Operon Coordination in Different Bacterial Hosts

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1970, P Copyright American Society for Microbiology Vol. 103, No. 2 Printed In U.S.A. Operon Coordination in Different Bacterial Hosts PO CHI WU, PETER F. JOHNSON, AND AUSTIN NEWTON Departments ofbiology and Biochemical Sciences, Moffett Laboratory, Princeton University, Princeton, New Jersey Received for publication 1 May 1970 Coordination of gene expression in the lac operon was compared in Escherichia coil and Salmonella typhimurium as an approach to detecting possible differences in protein synthesis or membrane structure between organisms. Either a wild-type F' lac proab episome or the same episome with a polar mutation in one of the lac genes was introduced into pro- derivatives of the two strains of bacteria. Activity assays showed that the (3-galactosidase levels were only slightly lower in the S. typhimurium cells than in E. coli cells, whereas the transacetylase levels were significantly higher in S. typhimurium for all of the lac markers tested. Galactoside transport activities were always comparable in the two strains of bacteria; this latter result indicates that the cell envelopes of E. coli and S. typhimurium do not differ sufficiently to affect the membrane-associated lac transport system. It was found, however, that the specific transport activity is very sensitive to culture age in both bacteria, and decreases rapidly in cultures past the mid-exponential phase of growth. Expression of the lac operon is sensitive to changes in a variety of genetic and physiological conditions in the cell. Thus, the level of lac gene products is reduced by catabolite repression (12), polar mutations in structural genes (14), and mutations in the promoter region of lac (9). Ballesteros-Olmo et al. (2) have also shown that the methylating capacity of the cell influences the kinetics of induction of the lac operon when it is carried by cells of Salmonella typhimurium, and Fox (6) has demonstrated a lipid requirement for normal induction of the f,-galactoside (lac) permease system in Escherichia coli. This latter activity is dependent upon the membrane-bound M protein (7). We propose that another way to examine the effect of cytoplasmic factors on gene expression is to compare the coordination of one operon in different bacterial species. If the genes of the lac operon in S. typhimurium are transcribed into one polycistronic messenger ribonucleic acid (RNA) as they are in E. coli (1), then a comparison of the relative amounts of lac operon proteins made in the two bacteria (when both carry an F'lac episome) should indicate whether genes in the specific messenger are translated with equal efficiencies in the two hosts. The lac permease activities should also reflect any differences in membrane structure between the two strains. There is physical evidence (18), for the R factors at least, that in E. coli and in S. typhimurium there is only one episome copy per chromosome. Some of the previous studies on the expression 318 of genes from E. coli in other bacterial hosts have utilized the episomes F'lIac (2, 3, 5), F'trp (4, 20) and F'pho (19). In the present study, we used an F'lac proab episome to introduce the lac operon into a lac-pro deletion strain of E. coli and a pro- deletion strain of S. typhimurium, which is normally lac. The f3-galactosidase (z gene product),,b-galactoside permease (y gene product), and galactoside transacetylase (transacetylase; a gene product) activities were measured in cells which carried an episome with a wild-type lac operon or one with a chain termination mutation in one of the structural genes (see Fig. 1). These latter mutations, which in E. coli allow distal gene expression from 0 to 100% of the wild-type levels (13, 14), were examined on the prospect that the polar effect in lac might be especially sensitive to any strain difference in protein synthesis. MATERIALS AND MEEHODS Media. Minimal medium contained M63 salts (15) supplemented with 5 mg of thiamine per liter and 0.2% of either glycerol or sodium citrate as the carbon source. Isopropyl-13-D-thiogalactoside (IPTG; Koch-Light Chemical Co.) was added to the glycerolsupplemented medium at a concentration of 5 X 10-4 M to induce the lac operon. This concentration of IPTG was found to induce fully both E. coli and S. typhimurium strains. Solid minimal medium was prepared by the addition of 2% agar (Difco) to the above liquid medium. MacConkey Agar (BBL) was used for identification of lac+ and lac- strains. Strains. Figure 1 shows a map of the lac operon

2 V4 V4ii 0 z y a spectrophotometer, and the dry weight of cells was calculated from a calibration curve which related dry weight to optical density at 650 nm. Protein concentra- UIIS X82 NG200 YA625 X90 NG0 tion was determined by the method of Lowry et al. w NG1116 YA596 YA623 NG' (11). Although growth of cells in unsupplemented FIG. 1. Map positions of chain termination muta- minimal medium should prevent segregation of tions in lac operon. Mapping of thoie z- mutations has episomes because of complementation between epi- relative positions somal and chromosomal genes to give the pro+ been described previously (14); theiir in the figure indicate their order i) relative map distances. with the mutations of z and y used in this study. The F- strains of E. coli which carry these lac mutations have been described (14). Other bacterial strains used were E. coli MX103S, an F-, Sm' strain which carries a lac-pro deletion; S. typhimurium ProB25, an F-, Sms, pro-b deletion mutant (obtained from J. Yourono); and E. coli E5014, a Sm' deletion mutant of lac which carries the F'lac proab episome (obtained from F. Jacob). F' lac PrOAB episomes which carry the z and y mutations (Fig. 1) were constructed by isolation of lac- homogenotes on MacConkey Agar from lac+ heterogenotes of the genotype Fm1ac-/F'Iac+proAB+- Each of the resulting lac- episomes was transferred to ProB25 and MX103S by the appropriate mating and isolation of pro+, Sm' recombinants. Construction of the partial diploids in S. typhimurium was facilitated by selection of pro+ recombinants on minimalcitrate plates which will support growth of the recombinant strains, but not of the parent strains. The resulting strains were verified by backcrosses against the parent E. coli strains and were designated as shown in Table 1. Assays. For determination of transacetylase activity, E. coli or S. typhimurium cells were grown, extracted, and assayed as described previously (13). Specific activity is expressed as nanomoles of acetyl- IPTG formed per minute per milligram of protein. j3-galactosidase activity was determined as described by Willson et al. (21), and the specific activity is expressed as nanomoles of o-nitrophenol formed per minute per milligram of protein. Galactoside permease activity was determined as described by Prestidge and Pardee (16). A 2-ml amount of an overnight culture of cells was inoculated into 10 ml of fresh medium which contained inducer, and this culture was incubated with shaking at 37 C until the optical density at 650 nm reached 0.5 to 0.8. The cells were collected by centrifugation, resuspended in 15 ml of minimal medium without IPTG, and grown for an additional 15 min. Uptake was assayed by incubation of 0.65 ml of the cell culture with 0.3 ml of a reaction mixture [containing 6.13 sag of thio-g-methyl galacto- of 14C-TMG per ml] side (TMG) per ml and 0.5,&c for 5 min at 25 C. After incubation, 0.7 ml of the mixture was filtered onto HA filters (Millipore Corp., Bedford, Mass.) and washed with 10 ml of M63 salts. The filters were counted in Bray's solution in a Packard liquid scintillation counter. The specific activity is expressed as nanomoles of TMG taken up per minute per milligram (dry weight) of cells, except where indicated otherwise. Cell growth was followed by measurement of turbidity at 650 nm in a Gilford the gene but not phenotype, cultures were frequently examined before assay to verify that episome loss was limited to less than 1% of the cell population. RESULTS The strains listed in Table 1 were constructed by introduction of the F'iac proab episome into the pro- derivatives of E. coli and S. typhimurium as described above (Materials and Methods). To determine whether the level of lac expression differs in E. coil and S. typhimurium, the specific activities for /3-galactoside permease and transacetylase were determined on these strains, and the values for each mutation in the two hosts were compared. The activity of each mutant was also expressed as the percentage of wild-type activity in that strain. These latter values would reveal whether the gradient of polarity (see 14) described by chain termination mutations in S. typhimurium cells is different from the one described by the same mutations in E. coli cells. /3-Galactosidase activities were determined on the wild-type, lac+ strains.,b-galactosidase. When the Mac+ strains F300 and F316 (see Table 1) were assayed for 13- TABLE 1. E. coli and S. typhimurium strains carrying F'lac proab episomes pro- lac- strains lac marker Cdn carrying episomes carried on cdn episomea assignmenta s E. coli typhimurium z+y+a+ Wild-type F300 F316 U 118 UAA F301 F317 co Frameshift F303 F319 X 82 UAG F304 F320 NG 1116 UAG F305 F321 NG 200 UAG F306 F322 YA 596 UAA F307 F323 YA 625 UAG F309 F325 YA 623 UAA F310 F326 X90 UAA F311 F327 NG 328 UAG F312 F328 NG 707 UAG F313 F329 a Mapping and codon assignment of UAA and UAG mutations has been described previously (13; see Fig. 1). The frameshift designation of w was determined by revertibility with drug, ICR191 (unpublished data; Malamy, personal communication). VOLE 103X1970 OPERON COORDINATION IN BACTERIAL HOSTS 319

3 320 WU, JOHNSON, AND NEWTON J. BACTERIOL. w >20.0 V LV ~~~~~~~ Ofl TIME (HOURS) FIG. 2. Lac permease activity as a functiont of cell growth. Overnight cultures of E. coli strain F 300 and S. typhimurium strain F 316 were each inoculated into 10 ml of minimal medium plus IPTG at an initial optical density ofapproximately The two cultures were incubated at 37 C, and samples were taken at the times indicated for determination of optical densities (O, S. typhimurium; *, E. coli) and fl-galactoside permease activity (V, 0. typhimurium; A, E. coli). The lac permease activities in this particular experiment are expressed as nanomoles of thio-n0-methyl galactoside taken up under the standard assay conditions (see Materials and Methods) divided by the optical density of the culture at 650 nm. In the case of permease activity, a curve is indicated only for the points which correspond to the culture of S. typhimurium cells. galactosidase activity, the difference between the specific activity in E. coli and in S. typhimurium was found to be small: an average of three assays gave 33,700 units/mg of protein for F300 and 29,500 units/mg of protein for F316. Mixing experiments showed that extracts of lay- E. coli (MX103S) and S. typhimurium (ProB25) host cepis do not contain factors which either stimulate or inhibit fb-galactosidase activity in cell extracts offscac+ strains of S. typhimurium or E. coli. Galactoside permease. Preliminary experiments showed that, under our conditions, the galactoside permease activity in strains of E. coli and S. Myphimurium is sensitive to culture age; specific activity is greatest in the mid-exponential phase of growth and decreases rapidly thereafter (Fig. 2). Thus, an effort was always made to harvest cells for assay in exponential phase at the cell density givingmaxieal permease activity. Under these conditions, reproducible values could be obtained for the permease activities. 1.6 The results of these determinations (Table 2) show that the polar mutations of z affect the expression of y to about the same extent in E. coli and S. typhimurium. Any differences in the wall or membrane structure of the two hosts are not evident from lac permease activities for these mutants or for the wild-type strains. Transacetylase. The most striking result from these assays was the finding that the specific activity determined for the transacetylase enzyme was consistently higher for an F'iac episome in S. typhimurium than it was for the same episome in E. coli. This difference was reproducible and was observed for the wild-type strains and all of the z- strains; the effect was more pronounced for the two y mutants examined (Table 3). Mixing experiments, similar to those described for the assay of 3-galactosidase (above), indicated that extracts of lac- E. coli and S. typhimurium cells contain no enzyme activators or inhibitors which could account for the differences in activities observed. Although the specific activity corresponding to each z mutation was higher in S. typhimurium cells than in E. coli cells, no strain ditterence was seen when the values were calculated relative to the respective iac+ parents (Table 3). The relative transacetylase values were somewhat higher in S. typhimurium for the two mutants of y (Table 3), but the gradient of polarity for the set of z TABLE 2. Comparison of galactoside permease activities ofe. coli and S. typhimurium strainsa Genotype of F' lac proab episome lac+... Z+y+ U 118 co... X NG 1116 NG YA YA YA X z+y- NG NG Specific activityb E. coli IRelative activity (%)b S. typhi- E. coli S. typhimureum Imursum a Strains used are described in Table 1 and Fig. 1. b Some variation was observed in the specific activities of a strain assayed at different times; the relative activity (percentage of the specific activity of the lac+ strain which is taken as 100) was reproducible. The specific activities are averages of at least two determinations.

4 VOL. 103, 1970 OPERON COORDINATION IN BACTERIAL HOSTS TABLE 3. Comparison of transacetylase activities of E. coli and S. typhimurium strains" Specific activity Relative activity (%)b Genotype of F' lac proab episome cols.. Y E S. E. coli typhimursum Ecoi murium lac U X NG NG YA YA YA X NG NG astrains are described in Table 1. b Relative transacetylase activity is expressed as a percentage of lac+ activity, which is taken as 100. mutations was identical in the two strains. It was also comparable to the one determined for haploid E. coli strains which carried the same mutations (see 14). DISCUSSION The above results show that expression of the z and y genes in the lac operon is coordinated in a similar fashion in E. coli and S. typhimurium. This is not surprising in view of how closely the two bacteria are related. Differences were noted, however, in the levels of transacetylase: relative to the (3-galactosidase activities, transacetylase activities were consistently higher in S. typhimurium than in E. coli. Results of the individual assays may be summarized as follows. (i) Levels of (#-galactosidase activity in S. typhimurium are only slightly lower than in E. coli (perhaps by 10%, see above). In contrast, similar experiments with Proteus have shown that when cells of this bacterial strain carry an F'lac episome the level of j3-galactosidase activity is drastically reduced (3, 5). (ii) The membrane-bound M protein functions equally well in #-galactoside transport in S. typhimurium and E. coli. This may indicate that the cell envelopes of the two bacteria are structurally very similar or, alternatively, that the binding of transport proteins in the membrane does not involve a "site" of great specificity. The assay of y gene function in bacteria more distantly related to E. coli than is S. typhimurium could help to evaluate these possibilities. 321 We did find, however, that the lac permease activity in both strains is sensitive to culture age. The finding of Randle et al. (17) that the phosphoglyceride composition of E. coli cells changes during growth is interesting in this regard. This suggests that at least part of the rapid decrease in specific lac transport activity of cells in the later stages of growth may be due to some alteration in the functional relationship between the M protein and the cell envelope. Direct measurement of y gene product (M protein) under these same conditions would indicate to what extent cessation of M protein synthesis contributes to the effect. Using a fatty acid auxotroph of E. coli (8), Fox (6) recently showed that a lipid component is important for normal induction of the lac transport system in E. coli. (iii) In contrast to the above results for I3- galactosidase and permease activities (i and ii), the levels of transacetylase activity are consistently higher in S. typhimurium than in E. co/i cells for all of the episomes tested. If it is assumed that the structural genes of the lac operon are transcribed into a single polycistronic messenger RNA in S. typhimurium, as they are in E. coli (1), then it might be concluded that translation of the a cistron in the lac messenger is relatively more efficient (compared with the z and y cistrons) in S. typhimurium than in E. coli. The enzyme activity measurements made here do not give any indication of which component in the translation step might be responsible for the effect on transacetylase activity. Among the types of effects that may be considered, however, is more efficient initiation of protein synthesis at the beginning of the messenger segment for the a gene in the S. typhimurium cytoplasm. Lodish (10), for example, has shown that the ribosomes of Bacillus stearothermophilus differ from those of E. coli in initiation of protein synthesis in vitro when the RNA from bacteriophage f2 is used as the messenger. (iv) The gradient of polarity described by lac- chain termination mutations in F- strains of E. coli (14) is not altered in S. typhimurium; polar z mutations reduce expression of the y and a genes coordinately in S. typhimurium cells to the same extent as they do in E. coli cells. As mentioned above, the transacetylase activities for the two y polar mutants are considerably higher in S. typhimurium, and the differences are evident when the activities are expressed as a percentage of the wild-type value. ACKNOWLEDGMENTS We are grateful to Joseph Yourno, who supplied several mutants of S. typhimurium used in these studies, and to T. H. Carter for critical reading of the manuscript. This work was supported by a grant from the National Science

5 322 WU, JOHNSON, AND NEWTON J. BACTERIOL. Foundation, by Biomedical Sciences Support Grant FR-07057, by the Eugene Higgins Trust Fund, and by facilities made available by the Whitehall and John A. Hartford Foundation to the Department of Biology. A. N. was supported by a Career Development Award from the National Institutes of Health. LITERATURE CITED 1. Attardi, G., S. Naono, J. Rouviere, F. Jacob, and F. Gros Production of messenger RNA and regulation of protein synthesis. Cold Spring Harbor Symp. Quant. Biol. 28: Baliesteros-Olmo, A., J. S. Kovach, P. van Knippenberg, and R. F. Goldberger Kinetics of induction of the lactose operon on an episome in Salnonella typhimurium. J. Bacteriol. 98: Colby, C., Jr., F. D. Martin, and A. S. L. Hu Catabolite repression of the synthesis of 6-galactosidase in Proteus Mirabilis F-lac. Biochim. Biophys. Acta 157: Creighton, T. E., D. R. Helinski, R. L. Somerville, and C. Yanofsky Comparison of the tryptophan synthetase a subunits of several species of Enterobacteriaceae. J. Bacteriol. 91: Falkow, S., J. A. Wohlhieter, R. V. Citarella, and L. S. Baron Transfer of episomic elements to Proteus. I. Transfer of F-linked chromosomal determinants. J. Bacteriol. 87: Fox, C. F A lipid requirement for induction of lactose transport in Escherichia coll. Proc. Nat. Acad. Sci. US.A. 63: Fox, C. F., J. R. Carter, and E. P. Kennedy Genetic control of the membrane component of the lactose transport system of Escherichia coli. Proc. Nat. Acad. Sci. US.A. 57: Gilbert, D. F., and P. R. Vagelos Fatty acid mutant of E. coli lacking a 6-hydroxydecanoyl thioester dehydrase. Proc. Nat. Acad. Sci. U.S.A. 58: Ippin, K., J. H. Miller, J. Scaife, and J. Beckwith New controlling element in the lac operon of E. colt. Nature (London) 217: Lodish, H. F Species specificity of polypeptide chain initiation. Nature (London) 229: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. L. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Nakata, D., and B. Magasanik The roles of inducer and catabolite repressor in the synthesis of,-galactosidase in E. colt. J. Mol. Biol. 8: Newton, A Reinitiation of polypeptide synthesis and polarity in the lac operon of Escherichia colt. J. Mol. Biol. 41: Newton, A., J. Beckwith, D. Zipser, and S. Brenner Nonsense mutants and polarity in the lac operon of Escherichia colt. J. Mol. Biol. 14: Pardee, A. B., F. Jacob, and J. Monod The genetic control of cytoplasmic expression of "inducibility" in the synthesis of B-galactosidase by E. colt. J. Mol. Biol. 1: Prestidge, L. S., and A. B. Pardee A second permease for methyl-thio-s-d-galactoside in Escherichia colt. Biochim. Biophys. Acta 100: Randle, C. L., P. W. Albro, and J. C. Dittmer Phosphoglyceride composition gram negative bacteria and changes in composition during growth. Biochim. Biophys. Acta 187: Rownd, R., R. Nakaya, and A. Nakamura Molecular nature of the drug-resistance factors of the Enterobacteriaceae. J. Mol. Biol. 17: Signer, E. R Gene expression in foreign cytoplasm. J. Mol. Biol. 12: Somerville, R. L Tryptophan operon of Escherichia coll: regulatory behavior in Salmonella typhimurium cytoplasm. Science (Washington) 154: Willson, C., D. Perrin, M. Cohn, F. Jacob, and J. Monod Non-inducible mutants of the regulator gene in the "lactose" system of Escherichia coli. J. Mol. Biol. 8: