v or 4 a u . I ‘. J 't’ ‘0 ,. ‘- i ,. s, ,. ,A --po-.. r r—n ”‘0 “v v. <- ‘ av ....~ .r..- qlfib—s 4.. _' ~?fl€r‘~ -'- I ....h . '...,.“ "u . v.1} I‘IJI nwvvl- 1’ ' '7' .1111“ 7‘ g Hit-9: .on w.--".-..4._~.. .W' -—.g-<.. Dissertatiun for the: Degree: of Ph! D. MICHlGAN STATE UNIVERSITY ROS’ELYN VAUEAN LITTLE . f 1.914,. » . ~ ‘ . . . . , ‘, .. . . ‘ . ,. :4 o. vrdnfgm—mv «. ur‘ u H ..L '3'" _. r>wlr~lrl~ A _,A,. . 0-. . M» N17 1-» "a Hwy“, A ,~.m: . "was". t" .... ,- . _., , '., . .'..r..,,. "1” '-.‘- !.>’<- y: :r'..) $21.7"? . ’p- - ' . . . u. .- 1- \- hrvrrv I W o -— 2?... 44 '.. . ‘ . v - .M.‘ v1 , ‘7‘: 2%: .. n»: . W .q'. ,_ ,0 ‘7 “"‘fiffmu, . ' , ‘M, n 4'“; ~-;- .1 .A' A »v 0'. I r '1 I‘ g. .3 f - ' “p.31 fin»? m, .,—,’.~>'qvu. ‘ >e 0",: w ('0 to m. .err . c u~m~ -v "‘-’-""£'"~ .— .. “-76- ‘—a-.~ “31¢,“ Havan— Jvm ac r—7v- .ro-vv v44- —» -‘ “2:53 This is to certify that the thesis entitled TEMPERATURE-DEPENDENT REGULATION BY Ca2+ OF MACROMOLECULAR SYNTHESIS IN YERSINIA PESTIS presented by Roselyn Valjean Little has been accepted towards fulfillment of the requirements for Ph.D. Microbiology and PuEIic Health degree in ‘6Z34QJ:77/3~w£«1hq_ Major professor Date Feb. 19, 1974 0-7639 ABSTRACT TEMPERATURE-DEPENDENT REGULATION BY Ca2+ OF MACROMOLECULAR SYNTHESIS IN YERSINIA PESTIS BY Roselyn Valjean Little Growth of wild-type cells of Yersinia pestis is known to be dependent at 37 C, but not 26 C, upon the presence of physiological concentrations of Ca2+. During logarthmic growth at 26 C without Ca2+ or at 37 C with Ca2+ (permissive conditions), and 37 C without Ca2+ (re- strictive condition), the increase in bacterial mass paralleled that of total deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. This increase in mass was also proportional to the rates of DNA, RNA, and protein synthesis under permissive conditions. At the restrictive condition, however, the rate of DNA synthesis rapidly decreased whereas the rates of RNA and protein syn- thesis essentially remained constant. A net loss of radio- activity occurred in cells cultivated under permissive conditions but not in those at the restrictive condition following a label-chase with uracil-S-3H and excess un- labeled uracil; static cells (restrictive condition) Roselyn Valjean Little maintained a constant level of radioactivity. These observa- tions indicate degradation of RNA during logarithmic growth and either rapid turnover or conservation of RNA during bac- teriostasis. The demonstration of an unstable RNA fraction in growing cells (26 C) but not in static cells (37 C) follow- ing rifampin treatment implicates the synthesis of stable mRNA during bacteriostasis. Upon shift from restrictive to permissive conditions, further increase in mass occurred which corresponded to an immediate increase in rates of pro- tein, but not RNA synthesis. Following return to 26 C, DNA synthesis was initiated after mass had doubled but synthesis of new DNA was not detected at 37 C, folloWing addition of Ca2+, even though mass almost tripled. TEMPERATURE-DEPENDENT REGULATION BY Ca2+ OF MACROMOLECULAR SYNTHESIS IN YERSINIA PESTIS BY Roselyn Valjean Little A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1974 DEDICATION To my parents who have given me understanding, encouragement, and support in all endeavors, present and past. ii ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. R. R. Brubaker ("Professor Bob") who has been an advisor, professor, and friend to me. His advice and guidance steered me in the right direction through what sometimes appeared to be an unfathomable maze. As a professor, his teachings are invaluable and as a friend, one could not have better. To the members of my committee, Dr. Ronald Patter- son, Dr. Loren Snyder, and Dr. John Boezi, thank you for the many helpful discussions and other beneficial experi- ences received from having interacted with you. I wish to extend special thanks to Dr. Ronald Patterson for the use of facilities and equipment in his laboratory used for a portion of this research. My appreciation is sincerely extended to Ms. Prudence Hall ("Prudy") for her technical assistance in some of these experiments. I wish to acknowledge the Equal Opportunity Program Fellowship Office, Department of Microbiology and Public Health, and National Institutes of Health, Bethesda, Mary- land, for financial assistance during my graduate study in the Department of Microbiology and Public Health. iii TABLE OF CONTENTS GENERAL INTRODUCTION . . . . . . . . . LITERATURE SURVEY . . . . . . . . . . The Yersinia pestis Model . . . . . Taxonomy . . . . . . . . . . Physiology . . . . . . . . . Virulence Determinants . . . . . Bacteriostasis . . . . . . . . Control of Macromolecular Synthesis . . Kinetics of Macromolecular Synthesis . Shift-Up Experiments '. . . . . Shift-Down Experiments . . . . . Interdependence of DNA, RNA, and Protein References . . . . . . . . . . ARTICLE . . . . . . . . . . . . . iv Page GENERAL INTRODUCT ION Wild type cells of Yersinia pestis provide a sim- ple and unique system for the study of cell division be- cause of their response to changes in temperature when cultivated in Ca++-deficient media containing 0.02 M Mg++. There is no Mg++- dependent requirement for Ca++ when yersiniae are cultivated at 26 C. At 37 C, however, these cells become static unless physiological levels of Ca++ (0.0025 M) are present (5,7,32,42). Virulence or V and W antigens are expressed by wild-type (VWAf) cells only under conditions favorable for bacteriostasis (7,32,48), and since these conditions simulate mammalian intracellular fluid this response may be an adaptive mechanism necessary for the survival of these organisms within the intracellu— lar environment of the host (7,32,42-43). Sr+++ and Zn+ + (0.0025 M) can substitute for Ca++ in relieving bacterio- stasis at 37 C (32) but the role of these divalent cations in cell division has not been defined. The response of Yersinia pestis to a change in the temperature of its environment mimics that of a DNA—A or C type temperature sensitive mutant of Escherichia coli; the latter are blocked in the initiation of DNA synthesis and cell division at the restrictive condition. Bacteriostasis in yersiniae, in contrast to that in temper- ature sensitive bacteria, does not result in the formation of filaments but the static cells instead attain a size about twice that of dividing cells (28)._ A significant difference does, however, exist between yersiniae and tem- perature-sensitive bacteria, and should be kept in mind in making analogies between the two. The conditional state of yersiniae does not result from an alteration at the genetic level which is only expressed at the restrictive temperature as is observed in a temperature sensitive mu- tant. The conditional state of yersiniae appears to be related to changes in genetic expression that are influ- enced by the environment of these cells since the tempera- ture sensitivity of this organism is only expressed during cultivation at the restrictive temperature in the presence of high concentrations of Mg++ (0.02 M) and the absence of physiological levels of Ca++ (0.0025 M). Accordingly, changes in genetic expression in yersiniae which are ef- fected by environmental conditions can be studied by com— paring the molecular mechanisms that are Operative in dividing and static yersiniae; this is the basis for the experimental research in this dissertation. LITERATURE SURVEY The Yersinia pestis Model Only a summary of the properties of the Yersinia pestis system will be given; for a more detailed discussion of this system see the review by R. R. Brubaker (7). Taxonomy Yersinia pestis, the causative agent of bubonic plague, was formerly classified as Pasteurella pestis. Following the recommendation by the Xth International Con- gress of Microbiology (1970), reclassification as Yersinia pestis placed the organism in a genus named for its discov— erer, Yersin (7,39,71). The genus Yersinia contains Z. pestis, X. pseudotuberculosis, and X- enterocolitica (7). Yersiniae show a taxonomic relationship with enteric bac- teria based on common antigens (7,49), common sensitivity to phages (7,26,50,70,76), similarities detected by Addi- + sonian analysis (7,72), acceptance by yersiniae of F-lac (7,55), and RTF (7,25,40) from Escherichia coli, sensitiv- ity of certain strains of E. coli to pesticin (a bacterio- cin produced by Y. pestis) (4,7,70), DNA homology (7,66), and G-C content (7,49,54). Physiology Yersinia pestis will grow within a temperature range of -2 C to 45 C, and will tolerate extremes of pH between 5.0 and 9.6 (7,73-74). For the cultivation of yersiniae in yitrg the optimum temperature is 28 C, and the Optimum pH is between 7.2 and 7.6 (7,73-74). The or- ganism is less fastidious in its nutritional requirements at 26 C than at 37 C. At 26 C there is a requirement for L-methionine and L-phenylalanine; L-isoleucine, L-valine, and glycine will enhance growth (7,11,20,33). At 37 C yersiniae are extremely sensitive to high concentrations of free amino acids (6-7), and require biotin, pantothenate, thiamin, and glutamic acid for growth (7). The fermentation of glucose in X. pestis takes place via the Embden-Meyerhof pathway and the fermentation prod- ucts are lactate, ethanol, acetate, and formate (7,21,69). Acetate is oxidized to CO2 by the tricarboxylic acid cycle in aerated cultures (7,68). The hexose-monophosphate path- way is not operative in this organism because of the ab- sence of glucose-6-phosphate dehydrogenase (1,7,19,57-58), and pentose is probably synthesized by the rearrangement of 3 C and 6 C fragments by transketolase and transaldolase (7). An adaptive Entner-Doudoroff pathway, and remaining enzymes of the hexose-monophosphate pathway metabolize gluconate (7,57). Virulence Deter- minants The five established determinants of virulence in X. pestis involved in the process of infection are V and W antigens, Fl antigen, pesticin I, pigmentation, and the de novo synthesis of purines (7). The expression of these virulence determinants in yitrg depends upon either the temperature of cultivation or the presence or absence of divalent cations. Virulence or V and W antigens are a protein and lipoprotein (7,48) respectively that are always produced toqether by wild type strains (VWA+) (7,9,12); avirulent mutants (VWA_) do not produce either antigen (7,9,12). Virulent organisms (VWA+) show a Mg++ dependent requirement for Ca++ at 37 C for growth whereas avirulent mutants (VWA-) do not (7); Ca++ dependence is, therefore, implicated in pathogenicity. V and W antigens are only expressed in yitrg under conditions favorable for bacteriostasis (7,32, 48). Fraction 1 antigen (F1) is a protein antigen asso- ciated with the envelope produced by wild type (FRA+) strains (7,23). Organisms that are FRA' lack the ability to produce fraction 1 antigen, and are of reduced virulence in guinea pigs Inn: not mice (7,10). The optimum tempera- ture for expression of F1 antigen is 37 C; at 26 C small amounts of the antigen are present in a bound state (7,23). Pesticin, a bacteriocin, is produced by wild type (PST+) cells of X. pestis simultaneously with fibrinolysin and coagulase (7). Loss of one of these determinants re- sults in the concomitant loss of the other two, and cells lacking these three determinants are of reduced virulence in mice (7). Coagulase and fibrinolysin are associated with invasive properties of the organism, and whether the role of pesticin itself is associated with virulence is not certain (7). Pesticin inhibits the growth of certain strains of z. pseudotuberculosis, E. coli, X. enterocolitica, and PST- strains of X. pestis (4,7,70). The optimum tem- perature for production of pesticin I is 26 C (7). Wild type cells of X. pestis that are PGM+ can ab— sorb exogenous hemin and basic aromatic dyes, and grow as pigmented colonies on solid media (7). Strains of X. pestis that are PGM- do not absorb pigments, grow as non- pigmented colonies, and are avirulent (7). Optimum condi- tions for the expression of pigmentation are a temperature of 26 C and a pH of 8.0 (7). Wild type yersiniae are able to carry out the de novo synthesis of purines and are virulent; purine auxo- trophs are of reduced virulence in mice, and the degree of reduction in virulence depends on the location of the meta- bolic block (7). A block prior to the formation of IMP causes only a slight reduction in virulence in mice (LD % 50 102 cells) whereas a block between IMP and GMP results in an LD50 of > 108 cells (7). The avirulence of purine auxotrOphs is thought to be due to the inability to obtain purines from the host (7). Bacteriostasis Bacteriostasis in Yersinia pestis occurs during cultivation at 37 C in Ca++ deficient media containing 0.02 M Mg++ but the regulatory mechanisms responsible for stasis are not known. Observations by Yang and Brubaker (7,80) show that static cells remain viable, and there is no difference in the membrane permeability of static and dividing cells based on permeation of L-isoleucine,oxygen uptake, or release of preloaded 32F. Based on total syn- thesis of macromolecules by the incorporation of labeled isotope, protein and RNA synthesis are essentially identi- cal in both static and dividing cells but DNA synthesis steps in static cells about 4 hours after the removal of Ca++ (7,80). This residual DNA synthesis in static cells is sufficient to complete the current round of chromosome replication since the DNA content of static cells is greater than that of dividing cells (7,80), and static cells re- turned to 26 C in the presence of mitomycin C were able to perform one division (80). In the absence of mitomycin C, static cells returned to 26 C underwent at least two syn- chronous divisions (80). Control of Macromolecular Synthesis An understanding of the regulation of macromolec- ular synthesis within the cell is critical for a definitive solution to the enigma of cell division because a cell di- vides subsequent to completion of a round of replication (14,30) or attainment of a critical ratio of cell mass to DNA (15). The regulatory mechanisms that govern the SYD- thesis of macromolecules within the cell are not entirely understood but the flow of genetic information is known to proceed in a unidirectional manner. The genetic informa- tion encoded in DNA molecules is transcribed by a DNA-de- pendent RNA polymerase into 3 classes of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Following transcription, mRNA is translated into protein on polyribosomal structures by interaction with charged tRNA molecules. Kinetics of Macro- molecular Synthesis Maa1¢e and Kjeldgaard (52) stated as a general principle of regulation that "the overall production of DNA, RNA, and protein is regulated by mechanisms that con- trol the frequencies with which the synthesis of individual nucleotide and amino acid chains are initiated." This principle has been substantiated not only by the classic "shift experiments" performed by Kjeldgaard, Maal¢e, and Schaechter (37) but also by Yoshikawa et al. (82), Helm- stetter and C00per (29), Bremer and Yuan (2), Manor et al. (53), and Winslow and Lazzarini (78). Shift-Up Experiments.--In shift-up experiments (37, 52) bacteria in balanced growth were transferred from a simple defined medium to an enriched medium resulting in ‘ an increase in growth rate. The physiological changes ac- companying a shift-up were analyzed. The main feature of the shift-up is the dissocia- tion of RNA synthesis from other cellular events. There is an immediate increase in the rate of RNA synthesis while other activities initially continue at the pre—shift rate. Mass synthesis increases rapidly with a lag of about 5 minutes before the post-shift rate in the enriched medium is attained. DNA synthesis continues at the pre- shift rate for 20 minutes before the definitive rate in the enriched medium is reached. The average number of nuclei per cell remains constant at about 1.5 for 35 minutes and increases to 3.0 by 50-55 minutes after the shift. Cell division is maintained at the pre-shift rate for about 70 minutes before an increased rate is observed. This dis- sociation pattern is interpreted by Maal¢e and Kjeldgaard (37,52) to implicate the operation of separate control mechanisms for macromolecular synthesis. 10 RNA synthesis following a shift-up is unique be- cause not only is the increase in rate immediate but also because the initial rate in broth exceeds the definitive rate. This observation was made using chemical assays and further study of this phenomenon was made following the kinetics of RNA synthesis by the incorporation of 32F or l4C-uracil. The results of these experiments showed that the rapid increase in RNA synthesis is maintained for 25- 30 minutes before the definitive rate characteristic for broth is reached. Slow growing cells were concluded to have a latent capacity for RNA production because of the immediate increase in the rate of RNA synthesis following a shift-up (52), and the large and instantaneous rate of RNA synthesis in the presence of high concentrations of chloramphenicol (24,44). Consideration of the components necessary for RNA synthesis led to the hypothesis by Maal¢e and Kjeldgaard (52) that the overall rate of RNA synthesis is governed by the frequency of initiation of new chains. The frequency with which new chains are in— itiated was postulated to be controlled at the DNA level by a general, nonspecific repression mechanism. That is, RNA synthesis depends on the availability of "receptive" DNA sites or derepressed operons. The kinetics of protein synthesis during the pe- riod following a shift-up was followed by the incorporation 11 of labeled amino-acids and the results indicated a lag of 20 minutes before an increase in the post-shift rate is at- tained. The correlation between rRNA and growth rate (38, 52), the number of ribosomes and growth rate (18), and the number of ribosomes and protein synthesis (77) were the bases for the conclusion by Maal¢e and Kjeldgaard that the post-shift increase in the rate of protein synthesis was due to an increased concentration of ribosomes in the en- riched medium. The autoradiography of DNA by Cairns (13), the semiconservative nature of DNA replication demonstrated by Meselson and Stahl (56), genetic evidence provided by Yoshikawa and Sueoka (81) resulting from transormation of DNA markers from Bacillus subtilis, Maal¢e and Kjeldgaard's autoradiograms of fast and slow growing cells (52), and the multifork replication pattern observed in rapidly growing cells by Oishi §t_gl. (61) and Pritchard and Lark (65) formed the bases for interpretations by Maal¢e and Kjeld- gaard (52) which are given in the following statements. (i) "Replication is normally initiated at a specific site and proceeds continuously through the entire length of the genome; at a given temperature, this unit process takes approximately the same time whatever the growth rate." (ii) "During slow growth, DNA synthesis is dis- continuous, and a full round of replication takes less than one division time." (iii) " At the growth rate 12 characteristic of glucose—minimal medium, synthesis is al- most continuous, and one round of replication can just be completed during the division cycleJ' (iv) "At higher growth rates, synthesis is also continuous, but less than one round of replication is accomplished between successive divisions; the necessary doubling of the total DNA content _of the cell is achieved by having more than one growing point per nucleus." Shift-Down Experiments.--A shift-down experiment (37,52) is the correlative of a shift-up experiment, and. involves the transfer of bacteria from an enriched medium to a simple defined medium accompanied by a decrease in growth rate. The effects of a shift—down on DNA synthesis and cell division are similar to that observed following a shift-up in that the pre-shift rates are maintained for some time before the post-shift rates characteristic of the new medium are attained. RNA and protein synthesis, however, stOp immediately following a shift-down. The immediate effects of protein and RNA synthesis are followed after about 60 minutes by a gradual increase in rates that leads to balanced growth at reduced rates that are char- acteristic of the new medium. The immediate cessation of protein and RNA synthesis after a shift-down were suggested by Maal¢e and Kjeldgaard to simulate the direct and indirect effects respectively of amino acid starvation (52). l3 Interdependence of DNA, RNA, and Pro- tein There is evidence for an interrelationship between the synthesis of DNA and that of protein and RNA. The overall cycle of macromolecular synthesis, therefore, ap- pears to be regulated by mechanisms that are interdepend- ent. Stated simply: DNA is needed as a template for the synthesis of RNA (mRNA, rRNA, and tRNA); RNA serves as a template (mRNA), workbench (rRNA), and amino acid carrier (tRNA) for the synthesis of protein; protein and RNA are required for the synthesis of DNA. DNA i RNA J. PROTEIN The role of DNA in RNA synthesis (35,75), and RNA is pro- tein synthesis (3,27,34,59-60,63) is well documented, and will not be discussed in this review. The involvement of protein and RNA synthesis in the synthesis of DNA is of current interest and will be considered. The requirement for protein synthesis in the in— itiation of DNA replication was first suggested by Maa1¢e and Hanawalt (51). Experimental verification of a re- quirement for protein synthesis to initiate replication, 14 but not to maintain it, was provided by Lark, Repko, and Hoffman (45). This evidence lends support to the postula- tion by Jacob, Brenner, and Cuzin (36) of a hypothetical protein initiator coded for by a specific region of the DNA template in their "replicon" model. This positive control of the initiation of replication is also embodied in the models of Helmstetter gt_al. (31) and Donachie and Masters (16). A negative control of replication is set forth in the model by Pritchard §t_§l. (64) in which a re- pressor of initiation is synthesized and diluted below a critical concentration during growth allowing initiation to take place. The demonstration of a period before initiation in which protein synthesis is not needed in E. coli 15 T- by Lark and Renger (46) indicates the involvement of some other event in initiation. The possible involvement of transcription in the initiation of DNA synthesis was first suggested by Dove gt_gl. (17) based on observations of the replication of phage 1. Subsequently, Brutlag, Schekman, and Kornberg (8) demonstrated the necessity for transcrip- tion in the conversion of M13 single-stranded DNA to the double-stranded replicative form and suggested the possible role of RNA as a primer for DNA synthesis. Stable replica— tion in the absence of protein synthesis observed by Rosen- berg et a1. (67) and by Kogoma and Lark (41) requires 15 tranScription for its establishment but not for its mainte- nance because it can continue in the presence of rifampicin or streptolydigin. This observation favors a model of rep- lication in which RNA is needed as a stable product rather than as a primer which would need to be synthesized before each round of replication (62). Lark (47) has demonstrated a requirement for RNA synthesis in E. coli 15 T- even after the requirement for protein synthesis has been satisfied. Initiation of replication was sensitive to the rifampicin or streptolydigin mediated inhibition of RNA synthesis at ~a time when it was insensitive to high concentrations of chloramphenicol. The requirement for RNA synthesis was not, therefore, an indirect consequence of blocking the synthesis of protein(s) needed for initiation. Based on this evidence, Lark has proposed a model for replication whereby an RNA core with assembled proteins constitute an initiation complex for DNA synthesis. 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The nature of the system catalyzing the synthesis of induced B-galactosidase. Cold Spring Harbor Symp. Quant. Biol. 26: 133-144. Oishi, M., Yoshikawa, H., and N. Sueoka. 1964. Syn- chronous and dichotomous replications of the Bacillus subtilis chromosome during spore germination. Nature (Lond.). 204: 1069-1073. Pato, M. L. 1972. Regulation of chromosome replication and the bacterial cell cycle. Ann. Rev. Micro- biol. 26: 347-368. Plesner, P. 1961. Changes in ribosome structure and function during synchronized cell division. Cold Spring Harbor Symp. Quant. Biol. 26: 159-162. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 22 Pritchard, R. H., Barth, P. J., and J. Collins. 1964. Control of DNA synthesis in bacteria. Symp. Soc. Gen. Microbiol. 19: 263-297. Pritchard, R. H., and K. G. Lark. 1964. Induction of replication by thymine starvation at the chromo- some origin in Escherichia coli. J. Mol. Biol. 9: 288-307. Ritter, D. B., and R. K. Gerloff. 1966. Deoxyribonu- cleic acid hybridization among some species of the genus Pasteurella. J. Bacteriol. 92: 1838- 1839. Rosenberg, B. H., Cavalieri, L. F., and G. Ungers. 1969. The negative control mechanism for Escherichia coli DNA replication. Proc. Nat. Acad. Sci. U.S.A. 63: 1410-1417. Santer, M., and S. Ajl. 1954. Metabolic reactions of Pasteurella pestis. I. Terminal oxidation. J. Bacteriol. 67: 379-386. Santer, M., and S. Ajl. 1955. Metabolic reactions of Pasteurella pestis. II. The fermentatiOn of glucose. J. Bacteriol. 69: 298-302. Smith, D. A., and T. W. Burrows. 1962. Phage and bac- teriocin studies with Pasteurella pestis and other bacteria. Nature (Lond.) 193: 397-398. Smith, J. E., and E. Thal. 1965. A taxonomic study of the genus Pasteurella using a numerical technique. Acta path. microbiol. scand. 64: 213-223. Sneath, P. H. A., and S. T. Cowan. 1958. An electro- taxonomic survey of bacteria. J. Gen. Microbiol. 19: 551-565. Sokhey, S. S., and M. K. Habbu. 1943a. Optimum and limiting temperatures for the growth of the plague bacillus in broth. J. Bacteriol. 46: 25-32. Sokhey, S. S., and M. K. Habbu. 1943b. Optimum and limiting hydrogen ion concentration for the growth of the plague bacillus in broth. J. Bacteriol. 46: 33-37. 75. 76. 77. 78. 79. 80. 81. 82. 23 Spiegelman, S. 1961. The relation of informational RNA to DNA. Cold Spring Harbor Quant. Biol. 26: 75-90. Stocker, B. A. D. 1955. Bacteriophages and bacterial classification. J. Gen. Microbiol. 12: 375-381. Wade, H. E., and D. M. Morgan. 1957. The nature of the fluctuating ribonucleic acid in Escherichia coli. Biochem. J. 65: 321-331. Winslow, R. M., and R. A. Lazzarini. 1969. The rates of synthesis and chain elongation of ribonucleic acid in Escherichia coli. J. Biol. Chem. 244: 1128-1137. Worcel, A. and E. Burgi. 1972. On the structure of the folded chromosome of Escherichia coli. J. Mol. Biol. 71: 127-147. Yang, Gene C. H. 1970 Inhibition of the synthesis of deoxyribonucleic acid in Yersinia pestis during production of virulence antigens. Doctoral Dissertation, Michigan State University. Yoshikawa, H., and N. Sueoka. 1963. Sequential replica- tion of Bacillus subtilis chromosome. Proc. Nat. Acad. Sci. U.S. 49: 559-566. Yoshikawa, H., O'Sullivan, A., and N. Sueoka. 1964. Sequential replication of the Bacillus subtilis chromosome. III. Regulation of initiation. Proc. Nat. Acad. Sci. U. S. 52: 973-980. ARTICLE TEMPERATURE-DEPENDENT REGULATION BY Ca2+ OF MACROMOLECULAR SYNTHESIS IN YERSINIA PESTIS BY R. V. Little and R. R. Brubaker (Manuscript to be published) 24 ABSTRACT Growth of wild-type cells of Yersinia pestis is known to be dependent at 37 C, but not 26 C, upon the pres- ence of physiological concentrations of Ca2+. During logarithmic growth at 26 C without Ca2+ or at 37 C with Ca2+ (permissive conditions), and 37 C without Ca2+ (re- strictive condition), the increase in bacterial mass paral- leled that of total deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. This increase in mass was also proportional to the rates of DNA, RNA, and protein synthesis under permissive conditions. At the restrictive condition, however, the rate of DNA synthesis rapidly decreased whereas the rates of RNA and protein synthesis essentially remained constant. A net loss of radioactivity occurred in cells cultivated under permissive conditions but not in those at the restrictive condition following a label-chase with uracil- 5-3H and excess unlabeled uracil; static cells (restrictive condition) maintained a constant level of radioactivity. These observations indicate degradation of RNA during loga- rithmic growth and either rapid turnover or conservation of RNA during bacteriostasis. The demonstration of an unstable RNA fraction in growing cells (26 C) but not in static cells 25 26 (37 C) following rifampin treatment implicates the synthesis of stable mRNA during bacteriostasis. Upon shift from re- strictive to permissive conditions, further increase in mass occurred which corresponded to an immediate increase in rates of protein, but not RNA synthesis. Following return to 26 C, DNA synthesis was initiated after mass had doubled but syn- thesis of new DNA was not detected at 37 C, following addi- tion of Ca2+, even though mass almost tripled. TEMPERATURE-DEPENDENT REGULATION BY Ca2+ OF MACROMOLECULAR SYNTHESIS IN YERSINIA PESTIS Wild-type cells of Yersinia pestis (a facultative intracellular parasite) fail to grow in vitro at tempera- tures characteristic of the mammalian hosts unless Ca2+ is + . . . . present. M92 potentiates the nutritional requirement for + . . . . . Ca2 which is not observed during cultivation at room tempera- ture. The concentrations of Ca2+ and Mg2+ that promote growth or bacteriostasis in vitro are identical to those reported to exist within mammalian extracellular (2.5 mM Ca2+; 1.2 mM MgZ+) and intracellular (no Ca2+; 20 mM Mgz+) fluids, respectively (13). Although this relationship may be coincidental, the response of yersiniae to Ca2+ is unique in nature and presumably reflects an adaptation to these distinct in yiyg environments (4). Ca2+ can serve as a cofactor for certain membrane- associated enzymes (8) and exoenzymes (17) and can also exist as a structural component of the gram negative cell surface (6,7,19). Previous attempts to equate bacteriostasis with loss of these functions were not successful. Ca2+- starved yersiniae were about twice the mass of normal or- ganisms but were otherwise similar in morphologY; both 27 28 types of cells exhibited comparable rates of oxidation, trans- 32P (4). These findings port, and retention of endogenous suggested that the cell membrane was not grossly altered dur- ing bacteriostasis. In this case, catabolic pathways and possibly anabolic reactions might also remain functional. In order to test this possibility, a comparative study of macromolecular synthesis was initiated. Preliminary experi- ments, as expected, failed to disclose the occurrence of significant deoxyribonucleic acid (DNA) synthesis during bacteriostasis. However, the apparent rates of ribonucleic acid (RNA) and protein synthesis per static organism closely approximated those detected per normal cell (20). The purpose of this report is to define the kinetics of DNA, RNA and protein synthesis in static and normal yer- siniae and to relate these findings to the accumulation of mass. MATERIALS AND METHODS Bacteria The live vaccine strain EV76 of Y. pestis was used throughout this investigation. Cells of this isolate ex- hibit the nutritional requirement for Ca2+ typical of wild- type organisms but are avirulent due to the mutational loss of a determinant said to regulate iron metabolism in yiyg. No genetic or phenotypic relationships exist between this 2 property and the expression of Ca +-dependence (4). Cultivation Modified Higuchi medium (20) was used in all experi- ments. The composition of this medium, which was also used as a diluent, is shown in Table l. The organisms were incubated for 36 to 48 hr at 26 C on slopes of blood agar base (Baltimore Biological Labs, Bal- timore, Md.), removed in diluent, centrifuged at 27,000 x g for 10 min at 5 C, resuspended, and added to 100 m1 of medium per 1 liter flask. Inoculation was performed to yield an optical density of about 0.08 at 620 nm which corresponded to 7.5 x 107 viable cells per ml. Flasks were aerated in a model G76 gyrotatory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 26 C (a permissive condition) for 3 to 5 hr until the optical 29 3O density approximated 0.1. By this time the cells had gener- ally commenced logarithmic growth and the cultures were either retained at 26 C, shifted to another shaker set at 37 C following addition of 2.5 mM CaCl (another permissive 2+ 2 condition), or shifted to 37 C without addition of Ca (restrictive condition). In some experiments, cultures were maintained under the restrictive condition just described for 10 hr and then shifted to one or the other permissive condition. Routine plating was performed on blood agar base and oxalate agar (12) in order to monitor the incidence of Ca2+- independent mutants in the cultures. In no case did the latter contribute significantly to the total bacterial popu- lation. Net Synthesis of Macromolecules Samples of 2.0 ml were periodically removed from cultures and immediately frozen in an alcohol-dry ice bath. Prior to analysis, the samples were thawed and then precipi- tated by addition of an equal volume of chilled 10% trichloro— acetic acid. After storage for 30 min in the cold, the pre- cipitates were collected by centrifugation, washed with 5% trichloroacetic acid, and then resuspended in 0.5 ml of 10% perchloric acid. Nucleic acids were quantitatively extracted (9) by treatment at 70 C for 10 min followed by overnight in- cubation at room temperature. After centrifugation, the 31 supernatant fluids were removed and the precipitates were again extracted by the same procedure. Both supernatant fractions were pooled and assayed for DNA and RNA by the diphenylamine method of Giles and Myers (10) and the orcinol procedure of Dische (5), respectively. Precipitates remain- ing after the second perchloric acid extraction were dis- solved by boiling in 1.0 m1 of 1 N NaOH for 10 min; protein in these samples was determined by the method of Lowry gt_al. (14). Standards were calf thymus DNA, yeast RNA, and Bovine serum albumin obtained from the Sigma Chemical Co. (St. Louis, Mo.). Kinetics of Macromolecular Synthesis The uptake of radioactive thymine or thymidine by yersiniae was not inducible thus carrier-free isotopes were used to determine the kinetics of DNA synthesis. In prelimi- nary experiments, a 3 min lag was observed and then linear incorporation into DNA was directly proportional to cell mass over the range encountered during growth under permissive conditions. The incorporation of thymine was about 4 times that of thymidine thus the former was used in pulse-labeling experiments; incorporation of thymine was not significantly influenced by the temperature of incubation. The kinetics of DNA synthesis were determined by add- ing 1.0 m1 of culture to tubes (20 x 150 mm) containing 0.1 m1 of an aqueous solution of carrier-free thymine-methy1-3H 4.— 77F 32 (10 u Ci). The tubes were aerated for 7 min in a water bath set at the same temperature as the parent culture. Incorpora- tion of isotope was stOpped by addition of 2 ml of cold 5% trichloroacetic acid and the samples were stored for 1 hr in an ice bath. Following centrifugation, the supernatant fluids were carefully decanted and the precipitates were resuspended in 0.2 ml of 5.5 N NaOH. The tubes then received 3 m1 of cold 5% trichloroacetic acid and, after 30 min in an ice bath, the precipitates were collected by filtration through membrane filters (Arthur H. Thomas, Co., Philadelphia, Pa.). After washing with an additional 20 m1 of 5% trichloro- acetic acid, the membranes were dried and prepared for count- ing. All solutions of trichloroacetic acid contained 0.004% nonradioactive thymine. Variations in growth were sometimes noted when exoge- runusuracilor L-histidine was added to the medium. In order to avoid these variables, carrier-free uracil-5-3H (10 u Ci per m1) and uniformly labeled l4 C-L-histidine (0.1 u Ci per ml) were used to determine the kinetics of RNA and protein synthesis, respectively. Histidine was chosen as a precursor of protein because this compound was the only noncataboliz- able amino acid not already present at high concentration in the medium. Under the conditions used in pulse-labeling ex- periments, the incorporation of radioactive uracil and histi- dine into trichloroacetic acid—insoluble material was linear and proportional to mass over the range encountered during 33 growth at permissive conditions. However, the incorporation of uracil during cultivation at 37 C was significantly greater than that observed at 26 C. RNA synthesis per unit of cell mass at 37 C was therefore greater than that determined at 26 C. In contrast, the incorporation of histidine per unit of cell mass was slightly greater at 26 C than at 37 C. The procedure used for determining the kinetics of RNA and protein synthesis was similar to that described for DNA except that pulses were of 5 and 6 min duration, respectively, and, following storage of trichloroacetic acid-precipitated material for 1 hr, the precipitates were directly collected on membrane filters. After drying, the membranes were placed in vials which received 10 ml of toluene base containing 0.4% 2,5-dipheny1- oxazole (PPO) and 0.005% 1,4-di-2-(5-phenyloxazoly1)-benzene (dimethyl POPOP). Radioactivity was determined in a Packard Tricarb scintillation counter. Radioisotopes were purchased from New England Nuclear (Boston, Mass.). Degradation of RNA Logarithmically growing cells (26 C) were radio- actively labeled in modified Higuchi medium containing uracil-5-3H (0.1 u Ci per m1) and unlabeled uracil (1 pg per ml) for 12 hr. Following this labeling period, excess cold uracil (5 pg per ml) was added and the label was chased for 4 hr. The cells were collected by centrifugation at 34 27,000 x g for 20 min at 5 C, washed once in 0.033 M potassium phosphate (pH 7.0), and resuspended in modified Higuchi medium containing unlabeled uracil (1 pg per ml) to yield an optical density of 0.1. Resuspended cultures were aerated under the restrictive and permissive conditions. Samples (1.0 ml) were removed from cultures at hourly intervals for 11 hr and treated in the same manner described for the kinetics of RNA synthesis. Rifampin Treatment Growing (26 C without Ca2+) and static (37 C without Ca2+) yersiniae were treated with sodium phosphate-ethylene dinitrilotetraacetic acid (0.1 M phosphate, 10-3 M EDTA, pH 6.8) as described by Bremer et_al.(3) to facilitate the up- take of rifampin (18). The EDTA treatment was terminated by dilution with 26 C or 37 C medium lacking Ca2+ (1 vol). At 4 min subsequent to the termination of EDTA treatment 1.0 m1 aliquots of the treated cells were added to tubes con- 14 taining 0.1 m1 of uracil-Z-C (10 u Ci) and 0.1 ml of rifam- pin (100 ug). After various periods of incubation (26 C or 14 and rifampin, 0.5 ml 37 C with aeration) with uracil-Z-C samples were removed and processed to obtain the alkaline hydrolySate of the acid precipitate following the procedure of Bremer gt_al. (3); this procedure completely excludes radioactive DNA. The alkaline hydrolysate (0.5 ml) containing the radioactive ribonucleotides was counted in 5 ml of Bray's 35 liquid scintillation fluid (2). Rifampin was purchased from Calbiochem (San Diego, Calif.). £43.55 Cell mass was determined as a direct function of op- tical density at 620 nm in 1 ml cuvettes with a model 2000 spectrophotometer (Gilford Instrument Labs, Oberlin, Ohio). Uninoculated medium was used as a blank. RESULTS ties: The increase in cell mass during cultivation at 26 C and at 37 C in the presence and absence of Ca2+ is shown in Figure 1. After a lag, the rates of increase were similar under the 2 sets of permissive conditions but the logarithmic growth phase was extended for about 2 hr during incubation at 26 C. After the first shift to restrictive conditions, mass doubled and then decreased Slightly towards the end of the determination. A significant lag again occurred after the second shift to permissive conditions; this phase was most prominent in the culture which received Ca2+ at 37 C. This determination of mass defines the experimental system used in subsequent investigations of macromolecular synthesis. The responses shown in Figure 1 were generally reproducible although some variation was noted upon initial shift to 37 C in the presence of Ca2+ and in the duration of the lag period following recovery from bacteriostasis at 37 C following addition of Ca2+. In order to facilitate direct comparisons, mass is shown in subsequent illustrations de- picting total synthesis and rates of synthesis of macro- molecules. 36 37 Figure 1.--Effect of Ca2+ and temperature on the growth of Y. pestis strain EV76. A series of cultures were i3- oculated and aerated at 26 C; one culture remained at this temperature throughout the experiment (.) . At the point shown by the first arrow, one culture received 2.5 mM CaCl and was shifted to 37 C (C)), and 32$ultures were Shifted to 37 C without addition of Ca (1)). One of the latter was maintained un- der this condition and, at the point shown by the second arrow, the second was shifted to 26 C with- out addition of Ca2+ (6) while the third culture was retained at 37 C after receiving 2.5 mM Ca2+ ((1)- ##wu * OPTICAL DENSITY 38 E I I I I I I g I0.000 :— : Q . ' I A: . 0 ° " .t- LOCO . ........ """"""""" I llllllll O.|OO II] I I IIIIIII llll 0.0lO I I I I I I I 4 8 |2l6 202428 HOURS O 39 Total Synthesis of Macromolecules Increases in net DNA, RNA and protein paralleled that of mass during growth under permissive conditions (Figure 2). Similar results were obtained during cultivation under re- strictive conditions indicating that bacteriostasis was not a function of gross change of macromolecular composition. Kinetics of Macromolecular Synthesis During the Onset of Stasis In control cultures incubated under permissive con- ditions, DNA synthesis closely paralleled mass until the cells approached the stationary phase whereupon a decrease became evident (Figure 3A, 3C). DNA synthesis under restrict- ive conditions also initially followed mass and then fell rapidly following the onset of bacteriostasis (Figure BB). Similarly, the ratio of RNA synthesis to mass re- mained nearly constant during cultivation under permissive conditions (Figure 4A, 4C). However, unlike the results ob- tained with DNA, RNA synthesis under restrictive conditions was maintained relative to mass following the onset of bac- teriostasis (Figure 4B). Constant ratios of mass to protein synthesis were also observed during growth under permissive conditions (Figure 5A, 5C). In contrast, protein synthesis under re- strictive conditions fell to a reduced but constant level relative to mass as the organisms became static (Figure SB). 40 Figure 2.--Total DNA, RNA and protein, as determined by chemi- cal assay, in cultures of Y.‘ estis strain EV76 cultivated at 26 C without Ca + (. , at 37 C with- out Ca2+ (o), and at 37 c with 2.5 mM Ca2+ (o). All cultures were incubated for 4 hr at 26 C with- out Ca2+ before shift to the indicated conditions. SHOOH SHnOH 41 OPTICAL DENSITY 8 8 8 O O O O I III'TII I llllllll I IIIIIII ro— .. A- .— 0)- _ m— _ 5" _ fi— _. 3; I lllllllI I IIIIII Protein (Hg/ml) RNA (Hg/ml) DNA(ug/ml) u 5 5 _ O O O 04 0 Rev ""I -_ "'l N :- :\\\.I.\L.n£ II I III»:- 8 er 0| 8/ / . tI 42 Figure 3.--Kinetics of DNA synthesis, determined by pulse- labeling with 3H-thymine, in cells of X. pestis strain EV76 cultivated at 26 C without Ca + (A), at 37 C without Ca2+ (B), and at 37 C with 2.5 mM Ca2+ (C). All cultures were incubated for 3 hr at 26 C without Ca2+ before shift to the in- dicated conditions: (0) , optical density; (0) , radioactivity. SHnOH SUflOH SHnOH 3| 43 OPTICAL DENSITY O ,0 '— 2 6 O o o 8 IIITAyI I IIIIIIrl IIIIII - o I> 7 I— —. I- -—1 J ITIIIIIII ||||||T I [Tlnll I IIIIIII] I IIIInI ’0 w ‘ I— —-I " ‘I P .PllllllI L lvlllIlJ l IIIIiL'II I IIII'Tr I lllllll] I IIIIIII I— 0 —I I om co, 0,, INCORPORATION 0F 3H-THYMINE (CPM) 44 Figure 4.--Kinetics of RNA synthesis, determined by pulse- 1abeling with 3H-uravil, in Cells of Y. pestis strain EV76 cultivated at 26 C withouE Caz; (A), at 37 c without Ca2+ (B), and at 37 c with 2.5 mM Ca2+ (C). All cultures were incubated for 4 hr at 26 C without Ca2+ before shift to the indi- cated conditions: (0) , optical density; (O) , radioactivity. SHflOH SHflOH SHnOH 2| 3| BI 45 OPTICAL DENSITY O .0 .- 8 O O I IIIILI I IIIIIIII I IIIIIII .. ]> .. I LllllllI 4 I llllllI I IIIATI T IIIIIIII TTJIII e w J - . _ _ IIIIIIIII vIIIIIIII lJllll I I IIL I IIIIIII I FIIIIII — 0 - I— _ llllllllI I ON O"I O. INCORPORATION 0F z’H-UR/Icu. (0PM) 46 Figure 5.--Kinetics of protein synthesis, determined by pulse- labeling with l4c-histidine, in cells of Y. pestis strain EV76 cultivated at 26 C without CaZI (A), at 37 c without Ca2+ (B), and at 37 c with 2.5 mM Ca + (C). All cultures were incubated for 5 hr at 26 C without Ca2+ before shift to the indicated conditions: (0), opitcal density; (.), radio- activity. -—_— SHnOH SHnOH SHDOH 2| 3| El 47 OPTICAL DENSITY O 0 "‘ . :__ o Q o o O O O I III HI] I Tllllll] I IIIIIII .. >— o T I 1111111] I IIIIIIII IIIIII I IIIAIII I IITIITTI IIIIIFII I- m —I I— — - a b I IIIIIIII 111111 I IIII ‘4 I III" II I [TUTTI] I IIIII L O '7 e -I o "' _I I— _ I I I IIIIII .1 I I III I I I IIIII om o“, o; INCORPORATION OF I‘IC-HISTIDINE (0PM) 48 Kinetics of Macromolecular Synthesis Upon Recovery from Stasis In the following experiments, sets of 3 parallel cul- tures were incubated for 10 hr under restrictive conditions; a culture was then shifted to each of the permissive condi- tions and the remainder was maintained as a control at 37 C without added Ca2+. Increase in mass and macromolecular synthesis were followed as the cells recovered from bacterio- stasis. Significant synthesis of DNA was delayed for about 8 hr following return of static organisms to 26 C (Figure 6). At this time, mass has approximately doubled and a rapid syn- thesis of DNA was maintained until a second doubling occurred. Thereafter, synthesis paralleled the increase of mass. New DNA was not produced in the control culture retained under restrictive conditions. Similarly, synthesis of DNA was not initiated in cells maintained at 37 C after addition of Ca2+ even though mass had almost tripled at an optical density of 0.8 when the experiment was terminated (not illustrated). Upon return to 26 C, the ratio of RNA synthesis to mass appeared to decrease initially (Figure 7A); this change was caused in part by the effect of temperature on uracil- transport noted previously. The decreased ratio observed following addition of Ca2+ to cells maintained at 37 C (Figure 7C) cannot be explained on this basis. A similar initial decrease in synthetic rate was also determined in control cells retained under restrictive conditions (Figure 7B). 49 Figure 6.—-Reinitiation of DNA synthesis, determined by pulse- 1abeling with 3H-thymine,in cells of Y. pestis strain EV76 upon shift from 37 C to 26 C in Ca +- deficient medium. The organisms had previously been maintained at 37 C for 12 hr: (()), optical density; (.), radioactivity. 50 3&9 wZ_E»I._.IIn no zo_._. :I :22: .5... _ .52: _ m >._._mzmo I_ e .1 .—| o l IIIIIIII I IIIIIIII F I lllllll IA Illlll] I IIIIII CDq _I —d _I O ' -4 — 1411111“ L I I 111111 I lllllll] fiAIIIIIIII IIIIIII 04 o —I d I IIIIIIII llJll'lI lilllLll V zOl 0m 6,, INCORPORATION 0F z’H-IJRACIIJCPM) 53 In contrast, the ratio of protein synthesis to mass increased following return to permissive conditions. This increase was more apparent following return to 26 C (Figure 8A) than after addition of Ca2+ to cells maintained at 37 C (Figure 8C). In both cases, however, the ratios eventually approached those observed during initial growth under per- missive conditions (Figure 5A, 5C). A further decrease in protein synthesis relative to mass wassobserved in control cultures maintained under restrictive conditions (Figure 8B). The results that have been given for the kinetics of macromolecular synthesis by pulse-labeling of DNA, RNA, and protein correspond to those Obtained by replotting theSe data to give the rates of nacromolecular synthesis during the onset Of stasis (Figure 9) and upon recovery from stasis (Figure 10). Degradation of RNA Following a 12 hr labeling period of RNA with uracil- 5-3H and a 4 hr chase with excess unlabeled uracil, loss of radioactivity was observed only under permissive conditions and not at the restrictive condition upon resuspension in unlabeled modified Higuchi medium (Figure 11). Rifampin Treatment Treatment of growing cells (26 C) with rifampin re- sulted in an immediate burst Of RNA synthesis which reached a maximum at 30 sec, and subsequently declined to a constant 54 Figure 8.--Kinetics of protein synthesis, determined by pulse- 1abeling with l4C-histidine, in cells of Y. pestis strain EV76 cultivated at 26 C without Ca?+ (A), at 37 c without Ca2+ (B), and at 37 c with 2.5 mM Ca2+ (C). All cultures were incubated for 10 hr at 37 C without Ca2+ before shift to the indicated conditions: (0) , Optical density; (.), radioactivity. SHnOH SHDOH SHDOH 3| 3|- 3| 55 OPTICAL DENSITY O .0 — 9 a 8 O O o I IIIIII I— — _ I IIIIIIII I IIIIIIII I III I f‘glllllll I IIIII _ m —I — . _ C b I ll’llllI4_1 IIIIIII I IIIIIII T IIIIT f IIIIIII IIIII ._ O _. I IIIIIIII 1111111 ON 00‘ 0‘. INCORPORATION OF "C-HISTIOINE (CPM) 56 Figure 9.--Rates of macromolecular synthesis during the onset of stasis in cells of Y. estis strain EV76 cultivated at 26 C without Ca (1), at 37 C without Ca2+ (2), and at 37 C with 2.5 mM Ca2+ (3). The data ShOWn in Fig. 3-5 was redrawn to illustrate the rates of DNA syn- thesis (A), RNA synthesis (B), and protein synthesis (C). 57 m1 000m .0000 OOON 000m 000.: ooodm ooodv ooodm OOOdm _ 7 d A I Ii OOO. OOON 000m .1an '00 19d (WdOIAllAllOVOIGVH Figure]lL--Rates of macromolecular synthesis upon recovery from stasis in cells of Y. pestis strain EV76 cultivated at 26 C without Ca + (1), at 37 C without Ca2+ (2), and at 37 C with 2.5 mM Ca2+ (3). The data shown in Figures 6-8 was redrawn to illustrate the rates of DNA synthesis (A), RNA synthesis (B), and protein synthesis (C). 59 000. 000m 008 000k 0000 000.. _ 000.0N 000.0? 000.00 000.00 N_ 000_ 000m 000m UNIT '00 led (WdOMIIAIIOVOlGVH |lll in!!! {I J 60 Figure 11.--Loss of trichloroacetic acid-insoluble radioactiv- ity, following a label-chase with uracil-5-3H and excess unlabeled uracil, in cells of Y. pestis strain EV76 during cultivation in unlabeled modi- fied Higuchi medium at 26 C without Ca2+ (.), at 37 C without Ca2+ (o), and at 37 c with 2.5 mM Ca2+ (C) . 9.0.0 IO I I r. - _ . L O O O O 2 4 m a m >._._>_._.0<0_o