Wiley & Sons, Place Published. Avery, L. & Kaiser, D. (1983). In situ transposon replacement and isolation of a spontaneous tandem genetic duplication. Mol. Gen. Genet, 191, 99-109. Baker, M. E. (1994). Myxococcus xanthus C-factor, a morphogenetic paracrine signal, is similar to Escherichia coli 3-oxoacyl-[acyl-carrier-protein] reductase and human 17B- hydroxysteroid dehydrogenase. Biochem. J., 301, 311-312. Baldus, J ., Green, B., Youngman, P. & Moran, C. J. (1994). Phosphorylation of Bacillus subtilis transcription factor SpoOA stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J. Bacteriol., 176, 296-306. Brandner, J. & Kroos, L. unpublished data. Brandner, J., White, J. & Kroos, L. unpublished data. Burton, 2., Gross, C., Watanabe, K. & Burgess, R. (1983). The operon that encodes the sigma subunit of RNA polymerase also encodes ribosomal protein 821 and DNA_ primase in E. coli K12. Cell, 32, 335-349. Busby, S. & Ebright, R. H. (1994). Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell, 79, 743-746. Butler, J. 8., Springer, M. & Grunberg-Manago, M. (1987). AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational auto control of its own gene (infC) in vivo. Proc. Natl. Acad. Sci. USA, 84, 4022-4025. Cheng, Y. & Kaiser, D. (1989a). dsg , a gene required for cell-cell interaction early in -- Myxococcus development. J. Bacteriol., 171, 3719-3726. Cheng, Y. & Kaiser, D. (1989b). dsg , a gene required for Myxococcus development, is necessary for cell viability. J. Bacteriol., 171, 3727-3731. Clark, H. R., Leigh, J. A. & Duglas, C. J. (1992). Molecular signals in the interactions between plants and microbes. Cell, 71, 191-199. 138 Clewell, D. B. & Weaver, K. E. (1989). Sex pheromones and plasmid transfer in Enterococcusfaecalis. Plasmid, 21, 175-184. Collado-Vides, J., Magasanik, B. & Gralla, J. D. (1991). Control site location and transcriptional regulation in Escherichia coli. Microbiol. Rev., 55, 371-394. Cumsky, M. G. & Zusman, D. R. (1979). Myxobacterial hemagglutinin: a development- specific lectin of Myxococcus xanthus. Proc. Natl. Acad. Sci. USA, 76, 5505-5509. Davis, J. M., Mayor, J. M. & Plamann, L. S. (1995). asgC 767 is an allele of the major sigma factor gene of Myxococcus xanthus. Absrr. of the 22nd Intl. Conf. on the Biol. of the Myxobacreria, 16. Davis, R., Botstein, D. & Roth, J. (1980). In Advanced bacterial genetics. eds), Pages, Cold Spring Harbor Laboratory Press, Place Published. Dillin, A. & Rine, J. (1995). On the origin of a silencer. Trends Biochem. Sci., 20, 231-235. Downard, J. & Kroos, L. (1993). In Transcriptional regulation of developmental gene expression in Myxococcus xanthus. (Dworkin, M.& Kaiser, D.s, eds), 183-199, American Society for Microbiology, Place Published. Downard, J ., Ramaswamy, S. V. & Kil, K. (1993). Identification of esg, a genetic locus involved in cell-cell signaling during Myxococcus xanthus development. J. Bacteriol., 175, 7762-7770. Downard, J. S., Kupfer, D. & Zusman, D. R. (1984). Gene expression during development of Myxococcus xanthus: analysis of the genes for protein S. J. Mol. Biol., 175, 469-492. Downard, J. S. & Zusman, D. R. (1985). Differential expression of protein S genes during Myxococcus xanthus development. J. Bacteriol., 161, 1146-1155. Dunny, G. M. (1990). Genetic functions and cell-cell interactions in the pheromone- inducible plasmid transfer system of Enterococcusfaecalis. Mol. Microbiol., 4, 689-696. Dworkin, M. & Kaiser, D. (1993). In Myxobacteria J]. eds), Pages, American Society for Microbiology, Place Published. Fawcett, T. & Bartlett, 8. (1990). An effective method for eliminating "artifact banding" when sequencing double-stranded DNA templates. Biotechniques, 9, 46-48. Fink, J. M. & Zissler, J. F. (1989). Defects in morility and development of Myxococcus xanthus lipopolysaccharide mutants. J. Bacteriol., 171, 2042-2048. Fisseha, M. & Kroos, L. unpublished data. Frasc‘h, S. C. & Dworkin, M. (1995). Tyrosine kinases with differential activity during development in Myxococcus xanthus. Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, 39. 139 Gill, R. E. & Bomemann, M. C. (1988b). Identification and characterization of the Myxococcus xanthus bsgA gene product. J. Bacteriol., 170, 5289-5297. Gill, R. E. & Cull, M. G. (1986). Control of developmental gene expression by cell-to- cell interactions in Myxococcus xanthus. J. Bacteriol., 168, 341-347. Gill, R. E., Cull, M. G. & Fly, S. (1988a). Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus. J. Bacteriol., 170, 5279-5288. Gill, R. E., Karlok, M. & Benton, D. (1993). Myxococcus xanthus encodes an ATP- dependent protease which is required for developmental gene transcription and intercellular signaling. J. Bacteriol., 175, 4538-4544. Gilman, M. & Chamberlin, M. J. (1983). DevelOpmental and genetic regulation of Bacillus subtilis genes transcribed by 628-RNA polymerase. Cell, 35, 285-293. Gold, L., Storrno, G. & Saunders, R. (1984). Escherichia coli translational initiation factor IF3: a unique case of translational regulation. Proc. Natl. Acad. Sci. USA, 81, 7061-7065. Gottschalk, G. (1986). In Bacterial Metabolism, 359 Pages, Springer-Verlag, Place Published. Grossman, A. D. & Losick, R. (1988). Extracellular control of spore formation in Bacillus subtilis. Proc. Nat. Acad. Sci. USA, 85, 4369-4373. Guespin-Michel, J ., Letouvet-Pawlak, B. & Petit, F. (1993). In Protein secretion in myxobacteria. (Dworkin, M.& Kaiser, D.s, eds), 235-255, American Society for Microbiology, Place Published. Guo, D., Bowden, G. & Kaplan, H. B. (1995). Analysis of the Myxococcus xanthus sasA locus a negative regulator of an A-signal-dependent gene. Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, l9. Hagen, D. C., Bretscher, A. P. & Kaiser, D. (1978). Synergism between morphogenetic mutants of Myxococcus xanthus. Dev. Biol., 64, 284-296. Hagen, T. J. & Shimkets, L. J. (1990). Nucleotide sequence and transcriptional products of the csg locus of Myxococcus xanthus. J. Bacteriol., 172, 15-23. gialdenwang, W. G. (1995). The sigma factors of Bacillus subtilis. Microbiol. Rev., 9, 1-30. Hao, T. & Kroos, L. unpublished data. Hartzell, P. & Kaiser, D. (1991). Function of MglA, a 22-kilodalton protein essential for gliding in Myxococcus xanthus. J. Bacteriol., 173, 7615-7624. Helmann, J. (1995). Compilation and analysis of Bacillus subtilis (SA-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res., in press, 140 Herskowitz, I. (1989). A regulatory heirarchy for cell specialization in yeast. Nature, 342, 749-757. Hodgkin, J. & Kaiser, D. (1977). Cell-to-cell stimulation of motility in nonmotile mutants of M. xanthus. Proc. Natl. Acad. Sci. USA, 74, 2938-2942. Hodgkin, J. & Kaiser, D. (1979b). Genetics of Gliding Motility 1n Myxococcus xanthus (Myxobacterales): Two Gene Systems Control Movement. Mol. Gen. Genet, 171, 177- 191. Igo, M. M. & Losick, R. (1986). Regulation of a promoter region that is utilized by minor forms of RNA polymerase holoenzyme in Bacillus subtilis. J. Mol. Biol., 191, 615-624. Inouye, M., Inouye, S. & Zusman, D. R. (1979a). Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus. Proc. Natl. Acad. Sci. USA, 76, 209—213. Inouye, M., Inouye, S. & Zusman, D. R. (1979b). Gene expression during development of Myxococcus xanthus: pattern of protein synthesis. Dev. Biol., 68, 579-591. Inouye, S. (1990). Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus. J. Bacteriol., 172, 80-85. Inouye, S., Franceschini, T. & Inouye, M. (1983a). Structural similarities between the development-specific protein S from a Gram-negative bacterium, Myxococcus xanthus, and calmodulin. Proc. Natl. Acad. Sci. USA, 80, 6829-6833. Inouye, S., Ike, Y. & Inouye, M. (1983b). Tandem repeat of the genes for protein S, a development-specific protein of Myxococcus xanthus. J. Biol. C hem, 258, 38-40. Jacobs, M., Eliasson, M., Uhlen, M. & Flock, J. I. (1985). Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res., 13, 8913-8926. Kaiser, D. (1986). Control of multicellular development: Dictyostelium and Myxococcus. Ann. Rev. Genet, 20, 539-566. Kaiser, D. & Crosby, C. (1983). Cell movement and its coordination in swarms of Myxococcus xanthus. Cell Motil., 3, 227-245. Kaiser, D. & Kroos, L. (1993). In Intercellular Signaling. (Dworkin, M.& Kaiser, D.s, eds), 257-284, American Society for Microbiology, Place Published. Kaiser, D. Kroos, L. & Kuspa, A. (1985). In Cell interactions govern the temporal pattern of Myxococcus development. eds), 823- 830, Cold Spring Harbor Laboratory, Place Published. Kalman, L. V., Cheng, Y. & Kaiser, D. (1994). The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 In vivo. J. Bacteriol., 176, 1434-1442. 141 Kaplan, H. B., Kuspa, A. & Kaiser, D. (1991). Suppressors that permit A-signal- independent development gene expression in Myxococcus xanthus. J. Bacteriol., 173, 1460-1470. Keseler, I. M. & Kaiser, D. (1995). The role of 054 in transcription of the developmentally regulated gene 4521 . Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, 20. Kil, K.-S., Brown, G. & Downard, J. (1990). A segment of Myxococcus xanthus ops DNA functions as an upstream activation site for tsp gene transcription. J. Bacteriol., 172, 3081-3088. Kim, S. K. (1991). Intercellular signaling in Myxococcus development: The role of C- factor. Trends in Genet, 7, 361-365. Kim, S. K. & Kaiser, D. (1990a). Cell alignment required in differentiation of Myxococcus xanthus. Science, 249, 926-928. Kim, S. K. & Kaiser, D. (1990b). Cell motility is required for the transmission of C- factor, an intercellular signal that coordinates fruiting body morphogenesis of Myxococcus xanthus. Genes Dev., 4, 896-905. Kim, S. K. & Kaiser, D. (1990a). C-factor: a cell-cell signaling protein required for fruiting body morphogenesis of M. xanthus. Cell, 61, 19-26. Kim, S. K. & Kaiser, D. (1990b). Purification and properties of myxococcus xanthus C- factor, an intercellular signaling protein. Proc. Natl. Acad. Sci. USA, 87, 3635-3639. Kim, S. K. & Kaiser, D. (1991). C—factor has distinct aggregation and sporulation thresholds during Myxococcus development. J. Bacteriol., 173, 1722-1728. Koga, H., Aramaki, H., Yamaguchi, E., Takeuchi, K., Horiuchi, T. & Gunsalus, I. C. (1986). camR, a negative regulator locus of the cytochrome P-450 cam hydrolyase operon. J. Bacteriol., 166, 1089-1095. Koga, H., Rauchfuss, B. & Gunsalus, I. C. (1985). P-450cam gene cloning and expression in Pseudomonas putida and Escherichia coli. Biochem. Biophys. Res. Commun., 130, 412-417. Koga, H., Yamaguchi, E., Matsunaga, K., Aramaki, H. & Horiuchi, T. (1989). Cloning and nucleotide sequence of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Psedomonas putida. J. Biochem., 106, 831-836. Kroos, L. & Cutting, S. (1994). In Intercellular and intercompartmental communication during Bacillus subtilis sporulation. (Pi ggot, P. J., C.P. Moran, J .& Youngman, P.s, eds), 155-180, Amer. Soc. for Microbiol., Place Published. Kroos, L., Hartzell, P., Stephens, K. & Kaiser, D. (1988). A link between cell movement and gene expression argues that modlity is required for cell-cell signaling during fruiting body development. Genes Dev., 2, 1677-1685. 142 Kroos, L. & Kaiser, D. (1984). Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into M yxocaccus xanthus. Proc. Natl. Acad. Sci. USA, 81, 5816-5820. Kroos, L. & Kaiser, D. (1987). Expression of many developmentally regulated genes in Myxococcus depends on a sequence of cell interactions. Genes Dev., 1, 840-854. Kroos, L., Kuspa, A. & Kaiser, D. (1986). A global analysis of developmentally regulated genes in Myxococcus xanthus. Dev. Biol., 117, 252-266. Kuspa, A. & Kaiser, D. (1989). Genes required for developmental signaling in Myxococcus xanthus: three asg loci. J. Bacteriol., 171, 2762-2772. Kuspa, A., Kroos, L. & Kaiser, D. (1986). Intercellular signaling is required for developmental gene expression in Myxococcus xanthus. Dev. Biol., 117, 267-276. Kuspa, A., Plamann, L. & Kaiser, D. (1992a). Identification of heat-stable A-factor from Myxococcus xanthus. J. Bacterial, 174, 3319-3326. Kuspa, A., Plamann, L. & Kaiser, D. (l992b). A-si gnallin g and the cell density requirement for Myxococcus xanthus development. J. Bacteriol., 174, 7360-7369. LaRossa, R., Kuner, J., Hagen, D., Manoil, C. & Kaiser, D. (1983). Developmental cell interactions of Myxococcus xanthus: analysis of mutants. J. Bacteriol., 153, 1394-1404. Laue, B. E. & Gill, R. (1994). Use of a phase variation—specific promotor of Myxococcus xanthus in a strategy for isolating a phase-locked mutant. J. Bacteriol., 176, 5341-5349. Lee, B., Lee, K., Robles, J. & Shimkets, L. J. (1995a). Developmental C-signaling of Myxococcus xanthus involves an extracellular NAD(P)-containin g protein. Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, 24. Lee, B.-U., Lee, K., Robles, J. & Shimkets, L. (1995b). A tactile sensory system of Myxococcus xanthus involves an extracellular NAD(P)+-containin g protein. Genes Dev., submitted, Lee, K. & Shimkets, L. (1994). Cloning and characterization of the sacA locus which restores development to Myxococcus xanthus C-signaling mutants. J. Baoterial., 176, 2200-2209. Lee, K. & Shimkets, L. (1995). Suppression of a signaling defect during Myxococcus xanthus development. J. Bacteriol., submitted, Lee, Y.-C., Miyata, Y., Terada, I., Ohta, T. & Matsuzawa, H. (1991). Involvement of NHz-terminal pro—sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli. Agric. Biol. Chem, 55, 3027-3032. Li, S.-F., Lee, B. & Shimkets, L. J. (1992). csgA expression entrains Myxococcus xanthus development. Genes Dev., 6, 401-410. Li, S.-F. & Shimkets, L. J. (1988). Site-specific integration and expression of a 143 developmental promoter in Myxococcus xanthus. J. Bacteriol., 170, 5552-5556. Li, S.-F. & Shimkets, L. J. (1993). Effect of dsp mutations on the cell-to-cell ' transmission of ngA in Myxococcus xanthus. J. Bacteriol., 175, 3648-3652. Lisser, S. & Margalit, H. (1993). Compilation of E. coli mRNA promoter sequences. Nucleic Acids Res., 21, 1507-1516. MacNeil, S. D., Calara, F. & Hartzell, P. (1994). New clusters of genes required for gliding motility in Myxococcus xanthus. Molec. Microbiol., 14, 61-71. McCleary, W. R., Esmon, B. & Zusman, D. R. (1991). Myxococcus xanthus protein C is a major spore surface protein. J. Bacteriol., 173, 2141-2145. Munoz-Dorado, J ., Inouye, S. & Inouye, M. (1991). A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a Gram-negative bacterium. Cell, 67, 995-1006. Nelson, D. & Zusman, D. (1983). Evidence for long-lived mRNA during fruiting body formation in Myxococcus xanthus. Proc. Natl. Acad. Sci. USA, 80, 1467-1471. O'Connor, K. A. & Zusman, D. R. (1989). Patterns of cellular interactions during fruiting-body formation in Myxococcus xanthus. J. Bacreriol, 171, 6013-6042. Omer, C. A., Lenstra, R., Litle, P. J., Dean, C., Tepperman, J. M., Leto, K. J., Romesser, J. A. & O'Keef, D. P. (1990). Genes for two herbicide-inducible cytochrome P-450 from Streptomyces griseolus. J. Bacteriol., 172, 3335-3345. Pabo, C. O. & Sauer, R. T. (1992). Transcription factors: structural families and principles of DNA recognition. Annu. Rev. Biochem., 61, 1053-1095. Parkinson, J. S. (1993). Signal transduction schemes of bacteria. Cell, 73, 857-871. Patel, N. V. & Omer, C. A. (1992). Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabloizing cytochromes P-450 in Streptomyces griseolus. Gene, 112, 67-76. Persson, B., Krook, M. & Jomvall, H. (1991). CharacteriStics of short-chain alcohol dehdrogenases and related enzymes. Eur. J. Biochem., 200, 537-543. Plamann, L., Davis, J ., Cantwell, B. & Mayor, J. (1994). Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus. J. Bacteriol, 176, 2013-2022. Plamann, L., Kuspa, A. & Kaiser, D. (1992). Proteins that rescue A-signal-defective mutants of Myxococcus xanthus. J. Bacteriol, 174, 3311-3318. Plamann, L., Li, Y., Cantwell, B. & Mayor, J. (1995). The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development. J. Bacteriol., 177, 2014-2020. Ptashne, M. (1987). In A genetic switch, Pages, Cell press and Blackwell scientific. 144 Pugsley, A. (1993). The complete general secretory pathway in gram-negative bacteria. Microbial. Rev., 57, 50-108. Rhie, H.-G. & Shimkets, L. J. (1991). Low-temperature induction of Myxococcus , xanthus developmental gene expression in wild-type and csgA suppressor cells. J. Bacteriol., 173, 2206-2211. Romeo, J. M. & Zusman, D. R. (1991). Transcription of the myxobacterial hemagglutinin gene is mediated by a 054-like promoter and a cis-acting upstream regulatory region of DNA. J. BaCterial., 173, 2969-2976. Romeo, J. M. & Zusman, D. R. (1992). Determinants of an unusually stable mRNA in the bacterium Myxococcus xanthus. Mol. Microbiol., 6, 2975-2988. Rosenberg, E., Keller, K. & Dworkin, M. (1977). Cell density-dependent growth of Myxococcus xanthus on casein. J. Bacteriol., 129, 770-777. Rosenbluh, A. & Rosenberg, E. (1989). Autocide AMI rescues development in dsg mutants of Myxococcus xanthus. J. Bacteriol., 171, 1513-1518. Rudner, D. 2., LeDeaux, J. R., Ireton, K. & Grossman, A. D. (1991). The spaOK locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J. Bacteriol., 173, 1388-1398. Sager, B. & Kaiser, D. (1993a). Spatial restriction of cellular differentiation. Genes Dev., 7, 1645-1653. Sager, B. & Kaiser, D. (1993b). Two cell-density domains within the Myxococcus xanthus. Proc. Natl. Acad. Sci. USA, 90, 3690-3694. Sager, B. & Kaiser, D. (1994). Intercellular C-signaling and the traveling waves of Myxococcus. Genes Dev., 8, 2793-2804. Sambrook, J., Fritsch, E. & Maniatis, T. (1989). In Molecular Cloning. eds), Pages, Cold Spring Harbor Laboratory Press, Place Published. Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain- terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Setlow, B., Magill, P., Febbroriello, P., Nakhimovsky, L., Koppel, D. & Setlow, P. (1991). Condensation of the forespore nucleoid early in sporulation of Bacillus species. J. Bacteriol., 173, 6270-6278. Shimkets, L. J. personal communication. Shimkets, L. J. (1990). Social and developmental biology of the myxobacteria. Microbiol. Rev., 54, 473-501. Shimkets, L. J. & Asher, S. J. (1988). Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus. Mol. Gen. Genet, 211, 63-71. Shimkets, L. J ., Gill, R. E. & Kaiser, D. (1983). Developmental cell interactions in 145 Myxococcus xanthus and the spoC locus. Proc. Natl. Acad. Sci. USA, 80, 1406-1410. Shimkets, L. J. & Kaiser, D. (1982a). Induction of coordinated movement of Myxococcus xanthus cells. J. Bacteriol., 152, 451-461. Shimkets, L. J. & Kaiser, D. (1982b). Murein components rescue developmental sporulation of Myxococcus xanthus. J. Bacteriol., 152, 462-470. Shimkets, L. J. & Rafiee, H. (1990). ngA, an extracellular protein essential for myxocaccus xanthus development. J. Bacteriol., 172, 5299-5306. Simons, R., Houman, F. & Kleckner, N. (1987). Improved single and multicopy lac- based cloning vectors for protein and operon fusions. Gene, 53, 85-96. Singer, M. & Kaiser, D. (1995). Ectopic production of guanosine penta-and tetraphosphate can initiate early developmental gene expression in Myxococcus xanthus. Genes and Dev., 9, 1633-1644. Sodergren, E. & Kaiser, D. (1983). Insertions of Tn5 near genes that govern stimulatable cell motility in Myxococcus. J. Mol. Biol., 167, 295-310. Stellwag, E., Fink, J. M. & Zissler, J. (1985). Physical characterizatin of the genome of the Myxococcus xanthus bacteriophage MX-8. Mol. Gen. Genet, 199, 123-132. Stephens, K., Hartzell, P. & Kaiser, D. (1988). Gliding motility in Myxococcus xanthus :mgl locus, RNA and predicted protein products. J. Bacteriol., 171, 819-830. Stephens, K. & Kaiser, D. (1987). Genetics of gliding motility in Myxococcus xanthus: molecular cloning of the mgl locus. Mol. Gen. Genet, 207, 256-266. Sternberg, P. W. & Horvitz, H. R. (1989). The combined action of two intercellular signaling pathways specifies three cell fates during vulval induction in C. elegans. Cell, 58, 679-693. Stock, J. B., Stock, A. M. & Mottonen, J. M. (1990). Signal transduction in bacteria. Nature, 344, 395-400. Teintze, M., Thomas, R., Furuichi, T., Inouye, M. & Inouye, S. (1985). Two homologous genes coding for spore-specific proteins are expressed at different times during development of Myxococcus xanthus. J. Bacteriol., 163, 121-125. Terada, 1., Kwon, S., Miyata, Y., Matsuzawa, H. & Ohta, T. (1990). Unique precursor structure of an extracellular protease, aqualysin I, with NH;- and COOH-terrninal pro- sequences and its processing in Escherichia coli. J. Biol. Chem, 265, 6576-6581. Thony-Meyer, L. & Kaiser, D. (1993). devRS, an autoregulated and essential genetic locus for fruiting body development in Myxococcus xanthus. J. Bacteriol., 175, 7450- 7462. Toal, D. R., Clifton, S., Roe, B. & Downard, J. (1995). The esg locus of Myxococcus xanthus encodes the E1 or and E113 subunits of a branced-chain keto acid dehydrogenase. Mol. Microbiol., 16, 177-189. 146 Udo, H., Munoz-Dorado, J., Inouye, M. & Inouye, S. (1995). Myxococcus xanthus, a Gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks secretion of B—lactamase by phosphorylation. Genes Dev., 9, 972-983. Weinberg, R. A. & Zusman, D. R. (1990). Alkaline, acid and neutral phosphatase activities are induced during development in Myxococcus xanthus. J. Bacteriol., 172, 2294-2302. Willey, J., Schwedock, J. & Losick, R. (1993). Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces caelicalor. Genes and Dev., 7, 895-903. Yang, C. & Kaplan, H. (1995). The Myxococcus xanthus wild-type sasB7 gene encodes a member of the histidine kinase family. Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, 53. Yanisch-Perron, C., Vieria, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences M13 mp18 and pUC19 vectors. Gene, 33, 103-1 19. York, K., Kenney, T., Satola, S., Moran, C. J., Poth, H. & Youngman, P. (1992). SpoOA controls the (SA-dependent activation of Bacillus subtilis sporulation-specific transcription unit spoIIE. J. Bacteriol., 174, 2648-2658. Zhang, W., Inouye, M. & Inouye, S. (1995). Reciprocal roles of two protein Ser/Thr kinase genes, pkn5 and pkn6 in Myxococcus xanthus development that are diversely located on the chromosome with a 46 bp distance. Abstr. of the 22nd Intl. Conf. on the Biol. of the Myxobacteria, 41. HICHIGRN STRTE UNIV. LIBRRRIES 11111111|||llllllllllll|11111111111lllllllHI 31293014107381