Vol. 173, No. 7

JOURNAL OF BACTERIOLOGY, Apr. 1991, p. 2265-2270

0021-9193/91/072265-06$02.00/0 Copyright © 1991, American Society for Microbiology

A GTP-Binding Protein (Era) Has an Essential Role in Growth Rate and Cell Cycle Control in Escherichia coli NATAN GOLLOP AND PAUL E. MARCH* Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854 Received 23 October 1990/Accepted 31 January 1991

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40°C but completely repressed at 27°C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTPbinding proteins as important regulators of cell growth and division may be ubiquitous in nature.

It is now widely recognized that a large family of similar proteins possessing GTPase activity are key regulators of cell growth and division. These proteins include factors required for protein synthesis, G proteins, and the ras oncogenes, among others. Only a few such GTP-binding proteins are known in Escherichia coli, including a membrane-associated GTP-binding protein (Era) which was shown to possess low-level intrinsic GTPase activity (1, 8, 16). The gene (era) for this protein has been cloned, sequenced, and located at min 55 on the E. coli chromosome within the rnc operon (1). Mutational analysis has shown that an intact era allele is required for growth on plates, but analysis of conditional mutants has not led to the delineation of any phenotype that suggests a function for Era (11, 16, 21). In order to understand the function of Era in vivo, we constructed several strains in which transcription of era is controlled by the phage X promoter PR. The strategy employed in mutant construction allows one to obtain a new strain by simply transforming bacteria with the vector harboring the disrupted gene. era mutants incubated at the nonpermissive temperature are nonviable and exhibit an elongated cell phenotype. MATERIALS AND METHODS Strains. The bacterial strains employed to construct conditional era mutants have been described elsewhere and are referred to in Table 1. Growth conditions. Cells were grown in Luria broth media (5 g of yeast extract, 10 g of tryptone [Difco], and 5 g of NaCl per liter). The media were supplemented with antibiotics at the following concentrations in micrograms per milliliter: ampicillin, 50; kanamycin sulfate, 50; and tetracycline, 15. Shift-down experiments were carried out in two water bath shakers with platforms rotating at the same speed. Growth was monitored turbidimetrically by using a KlettSummerson calorimeter containing a red 66 filter. Strategy of mutant construction. The basic strategy involved constructing a temperature-sensitive vector which would permit survival of recipients only when there was a single homologous recombination between the plasmid and *

the targeted gene while at the same time disrupting the target. The plasmid pNG187 was employed to disrupt era in the chromosome, as shown in Fig. 1. The key feature of this plasmid is that a truncated clone of era (era') encoding only the first 142 amino acids (out of 316 total residues) is expressed by a phage A mutant promoter PR (X3) (9). The X3 mutation reduces expression from PR by 90%. The plasmid also contains the temperature-sensitive phage X repressor c1857 (20). The origin of replication of pNG187 was derived from the low-copy-number plasmid pEL3 (2); thus, DNA replication of this plasmid is temperature sensitive. Transformation of the plasmid pNG187 into the recipient strain was performed at 30°C. Recombinants were obtained by selecting for kanamycin resistance at the nonpermissive temperature (42°C). Plasmid integration into the chromosome at era by single homologous recombination results in cells which grow at 42°C on Luria broth media containing kanamycin and which possess a disrupted rnc operon, as shown in the bottom line of Fig. 1. Candidates that grew stably on antibiotic-containing media at 42°C were isolated and checked for growth at 27°C. All strains were found to be cold sensitive for growth because of the repression of the synthesis of Era by the c1857 repressor. Some mutant strains TABLE 1. Strains employed to construct recombinants with pNG187a Parental strain

JM83 PAM162 PAM163

JFL126b JFL128b JFL127b a

Pertinent parental genotype

None

sulB26 lon-22 sulA27 lon-22 sulB2J Ion-100

Reference or source

17 12 12 J. Lutkenhaus

sulB14 lon-100 sulB9 lon-100

J. Lutkenhaus J. Lutkenhaus Parental strains listed were transformed with pNG187 and subsequently

screened for cold sensitivity as described in the Materials and Methods section. Mutant strains were grown at 40°C and later shifted to 27°C for 10 to 12 h. Cell morphology was examined by phase-contrast microscopy. The PAM and JFL derivative strains were not made recA nor transformed with pCQV2. The era phenotypes of all cold-sensitive strains were elongated. b These strains were derived from JFL125 (14) and were kindly provided by J. Lutkenhaus, University of Kansas Medical Center, Kansas City.

Corresponding author.

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FIG. 3. Immunoblotting of lysates from CTER411. Cultures initially grown at 40°C and then shifted to 27°C as described in the legend to Fig. 2. Samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h after the shift to 27°C (lanes 2 to 12, respectively). Lane 1 is partially purified Era. Samples were subjected to analysis by immunoblotting as described in Materials and Methods. were

FIG. 1. Strategy for one-step construction of conditional mutants in an essential gene. The key features of the plasmid pNG187 are that both plasmid DNA replication and repression of transcription from PR are temperature sensitive. Thus, at 42°C one may obtain the disruption of an essential gene by single homologous recombination (as shown here for era) because in the recombinant a functional copy of the gene is under the control of the PR promoter, which is derepressed. The resultant strain is cold sensitive if the targeted gene is essential. Abbreviations: PR, mutant (X3) A promoter; era', partial era; cI, temperature-sensitive X repressor gene (cI857); KanR, the complete kanamycin resistance gene (with its own promoter) derived from transposon Tn9O7.

were transduced with phage P1 lysate harboring an allele of recA disrupted by the Tetr gene. Kanr Tetr isolates were confirmed to be recA by ultrasensitivity to UV irradiation. These isolates were transformed with pCQV2 (19), which provides the temperature-sensitive repressor (cI857) in pBR322 and ensures that expression from the PR (X3) promoter is minimal. Immunoblotting. Cells were grown at either 40 or 27°C as described in the legend of Fig. 3. Samples were pelleted and boiled in sodium dodecyl sulfate (SDS), and equivalent protein amounts were analyzed by SDS-polyacrylamide gel

electrophoresis (17% polyacrylamide) followed by immunoblotting with anti-Era antiserum. Protein was transferred to 0.45-I,m nitrocellulose sheets by using a semidry electroblotting apparatus (Semi-Phor TE70, Hoefer Scientific Instruments). Detection of Era in Fig. 3 was performed with the Alkaline Phosphatase Immun-Blot Kit (Bio-Rad) accord-

ing to the manufacturer's instructions. The amount of Era present under various growth conditions was quantitated by immunoblotting and detection with the ECL system (Amersham). In this system, the antigen is detected by chemiluminescence and the image is produced on X-ray film. These data were then quantitated by densitometric analysis of the X-ray film. An additional advantage of the ECL system was that it was at least one order of magnitude more sensitive than the Immun-Blot Kit in the detection of Era. Preparation of purified anti-Era immunoglobulin G (8) and electrophoresis procedures (15) were described previously. Fluorescent staining. Cells were grown at either 40 or 27°C as described in the legend of Fig. 6 and then washed once with 0.1 M cacodylic acid, pH 7. Staining of DNA with bisbenzimide (Hoechst no. 33342) was carried out as de-

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CTER411 (started at 0 min) was grown to 5 Klett units (K. U.) (110 min), at which point one-half of the culture was shifted to 27°C. Growth was monitored for several hours at 40°C (0) and 27°C (0).

FIG. 4. Quantitation of the amount of Era present in cells grown at 27°C relative to that present in cells grown at 40°C. Cells were grown initially at 40°C, and then half of the culture was shifted to 27°C as described in the legend to Fig. 2. At 0, 1, 2, 3, 5, 7, and 9 h after the temperature shift, samples were withdrawn from both cultures, pelleted, and subjected to electrophoresis and immunoblotting (see Materials and Methods). The amount of lysate analyzed was normalized according to the optical density of the culture at the time of sampling. Era was visualized by using a chemiluminescent probe (see text). The amount of Era was quantitated by densitometric scanning of the resultant X-ray film. The ratio of the area of the peak generated by the Era band at 27°C to the area of the peak at 40°C was plotted versus time after the temperature shift.

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FIG. 5. Phase-contrast microscopy of CTER411 grown at 27°C. The number of hours after the shift to 27°C at which the samples were taken is indicated by the number at the lower right of each panel. Magnification, X2,000.

scribed previously (5) except that cacodylic acid was used in place of Veronal as the buffering system. Analytical methods. The total protein content was determined with Coomassie blue G-250 (4) by using the Bio-Rad Protein Assay and bovine serum albumin as a standard. DNA was determined by using the fluorescent dye bisbenzimide (Hoechst no. 33258) (7). RESULTS AND DISCUSSION Previous studies have demonstrated that conditional muera result in strains that are deficient for growth on agar plates incubated under nonpermissive conditions (11, 16). However, attempts to isolate nonleaky conditional mutants suitable for detailed phenotypic analysis tant alleles of

in liquid culture were not successful. Therefore, the physiological role of era was not elucidated. To overcome this problem, we constructed a new strain (Fig. 1) in which the transcription of era is controlled by the phage X promoterpR. The time required for depletion of Era from bacterial cells is a function of the amount of protein present at the permissive temperature, the half-life of the protein, and the level of repression of the PR promoter. To achieve Era expression at 40°C sufficient for normal growth and yet obtain complete repression at 27°C, the PR promoter mutation X3 was introduced into our constructions, resulting in a 90% reduction of expression compared with that of wild-type PR (9). In order to eliminate residual leakage of the promoter, the gene for the temperature-sensitive repressor (c1857) (20) was included on a multicopy plasmid (pCQV2) (19). Strain JM83

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FIG. 6. Phase-contrast (Nomarsky differential interference) (A and C) and fluorescence (B and D) micrographs of CTER411 cells 20 h after shift to 27°C (A and B) or of late-log-phase cells at 40°C (C and D). The cells were stained as described in Materials and Methods. The same field is shown for panels A and B and for panels C and D. Bar, 10 ,um.

was employed to obtain two mutant strains, CTER411 and CTER412, in which the production of Era is completely repressed at 27°C and derepressed at 40°C. Both strains grew normally at 40°C, with a generation time of 30 min. However, growth arrest was noted very soon after a temperature shift to 27°C in liquid culture (Fig. 2). In order to evaluate the degree of Era expression at the nonpermissive temperature, samples were taken 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h after the temperature shift for analysis by immunoblotting (Fig. 3). A reduction in the amount of Era was detected by 2 h after shifting to 27°C (Fig. 3, lane 4). In order to quantitate this reduction, immunoblotting was carried out by using the ECL detection system (Amersham). By using this approach, Era was visualized on X-ray film after exposure of the nitrocellulose containing the blotted protein. The relative amounts of Era were determined by densitometric analysis of the Era-containing bands. The data shown in Fig. 4 were generated by growing cells under the same conditions as described for Fig. 3. At 0, 1, 2, 3, 5, 7, and 9 h after shifting to 27°C, samples were removed from cells grown at 40°C as well as 27°C and were subjected to electrophoresis and immunoblotting. The quantitation of these results demonstrates that by 2 h after shifting cells to 27°C, the amount of Era produced is reduced to less than 10% of the amount present in cells incubated at 40°C. In these experiments, it was also determined that the amount of Era in the parental strain JM83 does not differ from the amount of Era produced in CTER411 at the permissive temperature by more than a factor of 2 (not shown). Viability of the mutant cells at the nonpermissive temperature was analyzed by comparing the

total number of cells present (as determined by quantitative microscopic analysis) to the number of CFU present. After 2 h, less than 10% of the cells present in the culture were able to form colonies when returned to the permissive temperature, and by 5 h, this number was reduced to less than 1%. It was apparent that relative amounts of Era were directly proportional to relative viability. During the first few hours of incubation at 27°C, cells gradually elongated. By 5 h, the mutant cells were noticeably longer, and they continued to elongate for the duration of the experiment (Fig. 5). Filaments that had grown for 13 h at the nonpermissive temperature were depleted of septa, indicating a disruption in cell division, probably at an early stage of the cell cycle prior to the formation of the initial constriction. Upon more extended incubation (more than 24 h), lysis of the filaments was observed. Many changes in growth conditions or in internal homeostasis (e.g., overproduction of some proteins) can cause elongation and filamentation of bacterial cells (13). The data presented in Fig. 2, 3, and 4 indicate that the filamentation phenotype may be a secondary consequence of an earlier defect because growth is significantly affected after 2 h at the nonpermissive temperature and filamentation is not apparent until 5 h. Elongate cells form as a result of the blocking of DNA replication, interruption in chromosome segregation, or inhibition of cell division at septation (13). In an attempt to distinguish which of these processes were blocked, chromosome segregation was analyzed by fluorescence staining of cells and visualization of the DNA by fluorescence microscopy (Fig. 6B and D). The mutant cells at 40°C have one

V 3GROWTH RATE VOL. 173, 1991

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FIG. 7. The strategy employed to demonstrate 1 that plasmidborne sequences [particularly ori(Ts)] do not cause filamentation when present at a nonessential locus in the chronnosome. The plasmid pIE110 harbors a HindIII fragment from thie lep operon which contains the Tn9O7 kanamycin resistance gene ((Kanr) cloned at the site of an internal BglII-BglII deletion withi Ln lepA. This fragment also harbors two-thirds of the lep gene on downstream flanking sequences. DNA synthesis of pIE110 is cont the same origin present on pNG187 (Fig. 1). Both plasm common parent pEL3 (2). Single homologous reconnbination between the chromosomal lepA locus and pIE110 gives rise to a new strain that is a partial merodiploid at the lep operon as shown in the bottom line. The recombinant chromosome contaims a disrupted copy of the lep operon and a wild-type copy separate(d by plasmidencoded DNA which originated from pEL3. Recoimbinants are selected in the manner described for pNG187, except that they are not cold sensitive. With JM83 as a parent, recombinr ants show no defects in growth or morphology when grown at any temperature.

rolled bh

defined cluster of DNA as shown in Fig. 6D. ikt 27°C, the filamentous cells have several nucleoids whic-h represent segregation of the chromosomes (Fig. 6B). It vvas possible that the phenotype shown in Fig. 5 and 6 resultLed from the presence of the plasmid-borne ori(Ts) in the c} hromosome. However, we have placed the same ori(Ts) iimmediately upstream of the rnc operon, creating a strain X which is a partial merodiploid at the lep operon (Fig. 7). Thlis strain has no defect in growth or morphology at any tiemperature. These data are in agreement with an earlier rep)ort that the origin of Rl plasmids can be placed in the c-hromosome without causing the phenotype observed here (1.8). Analysis of the DNA and total protein content has estab]lished that a constant ratio between total protein and total D)NA (29 + 4 ,ug of DNA per mg of protein) exists in CTE'R411 at all growth temperatures (these measurements wer e performed on cells incubated for up to 20 h at the no temperature). These findings support the conclussion that Era is required for cell growth and may play a role in the regulation of the cell cycle but is probably not directly involved with DNA replication. The importance of Era as a cell growth regulator is emphasized by the f;act that the reversal of the phenotype reported here requirred only the Era protein supplied by a plasmid harboring a fuinctional era allele without any of the region encoding flankiing genes. It must be pointed out that a mutation in era has bteen reported to be polar on the nonessential gene recO (21). 1Even though recO sequences were not required to restore viability to CTER411, it seemed possible that the era ph enotype required recA and recO as well. This possibility e can be ruled out because none of the PAM or JFL strains 4described in Table 1 are recA and they still show the era phe notype after selection of recombinants with pNG187. The fin ding that the

onpermissive

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amount of RNase III (encoded by rnc) and Era present in wild-type E. coli K-12 is small compared with that of other proteins is in agreement with the conclusion that Era is a regulatory protein (3, 8, 15, 22). Another possible explanation for the phenotype observed in Fig. 5 and 6 is that the SOS system is strongly induced, resulting in filamentation (6, 13). It should be pointed out, however, that strains CTER411 and CTER412 are recA mutants and should not invoke the SOS system in response to stress. To unambiguously demonstrate that the filamentation reported here does not require a functional stress response system, pNG187 was transformed into several additional strains. Strains with double mutations at sulA or sulB and Ion do not elongate during response to DNA damage (12), but wild-type E. coli does. However, all strains harboring the triple-mutant chromosome at sulA or sulil, lon, and era do form filaments, showing that the sulA-suiB SOS pathway is not involved in era filamentation (Table 1). The possibility remains that era has a function that is totally unrelated to cell division and that filaments arise as a secondary phenotype. Interestingly, however, it has been demonstrated in wild-type cells by immunoelectron microscopy that Era is localized at spatially separated sites on the internal face of the cytoplasmic membrane (8). These sites were observed at the cellular poles, the midpoint, and halfway between the poles and the midpoint. It was pointed out that this distribution of binding sites would be expected for a protein involved in septation. This observation supports the idea that Era may be involved with regulating cell division; however, since cells do not begin to elongate until Era levels are reduced by 95 to 99% in CTER411, these sites must be severely depleted before filamentation occurs. In most cases, the cell cycle is inhibited during the synthesis of the septa, but the initiation of septum formation can be observed as a cellular constriction. One mutation which exhibits a morphology similar to that of CTER411 growing at the nonpermissive temperature (filamentous, no septa, no constriction) is the temperature-sensitive mutation ftsZ (6, 13). ftsZ mutants are blocked in an early stage of cell division, after DNA synthesis and segregation but before the onset of septation. Whether this or any other E. coli cell division gene(s) is functionally linked to era is currently being investigated. It is of interest to note that a previous analysis of prokaryotic cell division has led to speculation that a GTP-binding protein may be an important regulatory element (10).

ACKNOWLEDGMENTS

The authors extend special gratitude to H. Geller for aiding us

with the preparation of photomicrographs and to A. St. John and G.

Avigad for critical reading of the manuscript. We thank all of the members of this laboratory for helpful discussions. This work was supported by the National Institutes of Health (grant GM40087-OlA1) and the New Jersey Commission on Cancer Research (grant 687-027). P.E.M. is a recipient of the American Cancer Society Junior Faculty Research Award (JFRA-208).

REFERENCES 1. Ahnn, J., P. E. March, H. E. Takiff, and M. Inouye. 1986. A

GTP-binding protein of Escherichia coli has homology to yeast Sci. USA 83:8849-8853. RAS proteins. Proc. Natl. Acad. E. Y. Machida, M.

Ledner, 2. Armstrong, K. A., R. Acosta, Pancotto, M. McCormick, H. Ohtsubo, and E. Ohtsubo. 1984. A

37 x

103 molecular weight plasmid-encoded protein is required

for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J. Mol. Biol.

175:331-347.

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3. Bardweli, J. C. A., P. Regnier, S. Chen, Y. Nakamura, M. Grunberg-Manago, and D. L. Court. 1989. Autoregulation of RNase III operon by mRNA processing. EMBO J. 8:3401-3407. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 5. Bukau, B., and G. C. Walker. 1989. AdnaK52 mutants of Escherichia coli have defects in chromosome segregation and plasmid maintenance at normal growth temperatures. J. Bacteriol. 171:6030-6038. 6. Donachie, W., and A. C. Robinson. 1987. Cell division: parameter values and the process, p. 1578-1593. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium, vol. 2. American Society for Microbiology, Washington, D.C. 7. Gallagher, R. R. 1988. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy, p. A3.9-A3.15. In F. M. Ausubal, R. Brent, D. D. Moore, J. D. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, New York. 8. GolHop, N., and P. E. March. Localization of membrane binding sites of Era in Escherichia coli. Res. Microbiol., in press. 9. Hawely, D., and W. R. McClure. 1980. In vitro comparison of initiation properties of bacteriophage X wild-type PR and X3 mutant promoters. Proc. Natl. Acad. Sci. USA 77:6381-6385. 10. Holland, I. B. 1987. Genetic analysis of E. coli division clock. Cell 48:361-362. 11. Inada, T., K. Kawakami, S. Chen, H. E. Takiff, D. L. Court, and Y. Nakamura. 1989. Temperature-sensitive lethal mutant of Era, a G protein in Escherichia coli. J. Bacteriol. 171:50175024. 12. Johnson, B. F. 1977. Fine structure mapping and properties of

J. BACTERIOL.

13. 14.

15. 16.

17.

18.

19. 20.

21.

22.

mutations suppressing the lon mutation in Escherichia coli K-12 and B strains. Genet. Res. 30:273-286. Lutkenhaus, J. 1990. Regulation of cell division in E. coli. Trends Genet. 6:22-25. Lutkenhaus, J., B. S. Sanjanwaler, and M. J. Lowe. 1986. Overproduction of FtsZ suppresses sensitivity of lon mutants to division inhibition. J. Bacteriol. 166:756-762. March, P. E., J. Ahnn, and M. Inouye. 1985. The DNA sequence of the gene (mc) encoding ribonuclease III of Escherichia coli. Nucleic Acids Res. 13:4677-4685. March, P. E., C. G. Lerner, J. Ahnn, X. Cui, and M. Inouye. 1988. The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth. Oncogene 2:539-544. Messing, J. 1979. A multipurpose cloning system based on the single stranded DNA bacteriophage M13. Recomb. DNA Tech. Bull. 2:43-48. Molin, S., and K. Nordstrom. 1980. Control of plasmid Rl replication: functions involved in replication, copy number control, incompatibility, and switch-off replication. J. Bacteriol. 141:111-120. Queen, C. 1983. A vector that uses phage signals for efficient synthesis of protein in Escherichia coli. J. Mol. Appl. Genet. 2:1-10. Roberts, J. W., and R. Devoret. 1983. Lysogenic induction, p. 123-144. In R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Takiff, H. E., S. Chen, and D. L. Court. 1989. Genetic analysis of the rnc operon of Escherichia coli. J. Bacteriol. 171:25812590. Watson, N., and D. Apirion. 1985. Molecular cloning of the gene for the RNA-processing enzyme RNase III of Escherichia coli. Proc. Natl. Acad. Sci. USA 82:849-853.