A ph-conditional mutant of Escherichia coli (fl-galactosidase/intracellular ph)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 74, No. 12, pp , December 1977 Genetics A ph-conditional mutant of Escherichia coli (fl-galactosidase/intracellular ph) MARK COLB AND LUCILLE SHAPIRO Department of Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York Communicated by Harry Eagle, September 22, 1977 ABSTRACI Mutants of Escherichia coli have been isolated that are able to grow on lactose at ph but not at ph 8.1. One of these mutants was analyzed and shown to ma in tie Zregion of the lactose operon. fl-galactosidase (#-D-gaIactoside galactohydrolase; EC ) activity in toluenized mutant cells at ph 8.0 was one-tenth that at ph. Enzyme purified to near homogeneity from the ph-conditional mutant similarly exhibited ph-conditional activity under conditions where wild-type enzyme was unaffected over a ph range of The phconditional fl-galactosidase was used in vivo as a probe for intracellular ph. We show that an internal ph of approximately is maintained through an external ph range of The phenotype of ph-conditional mutants was defined on medium with lactose as the sole carbon source. Under such conditions the gene product itself, f-galactosidase, is required to maintain intracellular ph, since such maintenance is clearly energy-dependent. Therefore, we were able to recover a phconditional mutant in a cytoplasmic gene product. We predict that with any phenotype independent of energy production, however, ph-sensitive mutants will be recovered only in surface elements. Since their introduction, conditional lethal mutants have been invaluable for the genetic analysis of essential cellular processes. A conditionally expressed mutation was first observed in 1943 in an apparently "pyridoxin-less" mutant of Neurospora sitophila (1). It was then demonstrated that growth of this mutant in the absence of pyridoxin was ph-sensitive (1). The first temperature-conditional mutants, also in Neurospora, were obtained only later (2). Kent and Lennarz isolated ph-sensitive growth mutants of Staphylococcus aureus with the idea that some would be altered in membrane lipid composition (3). Primary lesions were not identified, but some mutants were shown to be osmotically fragile and others exhibited lower lysylphosphatidylglycerol content (3). Most recently, missense mutations in the structural gene for the NADP-specific glutamate dehydrogenase of Neurospora crassa have been characterized (4). These mutations were shown to result in a loss of enzyme activity in vivo. In several of these mutants the in vitro enzyme activity, however, was shown to have an altered phdependent conformational transition (4). The purpose of the work reported here is to demonstrate that mutants conditional with respect to ph can be obtained in a chosen phenotype and to suggest a use particular to this class of mutants. The phenotype we chose for identifying ph-sensitive mutants in a given gene product was lactose utilization in Escherichia coli. Several mutants were isolated that were unable to grow on lactose at ph 8.15 but grew at a rate comparable to wild type at ph. These mutants efficiently utilized other carbon sources at both ph values. We describe here one of these mutants and present evidence that the ph-sensitive The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact phenotype is due to an altered 3-galactosidase (3-D-galactoside galactohydrolase; EC ). The in vivo activity of the mutant enzyme has been used to estimate intracellular ph and its variability with the external ph in a manner independent of the methods thus far used, i.e., accumulation of weak acid or base (5). The determination of intracellular ph and the study of its regulation are essential to understand chemiosmotically driven membrane functions. Included among these are phenomena as diverse as active transport (6, 7) and flagellar motility (8). MATERIALS AND METHODS Bacterial Strains and Growth Conditions. The E. coli strains used in this study are listed in Table 1. Bacteria were grown with shaking at 370 in M9 or modified M63 minimal medium, described in Miller (9), with either 0.2% glucose or 0.2% lactose as sole carbon source. Where required, media were supplemented with 20 mg of arginine, threonine, and leucine per ml. The modified M63 medium contained 0.1 mm MgSO4 to avoid precipitate formation at high ph. Media adjusted to ph 6.0 were further buffered with 80 mm 2-(N-morpholino)- ethanesulfonic acid and adjusted to the correct ph with KOH. Media adjusted to ph 8.0 were further buffered with 80 mm N-2-hydroxyethylpiperazine and media at ph 8.4 were buffered with tris(hydroxymethyl)methylaminopropane. The ph of the media was monitored throughout the growth periods depicted in Fig. 1. The ph of each culture was held within 0.1 unit of the set value. For the isolation of ph-conditional, lactose-negative mutants, tetrazolium indicator plates, adjusted to ph 8.0 (9), were used. Isolation of Mutants. E. coli E7074 was treated with 2- aminopurine as described (9). Mutagenized cultures were diluted and plated on lactose-tetrazolium agar at ph 8.0. Red colonies were picked and screened by streaking on lactose minimal agar at ph and 8.0. Growth curves of clones able to grow on lactose at ph but not at ph 8.0 were then determined in lactose and glucose minimal media. Clones showing ph-sensitive growth on lactose but not glucose were further characterized. The mutant strain MC42, characterized in this report, was picked from among 25,000 colonies grown from 100 separate mutagenesis tubes. From this lot, nine independent lac- mutants were recovered which were not ph-sensitive. Episome Transfer. Donor and recipient strains were grown on LB medium (9) with gentle shaking at 370 and then mixed in a ratio of 10 F':1 F-. The mating mixture was incubated for 2 hr at 370 without shaking and dilutions were plated on selective agar (9). P1-Mediated Transduction. Strains lysogenic for phage P1 (P1CM,c1100) were isolated and P1 lysates were prepared by Abbreviation: ZPhs, ph-sensitive /?-galactosidase gene.

2 5638 Genetics: Colb and Shapiro Proc. Natl. Acad. Sci. USA 74 (1977) Table 1. Bacterial strains E. coli Relevant strain phenotype Genotype Source or comment CSH22 Lac- F'1acZpro+thi/trpRA(1ac pro) M15 deletion in Z, CSH kit (Miller) CSH40 Lacyam F'lacZ+Y-pro+thi/A(1ac pro) CSH kit (Miller) E7074 Lacl- F'1acI-pro+/A(1ac pro)supe,thi J. Beckwith MC42 LaCZphS * As E7074 but lacz42 This laboratory MC43 LacZPhs F'lacZ42/A(lac pro) as in S90C This laboratory MC44 LacZ+ As RVnal but lacz+ P1-E7074 x RVnalV MC45 LacZPhs As RVnal but lacz42 P1-MC42 X RVnalV MC46 LacZ+/LacZphs F'lacZ+Y-/1acZ42 F'lac (CSH40) MC45t MC47 LacZ-/LacZPhs F'lacZ-Y+/lacZ42 F'lac (CSH22) MC4.51 RVnal Lac-,Nalr AlacX74,thi,nal M. Malamy S9OC Lac-,Strr A(lac pro) thistra J. Beckwith * LacZPhs, ph-sensitive,-galactosidase. t P1-mediated transduction. F'-mediated mating. thermal induction as described (9). The transductants were selected on lactose minimal agar at the permissive ph. Complementation Analysis. Complementation analysis was carried out by mating the MC45 transductant with CSH22 (F'Iac + I + Z -Y + ) and CSH40 (F'lac + I + Z + Y -). The donor in each case was counterselected with nalidixic acid and the F-ductants were distinguished from the recipient clones on plates containing 5-bromo-4-chloro-3-indolyl-f-D-galactoside, a substrate for 3-galactosidase that releases a blue dye on hydrolysis. This substrate does not induce the lac operon, therefore showing the degree of uninduced lac expression in a given colony. Since strain MC45 is constitutive (lad), it appears blue on the mating plates. Transfer to it of the F'lacI+ episome causes it to appear white on the mating plates. Several white F-ductant colonies were picked from each mating and their growth on lactose was characterized. Measurement of f,-galactosidase Activity. The in vivo activity of (-galactosidase was determined in M9 or modified M63 minimal medium with glucose as the sole carbon source. In experiments where in vivo and in vitro [-galactosidase activities were compared, cells were grown in M9 minimal medium because in M63 the activity of 3-galactosidase in toluenized cells was slightly unstable. The relative levels in the two media of in vivo (-galactosidase activity were the same at all ph values tested. To determine the in vivo activity, a 3.0-ml volume of cell culture ( OD660) was incubated at 37 with shaking in the presence of o-nitrophenyl-3-d-galactoside (0.83 mg/ml) for 2, 4, 6, and 8 min. The reaction was stopped by the addition of 1 M Na2CO3 (3 ml), followed by the immediate addition of one drop of toluene with mixing. Activities reported here assume 1.0 OD6o equals 108 cells. The suspension was centrifuged at 30,000 X g and the OD420 of the supernatant was determined. The in vitro activity of 4-galactosidase was measured in toluenized cells as described by Miller (9). 2- Mercaptoethanol enhances the activity of f-galactosidase. The high reducing activity in cells optimizes the reaction in vivo. The toluenized cell suspensions of mutant cultures were therefore brought to 0.25% mercaptoethanol for assay. RESULTS Characterization of ph-conditional growth on lactose The effect of ph on the growth of E. coli strain E7074 and the mutant strain MC42 on lactose minimal medium is shown in Fig. 1 A and B and Table 2. E7074 doubles in 65 min at ph and in 90 min at ph MC42 doubles in 280 min at ph and is unable to grow at ph The chromosome of the parent strain E7074 is deleted for the lac-pro region, which is carried on an F' episome. MC42 was mated with S90C (A[lacpro]StrA). The parent strain, mutant MC42, and the F-ductant grew equally well on glucose minimal medium at ph and 8.15 (data not shown). The F-ductant, MC43, grew on lactose at ph 7 with approximately the same generation time as MC42 and was unable to grow on lactose at ph 8.15 (Fig. 1; Table 2). The mutation is therefore carried on the F'lac. A P1 lysate grown on strain MC42 was used to transduce a lac deletion strain, RVnal. All lac transductants tested exhibited identical ph-sensitive growth, showing the mutation to be in the lac region (Table 2). One of these transductants, MC45, was compared to a control transductant, MC44 (RVnal transduced to lac + with a lysate grown on E7074). The relative rate of growth of these transductants at ph was similar to that observed with the respective lac donors, namely E7074 and MC42. The mutant strain MC42 and the transductant MC45 differ with respect to growth rate on lactose at ph 8.15 in that the mutant does not grow at all after the shift to restrictive ph, whereas the transductant MC42 grows at a steadily decreasing rate for some 10 hr (Fig. 1D). Complementation analysis was carried out by mating the MC45 transductant with CSH22 (F'lacI+Z-Y + ) and CSH40 (F'lac + Z + Y ). As is shown in Table 2, the F'Z + Y- of CSH40 complemented the mutation in strain MC45 and the F'Z-Y + of CSH22 did not. Therefore, the mutation is in the lacz gene of the lac operon. ph-dependent f3-galactosidase activity Activity of mutant and wild-type 0-galactosidase was assayed in toluenized cells of MC45 (lacz42) and MC44 (lacz + ) grown at various ph values (Fig. 2A). At its optimal ph, in these experiments, the mutant activity is 5% of the wild-type. The peak of activity is at neutrality and it drops about 10-fold, one ph unit to either side. The wild-type enzyme has virtually constant activity through this range. It should be noted that the mutant grows as well in lactose at ph 6.0 as at ph, though the activity of 0-galactosidase at ph 6.0 in toluenized cells is very low ḟl-galactosidase was purified to near homogeneity from MC45 and MC44 grown at neutral ph. The ph activity curve of the j-galactosidase purified from mutant strain MC45 (RVnal transduced to lacz111'sy+) was analogous to that obtained in toluenized cells (Fig. 2B). Enzyme purified from strain

3 Genetics: Colb and Shapiro Proc. Natl. Acad. Sci. USA 74 (1977) 5639 r B MC42 0o '40 ' C MC D MC ) j.01Ls ? Time, min FIG. 1. Effect of ph on growth in lactose minimal medium. The parent strain, E. coll E-1674 (A), the p)h-sensitive mutant. MC42 (B), the F-ductant (F'lacZPhs), MC43 (C), and the transductant, MC45 (D), were grown to late exponential phase in glucose minimal medium,centrifuged, and resuspended in M63 lactose minimal medium buffered at ph 6.0,, or Growth was measured spectrophotometrically. MC44 (RVnal transduced to lacz + Y+ ) showed constant activity over the ph range of Use of mutant enzyme as probe of intracellular ph We reasoned that in glucose-grown cells, where f-galactosidase is not required to maintain intracellular ph, the ph-conditional enzyme activity in MC45 (lac -Z phsy + ) could be used to estimate intracellular ph and its degree of variability with the external ph. We therefore measured the activity of 13-galactosidase in glucose-grown intact and toluenized cells. In the latter case the enzyme is functioning at the known external ph. The ratio of the activity in intact cells to that in toluenized cells Table 2. Effect of ph on growth in lactose minimal medium Relevant Doubling times, min E. coli strain characteristics ph 6.0 ph ph 8.15 E7074 F'lacZ+Y+1/(lac ) MC42 F'lacZPhs/A(lac) NG* MC43 FPlacZPIs/VA(lac) NG MC44 lacz+ Y MC45 laczph t MC46 F'1acZ+ Y/lacZPh MC47 F'lacZ- Y+/laCZPhS NG * NG = no growth. t Growth not exponential (see Fig. ID). is presumed to be the ratio of activity at the intracellular ph to that at the known external ph. An estimate of the intracellular ph can then be obtained by referring to the standard ph activity curve shown in Fig. 2A. In intact wild-type cells o-nitrophenylgalactoside hydrolysis catalyzed by f-galactosidase is permease-limited (11). This is based on the observation that toluenized cells hydrolyze o- nitrophenylgalactoside over 10 times faster than intact cells. Toluenization exposes the 3-galactosidase to a much higher substrate concentration than is achieved in vivo in the steadystate between influx and hydrolysis. In MC44 (laci-z + Y + ) activity of f-galactosidase in toluenized cells is over 10-fold higher at all ph values than the rate obtained in intact cells (Table 3) and is therefore permease-limited. MC44 and MC45 are isogenic except in Z. The possibility of a polar effect of the ph-sensitive Z gene (Zphs) on operon expression is considered unlikely because of two findings. The mutant enzyme migrates with the wild-type enzyme on sodium dodecyl sulfate/polyacrylamide gels and transacetylase activity was observed to be equal in strains MC44 and MC45. These results indicate that we are observing a similar level of gene expression in these operons and we therefore make the assumption that the permease activity is comparable in the two strains. In mutant MC45 (laczphsy+ ) the in vivo rate of 3-galactosidase activity at all ph values is less than one-fourth that observed in MC44 (lacz + Y+ ) (Table 3); that is, one-fourth the permease-limited

4 5640 Genetics: Colb and Shapiro Proc. Natl. Acad. Sci. USA 74 (1977) L1._ >-0) *- I C I 4-'.c CO (0 40 UZ C O (A ' oi5 C 6 ox >o. 4U) 4 0' ) 0-& io ' CT I ph ph FIG. 2. Effect of ph on the activity of f.-galactosidase. Cultures of MC44 (lacz+) and MC45 (lacphs) were grown to OD66o = 1.4 in minimal glucose medium at ph. Cells were collected by centrifugation and resuspended in M9 buffer adjusted to the indicated ph values. W-Galactosidase was assayed in toluenized cells by the method of Pardee et al. (10). Activity of the mutant enzyme was linear with time above ph 6.3 and below ph 8.3. The activity decayed during the reaction at the extremes of ph tested. The wild-type enzyme activity was linear at all ph values shown. (A) Activity in cells (1.0 ml of reaction mixture) of MC45 (lacphs) at 370 for 15 min using 15,ul of toluenized 10-fold concentrated culture and activity of MC44 (lacz+) at 37 for 6 min using 10,ul of toluenized 2-fold concentrated culture. (B) Activity of ti-galactosidase from MC45 (laczphs) purified through DEAE-cellulose chromatography. The activity of the mutant enzyme was significantly lower (10%) than the activity of the wild-type enzyme. The enzyme (7.2 Mg) was incubated at 370 for 20 min in Z buffer (9) at the ph values indicated. Enzyme purification and assay conditions were as described in Miller (9). rate. Hence, in the mutant strain the observed in vivo rate of o-nitrophenylgalactoside hydrolysis can be presumed to be f3-galactosidase-limited and therefore proceeds at an intracellular substrate concentration close to the limit that can be achieved by the permease. This would be above 2 mm, the external concentration, and is thus saturating for the mutant enzyme. The reaction rate proceeding at saturating substrate concentration reflects the intracellular ph. The significant decrease of in vivo activity of,b-galactosidase in wild-type strain MC44 at ph 8.2 (Table 3) reflects lowered permease activity at this ph (12). A similar change is not seen in MC45 because in this case permeation is not rate-limiting. Comparing the rates of hydrolysis in intact and toluenized cells of MC45, we estimate intracellular ph at external ph values of 6.9 and 7.8 At external ph 7.8 the in vivo rate is very close to that seen in toluenized cells (Table 3). Reference to the standard curve in Fig. 2A places the intracellular ph just under 7.9 or above 6.5. In these estimations the alkaline ph values are taken for the reasons described in the Discussion. At an external ph of 6.9 the rate of hydrolysis is 3.8-fold faster in toluenized cells than in intact cells, yielding an estimate of 8.0 for the intracellular ph. As expected, at ph 5.9 the in vitro activity is far below that observed in intact cells. The sensitivity of the estimation process is such that if the Table 3.,B-Galactosidase activity in vivo and in toluenized cells at various ph* ti-galactosidase activity, Assay nmol/min per 108 cells E. coli strain conditions ph 5.9 ph 6.9 ph 7.8 ph 8.2 MC45 (laczphs) In vivo MC45 (laczphs) Toluenized cells MC44 (lacz+) In vivo MC44 (lacz+) Toluenized cells * Cells were grown in M9 minimal medium. intracellular ph at an external ph of 5.9 were only 0.2 ph unit lower than that at external ph 7.8, activity at ph 5.9 would be approximately twice that at ph 7.8. In MC45 in vio activity is, however, remarkably constant through an external ph range of , indicating constancy of intracellular ph. At the alkaline extreme, 8.2, a slightly higher intracellular ph is indicated by a decrease in activity. However, this decrease scarcely accounts for the restrictiveness of this ph for growth on lactose. This phenotype must follow from the fact that maintenance of intracellular ph depends on the activity of the ph-sensitive molecule. This is not the case in the in vivo experiments described here, where glucose is the energy source. DISCUSSION A ph-sensitive mutant in lactose utilization has been isolated and the mutation has been shown to be in the structural gene for fl-galactosidase. The fl-galactosidase of this mutant shows very sharp ph-sensitivity in vitro. The wild-type Z gene on an F' episome complements the chromosomal ZPhs allele. We have used the mutant enzyme in vivo as a probe for intracellular ph. Our data indicate that this ph is either or over an external ph range of The alkaline values are almost certainly correct because of the following: (i) The ph optimum of the mutant enzyme is at neutrality. The in vivo activity of f3-galactosidase increases as the external ph is lowered from 8.2. Taking the alkaline estimate, this change reflects a lowering of internal ph in response to lowered external ph. If the internal ph were in the acidic range, an increase in enzyme activity would require an increase in intracellular ph in response to a lowered external ph. (ii) An alkaline interior ph would result from proton extrusion during respiration, as suggested by Mitchell (13-15). (iii) The phdependent growth phenotype of the mutant is more easily accounted for if intracellular ph is taken to be in the alkaline range (see below). (iv) Our estimate, ph , is in fairly close agreement with the range determined by Padan et al. (5) for respiring E. coli cells, over the external range The latter result was obtained by measuring intracel-

5 lular accumulation of 5,5-[ 14Cjdimethyl-2,4-oxazolidinedione, a weak acid. The small difference between these two estimates may reflect the different growth conditions used. Certainly, the observed constancy of in vivo activity through the external ph range argues strongly for the constancy of intracellular ph. In vivo activity is somewhat diminished at external ph 8.2. This indicates that close to 8.0 intracellular ph rises with external ph, a finding consistent with the work of Padan et al. (5). The change in activity at 8.2 is too small to explain the restrictiveness for growth on lactose. The likely explanation is that, in vivo activity was measured in glucose-growing cells where maintenance of intracellular ph is independent of (-galactosidase activity. However, with lactose as sole energy source, ph maintenance, clearly energy-requiring (5), is completely dependent on the mutant enzyme. After a shift to lactose medium at ph 8.15, any initial drift toward the ambient ph compromises the ability to restore intracellular ph via lactose utilization and allows further drift. Slightly above ph 8.0 the enzyme abruptly becomes inactive in vitro. It is likely that this "catastrophic" inactivation underlies the phenotype and not simply a lowered activity, since intracellular ph is already far from the peak of enzyme activity at permissive external ph. Using the same argument, we can account for the fact that the mutant grows as well on lactose at ph 6.0 as at ph, though ph 6.0 is highly restrictive for /-galactosidase activity in vitro. After a shift to lactose medium at ph 6.0, an internal drift toward the ambient ph from 7.8 would first be toward the peak of [-galactosidase activity. The enhanced activity would then permit maintenance of inside ph near its usual value. The mechanism of the tight regulation of intracellular ph observed in intact cells and, by Kaback's group, in respiring membrane vesicles (16), remains to be elucidated. It would be helpful to isolate and study mutants with altered ph regulation. The ph-sensitive lac mutant reported here may be used to select such mutants. Among pseudorevertants of MC45 able to grow on lactose at restrictive ph might be mutants that suppress the ph-sensitivity by maintaining a lower intracellular ph. Our original assumption was that intracellular ph is maintained tightly through a range of external ph. We expected, therefore, that ph-sensitive mutants would only be recovered in surface elements with a facet exposed to the outside. ph- Sensitive mutations would naturally arise in intracellular gene products, but an external ph shift would not expose the mutant phenotype. In this case, however, our selection did not exclude a mutant in Genetics: Colb and Shapiro a cytoplasmic gene product because the gene product itself was required to maintain the intracellular ph. This interpretation is supported by the fact that during growth on glucose at restrictive ph, intracellular /-galactosidase activity was only slightly affected. We therefore predict that with Proc. Natl. Acad. Sci. USA 74 (1977) 5641 any phenotype irrelevant to energy availability, allowing the usual constancy of intracellular ph, ph-sensitive mutants will only be recovered in surface elements. The Zphs gene introduced into any ph-sensitive mutant of E. coli can serve to verify that the intracellular ph has not changed after a shift to restrictive external ph, thus insuring the involvement of a surface element. For E. coli, we would recommend that ph 7.6 and 6.0 be adopted, respectively, as permissive and restrictive conditions for the selection of ph-conditional mutants. Since intracellular ph is about 7.8 and cells will not tolerate an outside ph much above 8.0, the restrictive ph ought to be acid to give a large margin between restrictiveness and permissiveness. As seen by the activity of the mutant enzyme in vivo, the ph range can be traversed with the strictest ph homeostasis. We thank M. Malamv for E. coli strains and helpful discussions and R. Schleif for critical reading of the manuscript. This investivation was supported by Grant GB 42545X from the National Science Foundation and Grant GM from the National Institutes of Health. M.C. is a Medical Science Training Program Fellow (National Institutes of Health Grant 5T5-GM 1674). L.S. is a recipient of a Hirschl Trust Award. 1. Stokes, J. L., Foster, J. W. & Woodward, C. R. (1943) Arch. Biochem. 2, Horowitz, N. H. (1948) Genetics 33, Kent, C. & Lennarz, W. J. (1972) Biochim. Biophys. Acta 288, Brett, M., Chambers, G. K., Holder, A. A., Fincham, J. R. S. & Wootton, J. C. (1976) J. Mol. 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