..:.... I... p ., nu -.. g .’ ) lllllllllllllllllHIHHHIUIIII!!!”lllllllllllllllllllllll 293 01402 7811 A \D “xii \1 This is to certify that the dissertation entitled Genetic and physiological characterization of a selenite-resistance determinant from an F-like plasmid of Stenotrophomonas maltophilia OROZ presented by Jonathan James Cagu iat has been accepted towards fulfillment of the requirements for Ph.D. Microbiology degree in Wt professor Juliu Jackson Date 9/22/95 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 fi_ ___-_.____.__ -, a _ ._____._ ‘ _. ___.__ LIBRARY Michigan State University PLACE N RETURN BOX to remove We checkout from your record. TO AVOID FINES return on or More dete due. DATE DUE DATE DUE DATE DUE MSU I. An Afflmetlve WOMEN-l OM 1m WM! GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF A SELENITE- RESISTANCE DETERMINANT FROM AN F-LIKE PLASMID OF STENOTROPHOMONAS MALTOPHILIA OROZ By Jonathan James Caguiat A DISSERTATION Submitted to Michigan State Univesity in partial fufillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1995 be in ABSTRACT GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF A SELENITE- RESISTANCE DETERMINANT FROM AN F-LIKE PLASMID OF STENOTROPHOMONAS MALTOPHILIA OROZ By Jonathan James Caguiat Stenotrophomonas maltophilia OROZ displayed growth resistance to several heavy metal salts when it was first isolated fiom mercury contaminated soil. Transformation of Escherichia coli HBlOl with genomic DNA from S. maltophilia OROZ yielded transformants containing pORl, a 100 kb plasmid that conferred resistance to Se(IV), Pb(II) and Hg(II). Cloning of HindIII fragments from pORl into pBR322 revealed that selenite-resistance was encoded by a 4 kb fragment in the recombinant plasmid, pLJ 100. Southern hybridizations of cloned pORl fragments to blots of agarose electrophoretic gels containing digestions of pORl resolved 60% of the physical map of pORl. Double restriction endonuclease analysis located the position of the uncloned fragments on the map. Transcription and translation in an in vitro expression system showed that the 4 kb HindIH fragment encoded a 35 kDa polypeptide. The nucleotide sequence of this fragment revealed that it contained a 2.2 kb segment identical to transposon, Tn1000; a 400 bp segment identical to repZA from repFIC; and open reading frames for a 3.7 kDa hypothetical polypeptide and a 35 kDa polypeptide not associated with Tn1000. The sequence of 15 N—terminal amino acid residues from the purified 35 kDa polypeptide matched the amino acid sequence of the 35 kDa open reading frame. Amino acid sequence comparison of this new selenite dissimilatory reduction polypeptide, SedR, to other known sequences did not reveal a relationship that implied its function. Growth experiments demonstrated that the selenite-resistant strains reduced selenite to eler thic vitl that cluti it enha it’dUi induc selem seleni elemental selenium. Earlier experiments suggested that glutathione, glutathione reductase, thioredoxin, and thioredoxin reductase may be involved in this pathway. Experiments with E. coli mutants for glutathione and thioredoxin biosynthesis and reduction showed that the wild type strain, which was already resistant to selenite, depended upon glutathione and thioredoxin reductase. The recombinant plasmid, pLJ 100, increased the enhanced resistance of the wild type and the mutant strains to selenite. The 4 kb insert may influence selenite resistance by encoding a redoxin that is reduced by other reductases, producing a reductase that reduces other redoxins or encoding a regulator that induces some other selenite-resistance pathway. In this study, I located the position of selenite-resistance on the physical map of pORl , sequenced a 4 kb fragment that conferred selenite-resistance and identified a 35 kDa polypeptide encoded by this fragment. Dedications To my wife, Tani Spielberg, and to my parents, Carlos and Julianna Caguiat. iv -—-e _—— ‘— - - _. _ Inc Dr. . degrt Dr P QUCSl Dr.) Atlar My ( Rich: Mr. ' Um. Acknowledgments I would like to thank all the people I worked with as a graduate student: Dr. Julius H. Jackson, my mentor, for giving me support and guidance to achieve my degree, Dr. Patricia A. Herring for her eagerness always to help me solve a problem or answer a question, Dr. Michael A. Winrow for teaching me new molecular techniques, Dr. Loren R. Snyder for providing me with space in his lab for a year while Julius was in Atlanta, My other committee members, Dr. Michael Bagdasan'an, Dr. Craig S. Criddle, and Dr. Richard C. Schwartz for their advice and Mr. Tony B. Grifin for helping me adjust to Atlanta during my year visit to Clark Atlanta University. LISl Ml MAT TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... ix LIST OF FIGURES ..................................................................................................... x INTRODUCTION... .................................................................................................... 1 MATERIALS AND METHODS ................................................................................. 12 Strains, plasmids and media ............................................................................... 12 Restriction endonucleases .................................................................................. 12 Total genomic preparations ................................................................................ 22 Plasmid preparations .......................................................................................... 22 Cloning of pORl fragments ............................................................................... 24 Transformations ................................................................................................ 25 Southern blot analysis ........................................................................................ 25 Plasmid mapping by digesting excised pORl fi'agments ...................................... 20 RESI Transfer of MRF’ Kan to HBlOl and MJ800 ..................................................... 28 Incompatibility analysis ...................................................................................... 29 DNA sequence determination ............................................................................ 29 In vitro protein expression ................................................................................. 30 In vivo protein expression using T7 RNA polymerase ........................................ 31 SDS polyacrylamide gel electrophoresis ............................................................. 32 N-terminal amino acid sequence determination of the SedR polypeptide ............. 33 Nucleotide and protein sequence analysis ........................................................... 34 Growth curves ................................................................................................... 35 Total protein assays ........................................................................................... 3 5 Bioremediation experiments ............................................................................... 35 RESULTS ................................................................................................................... 37 Growth curves of S. maltophilia, HBlOl, MJ800 and M1801 ............................ 37 Physical mapping and size determination of pORl ............................................. 37 Stability of pORl in S. mallophilia OROZ ......................................................... 42 In vitro expression of pORl fragments ............................................................... 46 vii Incompatibility experiments ............................................................................... 46 Nucleotide sequence determination of the 4 kb fi'agment .................................... 54 Sequence analysis .............................................................................................. 54 Sequence determination of the N—terminal regoin 35 kDa polypeptide ................ 59 Sequence analysis of SedR ................................................................................. 65 Deletion analysis of the 4 kb, HindIII fragment from pORl ................................ 65 Growth experiments of X2642, MJ 800 and MJ801 in selenite ............................ 69 Cysteine requirement for selenite-resistance ....................................................... 72 Genetic investigation of the role played by glutathione, glutathione reductase, thioredoxin and thioredoxin reductase in selenite-resistance ............... 75 Bioremediation experiments using S. maltophilia and MJ800 ............................. 75 DISCUSSION ........................................................................................................... 79 LIST OF REFERENCES .......................................................................................... 85 P‘ L Ta' .fl Table 10. LIST OF TABLES Page Dependence of Stenotrophomonas malrophilia OROZ upon reduced glutathione for growth in LB broth containing 10 mM selenite ........................... 10 Bacterial strains and plasmids used in this study ................................................. 13 Influence of pORl and pLJ 100 on cell grth in the presence of selenite ........... 38 Fragments sizes generated by restriction endonuclease digestions of pORl ........ 4O Incompatibility experiments using the F ' episome from M109 and pLJ 193 that contains a 13 kb BamHI fragment from pORl ................................ 51 Incompatibility experiments with selection for neither plasmid ............................ 52 Incompatibility experiments with selection for one of the plasmids ..................... 53 Influence of selenite on HBlOl strains containing plasmids with different segments of the 4 kb HindIII fragment ............................................................... 68 Cysteine requirement for growth on minimal plates containing selenite ............... 74 Growth of glutathione, glutathione reductase, thioredoxin and thioredoxin reductase mutants in 40 mM selenite ............................................... 76 ix Fiqu 10- In LIST OF FIGURES Figure Page 1. Possible reactions of selenate selenite and selenide with glutathione. .................. 4 2. Association of a 100 kb plasmid, pORl, conferring the transformation of Hg(II), Se(IV) and Pb(II) .............................................................................. 7 3. Physical map of the 4 kb, HindIII insert in pLJ 100 ............................................. 9 4. Proposed pathway for Se(IV) reduction in Stenotrophomonas maltophilia OROZ and E. coli .............................................................................................. 11 5. Agarose gel of pORl digestions ......................................................................... 39 6. Southern analysis of pORl digestions using the 4 kb, HindIII fi'agment from pORl as a probe ....................................................................................... 41 7. Physical mapof pORl ....................................................................................... 43 8. Agarose gel used to map two 26.4 Acc651 (Kpnl), a 21.5 kb BgIII and two 16.5 kb BglII fragments ..................................................................................... 44 9. HindIII digestions of Stenotrophomonas maltophilia OROZ plasmid bands ....... 45 10. In vitro transcription and translation of cloned pORl fragments ......................... 46 X 12 23. t 24. p ll. 12. 13. 14. 15. 16. 17 18. 19. 20. 21. 22. 23. 24. In vitro transcription and translation of the 4 kb HindIII insert in pLJ 100 ........... 48 Incompatibility testing and analysis for pORl and F ........................................... 50 Sequence features of the 4 kb, HindIII fi'agment from pORl .............................. 55 Nucleotide sequence of the 4 kb, HindIII, fragment from pORl ......................... 56 Putative nucleotide and amino acid sequences of SedR ...................................... 6O Hypothetical 3.7 kDa polypeptide from the 4 kb HindIII inseret in pLJ 100 ........ 62 Recombinant plasmids used for in vivo expression of SedR ................................ 63 SDS polyacrylamide gel of the expressed SedR polypeptide ............................... 64 Blast analysis of the SedR polypeptide sequence ................................................ 66 Deletions analysis of the 4 kb, HindIII fi'agment ................................................. 67 Correlation of turbidity to protein mass for X2642 grown in the absence of selenite .................................................................................... 70 Influence of 40 mM selenite on the grth of X2642 (pBR322), M1800 (pORl) and M1801 (pLJlOO) ................................................................. 71 SDS polyacrylamide gel of extracts from X2642 (pBR322) grown in the absence of selenite and MJ801 (pLJ 100) grown in 40 mM selenite ............... 73 Removal of selenite fi'om LB broth containing 20 mM selenite ........................... 78 25. Comparison of the HindIII (A) and BamHI (B) physical maps of pORl and the F-plasmid .............................................................................................. 80 xii 91165 c mineral knovm Selenat; llcReak- fl 0],, 1 Steinber Selenjde Selem'de (”Gillies INTRODUCTION Selenium is a group VIA element similar to sulfilr and tellurium. It exists in the +6, +4, 0 and -2 oxidation states as selenate (SeO42'), selenite (SeO32‘), elemental selenium (Seo) and selenide (I-ISe'), respectively. The presence of each depends upon the redox potential and pH (Geering et al., 1967; McNeal and Balistrier, 1989; Elrashidi er al., 1989; Masscheleyn et al., 1991). Selenate exists under highly oxidizing conditions, whereas selenite exists under mildly oxidizing conditions (Elrashidi et al., 1987). Although both are soluble, selenate tends to be more mobile (Alemi et al., 1991) and more available for biological absorption because selenite has a much higher affinity for metal oxides than selenate in soil (Christensen et al., 1989; Balistrier and Chao, 1990; Zhang and Sparks, 1990). Elemental selenium and selenide exist under reducing conditions. Elemental selenium exists as a red or black crystal. Selenide is present as hydrogen selenide gas, methyl selenide gas (Chan et al., 1976) metal selenide ores or organic selenide (Stadtman, 1990; Heider and Bock, 1993). Since elemental selenium and selenides are insoluble, they are not available for biological absorption. Microbes play a major role in the selenium cycle (Shrifi, 1964). There are three types of overlapping reactions in this cycle: oxidation and reduction, immobilization and mineralization, and methylation (Doran, 1982). First, several genera of bacteria are known to oxidize and reduce each inorganic species of selenium. Strains which reduce selenate and selenite to elemental selenium under aerobic conditions (Levine, 1925; McReady et al., 1965; Weiss et al., 1965; Burton et al., 1987; Lortie et al., 1992; Maiers et al., 1988) and by anaerobic respiration (Oremland et al., 1989; Rech and Macy, 1992; Steinberg et al., 1992) are the most common. Micrococcus lactilyticus reduces selenite to selenide (W oolfolk and Whiteley, 1962), Ihiobacillus fenooxidans oxidizes copper selenide to elemental selenium (Torba and Habashi, 1972) and Bacillus megaterium oxidizes elemental selenium to selenite (Sarathchandra and Watkinson, 1981). Secondly, I ino enxi phos 2 bacteria immobilize inorganic selenium by incorporating it into organic compounds and mineralize organic selenium by converting it back to inorganic selenium. Thirdly, they methylate inorganic and organic forms of selenium to form dimethyl selenide and dimethyl diselenide which diflirse into the atmosphere (Doran and Alexander, 1977; Thompson- Eagle and Frankenberger, 1989; Chan et al., 1976). Selenium enters the environment through industrial processes (Haygarth, 1994). It is used in staining glass or masking the color of iron oxides in glass; in the pigmentation of plastics, paints and ceramics; as an antioxidant in inks, vegetable oils and lubricants; and in the treatment of fungal infections and dandruff (N ewland, 1982). Selenium is released into the atmosphere by the combustion of coal and oil (Nriagu, 1988), and leeches into the environment from stored coal (Yang et al., 1983) or during the mining and refining of phosphate, uranium, copper, lead and zinc (World Health Organization, 1987). Agricultural activity in arid regions that contain high levels of naturally occurring selenium can cause serious problems. The selenosis of migratory birds in the Kesterson National Wildlife Refiige in the San Joaquin Valley in California is a well known example (Presser and Ohlendorf, 1987; Tanji et al., 1986). Marine sedimentary rock originating from volcanic dust or eroded igneous rock that contained high concentrations of selenium was deposited in this area during the Cretaceous period (Trelease and Beath, 1949; Davidson and Powers, 1957; Presser and Ohlendorf, 1987; Presser, 1994). Because the region is dry and a layer of clay is located immediately below the soil, runoff from the irrigation of farm land collects underneath the fields and evaporates to concentrate selenium and other salts that leach fiom the soil. A subsurface drainage system was developed to remove this water and direct it to the San Francisco Bay via the San Luis Drain. However, politics and fimding restrictions blocked the completion of this project (Marshall, 1985). Since it was built only as far as the Kesterson reservoir, the water from this drain was diverted into the wildlife refuge. Subsequently, several species of migratory birds were poisoned (Ohlendorf el al., 1986; Ohlendorf, 1989). 3 Animal and humans suffering from selenium poisoning exhibit various symptoms. Livestock feeding on selenium accumulating plants contract "alkaline disease," which is characterized by emaciation, lameness and tail, hair and hoof loss (Trelease and Beath, 1949). Humans also lose their hair and nails (Yang et al., 1983). Birds experience muscular atrophy, weight loss and embryo deformities (Ohlendorf et al., 1986; Ohlendorf, 1989). Bacteria sensitive to selenite exhibit complete grth inhibition. Many resistant strains demonstrate a grth curve with an increased lag phase (Leifson, 193 6; McReady etal., 1965). The mechanisms for selenium toxicity are not well understood. Elemental selenium and selenate are not toxic because they are not highly reactive. Selenate is toxic only once it has been converted to selenite (Martin, 1973). Selenite may interfere with protein function by oxidizing sulflrydryl groups to form disulfides (RS SR) and unstable selenosulfides (RS-Se-SR) (Ganther, 1971; Martin, 1973; Doran, 1982; Nakagawa, 1988). Figure 1 demonstrates how selenite may become toxic in the presence of reduced glutathione (GSH) (Whiting et al., 1980; Shamberger, 1985). It reacts with reduced glutathione to form selenodiglutathione (GSSeSG) (2). This compound is converted to selenoperoxide (GS Se') by reacting with one of the following: another molecule of reduced glutathione (3); glutathione reductase and NADPH (4) (Ganther, 1968; Ganther, 1971); or thioredoxin, thioredoxin reductase and NADPH (Ren et al., 1993; Bjomstedt et al. 1992; Holrngren and Kumar, 1988). GSSe' reacts with reduced glutathione (7) or glutathione reductase and NADPH (6) to generate selenide (HSe'). Seko et a1. (1988) observed that HSe‘ reacts with oxygen to form elemental selenium and a superoxide ion (05 ), a fi'ee radical, that may damage DNA (Shamberger, 1985) or lipids (Seko et al., 1988). Cell growth may also be inhibited by depletion of NADPH and competitive inhibition of thioredoxin reductase (Bjomstedt et al., 1992; Kumar et al., 1992). When Escherichia coli thioredoxin reductase and thioredoxin, or calf thymus thioredoxin are added to GSSeGS in the presence of oxygen, a high level of non-stochiometric NADPH GS HSeO,’ + 2 GSH —> HSeO,’ + GS-SG + H20 HSeO,’ + 4 GSH -—--—> GS-Se-SG + GS-SG + OH' + 2 H20 GS-Se-SG + GSH GS-Se' + GS-SG + IT glutathione reductase GS-Se-SG + NADPH -——> GS-Se' + GSH + NADP+ GS-Se' + HZO <——= Se0 + GSH + OH' glutathione reductase GS-Se’ + NADPH + H20 -—-—-> HSe' + GSH + NADP+ + OH' GS-Se' + GSH HSe' + GS-SG HSe’ + [O] -—-—> Se° + OH' HSe’ + [0] + 2 GSH ———> HSe' + GS-SG + up (1) (2) (3) (4) (5) (6) (7) (8) (9) Figure 1. Possible reactions of selenate, selenite and selenide with glutathione (Whiting etal., 1980; Shamberger, 1985). oxidatior oxidizes 10 iCSIOi may int selenomc 1957; TL‘ likely an S non-cova ()l'agner 1978; Di selenocys proteins Stadtman 0f eukan deiodinas. the active formate (1 (Cone e: 1 Ti “11h four SdeSa: “121988), 101m 311m SelenOCystl [MAS (14 5 oxidation occurs. Since the conversion of HSe' to elemental selenium is slow (8), it oxidizes and starts the cycle again. Subsequently, large quantities of NADPH are required to restore thioredoxin and thioredoxin reductase to their reduced state. Finally, selenium may interfere with protein firnction by replacing SUlfill' (Shrifi, 1954). Since selenomethione does not inhibit growth when it replaces methionine (Cowie and Cohen, 1957; Tuve and Williams, 1961; Frank et al., 1985), selenocysteine appears to be the most likely cause of toxicity (Heider and Bock, 1994). Selenium is also an important element in animal and bacterial metabolism. It exists non-covalently bound to the active centers of some bacterial xanthine dehydrogenases (Wagner and Andreesen, 1970) and nicotinic dehydrogenases (Imhofl‘ and Andreeson, ‘ 1978; Dilworth, 1982). It is covalently bound to some tRNAs (Wittwer, 1983), selenocysteine and selenomethionine. Selenomethionine is randomly incorporated into proteins and does not play an important role in protein function (Sliwkowski and Stadtman, 1984). Selenocysteine, on the other hand, is a key residue in the active centers of eukaryotic glutathione peroxidase (F orstrom et al., 1978) type I iodothyronine deiodinase (Behn er al., 1990) and plasmid protein P (Burk, 1991). It is also present in the active centers of prokaryotic hydrogenase (Rieder et al., 1984; Muth er al., 1987), forrnate dehydrogenase (Jones et al., 1979; Zinoni et al., 1986), and glycine reductase (Cone et al., 1976). The E. coli pathway for selenium incorporation into selenocysteine is associated with four genes: selA, seIB, selC and selD (Bock et al., 1991; Heider and Bock, 1994). SelC is a specific tRNA which possesses the opal termination code (UCA) (Leinfelder er al., 1988). This new tRNA is charged with a serine residue. SelA converts seryl-tRNA to form aminoacrylyl-tRNA (Forchhammer and Bock, 1991). SelD adds HSe' to form selenocysteyl-tRNA and also supplies selenide for the synthesis of selenium containing tRNAs (Leinfelder er al. 1990). Finally, SelB is a translation factor that incorporates selenocyf codon (Z A understol cell by tl sulfate ll system ( selenide resistant showed' (Gerrard mimic 51 different 6 selenocysteine into a growing polypeptide chain (Forchhammer et al., 1989) at a UGA codon (Zinoni et al., 1986; Zinoni et al., 1987). Although the mechanism for the incorporation of selenium into cell protein is well understood, the pathways for its transport and reduction are not clear. Selenate enters the cell by the sulfate transport system (Brown and Shrifi, 1980). Selenite may enter by the sulfate transport system (Lindblow-Kull et al., 1985) or a selenite specific transport system (Brown and Shrifi, 1982). Once inside the cell, both oxyanions are reduced to selenide or elemental selenium. McCready et al. (1965) and Levine (1925) observed that resistant strains accumulated elemental selenium inside the cells. Electron microscopy showed that it collects on the cell wall and membrane but not in the cytoplasm of E. coli (Gerrard et al., 1974). The discrepancies in these results and the ability of selenium to mimic sulfur suggest that selenium metabolism is probably complex and involves several different pathways. Pseudomonas maltophilia Oak Ridge Research Institute strain 02 (ATCC 53 510) was isolated from a mercury contaminated site in Oak Ridge, TN in 1986 (N. Revis, personal communication). This designation was changed by Swings et a1. (1983) to Xanthomonas maltophilia. Recently, it was renamed Stenotrophomonas maltophilia (Palleroni and Bradbury, 1993). S. maltophilia OROZ displayed a capacity for the chemical transformation of several difl‘erent heavy metal salts. It reduced Se(IV), Hg(H) and Au(III) to their elemental states and formed insoluble complexes with Pb(II), Cd(II), Ag(I) and Cr(III). Initial agarose electrophoretic gels of total genomic DNA from S. maltophilia OROZ demonstrated that it contained a single 100 kb plasmid band designated as pORl (Figure 2, lane 1). The two smaller plasmid bands seen in this gel were not detected in earlier gels. Total genomic DNA from S. maltophilia OROZ was transformed into E. coli strain HBlOl. Selenite-resistant transfonnants contained pORl (Fig. 2, lane 3) in the new strain MJ800, which also demonstrated resistance to Pb(II) and Hg(II). After purifying pORl fi'om MJ800 using a modified alkaline lysis protocol (Kado and Liu, é— pOR1 <—-Chromosomal DNA Figure 2. Association of a 100 kb plasmid, pORl, conferring the transformation of Hg(H), Se(IV) and Pb(H). Lane 1: Stenotrophomonas maltophilia OR02 total genomic DNA. Lane 2: Purified pORl from MJ800. Lane 3: Total genomic DNA from MJ800. 1C transfomiL of selenii: HdeII fr strain WEI 5.) reduce sel maltophilit sulfoximinrl Meister. 1" mallophzhr. merry was selenium. ( intermediau and glutathi Wereadded Slate. Thus, In th P0R1,5equ( 90” and iden 8 1981; Crosa et al., 1994), it was digested with HindIII, ligated into pBR322 and transformed into HBlOl. Agarose gel electrophoresis of small scale plasmid preparations of selenite-resistant colonies showed that selenite-resistance was located on a 4 kb, HindIII fragment from pORl (Fig. 3). This recombinant plasmid was called pLJ 100 in strain MJ801. S. maltophilia OR02 and E. coli may use glutathione and glutathione reductase to reduce selenite to elemental selenium (Blake et al. unpublished). In Table l, S. maltophilia OR02 grew in the two control cultures containing selenite or buthionine- sulfoximine, an inhibitor of the glutathione reductase synthesis pathway (Griflith and Meister, 1979) However, it failed to grow in the presence of both chemicals. Thus, S. maltophilia required glutathione for resistance to selenite. Stopped flow spectrophoto- metry was used to predict a possible pathway for the reduction selenite to elemental selenium. Glutathione and HZSeO3 reacted to form GS-Se-SG with the production of the intermediates shown in Figure 4 (Blake er al., personal communication). When NADPH and glutathione reductase or cell extracts fi'om S. maltophilia OR02 grown in selenite were added, elemental selenium was released and glutathione was returned to its reduced state. Thus, glutathione reductase may be involved in the bacterial reduction of selenite. In this study, I localized the position of selenite-resistance on the physical map of pORl, sequenced the 4 kb HindIII fragment which conferred resistance to selenite in E. coli and identified a 35 kDa polypeptide, SedR, encoded by this fragment. Figure 3. Physical map of the 4 kb, HindIII insert in pLJIOO. This cloned fragment was ligated into pBR322 and confers resistance to Pb(II) and Se(IV). Abbreviations: B, BamHI; Bg, BgIII; E, EcoRl; H, HindIII; K, Kpnl; P, PstI; and S, SphI. 10 Table 1. Dependence of Stenotrophomonas maltophilia OR02 upon reduced glutathione for grth in LB broth containing 10 mM selenite. Grth conditions Strain Grth Selenite BSOa S. maltophilia OR02 + - + S. maltophilia OR02 - + + S. maltophilia OR02 + + - aB SO, L-buthionine-[S,R]-sulfoximine, irreversibly inhibits y—glutamylcysteine synthetase in the pathway for glutathione (GS) biosynthesis. 11 H,SeO, “.04 l SeO2 “"1 l GSSeO,H GSH .1... Di GS-SeO-SG GSH GSOH GS-Se-SG GSH H.021 GSSG 2 NADPH g'm‘hi‘m _> (- 2 NADPH ' rEdictise' 2 NADP’ Hf (J ‘7 2 NADP‘ +H‘ GSH v 2 GSH GS-SeH r s GSH 2 GSH + Se° Figure 4. Proposed pathway for Se(IV) reduction in Stenotrophomonas maltophilia OR02 and E. coli. MATERIALS AND NIETHODS Strains, plasmids, and media. The strains and plasmids used in this study are listed in Table 2. M—9 minimal medium (Ausubel et al., 1992) for E. coli contained 0.6% (w/v) Na2I-IPO4, 0.3% (w/v) KHZPO4, 0.1% (w/v) NH4Cl, 0.055% (w/v) NaCl, 1 mM MgSO4 and 0.5% (w/v) D-glucose. Luria Bertani broth (LB broth) contained 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 0.5% (w/v) NaCl and 1 mM NaOH in distilled water (Ausubel et al., 1992). MacConkey agar was obtained from Difco Laboratories and prepared according to the manufacturer's instructions (Holt and Krieg, 1994). Terrific broth contained 1.2% (w/v) bacto-tryptone, 2.4% (w/v) bacto-yeast extract, 0.4% (v/v) glycerol, 17 mM KH2P04 and 72 mM KZI-IPO4 dissolved in distilled water (Tartof and Hobbs, 1987). All agar plates contained 1.6% (w/v) agar. When required, media was supplemented with 0.5 mg % (w/v) thiamine, 0.4 mM L-amino acid(s), 100 ug/ml Ampicillin, 60 ug/ml kanarnycin and 40 mM selenite. Cultures were grown at 37 0C with aeration in a baflled flask placed in a New Brunswick Scientific Co., Inc. gyrotory water bath shaker at 200 rpm. Restriction endonucleases. Restriction endonucleases were obtained from Promega. Acc651, BglII, HindIII, SacI, EcoICRI and Sphl were stored in 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, 50 mM NaCl, 0.1 mM ethylenediarninetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.5 mg/ml bovine serum albumin (BSA), and 50% (v/v) glycerol. BamI-II was stored in 10 mM Tris- HCl, pH 7.4, 300 mM KCI, 0.1 mM EDTA, 1 mM DTT, 0.5 mg/ml BSA and 50% (v/v) glycerol. EcoRI and PstI were stored in 10 mM Tris-HCl, pH 7 .4, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.15% DTT, 0.5 mg/ml BSA and 50% (v/v) glycerol. Acc651 and Bng required a 10 x digestion bufl‘er containing 60 mM Tris-HCl, pH 7.9, 60 mM MgCl2, 1.5 M NaCl and 10 mM DTT. 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Bo: 8:: 8:88: 88 e. 8.: 2: 88888 20:: :5: 8:: 8:88: 8:: e. o: 2: 88888 22:: :5: 8:: 8888: :88: e. 8.: 2: 88888 20:: :5: :5: 8:88: i=8: e. 8.2 2: 88888 2B: :5: 8o: 8:88: 882.. :2 w: 2: 88888 20:: :8: :8: 82:8: :88: :2 S 2: 88888 202: :8: 8o: 8:88: 882.. e. on 2: 88888 20:: 8:3: :83: 8:3: m::m: :23: :23: 85: :23: 53: 2:3: 223: 858:3— mowmtoaoasno Ego—oy— 2883 :0 £85 3.88: a 28.: 9.253 N 03...: 20 o: 2:2: o: 2:2: o: 2:2: 2 2:2: 2 2:2: 32233:: 28:22:02 as: :33. 22 28:22:82 :8: 8%.: 2 8:3: 2 2:2: 2 28: 2oz: :_ :8: 88888 :22 :23: 2: :8: 82:8: 82288:: 82:: 8 :20 82 h: 2: :8 atom 2:58:00 :22: 8: 3: 05 80:: 2.253.: ESMEHEE 82:: 8 2:8: :8 82:: mew—3:8 :62: 2: 3: 2: EB: 2858:: 28553.5 :5: :8: 8888: :88: e. v 2: 88888 1:: 20: :8: 8888: :88: :2 v 2: 88888 75: :08an 0:82:30: <21: E. .3»: 0:30:53 <72 E. .120: 8:3: :2 8:28: 3:: 8:3: :_ 8:22V :82 8:3: 8 8:22U :8: 8:3: _ 83: 8:3: ::3: 2:3: YE: 3:0: mo::2: 8:3: 8:3: 8:80.20“ 8282:8835 2.26.3: 2833 :o 53% 8.882 : 28.: «32:00» N 232.5 21 252:2 2o: - .255 856602852020 .. 295:0 005668-982: - :3va 35668836250 - :9va 85668-52 - :9me 005668.526 - :69: 3566822228 - :Cfivom :: 2:2: 82:: 2 82:: :o E: 22228 252:2 $222535 8 :3: :63: o: 2:2: 2 :mo :9 w m 2: :8 22m 2.25:8 22:2: 28:22:22 3:3: 3:883: 86668555 255—9: 3:62: :0 £85 €282 : 22: 22 EcoRI and Pstl required a 10 x digestion buffer containing 900 mM Tris-HCl, pH 7.5, 100 mM MgC12 and 500 mM NaCl. SacI required a 10 x digestion buffer containing 100 mM Tris-HCl, pH 7.5, 70 mM MgC12, 500 mM KCl and 10 mM DTT. SphI required a 10 x digestion buffer containing 100 mM Tris-HCI, pH 7.4, 100 mM MgC12 and 1.5 M KCl. EcoICRI required a 10 x digestion bufl‘er containing 60 mM Tris-HCl, pH 7.5, 60 mM MgC12, 500 M NaCl and 10 mM DTT. Total genomic preparations. Total genomic DNA was prepared using a method described by Ausubel et al. (1992). Cells from 100 ml of E. coli and 50 ml of S. maltophilia were harvested at 14,500 x g and resuspended in 9.5 ml of TE solution which contained 10 mM Tris-HCl, pH 8.0 and 0.5 mM EDTA. The suspension was mixed with 0.5 ml of 10% (w/v) sodium dodecyl sulfate (SDS) and 50 ul of 20 mg/ml proteinase k. This mixture was incubated at 37 0C for 1 hour. The preparation was then mixed with 1.8 ml of 5 M NaCl and 1.5 ml of of a solution containing 10% (w/v) cetyltrimethylammonium bromide (CTAB) and 0.7 M NaCl. After incubating the preparation at 65 °C for 20 min, it was extracted once with 5 ml of 24:1 (v/v) chloroformzisoamyl alcohol and a second time with an equal volume of 1:1 (v/v) phenol and chloroform. The DNA was then precipitated with 0.6 volumes of isopropanol, centrifirged at 20,000 x g, gently mixed with 5 ml of 70% (v/v) ethanol, centrifuged at 20,000 x g, dried under a vacuum after discarding the ethanol and resuspended in 1.5 ml of TE containing 5 ug/m] of RNase. Plasmid preparations. Purification of pORl was performed by a modified alkaline-lysis protocol (Kado and Liu, 1981 and Crosa et al., 1984). After growing MJ800(pORl) in LB broth containing 40 mM selenite for 1-2 days at room temperature in a shaking water bath, 0.3 g of harvested wet cells were resuspended in 2 ml of TE (pH 8.0), mixed with 5.5 ml of lysing solution (3.4% (w/v) SDS and 0.032 M NaOH dissolved in TE) and incubated at 65 °C for 45 min. The lysed cells were gently mixed with 0.412 ml of 2 M Tris-HCl, pH 7.2 and 2 ml of 5 M NaCl. Afier incubating the preparation on ice in the refrigerator overnight, it was centrifuged at 20,000 x g for 20 min, and the 23 supernatant was poured through cheese cloth into a new tube. Plasmid DNA was precipitated with 0.6 volumes of isopropanol for 20 min at room temperature, centrifuged at 20,000 x g for 15 min, gently mixed with 5 ml of 70% (v/v) ethanol, centrifirged at 20,000 x g for 10 min, dried under a vacuum afier discarding the ethanol and resuspended in 1 ml of TE containing 5 ug/ml RNase. Small scale plasmid preparations were performed using an alkaline lysis procedure (Bimboim and Doly, 1979). The strain harboring the desired plasmid was grown overnight in 5 ml of LB broth containing the appropriate antibiotic(s). Cells from 1.5 ml of culture were harvested at 14,000 x g for l min, resuspended in 0.1 ml of TE, gently mixed with 0.2 ml of lysing solution (1% (w/v) SDS and 0.2 N NaOH dissolved in TE) and incubated on ice for 5 minutes. Cell debris and SDS were precipitated by adding 0.15 ml of 3 M potassium acetate (pH 5.2) and incubating the sample on ice an additional 5 min. After centrifuging the preparation at 14,000 x g for 15 min, the supernatant was poured into a new tube. Plasmid was precipitated with 1 ml of 95 % (v/v) ethanol, centrifuged at 14,000 x g for 15 minutes, mixed with 0.5 ml of 70 % (v/v) ethanol, centrifuged at 14,000 x g for 5 minutes, dried under a vacuum after discarding the ethanol and resusupended in 30 ul of TE containing 5 ug/ml of RNase. Restriction enzyme digests of the plasmid contained 5 u] of plasmid, 2 ul of 10 x digestion buffer, 0.5 ul of the appropriate enzyme (5 U) and 12.5 ul of distilled water. Digestions were mixed with 10 x loading buffer, incubated at 37 °C for 1-2 hr and separated on a 0.8% agarose gel. All gels were stained for 15 min at room temperature in water containing 0.5 rig/ml ethidium bromide. Large quantities of DNA were purified using Promega's Maxipreps (Sambrook eta]., 1989). A 250 ml culture harboring the desired plasmid was grown in LB-broth containing the appropriate antibiotic at 37 °C overnight in a shaking water bath. The cells were harvested at 14,000 x g for 15 min, resuspended in 15 ml of resuspension buffer (50 mM Tris-HCl, pH7 .5, 10 mM EDTA and 100 mg/ml RNase) and mixed with 15 ml of lysing solution (0.2 M NaOH, and 1% SDS). When the solution cleared, it was neutralized with 15 ml of 2.55 M potassium acetate (pH 4.8), cc isopropa mixed packed i adding 1: EDTA 211 through followed The vacu was drier resin by centrifug C oprRl Ll] of 10 Magic C into the “'35 pus} an Epper 24 4.8), centrifuged at 14,000 x g for 15 minutes and poured through cheese cloth into a new tube. The DNA was precipitated at room temperature for 20 minutes with 0.6 volumes of isopropanol, centrifuged at 14,000 x g for 15 minutes, resuspended in 2 ml of TE and mixed with 10 ml of maxiprep resin. Using a Promega vacuum manifold, the resin was packed into a maxiprep column. Resin remaining in the centrifirge tube was removed by adding 12 ml of column wash solution (200 mM NaCl, 20 mM Tris-HCl, pH7.5, 5 mM EDTA and 50% (v/v) ethanol) to the tube, decanting it into the column and pulling it through with the vacuum manifold. An additional 13 ml of column wash solution, followed by 5 ml of 80% (v/v) ethanol were pulled through the column to wash the resin. The vacuum was applied for 15 extra min to remove as much liquid as possible. The resin was dried at 1,300 x g for 5 min in a clinical centrifuge. The DNA was eluted from the resin by incubating it with 1.5 ml of distilled warm (65 °C) water for 1 min and centrifirging the column for 5 min in a clinical centrifiige at 1,300 x g. Cloning of pORl fragments. Acc6SI (KpnI), HindIII, Sac] or BamHI digestions of pORl containing 2 pl (20 U) restriction endonuclease; 88 ul (0.9 ug) of pORl; and 10 ul of 10 x digestion bufi‘er were incubated overnight at 37 °C and mixed with Promega's Magic Cleanup kit resin. The resin was packed into a Cleanup kit column by pushing it into the column with a 3 ml syringe; washed with 2 ml of 80% (v/v) isopropanol, which was pushed through the column with the syringe; and dried by centrifuging the column in an Eppendorf tube for 20 seconds at 14,000 x g. Plasmid fi'agments were eluted fi'om the resin by incubating it with 30 u] of distilled warm (65 °C) water for 1 minute and centrifuging the column in a new tube at 14,000 x g for 20 seconds. Ligations (Maniatus et al., 1989) consisted of 15 ul (0.4 ug) of digested pORl, 2 ul of 10 x ligation buffer (300 mM Tris-HCl, pH 7.8, 100 mM MgCl2, lOOmM DTT and 5 mM ATP), 2 ul of pUC19 (50 ng) and 1 u] of T4 DNA ligase (3U), which was stored in 10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol. Eschericia coli strain DHSor was transformed with 15 ul (0.34 pg) of ligase reaction, and 100 u] of the Ir; ampic ampic appro al. 15 desire -80 °( (Hana LB bn 10 ml Iransfc 0.01 h gently incuba nixed in V011 l4.00( plates I~B brr BamH pufifie. Were i bromp} gel [0 i 25 the transformation reaction was plated on McConkey agar plates containing 100 ug/ml of arnpicillin. White transforrnants were grown in 5 ml of LB broth containing 100 ug/ml of arnpicillin. Small scale plasmid preparations fiom 1.5 ml of culture were digested with the appropriate enzyme, mixed with 10 x loading buffer (20% (w/v) ficoll, 0.1 M EDTA, pH 8.0, 1% (w/v) SDS, 0.25% (w/v) bromphenol blue and 0.25% xylene cyanol (Ausubel et al., 1989)) and electrophoresed through a 0.8% (w/v) agarose gel. Cultures containing the desired recombinant plasmid were mixed 1:1 (v/v) with 50% (v/v) glycerol and stored at -80 °C. Transformations. Competent cells were prepared using a modified CaCl2 method (Hanahan, 1983). A 100 ml culture containing the desired strain of E. coli was grown in LB broth to an optical density of 0.4. Cells were centrifuged at 3,000 x g, resuspended in 10 ml of 0.15 M NaCl, centrifuged at 3,000 x g again, resuspended in 1 ml of transformation buffer (15% (v/v) glycerol, 0.1 M CaClz, 0.01 M Tris-HCl, pH 8.0 and 0.01 M MgClz), and flown at -80 °C. The cells were thawed on ice, and 0.1 ml was gently mixed with the 0.03-0.5 pg of plasmid DNA. The transformation mixture was incubated on ice for 30 min, heat shocked for 2 min at 42 °C, incubated on ice for 15 min, mixed with 1 ml of LB-broth, and incubated at 37 °C for 45 min. Cells were either plated in volumes of 0.1 ml, or the entire transformation was plated by centrifuging them at 14,000 x g for 10 sec, resuspending them in residual supernatant and spreading them on plates with the appropriate selection. Single colonies were inoculated into 5 ml cultures of LB broth and grown overnight for small scale plasmid preparations. Southern blot analysis. BglII, Acc651 (KpnI), HindIII, EcoICRI (SacI), and BamHI restriction endonuclease enzyme digestions consisting of 25 ul (0.25ug) of purified pORl, 3 1,11 of 10 x digestion bufi‘er, and 2 ul (approximately 20 U) of enzyme were incubated overnight at 37 °C and separated on a 0.8% agarose gel until the bromphenol blue dye migrated 11.5 cm. The fractionated DNA was transferred fi'om the gel to a Biorad Zeta Probe membrane (Reed and Mann, 1985, Sambrook et al., 1989) using Bl in 0.2 h" 800 M min; ant and soa filter pa on the , 1982): t and the the Trar transfer overnjg} remover stratalinl I Restricti of DNA incubate Sea Plac bands w Was mix c0111mm I iSOprOpa in an El inc“batin Eppendo inihe 0pf 26 using Biorad's Trans-Blot Cell apparatus. The gel was soaked in 0.25 M HCl for 10 min; in 0.2 M NaOH and 0.5 M NaCl for 30 min, twice; in 5 x TAE buffer (20x TAE contained 800 mM Tris-HCl, 400 mM acetate, 20 mM EDTA and glacial acetic acid, pH 7.4) for 10 min; and in 0.5 x TAE bufi‘er for 10 min. The membrane was cut the same size as the gel and soaked in 0.5 x TAB for 10 min. Two pads from the apparatus and two sheets of filter paper the same size as the gel were briefly soaked in 0.5 x TAE. The gel was placed on the gel holder from the Trans-Blot Cell apparatus in the following order (Danner, 1982): the first pad, the first sheet of filter paper, the gel, the second sheet of filter paper and the second pad. After removing all air bubbles, the gel holder was closed and placed in the Trans-Blot tank so that the membrane faced the cathode. The tank contained 0.5 x transfer bufi‘er, cooled to 4 0C. The DNA was tranferred to the membrane at 40 V overnight and was completed at 80 V for 1 hr the following day. The membrane was removed from the gel holder; washed with 1 x TAE buffer; treated in a Stratagene UV stratalinker 2400 to fix the DNA to the membrane; and air dried. Fragments were isolated for nick translation using the following protocol. Restriction endonuclease digestions of the recombinant plasmids containing 43 pl (20 pg) of DNA, 5 pl of 10 x digestion buffer, and 2 pl (20 U) of restriction endonuclease were incubated at 37 °C for 2 hr and separated on a 0.8% agarose gel using FMC Bio Products' Sea Plaque agarose. The gel was stained with ethidium bromide and the desired fragment bands were excised from the gel. Up to 500 pl of melted agarose containing the fragment was mixed with 1 ml Promega's PCR Prep resin. The resin was packed onto a PCR Prep column by pushing it through the column with a 3 ml syringe, washed with 2 ml 80% (v/v) isopropanol which was pushed through the column, and dried by centrifuging the column in an Eppendorf tube for 20 sec at 14,000 x g. DNA was eluted from the resin by incubating it with 50 pl of double distilled water for 1 minute and centrifuging it in a new Eppendorf tube at 18,500 x g for 20 sec. The sample was incubated at 65 °C for 30 min in the opened Eppendorf tube to concentrate the DNA and remove excess isopropanol. 27 DNA fragments were labeled by using the following 50 pl reaction from Promega's nick translation system (Rigby et al., 1977 and Sambrook et al, 1989): 3 pl of deoxyadenosine triphosphate (300 mM), 3 pl of deoxyguanosine triphosphate (300 mM), 3 pl of deoxythimidine triphosphate (300 mM); 5 pl of 10 x nick translation buffer (500 mM Tris-HCl, pH 7.2, 100 mM MgSO4 and lmM DTT); 0.5 to 1.0 pg of DNA dissolved in 23 pl of water; 5 p1 of DNA polymerase/DNaseI mix (DNA polymeraseI [1 U/pl], 0.2 ng/pl DNaseI, 50% (v/v) glycerol, 50 mM Tris-HCl, pH 7.2, 10 mM MgSO,,, 0.1 mM DTT and 0.5 mg/ml nuclease free BSA); and 7 pl of [or-32F] deoxycytidine (70 mCi at 400 Ci/mmol and 10 mCi/ml). Afier incubating the reaction for 1 hr at 15 0C, 5 pl of 0.25 M EDTA (pH 8.0) were added to stop the reaction. To measure the percent incorporation, 1 pl of the reaction was added to 99 pl of 0.2 M EDTA (pH 8.0), and 3 pl of this dilution was placed, in duplicate, on Angel 934 AH fiber filters. The filters were dried under a heat lamp. One was placed in a scintillation vial containing 10 ml of scintillation fluid. The other filter was washed twice for 5 min in 50 ml of 0.5 M sodium phosphate (pH 6.8), dried under a heat lamp and placed in another scintillation vial. Percent incorporation was calculated by dividing the counts per minute (cpm) of the washed filter by the cpm of the unwashed filter and multiplying this quotient by 100. Fragments with greater than 30% incorporation were used for hybridization. The labeling reaction was diluted with 0.45 ml of TE, concentrated for 5 min at 3000 x g in an Amicon Microcon, diluted with 0.45 ml of TE, boiled for 5 min and added to a hybridization bottle containing a blot and hybridization buffer. Hybridizations were achieved using Bellco's rnicrohybridization oven and a protocol (Church and Gilbert, 1984) modified for Biorad's Zeta Probe membrane. The membrane, rolled tightly in a Bellco nylon mesh, was placed in a hybridization bottle containing 0.2 x SSC bufi‘er. The mesh and membrane were unraveled against the wall of the bottle so that all air bubbles were eliminated. Afier decanting the SSC buffer, 40 ml of hybridization bufi‘er (lmM EDTA, 7% (w/v) SDS, and 0.5 M NaHPO4 [0.5 M sodium], pH 7.2, replace: The blc EDTA in 1%( mappe develop were n consisti U) of r samples sammes distilled (WV) 5. was srai Slabs we 30 H1 or Aller im PCR Pr: 1 transferr. Kan Cells °C. The SirepmmI selffnite ar Km) Wen 28 pH 7 .2) were added, and the blot was incubated at 65 °C for 15 min. The buffer was replaced with 40 ml of flesh hybridization buffer, and the denatured probe was added. The blot was incubated overnight at 65 0C; washed in 150 ml of 5% (w/v) SDS, 1 mM EDTA and 40 mM NaHPO4 (40 mM sodium), pH 7.2 at 65 0C for 1-2 hr, twice; washed in 1% (w/v) SDS, 1 mM EDTA, and 40 mM NaHPO4, pH 7.2 at 65 0C for 1-2 hr, twice; wrapped in plastic; and placed on film in an autoradiogram cassette. The film was developed after exposing it overnight at -80 °C. Plasmid mapping by digesting excised pORl fragments. Uncloned fi'agments were mapped using a modified technique described by Danna (1980). Digestions consisting of 897 pl (9 pg) of purified pORl, 100 pl of 10 x digestion bufl‘er and 3 pl (30 U) of restriction enzyme were incubated ovemight at 37 °C, divided into two 500 pl samples and treated separately with Promega's Clean Up system. The DNA from both samples was eluted into the same tube with 100 pl (50 p] for each cleanup reaction) of distilled warm (65°C) water, mixed with 10 x loading bufi‘er and fi'actionated on a 0.8% (w/v) Sea Plaque agarose gel until the bromphenol blue dye migrated 11.5 cm. The gel was stained with ethidium bromide and selected bands were excised from the gel. The gel slabs were melted at 65 °C and used in the following digestions: 100 p1 of melted gel slab, 30 pl of 10 x digestion bufi‘er, 168 pl of water and 2 pl (20 U) of restriction enzyme. After incubating the digestions overnight at 37 0C, the DNA was purified using Promega's PCR Preps system and separated on a 0.8% (w/v) agarose gel. Transfer of MRF' Kan to H3101 and M1800. The F' episome, MRF' Kan, was transferred from XLl-Blue MRF’ Kan to HB101 and M1800 by mating. XLl-Blue MRF’ Kan cells were mixed on an LB plate with HBlOl or M1800 and incubated for 2 hr at 37 °C. The cells were scraped ofi‘ the plates and streaked onto an LB plate containing streptomycin and kanamycin for mating with HB101 and onto an LB plate containing selenite and kanamycin for the mating with M1800. HBlOl(MRF' Kan) and M1800(MRF' Kan) were named M1840 and M1841, respectively. transfl M-9 r hi9n supple the in strain plasmi 10-7 ar 50 wer similar by 10" by 10" then pl; 29 Incompatibility analysis. The cloned pORl fragments inserted into pUC19 were transformed into 1M109 using the method described above, except 1M109 was grown in M-9 minimal medium. All transformation reactions were spread on two types of plates: M-9 minimal medium plates supplemented with ampicillin and M-9 minimal medium plates supplemented with ampicillin and 0.4 mM proline. Figure 12 illustrates the protocol for the incompatibility experiments with selection for neither plasmid (Berquist, 1987). A strain containing the competing plasmids was grown overnight under selection for both plasmids, diluted 10'6 in medium without selection, grown overnight, diluted by 10'6 or 10'7 and plated on medium without selection. To identify colonies which lost a plasmid, 50 were spotted onto a plate containing selection for each plasmid. Incompatibility experiments with selection for one of the competing plasmids was similar to the protocol above. The strain containing the competing plasmids was diluted by 10'6 in medium containing selection for one of the plasmids, grown overnight, diluted by 10'6 or 10'7 and plated on medium with selection for same plasmid. Colonies were then plated onto a plate containing selection for the other plasmid. DNA sequence determination. The 4 kb HindIII fi'agment from pORl was subcloned between the SP6 and T7 phage RNA polymerase promoters in plasmid, pSP73 (Krieg and Melton, 1987), to create plasmid pL1200 for sequencing (Fig. 13). A restriction endonuclease digestion containing 1 pl (1 pg) of pSP73, 10 pl (1 pg) of pLJ 100, 3 pl of 10 x digestion buffer, 14 pl of distilled water and 2 pl (20 U) of HindIII was incubated for 3 hr at 37 0C and treated with Promega's Clean Up system. The eluted sample was ligated at 4 0C for 12-15 hr in a reaction containing 10 pl (0.4 pg) of digested pSP73 and pL1100; 2 p1 of 10 x ligation bufi‘er; 7 p1 of distilled water; and 1 pl (3U) of T4 DNA ligase. Transformation of DHSa with 10 pl (0.2 pg) of the ligated DNA yielded pL1200. A large scale preparation was performed on a 250 ml culture of M1821 (pL1200) using Promega's Wizard Maxiprep system. This purified DNA was used to create Sphl, EcoRI, BamHI, PstI, and Acc651 (Kpnl) deletions in the 4 kb insert of pL1200 (Figure pl (6 l reactic plasmi Terrific they w« 260 nm primer 1 den't'atii promote the 4 kb primers Universit anthesizr base anal dissolved and 12 p Biochemjs Catalyst 8 PTOducts. In Sl'SIem (Z Cloned p0 WA in a 3O 13). Restriction endonuclease digestions containing 10 pl (6.8 pg) of pL1200, 2 p1 of 10 x digestion buffer, 6 pl of distilled water and 2 pl (2 U) of restriction endonuclease were treated with Promega's Clean Up system. The products were ligated in reactions containing 5 p1(0.6 pg) of DNA, 5 pl of 10 x ligation buffer, 38 p1 of distilled water and 2 pl (6 U) of T4 DNA ligase. Transformation of DHSa with 5 pl (0.06 pg) of each ligation reaction yielded strains with the desired deletions in the pL1200 insert. To obtain these plasmids for sequencing, DHSa containing the desired plasmid was grown overnight in Terrific broth at 37 °C. After purifying plasmids with the small scale plasmid preparation, they were treated with Promega’s Clean Up system. Preparations which demonstrated a 260 nm2280 nm optical density ratio between 1.8 and 2.0 were used for sequencing. Dyed primer reactions were used to determine partial sequences of the inserts in pL1200 and its derivative deletion plasmids. This reaction used dyed primers fi'om the SP6 or T7 promoter; nucleotide bases; and nucleotide base analogs (Sanger et al., 1977) to amplify the 4 kb insert by the polymerase chain reaction (PCR). Dyed terminator reactions used primers synthesized by the Macromolecular Structural Facility at Michigan State University with a Perk and Elmer Applied Biosystems model 394 oligonucleotide synthesizer. These reactions contained the synthesized primers, nucleotide bases and dyed base analogs in PCR reactions. Dyed primer reactions contained 3 pg of plasmid dissolved in 15 pl of distilled water. Dyed terminator reactions contained 2 pg of plasmid and 12 pmol of primer dissolved in 20 pl of distilled water. At the MSU-DOE-PRL Plant Biochemistry Facility, automated flourescent sequencing was performed using the ABI Catalyst 800 for Taq cycle sequencing and the ABI 373A Sequencer for the analysis of products. . In vitro protein expression. Promega's S30 coupled transcription and translation system (Zubay, 1973 and Zubay, 1980) was used to express the polypeptides encoded by cloned pORl fiagments. The 50 pl reaction contained the following: 2 pg of plasmid DNA in a volume of 12 pl of water, 20 p1 of premix minus methionine (1.25 mM all 20 amin0 m'phosr glutamz (Mash [ethane 35S me the rear adding harvesu of load blue, 2( 10 pl w fragmer orientat asmall of 10 x (stored BOGhrir. Promeg for] hr nut Zn Plasmid 118 11g: ”indln. digested DU 1 00‘ 31 amino acids, except methionine; 5 mM adenosine triphosphate; 1.25 mM cytosine triphosphate, guanosine triphosphate and uridine triphosphate; 525 mM potassium glutamate; and 50 mM phosphoenol pyruvate), 2 pl (80 Units) of RNase inhibitor (RNasin) from Promega (stored in 20 mM N-[2-hydroxyethyl]piperizine-N’- [ethanesulfonic acid], pH 7.6; 50 mM KCl; 8 mM DTT and 50% (v/v) glycerol), 1 pl of 35S methionine (1200 Ci/Mmol at 10 mCi/ml) and 15 pl of S30 extract. Afier incubating the reaction for 2 hr at 37 °C, the protein from 10 pl of the reaction was precipitated by adding it to 40 pl of acetone and incubating it on ice for 15 min. The protein was harvested at 18,500 x g for 5 min, dried under a vacuum for 15 min, resuspended in 20 p1 of loading buffer (100 mM Tris-HCl, pH 6.8; 4% (w/v) SDS, 0.2% (w/v) bromphenol blue, 20% (w/v) glycerol and 8% (w/v) B-mercaptoethanol) and boiled for 5 min. Then, 10 pl were fractionated on a 12% SDS polyacrylamide gel. In vivo protein expression using T7 RNA polymerase. The 4 kb HindIII fiagrnent was subcloned fi'om pLJ 100 into pT7-4 (Tabor and Richardson) in both orientations to give plasmids, pL1270 and pL1271 (Fig. 17). Purified pT7-4 plasmid from a small scale preparation of M1830 (pT7-4) was mixed with 88 p1 of distilled water, 10 pl of 10 x digestion bufi‘er, 1 pl (10 U) of HindIII and 1 p1 (0.05 pg) of DNase-free RNase (stored in 10 rnM Tris-HCl, pH 7.0, 50 rnM CaC12 and 50% (v/v) glycerol) from Boehringer Mannheim. The plasmid was digested for 1 hr at 37 °C and purified with Promega's Clean Up system. After treating it with 4 units of shrimp alkaline phosphatase for 1 hr at 37 0C in a reaction containing 100 mM glycine-NaOI-I, pH 9.6, 1 rnM MgClz, 1 mM ZnC12 and 1 mM p-nitrophenyl phosphate; it was incubated for 30 min at 65 0C. Plasmid pLJ 100 was digested at 37 °C for 1 hr with HindIII in a reaction containing 10 pl (1.8 pg) of DNA, 34 pl of distilled water, 5 pl of 10 x digestion buffer, and 1 pl (1 U) of HindIII. Afier treating the digestion with Promega's Clean Up system, it was mixed with digested pT7-4 for 12-15 hr at 15 0C in a ligation reaction containing 8 pl (0.4 pg) of pLJlOO, 5 pl (0.3 pg) of pT7-4, 2 p1 of 10 x ligation buffer, 3 pl of distilled water and 2 pl (6 U)‘ reactior cloned 1111832 with thr pl of 11 with 5 t uith p1. transfor three str of 600 r Harvest: mM Tris and 0.01 lTactiona S Phoresis Protean 1 Wiped W11 32 (6 U) of T4 DNA ligase. Transformation of DHSa with 10 p1 (0.035 pg) of the ligation reaction yielded a transformant containing pL1270 (Fig. 17). The 4 kb fragment was cloned in the opposite orientation by performing a small scale plasmid preparation on M1832 (pL1270), digesting the preparation with HindIII as described for pT7-4, treating it with the Clean Up system and religating it in a reaction containing 17 pl of DNA (2 pg), 2 pl of 10 x digestion bufi‘er and 1 pl (3U) of T4 DNA ligase. Transformation of DHSa with 5 pl (0.5 p1) of ligation reaction yielded pL1271 (Fig. 17). 113101 was transformed with plasmid, pGP1-2 (Tabor and Richardson, 1985) to give M1831. M1831 was transformed with pL1270, pL1271 and PT7-4 to give M1834, M1835 and M1836. All three strains and M1831 were grown at 30 °C to an optical density of 0.25 at a wavelength of 600 nm, incubated for 30 min at 42 °C, and grown at 37 °c for an additional 90 min. Harvested cells fi'om 6 ml of culture were resuspended in 0.1 ml of cracking buffer (60 mM Tris-HCl, pH 6.8, 1% (w/v) SDS, 1% (v/v) B-mercaptoethanol, 10% (v/v) glycerol and 0.01% (w/v) bromphenol blue). Afier boiling the samples 5 min, 30 pl were fractionated on a 12% SDS polyacrylamide electrophoretic gel. SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electro- phoresis (Ausubel et al., 1992 and Laemmli, 1970) was performed using a Bio-Rad Protean II Slab Cell apparatus. Two thoroughly cleaned glass plates and two spacers wiped with ethanol were assembled in the apparatus. The following filtered, resolving gel reagents for a 12% gel were mixed and degassed for 15 min: 12.25 ml of distilled water; 14.0 ml of 30% (w/v) acrylamide and 0.8% (w/v) bisacrylamide; and 8.75 ml of 4 x Tris- HCl/SDS (1.5 M Tris-HCl, pH 8.8 and 0.4% SDS). The solution was gently mixed with 0.116 ml of 10% (w/v) ammonium persulfate and 0.020 ml of TEMED and poured between the plates to a level of 4 cm from the top. After layering 1 ml of water saturated sec-butanol on the top of the running gel, it was allowed to polymerize for 1 hr at room temperature. The sec-butanol was decanted. The gel was rinsed with distilled water and dried. The following stacking gel reagents were mixed and degassed for 15 min: 9.15 ml of wa Tris-I vrith ( migral bromp B-lactc and m} at app respect in a so dried ir at -80 ' acetic a brilliant Water ft 88% (V, PhOIOgr; 1‘ H30"€ste POIl’pept; (WV), 0. nor am, P01381231; thiogb’col‘ 33 of water; 0.95 ml of 30% acrylamide and 0.8% (w/v) bisacrylamide; and 3.75 ml of 4 x Tris-HCl/SDS (0.5 M Tris-HCl, pH 6.8 and 0.4% SDS). The solution was gently mixed with 0.15 ml of 10% (w/v) ammonium persulfate and 0.015 ml of TEMED, and poured on top of the running gel. After the stacking gel polymerized, the Bio-Rad Protean H Slab Cell apparatus was assembled with the upper and lower reservoir buffers both containing 25 mM Tris-HCl, pH 8.3, 200 mM glycine and 0.1% (w/v) SDS. Protein samples migrated through the stacking gel at 100 V and the running gel at 200 V until the bromphenyl blue dye reached the bottom of the running gel. Protein standards, lysozyme, B-lactoglobulin, carbonic anhydrase, ovalbumin, bovine serum albumin, phosphorylase B and myosin (H-chain), were obtained fiom Bethesda Research Laboratories and migrated at apparent molecular weights of 15.4, 18.1, 28.3, 43.3, 69.8, 105.1 and 215.5 kDa, respectively. For autoradiography, the running gel was fixed for 1 hr with gentle shaking in a solution containing 50% (v/v) methanol, 3% (v/v) glycerol, 10%(v/v) acetic acid; dried in a Bio-Rad model 583 gel drier at 80 °C for 2 hr; and exposed to film, overnight, at -80 0C. For coomassie staining, gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid, and 40% distilled water for 30 min and stained in 0.05% (w/v) coomassie brilliant blue (Bio-Rad), 50% (v/v) methanol, 10% (v/v) acetic acid and 40% distilled water for 4 hr. They were destained with 7% (v/v) acetic acid, 5% (v/v) methanol and 88% (v/v) distilled water for 2 hr. After rinsing them with distilled water, they were photographed. N-terminal amino acid sequence determination of the SedR polypeptide. Harvested M1836 (pGP1-2 and pL1271) cells that were induced to synthesize the SedR polypeptide were resuspended in 100 pl of 2 x sample buffer (0.2 M sucrose, 6% SDS (w/v), 0.125 mM Tris-HCl, pH6.9, 4 mM EDTA, 0.5% (w/v) bromphenol blue and 286 rnM B-mercaptoethanol), incubated for 15 min at 65 °C and separated on a 12% SDS polyacrylamide electrophoretic gel. The upper electrode buffer contained 0.1 rnM sodium thioglycolate. After electrophoresis, polypeptides were transferred from the running gel to 34 a sheet of Biorad’s polyvinylidene diflouride (PVDF) membrane (Speicher, 1989) using Biorad's Trans-Blot Cell apparatus. The apparatus was assembled as mentioned in the protocol for DNA transfer, except Towbin buffer (Towbin et al., 1979), which contained 25 rnM Tris, 192 mM glycine and 20% (v/v) methanol, was used as the transfer buffer. Polypeptides were transferred to the membrane at 30 V for 15-20 hr. The polypeptides on the membrane were then stained with 40% (v/v) methanol and 0.025% (w/v) coomassie blue R for 15 min and destained with 50% (v/v) methanol for 5 min. The SedR polypeptide was excised, and the first 15 N-terminal amino acid residues were sequenced by Edman degredation (Edman and Begg, 1967) using a Perk and Ehner Applied Biosystems model 494 protein/peptide sequencer (Matsudaira, 1987). This work was performed by the Macromolecular Structure Facility at Michigan State University. Nucleotide and protein sequence analysis. The Macintosh program, Amplify (Engels, unpublished), was used to predict whether a primer selected from a known nucleotide sequence in a fragment would amplify an unknown segment of the fragment in the polymerase chain (PCR) reaction. Adjacent nucleotide sequences were identified using a nucleotide sequence comparison program from Intelligenetics Geneworks (Smith et al., 1981; Smith and Waterman, 1981). This program located identical stretches between nucleotide sequences so that a complete sequence could be assembled from partial overlapping sequences. Nucleotide and polypeptide sequences were analyzed by a basic local alignment search tool (Blast) (Altschul et al., 1990) at the National Center for Biotechnology Information (NCBI). This program compared nucleotide and polypeptide sequences to other known sequences and identified segments that were similar or identical to segments of the known sequences. Information on using Blast was obtained by sending electronic mail to b1ast@ncbi.nlm.nih. gov with the word HELP in the body of the message. Sequences were examined for open reading frames (Tzagoloff, 1982) using a program fi'om Intelligenetics Gene Works for the Macintosh. 35 Growth curves. LB Broth was inoculated 1:100 with an overnight culture containing X2642 (pBR322), M1800 (pORl) or M1801 (pLJlOO). These new cultures were grown at 30 °C in a baffled flask at 200 rpm. Turbidity was measured every hour using a Klett Summerson Colorimeter with a no. 59 filter. After 2 hr, 1 M sodium selenite was added to give a final concentration of 40 mM selenite. For every hour from 3 hr to 12 hr, 25 ml of cells were harvested at 14,000 x g; resuspended in 1 ml of a bufi‘er containing 10 rnM potassium phosphate and 1 mM EDTA, pH 7.1; sonicated; and centrifuged at 20,000 x g. The supernatant was poured into new tubes, fi'ozen at -20 °C, and total protein was determined at a later date using a Bradford assay (1976). Both the cells and the elemental selenium which became associated with the cells contributed to turbidity. Thus, the same growth experiment was performed for X2642 (pBR3 22) in the absence of selenite to establish a linear correlation between turbidity and total protein for cells not associated with elemental selenium (Fig. 21). This standard curve was used to determine the expected turbidity attributed to cells alone for strains grown in selenite (Fig. 22). Total protein assay. Total protein fi'om cell extracts were determined using a method described by Bradford (1976). Bradford reagent which, was obtained from BioRad, contained 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol and 8.5% (w/v) phosphoric acid. Standard assays contained 5 ml of Bradford reagent and 10 to 100 pg of BSA dissolved in 1 ml of 10 mM potassium phosphate and 1 mM EDTA, pH 7.1. Absorbance was measured at 595 nm. A standard curve demonstrating a linear correlation between absorbance and protein was used to determine the amount of protein in an unknown sample. Bioremediation experiments. The dialysis tubing experiments (Komori et al., 1990) were assembled as shown in Figure 20. The top of a no. 14 rubber stopper was removed with a saw so that it fit firmly inside the top of a 600 ml beaker. Five holes that could firmly hold 2 ml conical, screw cap tubes (Fisher) were drilled evenly around the circumference of the top. Another hole was drilled through the center for a bubble tube. 36 The bottoms of five 2 ml conical tubes were removed. Four Spectral/Por 4 dialysis tubes with a molecular weight cut ofi‘ of 14,000 (Baxter) were sealed at one end and fit around the neck of four of the screw cap tubes that were inserted into one of the holes around the circumference of the stopper. The fifth hole also contained a screw cap tube, but it was used to withdraw samples from the medium. The bubble tube was inserted through the hole in the center of the stopper. The 600 ml beaker was filled with 350 ml of LB broth, and the assembled stopper was placed firmly in the beaker. Aluminum foil was placed around the top of the beaker to seal it, and the whole apparatus was sterilized in an autoclave. Three systems were assembled. To each system, sodium selenite was added to a concentration of 10 mM. Separate cultures of S. maltophilia OR02, M1800 (pORl) and HBlOl were grown overnight in 250 ml of LB broth at 25 °C, harvested at 3000 x g, and resuspended in 40 ml of LB broth. Samples of 10 ml were added to dialysis bags immersed in the medium and allowed to grow at 25 °C. Oxygen was introduced to each system by bubbling sterile air through the bubble tubes. Selenite concentrations were measured 12 and 24 hr later. Selenite concentrations were measured by mixing 0.5 ml of 1 M sulfuric acid and 0.5 mM 2-mercaptobenzimidizole with 0.5 ml of LB broth containing selenite. Afier allowing the reaction to incubate at room temperature for 2 hr, the formation of a selenite/2-mercaptobenzimidizole complex was measured at 318 nm (Blake, personal communication). The amount of selenite in a sample was determined from a standard curve that established a linear correlation between the amount of selenite and absorbance at 318 nm. Non-sterile batch culture experiments were performed using S. maltophilia OR02. An overnight culture of S. maltophilia OROZ grown in sterile M-9 minimal medium was diluted 1:100 in non-sterile M-9 minimal medium containing 100 rnM selenite, 0.5% acetate and 0.4 mM cysteine. A similar culture that did not contain S. maltophilia OR02 was also started. Both cultures were aerated at 25 0C in baflled flasks at 200 rpm. RESULTS Growth curves of S. maltophilia OR02, HBlOl, M1800, and MJ801. To establish a definition for selenite resistance, the ability of each strain to grow in LB broth containing 40 mM selenite was followed with a Klett Summerson colorimeter using a no. 59 filter. Each strain was introduced to selenite during early log phase and turbidity was measured every half hour. In addition to the cells, the elemental selenium, which precipitated during the grth of these strains, also contributed to the turbidity. Thus, the apparent grth rate, p (doublings/hr), was calculated for each strain grown in the presence and absence of selenite of selenite (Table 3). Even with the contribution of elemental selenium to turbidity, HB 101 failed to grow in selenite. It demonstrated an apparent growth rate of less than 0.15 doubling/hr. S. maltophilia, M1800 (pORl) and M1801 (pLJ 100) grew in selenite at apparent growth rates of 0.83, 0.75 and 0.67 doublings/hr, respectively. Thus, pORl and the 4 kb HindIH insert in pLJ 100 conferred selenite-resistance in HB101. Physical mapping and size determination of pORl. The physical map of pORl was constructed using BgIII, Acc651 (KpnI), HindIII, SacI (EcoICRI) and BamHI restriction enzymes. Digestions of pORl in Figure 5 were fractionated by agarose gel electrophoresis, and the length and number of each fragment produced was determined to calculate a size of 100 kb for pORl (Table 4). The gel was Southern blotted to a nylon filter and cloned Acc651, HindHI, SacI and BamI-II fragments were used as probes in hybridizations to identify adjacent pORl fragments (Fig. 6). In lane 2 of Figure 6, the 4 kb HindIII fi'agment hybridized to the 4.3 kb, 2.8 kb and 1.3 kb BgIII fragments. Thus, these three fragments were adjacent, with the probe containing the complete 1.3 kb BglII fragment and a portion of the other two fi'agments. The 16.5 kb and 21.5 kb BglII fragments also displayed weak signals, but digestions of these fragments with HindIII and a digestion of the 4 kb HindIII fragment with BgIII revealed by agarose gel 37 38 Table 3. Influence of pORl and pLJ 100 on cell growth in the presence of selenite. Organism Plasmid aApparent growth rate p (doublings/hr) - Selenite + seleniteb S. maltophilia OROZ pORl 1.6 0.83 E. coli HB101 1.2 < 0.15 M1800 pORl 0.81 0.75 M1801 pL1100 0.71 0.67 aSelenite (40 rnM) was added to early log phase cultures growing at 30 0C. The grth rate p = 1/ g, where the generation time g = ln2/k and k is the instantaneous grth rate constant. bThe contribution of selenite to turbidity was included in these calculations. 39 .NOMO uaERoacE 68858308628“. Eoc <20 880.6% 88 ”2 83 .583 832 52.2 <29 0888 Ede a 83 <26 8888 89 SE: .88 .m 8286 58.5 a 83 <29 3 8886 :8 s 83 .208 @0888. E83 8 83 .558 868% 38% um 83 .208 8886 58$ 3. 83 .208 868% :28: 688. ”m 83 .208 888mm. Ema ”N 83 SS 3 8688 38$ 3 83 88688 Eon .8 .8 888... .m 8.68 or m whmm V m N F 40 Table 4. Fragment sizes generated by restriction endonuclease digestions of pORl. Fragment sizes (kb) BgIII Acc651 (KpnI) HindIII EcoICRI (SacI) BamI-II 21.5 26.4 33 23.6 44 16.5 26.4 23.6 15.0 26.2 16.6 19.0 11.7 14.0 13.4 10.2 7.4 10.0 13.6 6.1 8.8 7.2 4.3 11.8 4.3 7.5 6.2 4.0 8.0 2.5 4.0 3.5 2.7 6.7 1.3 3.7 2.4 2.7 5.7 1.3 2.8 2.2 2.5 2.4 1 2.4 2.2 2.3 1.9 2.1 1.3 1.4 1.3 101.1 100.7 99.9 100.8 100.1 The sum of the fi'agments sizes are noted at the bottom of the table. 41 .NOMO 6.9223835 moaofiotuohozsh 88m <75 o6co=ow .899 A: 054 .CMOE 08:2 89¢ <75 8.209% 58,—. ”a 051— .29 8:88 89 2:92 .88 .9 8688 9682 ”w 83 <29 .2 .8588 89 s 83 .358 8688 E89 8 83 .298 8686 986. ”m 83 .298 8888 998.5 a. 83 .358 8688 988 :88. a 83 .298 89.88 Ewe ”N 83 <29 & 6860va 53.5 A 054 .9989 a 5 $59 88m “585$ 3625 8— v 86 $6: mcoumowE 359 .«o 6.6.55 Eofisom .w 9.5»:— o_. m m n_o m V m N? 42 electrophoresis that none of the resulting fragments were similar in size. The 4 kb HindIII probe was not located near these two fragments. The location of other pORl fragments was determined by repeating this experiment with the cloned pORl fragments shown in Figure 7. Approximately 60% of the map was constructed by using cloned pORl fragments as probes in hybridizations. The rest of the map was completed by using double restriction enzyme digestions. For example in Figure 8, lane 1 contained two 26.4 kb Acc651 (KpnI) fragments digested with Bng, lane 4 contained one 21.5 kb BgIII fragment digested with Acc651 and lane 6 contained two 16.5 kb BgIII fragments digested with Acc6511. The restriction enzyme digestion products of 11.2 and 10.3 kb in lane 1 and 4 suggested that the 21.5 kb BgIII fragment was cut in half by Acc651 and contained a segment of each 26.4 kb Acc651 fragment. The digestion products of 16.1 and 15.2 kb in lanes 1 and 6 suggested that the other half of each 26.4 kb Acc651 fragment contained a part of one of the 16.5 kb BglII fragments. Stability of pORl in S. maltophilia OR02. Under laboratory conditions, it appeared that pORl was converted to smaller plasmids in S. maltophilia OR02. In Figure 5, lane 10, pORl was no longer detectable, and four smaller plasmid bands that were present were not in the original gel electrophoretic profiles of S. maltophilia OR02. In addition, the 4 kb HindIII fragment hybridized to pORl from E. coli in lane 9 of Figure 6 and to two smaller plasmid bands from S. maltophilia OR02 DNA (lane 10) but not to a 100 kb plasmid band fi'om S. maltophilia OR02. The four small S. maltophr’lia OR02 plasmid bands were excised from an agarose gel and digested with HindIII (Fig. 9). Plasmid bands A and C both yielded fragments of 4.9 kb and 3.7 kb, Plasmid bands B and D both yielded fragments of 3.6 kb. Since the 4 kb HindIII fragment from pORl hybridized to plasmid bands A and C, these plasmids contained a homologous sequence. No relationship could be established between the plasmids in bands B and D. 43 38w .m 8“ as? J. WEE: .m ”Ema d ”Baum .m 8283058.. .808me 20“‘ some @558an 8:: 2: 323 town: 0.8 EoEmSm coco—o some 3 @385 823258 mo 88m .29“ we use 339353 .b 9...»:— :‘gfi 3 RM 78% E v E: Figure 8. Agarose gel used to map two 26.4 kb Acc651 (KpnI), a 21.5 kb Bng and two 16.5 kb BgIII fragments. Lane 1: BgIII digested 26.4 kb Acc651 fragments. Lane 2: BglII digested pORl plasmid. Lane 3: HindIII digested 7» DNA. Lane 4: Acc651 digested 21.5 kb BgIII fragment. Lane 5: Acc651 digested pORl plasmid. Lane 6: Acc651 digested 16.5 kb BglII fragments. Lane 7: PstI digested A DNA. 45 12345678 chromosomal DNA -—> plasmid band A —> ._ plasmid band B -> ‘ plasmid band C -—> -~ plasmid band D -> --' Figure 9. HindHI digestions of Stenotrophomonas maltophilia OR02 plasmid bands. Lane 1: S. maltophilia OR02 total genomic DNA. Lane 2: S. maltophilia OR02 total ge- nomic DNAdigested with HindIII. Lane 3: Plasmid band A digested with HindIII. Lane 4: Plasmid band B digested with HindIII. Lane 5: pORl digested with HindIII. Lane 6: Plas- mid band C digested with HindIII. Lane 7: Plasmid band D digested with HindIII. Lane 8 1 DNA digested with HindIII. 46 In vitro expression of pORl fragments. The size and number of detectable polypeptides encoded by each pORl fragment was determined by introducing each into an in vitro transcription and translation system (Zubay, 1973) and separating the products with a 12% SDS polyacrylamide gel. A fragment was considered to encode a polypeptide if its electrophoretic profile contained a band different in size from those in the profile of the pUC19 or pBR322 control vectors (Figure 10). The 11.8 kb SacI fragment produced 44.6, 38.0, 33.0, 28.2 and 25.1 kDa polypeptides. The 7.8 kb Acc651 fragment encoded 72.8, 33.8 and 15.6 kDa polypeptides. The 3.5 kb Acc651fiagment generated a 16.3 kDa polypeptide. The 13.4 kb BamI-II fragment produced 37.0, 34.5, 25.5, 24.7, 16.9 and 15.6 kDa polypeptides. The 11.4 kb HindIII fragment generated 233.3, 213.1, 69.3, 56.2, 37.9, 35.3, 33.8, 32.8, 25.5 and 20.7 kDa polypeptides. The 7.2 kb Acc651 fiagment encoded 33.4 and 25.5 kDa polypeptides. The 8.0 kb SacI fragment produced 76.6, 36.1, 32.3 and 20.3 kDa polypeptides. The 4 kb HindIII fragment generated a 35.3 kDa polypeptide. This was the only consistent band observed in all other expression experiments with this fragment (Fig. 11). The other bands observed in Figure 10 for the 4 kb HindIII fiagment were not detected previously. The calculated size of the polypeptide(s) produced by each fiagment are presented below the line which represents each fragment in Figure 7. All polypeptides encoded by these fragments were probably not detected by this system. Two dimensional gel electrophoresis would distinguish between some of the pORl polypeptides which are similar in size to those of pUC19 and pBR322. Incompatibility experiments. Preliminary nucleotide sequence data showed that the 4 kb HindIII fragment fi'om pORl contained a segment from the transposon, Tn1000, which was originally discovered in the E. coli F -plasmid. To determine if pORl was related to the F-plasmid, incompatibility tests were conducted using pUC19 and JM109. The proA and proB genes were deleted in JM 109 so that proline synthesis depends upon an F' episome containing proA+ and proB+. JM109 was transformed with each 47 N N N N §§§§§§§§§§ m 3 m 3 m 3 In 3 m 3 a. a. n a. a a. O- D- 9 °- 215kDa—',_ag_—g-gggr-s“=8 105kDa—‘ i V l 70kDa—l -- I 43kDa— T I _ 5.. 23 kDa— r» ”i. i, ”kDa—fl ' Figure 10. In vitro transcription and translation of cloned pORl fragments. Lane 1: 11.7 kb 5001 fragment. Lane 2: 7.4 kb Acc651 (Kpnl) fragment. Lane 3: 3.5 kb Acc651 (Kpnl) fragment. Lane 4: 13.4 kb BamHI fragment. Lane 5: pUCl9. Lane 6: 11.8 kb HindIII fragment. Lane 7: 7.2 kb Acc651 (KpnI) fiagment. Lane 8: 7.7 kb SacI fragment. Lane 9: No DNA. Lane 10: 4 kb HindIII fragment. Lane 11: pBR322. The 4 kb HindIII fragment was the only one cloned in pBR322. All other fragments were cloned into pUCl9. 48 18 kDa — ‘ 14 kDa — Figure 11. In vitro transcription and translation of the 4 kb, HindIII insert in pLJlOO. Lane 1: pLJlOO. 2: no plasmid 3: pBR322. 49 recombinant of pUC19 containing a pORl insert and spread on minimal ampicillin plates supplemented with and without proline. All transforrnants grew on both plates, except for the one carrying pLJ 193 which contained a 13.4 kb BamI-II fragment fiom pORl. It grew on the ampicillin plate supplemented with proline but failed to grow on the ampicillin plate lacking proline. However, it grew when a transformant from the ampicillin and proline plate was streaked onto an ampicillin plate without proline. To investigate this result more closely, the following incompatibility experiment was performed (Fig. 12). Overnight cultures of M1846 (pLJ 193) and M1847 (pUCl9) were diluted by 10'6 in minimal medium with proline (selection for neither plasmid), grown overnight and plated on minimal medium plate with proline. Fifty colonies fi'om these plates were placed on minimal medium plates with ampicillin and proline (selection for pUC19 and pLJ 193) and on minimal medium plates lacking ampicillin and proline (selection for the episome). All colonies of M1847 (pUCl9) grew on both plates. All colonies of M1846 (pLJl93) grew on the ampicillin and proline plate, but none grew on the plate lacking ampicillin and proline (Table 5). The F' episome was eliminated in the presence of pLJ 193 but was retained in the presence of pUCl9. To determine if the 13 kb insert in pLJ 193 contained an incompatibility determinant for pORl, the same experiment was performed for M1844 (pORI and pLJ 193) and for M1845 (pORl and pUC19). Both strains retained selenite-resistance, but none of the M1844 colonies and 74% of the M1845 colonies retained ampicillin-resistance (Table 6). When this experiment was repeated with selection for one of the plasmids during competition (ampicillin or selenite), all M1844 and M1845 colonies retained selenite-resistance and 74% of the colonies from both strains retained ampicillin-resistance (Table 7). It was not clear whether pORl out competed the CoelEl incompatibility determinant from pUC19 or an incompatibility determinant fi'om the 13 kb BamI-II insert in pLJ 193. .m .83 Smog .8.— anbaea 1:: 9:38 5:32.353:— .~— 9...»?— .2: 32.. 2:95 + nE< 82¢ 23:6 65.0.:— o=__o.a oEomoEoEo 8.2823 oz .cozomzmm oz :8 .m D w. ill 93 + 8m... .9555. 2:33» B. 8:13 8:2... 51 Table 5. Incompatibility experiments using the F’ episome fiom M109 and pLJ 193 that contains a 13 kb BamHI fragment from pORl. Percent colony growth M-9 medium M1846(F'+pLJ 193) M1847(F' episome’erC 19) Minus proline 0 100 Proline + ampicillin 100 100 52 Table 6. Incompatibility experiments with selection for neither plasmid. Percent colony grth of HB101 strains pUC19 pLJl93 pORl MRF' Kan pORl pORl pORl + + + LB broth pLJ 193 pUCl9 MRF' Kan Ampicillin 100 100 NA NA 0 74 NA Selenite NA NA 100 NA 100 100 100 Kanamycin NA NA NA 100 NA NA 100 NA - not applicable 53 Table 7. Incompatibility experiments with selection for one of the plasmids. Percent colony grth of I-IBlOl strains pORl pORl pORl + + + LB broth pLJ 193 pUCl9 MRF’ Kan Ampicillin 74 74 NA Selenite 100 100 100 Kanamycin NA NA 100 NA - not applicable 54 Two plasmids are defined as incompatible only if elimination is reciprocal during competition. Since elimination was not reciprocal in the incompatibility experiments above, pORl was tested directly by mating it with a strain that contained an F’episome. An inconsistent pro marker in M1800 (pORl) made it difficult to transfer the F ' episome from 1M109 to M1800. Thus, M1800 was mated with XLl- Blue MRF' Kan, which possessed a kanamycin-resistance marker on the episome, MRF' Kan. This new strain, M1841, retained selenite and kanamycin resistance in both types of incompatibility experiments, without selection for either plasmid and with selection for one of the plasmids (Table 6 and 7). These results suggested that pORl may not contain an incF determinant, may contain a CoelEl replication origin, or carries and uses an additional replication origin unrelated to the one fi'om the F -plasmid. Nucleotide sequence determination of the 4 kb fragment. The nucleotide sequence of the 4 kb, HindIII fragment from pORl was determined using the strategy shown in Fig. 13. It was subcloned between the T7 and SP6 phage promoters in plasmid, pSP73, to create the recombinant plasmid, pL1200. Dyed primers for the T7 and SP6 phage promoters were used to sequence the insert at both ends. Since each reaction was accurate through 300 bp only, deletions in pL1200 were constructed using the restriction endonuclease sites shown in Figure 13, and the sequence was determined fi'om these sites using the dyed SP6 primer. There were not enough restriction enzyme sites in the 4 kb fiagment to obtain a complete sequence. The Macintosh program, Amplify (Engels, 1993), helped predict primers which had a strong potential to amplify new segments of the 4 kb insert in a polymerase chain reaction (PCR). These primers were synthesized and used with dyed base analogs to complete the sequence of the pL1200 insert. The nucleotide sequence of the 4 kb fragment is shown in Figure 14. Sequence analysis. The DNA sequence was analyzed by a basic local alignment search tool (Blast) at the National Center for Biotechnology Information (N CB1) (Altschul et al., 1990) This program compared it to other known DNA sequences (Fig. 13). A 2.2 55 i 1kb i H P E B K s H E 1 I | 3|“ l 1 1391:, MOO pLJ203 pLJ202 pLJ201 pLJ204 pLJ205 ~ repZA Tn1000 EQ< x > Figure 13. Sequence features of the 4 kb, HindIII fragment from pORl. The location of a 35 kDa open reading frame, a 3.7 kDa hypothetical open reading fi'ame, a portion of rep2A and a 2.2 kb segment of Tn1000 are noted below deletions used to sequence this fragment. The open reading frames are not in the same frame. Abbreviations: BamHI (B); BgIII (Bg);EcoR1 (E); Hindm (H);Kpn1 (K);Pst1 (P); and SphI (S). 56 Figure 14. Nucleotide sequence of the 4 kb, HindIII fiagment from pORl. AAGCTTTCAG CAATTCAGTT TAATATATTC ATGATTCAAT TATCTCAATT TCAAAACAGG TGACTTTATG TCTGTGATAA CGCACTACAA GTGATGTTAC GAATTAATTA TATTCTCACC CTGAAGATAT GAACGGAACG GAAGATACCA ATTTCGGTAT ACCTACATAA CTACAATTAT ACGAAGAAAC TTATCATCAC GTCTAAAAAT AAATATTATA TAATTTTCGA TTGGTATGAA CTTCAAGTCA TGTGATGTGT CAAGGGTAAT TTCTCCGGAG AGTAGAGAAT TTTGGTGATC GGAGATAATT TCTGGCAACG ATAAGCCGGA AGCTCCCAAC GGCCCAGCGC CGTTGTCCGT GTCAGGATCC TTTACACGCC AAGAGCGAAT TCATCTCATC CCAGTACTCC CAAGCCGGGG TACCCGCAGT CTTCCAGTAC AGTACTGATC TGTTAGATCC CCACTTCTCC GGGCATTGGC GATGGCGGGT CTTCAGCCAT CACTGACATG CCGTTAAATC ATGGTGTCTC TTACAGATAT TCCAGCTGAA CGACAGACAG TCTCCCCAGC GCGTTCCACC GAGGCGTTTC CGAACCATTC CACCTTAAAA ATTGCAGAAA CATGAGACTC TGTAAATATT ACTGAAGGGG CTCGTGCCAA GGGAGATGTT TTTTAATAGG CATATATTAC TATTGCGTTA TAACCCCAAT GATTAAAAAT TCTTACCTGT GATAAGACTT GTCTTCTTTA GGGATATGCT AACAATGTTC TGATTTACCC AGGCAGGTTA CGCTACCTGC CAGAACCACC CCATTTTCAT GTGCGCAAAA ATAAGAAAAA CCCCCACTAT AAATTCCTAA TGTCATGGCG AAGGCTGGGA GGCGCGCATA GTCACCAGTA CGTTGTTTAT TGAATGTATA GCGATATCTT GCTTCCTTCA CCCCAGTTGA AACAAATCGC GCCGACGATA CTACTTCCAT GAGTTCTGAC CCACTGACTC GTAGTTTGCC GGAAAACTGA GTCTGTCATG AAAAATCGAA AGATACGAAG TGAGTTGATG ACCTCCCCAA GCTGACCAGC CAGTGCTGGT CAATACCGCA CGCAAACTCA TACCGGTGGG TTCCTGTTTC GAGCTGCCGC TAGCCAGATG GAATACGCTG GATCACTGTT AAACATAGTG CAGGGACCGG AGTTCTGCCA ACAAATTCCA ATATACTTTA TTTATGAATA GAATTTCCCT TATTCTGTAA AAAATGAAAT TTTTTAAGAT TCTGTTTTAC CAACCGATCT AAAAATTCAG GATAACAAAG AATCTTCCGC GATATCGAAA GTCTTGCAGT AGACGTATCA GAGCTTTCAC CATGATATAG AAACCCCCCT TATGATTGAA AGATGCATTT CATATAGCGG TTGATATGAA CCTGTCCGAT AACCTCCTTT CAATGGAAGT ATTTAAATCT GTCACCAAAC TATTTACATT ACAAAACAAC TATGGGGTTT ATTTAAATTT ATGGCCCAAC TCAATCTATA TATAGAGCGT TCCTTCCTGC .ACGAGCCACA ATCTTCATCA AATTGCCTGT GCATGAATTG TCCGCTTTTG GATTTATTCA TACCCCAGTA ATTTCACCTG TCCCGTAATG TTATACCAGG GTCTTCACTG CGACCAGCAA GTTTCTGCAC ATATTGTGCT CATAAACTGA TGTGTAAATC AGTAGCTTGT TCCAGACCAA ATTCTTTTCA TTGCACACCT AGTCTGCCAC TTCAAGCCAG AATAACCTGC TGATAATATG GATAATGAAT 57 ATAATAGAAA TCTATCATCC CTATAAAAAT GCCTCCTATC ATGAGATTTC TATATAAAAA TTAAAGCCTA CAGTTGTTTC ATAACGCAAA ATGGTAGTGC TATCTTATTT ATGGTTTTTT GGGAGTGTTT TTTCAACTAA GAAATAATAA ACTTGCAAAA AAAGCAATAG CATATAGATG AGAGGAGTTA GTACGAAAAT GTGGTTCTGA TTTCCTTAAA TTGTTAAACA GAATTCAAAG TTCTTATTTA ATTGCCCATT AATAACGTAG AAATCAACAT CGACAGGTTG GAGGGCCAAT AGAGCTCTCA ATATTGATAT ATTTCCTTTT GTTCCACAAG CCTTCACGAT GCGTGGCGGC TCAATATAAT GCCAGTCCTG TACCCAACTT ACAGATCTGC CCCGCCAGAA GCCAGAACAG GATTCAGCCC TACCGGGTAC TGATGCCTCG GTGTGACAAA GCTCCAGTGT GAAGGTAATT TTATCAGCGG TGTGCAAATC CTGTCTGGGC TTGATCCGAT CTGATAGTCA AATCGTGATG TTACGTCCAT TCTTCACCTT ATGTTCCCGG ACGCTGTATC TTCAGGCGCA CGCAGCCAGT AAACAAATGC TCACATTAGA AAAGCTAAAC TGAAATAGAG CAAGCGAAGA ACTTAACGTA CCTACAACCT ATCTAAAGGG AAATGGAAAT TTTGGCACTA GTGTGGACAT TATTACGATA ACATTTTATT AGAAGAACTT TGTTTATTTA CATACCTTTC TTTAACAGAT TTATTGCTGA AATATTTAAA TGTTAGCTTC TTTTTTTAAT ATTGATATCA ACAAAAAGAT GAGGAGAAAA TACCTGTTCT TTGGGGTAAA CTGGCGAAGT GCTGTCAGAA ACTTTTCAGA GGAACGAAAA GTTCCCCTTT GCCCGTTGCA CCCATTACGG AACCCACTGA AACGCTTTCT CTTCTCCTCG TGAGGAGATA ATGGGCGCGA CAGCGAACCA ATATCCTCGG CACCGCCTCA ACCGAAAATA TGTCTGCTGC CACAATGCCA TCCAGAACCA GCGCATGCCG GGACTGAAAA CTGTTTCACC TTCCAGTCCG TTGCGCTCGA ATCTATCTCA TATATAAGAC GGGATGGATA CCACGGCTTT CGACAGGATG GCAACAACTT CGGCGCAGTG CGTCCCTCCG TCATCAAGGA ACATGTTCAC CAAGGTAATG TGGGGATGAT CTAATTGATT ATAGAGTTAT AACAGAAGTA AAAAGCGATC AAAAATATCA GAGAGTATGG ATTGCTGGAT GATTCTGACA ATCAACAAAC AACATTAATC AAAGTTTTCG TACATAATAA TCGCTTAATG TCCTATGTAA ATAATAGCAA TGGTATAGAA AGGCATTGCA ATTAAAACAA ATCACCTGGT GCGAAGATTA TAAGGGTTTA ATAAGGCTTA TAATGTTAAA TTTCTTTGCT TCCATTGCGA GAAAGCACTG ATAGGGGCAG CGTACGTTAA TAAAATATCC TCAGGGGGAG GCGCAATCCA TTAGTAACCA GATCTCTCCG ATTAAGCTGC TAGCGTTTTG GTTTTCAGTA GCGGTTCGAT GCCAACTTAT CCTGCATCGG ATATCGCTGC TCAAGAAGTC TGGAACCCAG AAATATTTTC TCAGCTGATG TCAACCAGGC CAACTGAGCC ATATTACAGG GCACCAGATT AGTAACAGTT GGAACCAATG TTTACCTTCA CCAGGTCTCA TCCCAGTGTC CTCGCGAGGA CATCCTGAAG CATCGTATAC TGCGCTTTTT CTGCCGGTGC 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 Figure 14 (cont'd) . CGCACGGAAA CTCAACCATT TACAGATTCT GACATCTGTA ATCAAGATCT AATGTCCAGA TGCTTTAACG TGCAGGTCCC GCAACACCGC TCGTCATGGA.AATTATTGTT GCCAGTCTGT TTTTGGGTAC TTATCATCCA GCAACAATGC TTCAGTGTCC TGAGTCTTTT ATTAGCATAT CAAGCACATC AATGCAGTAA GTACAGCAAG 58 CGGAAGAAAA CGTTTAACCC GACCGTACTG CTGTGGACGA GCAAGTTCAT TAACCTTGCT GCACTTGAAG ATAACCTGCC TGAGATCTGG ACATGTCCGC GCCAGTAACA.ATGCGGCCTG TTTTTGCCCG GTTTTCTTTT CTTCGCGGAT AACGGCATCG TCTAATGCCG TTATTTCCTG CTT 3660 3720 3780 3840 3900 3960 3993 59 kb SacI/HindIII portion, bases 1764-3995, was identical to the complement of bases 3771-5981 of transposon, Tn1000. This segment of Tn1000 contained the C-terminal end of the transposase, mpA (Broom et al., 1993). Bases 1411-1764 were identical to the rep2A gene from the repFlC replication origin. Previous research demonstrated that Tn1000 interrupted a repFIC replication origin in the F-plasmid (Berquist et al., 1986; Saadi et al., 1987; Willetts and Skurray, 1987). Thus, Tn1000 may also interrupt a repFIC replication origin in pORI. Analysis of the 4 kb fiagment using IntelliGenetics, Gene Works (Tzagolofi‘, 1982), predicted that the other 1.4 kb SacI/HindIII segment contained open reading fi'ames for a 35 kDa polypeptide (Fig. 15, row 2) from base pair 132 to 1067 and a 3.7 kDa polypeptide (Fig. 16) fi'om base pair 1102 to 1203. There were no apparent -35 and -10 promoter sequences for these open reading frames. Sequence determination of the N-terminal region of the 35 kDa polypeptide. A level of expression higher than the one observed for the in vitro transcription and translation system was required to obtain enough of the 35 kDa polypeptide for amino acid sequencing reactions. The 4 kb insert fi'om pLJ 100 was subcloned in both orientations into plasmid, pT7-4, to obtain expression of both strands from the T7 phage promoter located on this vector. The two new recombinant plasmids, pL127O and pL1271 (Fig. 17), were transformed into HBlOl possessing pGP1-2 (Tabor and Richardson, 1985), a plasmid which encoded T7 RNA polymerase at 42 °C. Afier inducing expression of the 4 kb insert with T7 RNA polymerase, the samples were separated on a 12% SDS polyacrylamide gel (Fig. 18). Lane 3 revealed that pL1271 in M1836 encoded a 35 kDa polypeptide, which was not detected in Lanes 1, 2 and 4. These lanes contained extracts from M1835 (pGP1-2 and pL1270), M1834 (pGP1-2 and pT7-4) and M1831 (pGP1-2), respectively. M1834 synthesized a 32 kDa polypeptide not encoded by the other plasmids. The 12% SDS polyacrylamide gel of the polypeptides expressed in viva was electroblotted to a PVDF membrane (Speicher, 1989). After staining the blot, the band containing the 35 kDa polypeptide was excised, and the sequence of 15 N-terminal amino 60 Figure 15. Putative nucleotide and amino acid sequences of SedR. The first row is the nucleotide sequence, the second row is the amino acid sequence predicted by Intel- ligenetics, Geneworks, and the third row is the partial N-terminal sequence determined from the purified polypeptide. 61 AAGCTTTCAGCACCTTAAAAACAAATTCCAATAATAGAAAAAACAAATGCCAAGGTAATG CAATTCAGTTATTGCAGAAAATATACTTTATCTATCATCCTCACATTAGATGGGGATGAT TAATATATTCCATGAGACTCTTTATGAATACTATAAAAATAAAGCTAAACCTAATTGATT M R L F M N T I K I K L N L I D Y M N T I K I K L N L I D Y ATGATTCAATTGTAAATATTGAATTTCCCTGCCTCCTATCTGAAATAGAGATAGAGTTAT D S I V N I E F P C L L S E I E I E L L D S TATCTCAATTACTGAAGGGGTATTCTGTAAATGAGATTTCCAAGCGAAGAAACAGAAGTA S Q L L K G Y S V N E I S K R R N R S I TCAAAACAGGCTCGTGCCAAAAAATGAAATTATATAAAAAACTTAACGTAAAAAGCGATC K T G S C Q K M K L Y K K L N V K S D L TGACTTTATGGGGAGATGTTTTTTTAAGATTTAAAGCCTACCTACAACCTAAAAATATCA T L W G D V F L R F K .A Y L Q P K N I I TCTGTGATAATTTTAATAGGTCTGTTTTACCAGTTGTTTCATCTAAAGGGGAGAGTATGG C D N F N R S V L P V V S S K G E S M A CGCACTACAACATATATTACCAACCGATCTATAACGCAAAAAATGGAAATATTGCTGGAT H Y N I Y Y Q P I Y N A. K N G N I .A G C GTGATGTTACTATTGCGTTAAAAAATTCAGATGGTAGTGCTTTGGCACTAGATTCTGACA D V T I A L K N S D G S A. L A L D S D R GAATTAATTATAACCCCAATGATAACAAAGTATCTTATTTGTGTGGACATATCAACAAAC I N Y N P N D N K V S Y L C G H I N K L TATTCTCACCGATTAAAAATAATCTTCCGCATGGTTTTTTTATTACGATAAACATTAATC F S P I K N N L P H G F F I T I N I N P CTGAAGATATTCTTACCTGTGATATCGAAAGGGAGTGTTTACATTTTATTAAAGTTTTCG E D I L T C D I E R E C L H F I K V F G GAACGGAACGGATAAGACTTGTCTTGCAGTTTTCAACTAAAGAAGAACTTTACATAATAA T E R I R L V L Q F S T K E E L Y I I R GAAGATACCAGTCTTCTTTAAGACGTATCAGAAATAATAATGTTTATTTATCGCTTAATG R Y Q S S L R R I R N N N V Y L S L N D ATTTCGGTATGGGATATGCTGAGCTTTCACACTTGCAAAACATACCTTTCTCCTATGTAA F G M G Y A E L S H L Q N I P F S Y V N .ACCTACATAAAACAATGTTCCATGATATAGAAAGCAATAGTTTAACAGATATAATAGCAA L H K T M F H D I E S N S L T D I I A T CTACAATTATTGATTTACCCAAACCCCCCTCATATAGATGTTATTGC T I I D L P K P P S Y R C Y C 60 120 180 17 13 240 37 15 300 57 360 77 420 97 480 117 560 137 620 157 680 177 740 197 800 217 860 237 920 257 980 277 1040 297 1067 312 62 .ACCTACATAAAACAATGTTCCATGATATAGAAAGCAATAGTTTAACAGATATAATAGCAA CTACAATTATTGATTTACCCAAACCCCCCTCATATAGATGTTATTGCTGATGGTATAGAA .ACGAAGAAACAGGCAGGTTATATGATTGAAAGAGGAGTTAAATATTTAAAAGGCATTGCA M I E R G V K Y L K G I A TTATCATCACCGCTACCTGCAGATGCATTTGTACGAAAATTGTTAGCTTCATTAAAACAA L S S P L P A D A F V R K L L A. S L K Q GTCTAA V 1020 1080 1140 13 1200 33 1206 34 Figure 16. Hypothetical 3.7 kDa polypeptide from the 4 kb HindIII insert in pLJlOO. 63 ind Ill 'gl II 0.20 T7 LJ 270 Bla p 6.40 Kb \ Bgl II 1.70 Bam HI 2.00 Hind Ill 4.00 EcoR l 2.60 ind Ill {‘2 T7 pLJ Z71 coRl 1.40 6.40 Kb Bam HI 2.00 B I ll 2. Hind "14.00 9 3° Bgl II 3.80 Figure 17. Recombinant plasmids used for in vivo expression of SedR. 23 kDa— 18 kDa— Figure 18. SDS polyacrylamide gel of the expressed SedR polypeptide. Lane 1; M183 5 (pGPl-Z and pL1270), Lane 2: M1834 (pGP1-2 and pT7-4). Lane 3: M1836 (pGP1-2 and pL1271). Lane 4: M1831 (pGPl-2). 65 acid residues was determined. It matched the open reading fiame predicted by Gene Works, except translation started four residues downstream of the predicted start site (Fig. 15, row 3). This new polypeptide was designated as the selenite dissimilatory reductase polypeptide or SedR. Sequence analysis of SedR. Blast analysis (Altshul et al., 1990) of the SedR amino acid sequence did not suggest a possible function for this polypeptide (Fig. 19). Residues 1-83 were 67% similar to residues 1-83 in the YAHA protein, a truncated, hypothetical polypeptide located near the E. coli genes encoding proteins for the synthesis of choline glycine betaine, which is involve in osmoregulation (Larnark et al., 1991). A segment from residue 25-75 also was 66% similar to residues 143-193 of the UvrC polypeptide which is involved in excision repair in E. coli (Sharma et al., 1986). The same segment of SedR was 62% similar to residues 151-201 of the RcsB polypeptide which is involved in the regulation of colonic acid and capsule synthesis in E. coli (Stout and Gottesman, 1990). The regions of RcsB and UvrC similar to SedR contained helix turn helix motifs, which are involved in DNA binding, located from residues 42-64 in the SedR polypeptide. Deletion analysis of the 4 kb, Hindlll fragment from pORl. To determine if sedR, the 3.7 kDa hypothetical polypeptide or some other segment of the 4 kb HindIII fragment fi'om pORl was responsible for selenite-resistance, the deletions shown in Figure 20 were constructed, transformed into I-IB 101 and tested for selenite-resistance (Table 8). Each strain was introduced to 40 mM selenite during early log phase. Turbidity was measured 22 to 30 hours later. The positive control, M1801 (pLJlOO), was the only strain that exhibited resistance to selenite. M1848 (pL1280), M1849 (pL1281) and M1852 (pLJ307), did not grow much better than X2642 (pBR322), and M1850 (pL129l) and M1851 (pL1294) failed to grow as well as M1853 (pUCl9). Plasmids, pL1270 and pL1271 (Fig. 17), were ideal for making more deletions in the 4 kb fragment. Before introducing the deletions, they were tested for selenite resistance in strains M1832 66 >sp|P21514|YAHA;ECOLI HYPOTHETICAL PROTEIN IN BETT 3'REGION (FRAGMENT). >pirlSlO897|510897 hypothetical protein (betT 5' region) - Escherichia coli (fragment) >pir|315178|515178 hypothetical protein - Escherichia coli >gp|X52905lECBET_1 Escherichia coli betT, betI, betB and betA genes. [Escherichia coli] Length = 126 Score = 170 (78.7 bits), Expect = 3.3e-17, P = 3.3e-17 Identities = 35/83 (42%), Positives = 56/83 (67%) Query: 1 MNTIKIKLNLIDYDSIVNIEFPCLLSEIEIELLSQLLKGYSVNEISKRRNRSIKTGSCQK 60 MN+ ++ L ++ + V++ P +SE E LL L++G SV EIS+ RNRS KT 5 QK Sbjct: 1 MNSCDFRVFLQEFGTTVHLSLPGSVSEKERLLLKLLMQGMSVTEISQYRNRSAKTISHQK 60 Query: 61 MKLYKKLNVKSDLTLWGDVFLRF 83 +L++KL ++SD+T W D+F ++ Sbjct: 61 KQLFEKLGIQSDITFWRDIFFQY 83 >gp|X03691|ECUVRC_2 E. coli uvrC gene for DNA repair. [Escherichia coli] Length = 211 Score = 101 (46.8 bits), Expect = 1.4e-05, P = 1.4e—05 Identities = 20/51 (39%), Positives = 34/51 (66%) Query: 25 LSEIEIELLSQLLKGYSVNEISKRRNRSIKTGSCQKMKLYKKLNVKSDLTL 75 LSE B++++ + KG VNEIS++ N S KT + + +++ KLN+ D+ L Sbjct: 143 LSERELQIMLMITKGQKVNEISEQLNLSPKTVNSYRYRMFSKLNIHGDVEL 193 helix turn helix motif >sp|P376>splP14374IRCSB_ECOLI REGULATOR OF CAPSULE SYNTHESIS B COMPONENT. >pirlJV0068|BVECCB rcsB protein - Escherichia coli >gp|M28242|ECORCSBC_2 capsule synthesis regulator component B [Escherichia coli] >gp|L11272lECORCSC_2 rcsB gene product [Escherichia coli] Length = 216 Score = 101 (46.8 bits), Expect = 1.4e-05, P = 1.4e—05 Identities = 24/51 (47%), Positives = 32/51 (62%) Query: 25 LSEIEIELLSQLLKGYSVNEISKRRNRSIKTGSCQKMKLYKKLNVKSDLTL 75 L5 E E+L +G+ V EI+K+ NRSIKT S QK KL V++D+ L Sbjct: 151 LSPKESEVLRLFAEGFLVTEIAKKLNRSIKTISSQKKSAMMKLGVENDIAL 201 helix turn helix motif Figure 19. Blast analysis of the SedR polypeptide sequence. Helix turn helix motifs pres- ent in UvrC and RcsB are underlined. 67 I lkb J r l H P E S B Bg B H l l I I I I IiI pL1291 pL1281 and pL1294 pL1307 pL1280 Figure 20 Deletions analysis of the 4 kb, HindIII fragment. The fi'agments fi'om pL1280, pL1281 and pLJ307 were cloned into pBR322. Plasmid, pL1281, contains the open reading frames for SedR and the hypothetical 3.4 kDa polypeptide. Plasmids, pL1291 and pL1294, were cloned under the control of the lac promoter in pUCl9. Lines show regions remaining in the deletion. 68 Table 8. Influence of selenite on 113101 strains containing plasmids with different segments of the 4 kb HindIII fragment. Strain Plasmid Turbidity (Klett Units) M1801 pLJ 100 330 M1830 pT7-4 78 M1832 pL1270 86 M1833 pL127l 89 X2642 pBR322 59 M1848 pL1280 82 M1849 pL1281 71 M1852 pLJ307 3 M1853 pUC l 9 64 M1850 pL1291 27 M185 1 pLJ 294 2 69 (pL1270) and M1833 (pL127l) (Table 8). Neither strain demonstrated resistance to selenite. Thus, it appeared that the whole 4 kb fragment and pBR322 are required for the expression of selenite resistance. In pL1291, sedR was cloned under the control of the lac promoter in pUC19, and in pL1294, both sedR and the hypothetical 3.7 kDa polypeptide were cloned under the control of the lac promoter. M1850 (pL1291), M1851 (pL1294) and M1853 (pUCl9) were grown to a turbidity of 90 Klett Units and introduced to IPTG to induce expression from the lac promoter. Alter 1 hr of expression, selenite was added. M1850 and M1851 did not demonstrate an immediate ability to reduce selenite by producing a red color. These experiments suggested that SedR and the hypothetical 3 .7 polypeptide may not play a direct role in selenite-resistance. Growth experiments of X2642, MJ800 and MJ801 in selenite. In earlier experiments, the grth of X2642 (pBR322), M1800 (pORl) and M1801 (pLJ 100) in LB broth containing 40 mM selenite was followed by measurements of turbidity with a Klett Summerson colorimeter. Both the cells and the elemental selenium which became associated with the cells contributed to the turbidity. To determine the turbidity attributed to cells alone, turbidity and total protein at each time point were measured for X2642 grown without selenite (Fig. 21A) and for the three strains in the presence of selenite. A plot of total protein versus turbidity for X2642 grown without selenite was used to establish a linear correlation of turbidity to protein mass (Fig. 21B). The expected turbidity for each strain grown in selenite was then determined from the total cell protein measured at each time point. Figure 22 shows a plot of turbidity versus time and expected turbidity vs time. Normally, bacteria growing in logarithmic phase follow the equation, dx E- 2 Inc, where x is cell mass or turbidity, t is time and k is the instantaneous growth rate constant. However, the growth of cells exposed to selenite followed 5% = C , where C is constant. Not all the cells in the population survived to reproduce. By integrating this 7O 700 , , , . , soo soo _ A /.71 - 250 .5 50° ' . -/ 200 m .8 / / ‘ 8 o 400 - ' . ‘ 9 Li: o/ / - 150 D 4.} o—i 300 '- ' 4—3 .3 // - 100 3 O o . M e- 200 - /./ 100 - o/:/ — 50 ./.4/ O '/I I I I I 0 2 4 e a 10 12 14 Time (Hours) 300 B . 3 200 - Cl :3 4.: +3 3 M 100 " o . l l 41 J l l o 100 200 300 400 500 600 Total Protein Figure 21. Correlation of turbidity to protein mass for X2642 grown in the absence of selenite. A: Growth of X2642 measured by I turbidity and C protein mass. B: Linear correlation between turbidity and protein mass. 71 500 400 —- m , +3 / '~ :5 300 — . , l- / 3 ' v Q) 200 r—— // fl :4 ~ . . 100 14 Time (hrs) Figure 22. Influence of 40 mM selenite on the growth of X2642 (pBR322), MJ800 (pORl) and M1801 (pLJ 100). Symbols: I X2642 - observed; [3 X2642 - expected; 0 M1801 - observed; 0 M1801 - expected; V M1800 - observed; V M1800 - expected. 72 Xt—x: equation and solving for C, C = which is the slope of a linear curve. Linear .— t2 regression for the time points between 5 and 8 hours demonstrated that M1800 (pORI) grew at a C value of 19 Klett units/hr with an R2 of 0.997 and MJ801 (pLJ 100) grew at a C value of 29 Klett units/hr with an R2 of 0.995. X2642 (pBR322) was completely inhibited and reduced little selenite. HBlOl containing pLJ 100 or pORl exhibited inhibited growth, but began reducing selenite within one hour after it was added and was most active during stationary phase. Protein extracts from hours 3, 5, 7, 9 and 11 of the growth curves for X2642 grown without selenite and MJ801 grown in 40 mM selenite were elecotrophoresed through a 12% SDS polyacrylamide gel (Fig. 23). Throughout the growth curve, X2642 produced a 25 kDa polypeptide that was not produced by M1801, and M1801 synthesized a 42 kDa polypeptide that was not made by X2642. During stationary phase, X2642 also encoded a 51 kDa polypeptide which was not generated by M] 801. Other experiments demonstrated that when X2642 was grown in 40 mM selenite, it still contained the 25 kDa polypeptide but not the 42 and 51 kDa polypeptides. When M1801 was grown in the absence of selenite, it encoded the 51 kDa polypeptide but did not contain the 25 and 42 kDa polypeptides during stationary phase (data not shown). Cysteine requirement for selenite-resistance. S. maltophilia OR02, HBlOl, M1 800 and M1 801 did not grow on M-9 minimal medium containing selenite. Since glutathione was shown to be necessary for selenite resistance, three amino acids, glutamate, cysteine and glycine, which constitute glutathione were used as supplements. Table 9 shows that each strain did not grow on minimal medium plates unless they were supplemented with 0.4 mM cysteine. When the other two amino acids were present and cysteine was absent, none of the strains grew on minimal plates containing 20 mM selenite. 73 3 5 7 9 11 N N N N §§§§§§§§§§ m 3 m 3 m 3 m 3 m 3 Q. a. a. O. D. Q a. Q D. D. 215kDa——a_“ a—gga 105kDa— " ' -.- . 70kDa—l . 43kDa— 28 kDa -—g 9'". «.Ai m " 18kDa—«i a; 9 I?" a: a Figure 23. SDS polyacrylamide gel of extracts from X2642 (pBR322) grown in the absence of selenite and M80] (pLJ 100) grown in 40 mM selenite. Samples from 3, 5, 7, 9 and 11 hours during the growth of each strain were separated on a 12% polyacrylamide electrophoretic gel. m~ ;‘z 74 Table 9. Cysteine requirement for growth on minimal plates containing selenite. Growth Minimal Plates with S. maltophilia OR02 HBlOl MJ800 M1801 Cysteine + + + + Selenite - - - - Cysteine + Selenite + + + + S. maltophilia, HB101, MJ800 (pORl) and MJ801 (pLJlOO) were streaked on M-9 minimal salts plates containing 0.5% glucose, 20 mM selenite and 0.4 mM cysteine. Growth was detected by the formation of colonies. 75 Genetic investigation of the role played by glutathione, glutathione reductase, thioredoxin and thioredoxin reductase in selenite-resistance. To determine the requirement of glutathione and glutathione reductase for selenite reduction, strains of E. coli with mutations in the genes for glutathione synthesis, gshA (y- glutamycysteine synthetase) and gshB (glutathione synthetase), and glutathione reductase, gar (Oden et al.,1994), were transformed with pBR322 and pLJ 100. E. coli strains with mutations in thioredoxin, IrxA, and thioredoxin reductase, tpr, (Oden et al., 1994), were also tested because they have been found to reduce selenite to elemental selenium (Holmgren and Kumar, 1988). Each strain was introduced to 40 mM selenite in early log phase and measured for growth after 24 hours with a Klett Summerson calorimeter. The wild type strain containing pBR3 22 already appeared to be resistant to selenite because it displayed a turbidity of 210 Klett units (Table 10). Both glutathione mutants (gshA and gshB) containing pBR3 22 and the thioredoxin reductase mutant (tpr) containing pBR322 did not grow well. They exhibited turbidities of 30, 26 and 30 Klett units, respectively. These results suggested that the wild type strain relied on glutathione and thioredoxin reductase, instead of glutathione reductase, to convert selenite to elemental selenium. The glutathione reductase (gar) and the thioredoxin (trxA) mutants containing pBR3 22 maintained some resistance to selenite with turbidities of 170 and 119 Klett units. All strains containing pLJ 100 exhibited an enhanced ability to grow in the presence of selenite. Selenite-resistance conferred by pLJlOO did not appear to depend upon any of the genes tested above. Perhaps, the 4 kb insert fi'om pLJ 100 is involved in some other pathway used to reduce selenite to elemental selenium. Bioremediation experiments using S. maltophilia OR02 and MJ800. Dissolved sodium selenite is clear in solution. When cells convert it to elemental selenium, it formed a red precipitate which became associated with the cells. To determine if S. maltophilia OR02, MJ800 (pORl) and HE 101 could remove selenite from LB broth under sterile conditions, each strain was grown overnight, harvested, resuspended and 76 Table 10. Growth of glutathione, glutathione reductase, thioredoxin and thioredoxin reductase mutants in 40 mM selenite. Turbidity Relative Turbidity Strain Mutant pBR3 22 pLJ l 00 pLJ 1 00/pBR3 22 JF1070 w.t. - AB1157 210 308 1.5 JF420 gor 170 315 1.9 II" 2200 gshA 30 196 6.5 JF2201 gshB 26 147 5.7 JF432 tpr 30 187 6.2 JFZO62 trxA 119 189 1.6 JF1097 trxA and gshA 52 135 2.6 JF2014 trxA and gar 89 1180 2.0 IrxA - thioredoxin tpr - thioredoxin reductase gar - glutathione reductase gshA - y-glutamylcysteine synthetase gshB - glutathione synthetase 77 placed in dialysis bags which were immersed in liquid medium containing 10 mM selenite. After 24 hours of growth under aerobic conditions, the dialysis bags contained red cells that accumulated elemental selenium (Fig. 24). The LB broth outside the bags was still yellow, but there was no measurable decrease in selenite concentration. Since LB broth is expensive to use as a nutrition source, M-9 minimal medium containing cysteine was used in a non-sterile batch reactor to determine whether S. maltophilia could remove selenite from water containing 100 mM selenite. This level of selenite was supposed to prevent the reactor from becoming contaminated by inhibiting the growth of other bacteria. If this experiment was successfirl, S. maltophilia OR02 could be used in a sequencing batch reactor to remove selenite from a continuous flow of water. A sequencing batch reactor consists of a series of batch reactors. While S. maltophilia OR02 removes selenite in one reactor from contaminated water supplemented with M-9 salts and cysteine, other batch reactors are filling. When the reactor finishes removing the selenite and the cells settle, the water is drained off the top of the cells and the reactor is filled again. The initial batch culture experiment was not successful. Both the control (no added bacteria) and the experimental culture did not grow well. Due to a lack of time, this work was not pursued further. 78 Figure 24. Removal of selenite from LB broth. Each strain, S. maltophilia OR02 (l), MJ800 (2), and I-IBlOl (3), was grown ovenight in 250 ml of LB broth, pelleted and resuspended in 40 ml of LB broth. Samples of 10 ml were added to dialysis bags immersed in LB broth containing 10 mM selenite. DISCUSSION The physical map of pORl from S. maltophilia OR02 reveals a size of 100 kb. Experimental tests for F incompatibility are inconclusive. However, the BamHI and HindIII maps of pORl and the F-plasmid (Skurray et al., 1977; Childs et al., 1977; Ohtsubo and Ohtsubo, 1977; Johnson and Willetts, 1980; Cheah and Skurray, 1986) are highly similar (Fig. 25). The fragments created by digestions of both plasmids with both enzymes are similar in size and present in the same order. The transposon, Tn] 000, appears to be located in a similar position on each map. In addition the 13 kb BamI-II fiagment from pORl , suspected of containing an incompatibility determinant for the F- plasmid, is located in a position corresponding to the incompatibility determinant of the F- plasmid. Finally, Tn1000 interrupts repFIC in the F-plasmid (Berquist et al., 1986; Saadi et al., 1987; Willetts and Skurray, 1987) and may interrupt repFIC in pORl. Hybridizations using a probe for repFIA (Courtuier et al., 1980) may verify the presence of an F—plasmid replication origin in pORl. If pORl contains a replication origin for the F-plasmid, Stenotrophomonas maltophilia is an unusual host. The F -plasmid can move by conjugation to other genera of bacteria and replicate, but it is not well maintained in the absence of selection. It transfers to Proteus (Falkow et al., 1964, Datta and Hedges, 1972), Erwinia Chrysanthemi (Chatterjee and Starr, 1972), Pseudomonas flourescens (Mergeay and Gertis, 1977) Pseudomonas aerugw'nosa (Guiney, 1982) and Legionella pneumophila (VVrater et al., 1994). It also recombines with itself (Palchaudhuri and Maas, 1976), pBR322 (Guyer, 1978), and the chromosomes of E. coli (Davidson et al., 1974) and Pseudomonas syringae (Leary et al., 1984). The instability of pORl in S. maltophilia and of the F -plasmid in genera of bacteria other than E. coli may explain why the F- plasmid is not observed in natural bacterial isolates. A partial polypeptide map of pORl is also presented in this study. SDS polyacrylamide gel electrophoresis of polypeptides expressed by cloned pORl fi'agments 79 80 .833 a :85 e. v 2% 85:3. 2: ace 9.: 883 83853 a 2% see 82.; .52 .8350 23 83:5 use E... as e .228 Based 2: e5 2% .8 as: noise Ev Essa es. 3 52$ 230 8.89:8 .3 ea»...— '82,; , . ., a- e. n: e. S Q E. e. 2 So 9: .3 28.2 a K95 ab 36 md 3.0 3.0 m: -mmSel... How e. «.2 fl 3 e. S 8. SN 3 3 2. 9. sea E95 2 2 S m 823. , a- Q R e. e. 3 e. .2 e. M; e. .3 2 2&3 a has as 3. 3 3 3 2 2 e: no 2.0 3 m: -wmmmmw com . . On 9. 92 8.2 e. as e: e. a. e. e. 4.: e. fl 3: a E a has 2. 2 Z 2 Z . 81 in an in vitro transcription and translation expression system (Zubay, 1973) demonstrates that pORl encodes at least 32 polypeptides ranging in size from 15.6 to 233 kDa. A 4 kb, HindIII fragment from pORl confers resistance to selenite. Comparison of the nucleotide sequence of this fragment to other nucleotide sequences by blast analysis (Altschul et al., 1990) shows that a 2.2 kb segment is identical to Tn1000 (Broom et al., 1993); a 400 base pair segment, adjacent to Tn1000, is identical to rep2A fiom the repFIC replication origin (Berquist et al., 1986; Saadi et al., 1987; Willets and Skurray, 1987); and a 1.4 kb segment, unrelated to Tn1000 and repFIC, contains Open reading frames for a 35 kDa polypeptide, SedR and a 3.7 kDa hypothetical polypeptide. Comparison of the amino acid sequences to other known amino acid sequences does not reveal a possible firnction for either polypeptide. Polyacrylarnide gel electrophoresis of protein encoded by this fi'agment in vitro using a transcription and translation system (Zubay, 1973) and in viva using a T7 RNA polymerase expression system (Tabor and Richardson, 1985) confirms the existence of sedR. The sequence of the 15 N-terminal amino acid residues fiom this polypeptide verifies its origin on the 4 kb fiagment. The percentage of acrylamide used to detect SedR is not high enough to resolve a 3.7 kDa polypeptide. Thus, it is unknown if the hypothetical 3.7 kDa polypeptide is encoded by the 4 kb fi'agment. The complete 4 kb insert in pLJ 100 appears to be required for the expression of selenite-resistance. Deletion of Tn1000 and of both polypeptides eliminates selenite- resistance. M1832 and M1833 that contain subclones of the 4 kb fragment in pT7-4 in both orientations, does not exhibit selenite-resistance. Expression of sedR and the 3 .7 kDa hypothetical polypeptide using the lac promoter in pUC19 also fails to induce selenite-resistance. Hence, the vector from pLJlOO, pBR322, appears to play a role in resistance to selenite. Even HB101 cells transformed with pLJ 100, purified from selenite- resistant cultures of MJ801, fail to exhibit resistance to selenite, immediately. To obtain a selenite-resistant colony, a transformant must be grown overnight at 37 °C, incubated overnight at room temperature and plated without dilution onto an LB plate containing 82 selenite. Then, only a few selenite-resistant colonies appear. This inconsistent expression may be explained by a poor ability of the gene products fi'om the 4 kb fragment to interact with E. coli proteins involved in selenite-resistance. Introduction of this fragment into a selenite-sensitive strain of S. maltophilia or Pseudomonas may give better expression. The influence of the 4 kb fragment on selenite resistance is unknown. Experiments using glutathione, glutathione reductase, thioredoxin and thioredoxin reductase mutants suggest that E. coli strain AB1157 uses glutathione and thioredoxin reductase, instead of glutathione and glutathione reductase, for resistance to selenite. Since thioredoxin can reduce selenite to elemental selenium (Holmgren and Kumar, 1988), this result is not surprising. However, all mutants carrying the 4 kb fragment exhibit enhanced resistance to selenite. Thus, pLJ 100 did not appear to require any of the above components to confer selenite-resistance. This plasmid may influence selenite-resistance by producing a new type of redoxin that is reduced by several different reductases, encoding a reductase that reduces several difi‘erent redoxins or producing a regulator that induces some other pathway. Selenite-resistant strains of bacteria may use three mechanism to relieve the toxicity of selenite. They may reduce it to elemental selenium (McReady et al., 1965), prevent it fi'om entering the cell (Weiss et al., 1965) or incorporate it into selenomethionine (Scala and Williams, 1962), which does not interfere with protein function when it replaces methionine (Cowie and Cohen, 1957; Tuve and Williams, 1961; Frank et al., 1985). The growth experiments on MJ800 (pORI), MJ801 (pLJlOO) and X2642 (pBR3 22) demonstrate that the 4 kb fiagment confers selenite-resistance by reducing it to elemental selenium. X2642 (pBR322) inoculated into LB broth containing 40 mM selenite fails to grow and reduce selenite to elemental selenium. The selenite resistant strains, M1800 (pORl) and MJ801 (pLJ 100) grow and reduce selenite in LB broth containing 40 mM selenite. Therefore, to grow in a rich medium containing selenite, HB101 must reduce it to elemental selenium. The selenite reducing activity was the 83 highest during stationary phase. This observation may be explained by the presence of high levels of glutathione in E. coli during this phase (F ahey et al., 1978). It is unknown if these resistant strains exclude selenite from cell or incorporate it into selenomethionine. Selenite may inhibit cell growth by oxidizing sulflrydryl groups to form unstable selenosulfides (RSSeSR) (Ganther, 1971; Martin, 1973; Doran, 1982; Nakagawa, 1988); by reacting with glutathione, glutathione reductase, thioredoxin, thioredoxin reductase and NADPH to generate deleterious oxygen fi'ee radicals (Seko et al., 1988); or by replacing sulfiir in cysteine to form selenocysteine, which interferes with protein synthesis (Shrift, 1954; Heider and Bock, 1993). This last idea is confirmed by selenite-resistant, cysK, E. coli mutants (F irnmel and Loughlin, 197 7) and previous research demonstrating that cysteine and other sulfirr containing compounds relieve the growth inhibition of some strains grown in minimal medium containing selenate (Oremland, 1994) and selenite (F els and Cheldelin, 1949). The cysteine requirement for S. maltophilia OR02, M] 800 (pORl) and MJ801 (pLJ 100) to grow on M-9 minimal medium plates containing selenite is also in agreement with these studies. When cysteine and selenite are present in M-9 salts medium, the cells use the available cysteine, instead of inadvertently incorporating selenium from selenite into cysteine. The cysteine requirement may also be explained by the need to produce large quantities of glutathione to reduce selenite to elemental selenium. Since cysteine is a major component of glutathione, the cell may not be able to produce cysteine rapidly enough to synthesize the glutathione needed to detoxify selenite. Nevertheless, the pathway for selenite-resistance encoded by pORl and pLJl 00 cannot compensate for the absence of cysteine in M-9 salts medium containing selenite. Extracts of M1801 (pLJlOO) grown in 40 rnM selenite contain an extra 43 kDa polypeptide not observed in X2642 (pBR322) extracts from cells grown in the absence of selenite and lack a 25 kDa and a 51 kDa polypeptide present in X2642 extracts from cells grown in the absence of selenite. The roles these polypeptides play in selenite-resistance is 84 unclear. Obtaining a short N-terminal amino acid sequence of these polypeptides may indicate a possible identity and mechanism for selenite-resistance. In the bioremediation experiments, S. maltophilia OR02, M1800 (pORl) and HB101 inside dialysis tubing immersed in LB broth containing selenite reduce the selenite which diffuses into the tubing and sequester it as elemental selenium. Although S. maltophilia OR02 does not grow in non-sterile, M-9 minimal salts medium containing 0.5% acetate and 100 mM selenite, it may grow when glucose is present as a carbon source or when selenite is present at lower concentrations. Because selenate is the form of selenium that contaminates water under oxidized conditions (Masscheleyn, 1990), most studies have concentrated on using strains that reduce selenate to elemental selenium using aerobic (Lortie er al., 1990) and anaerobic (Macy, 1994; Steinberg et al., 1992; DeMoll- Decker and Macy, 1993) bacteria. For bioremediation using S. maltophilia OR02, it probably must work in conjunction with another strain that reduces selenate to selenite to effectively remove selenium fiom contaminated water. To obtain an efiicient system for selenium bioremediation, a clear understanding for the genetic and biochemical processes are important. The mechanism of bacterial resistance to selenite appears to be complex and may involve several pathways. This work establishes a genetic background which begins to explore the mechanisms for selenite- resistance in bacteria. 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