Vol. 168, No. 2JOURNAL OP BACTERIOLOGY, Nov. 1986, p. 486-4930021-9193/86/110486-08$02.00/0Copyright © 1986, American Society for Microbiology

Starvation Proteins in Escherichia coli: Kinetics of Synthesis andRole in Starvation Survivalt

R. GENE GROAT, J. E. SCHULTZ, E. ZYCHLINSKY, A. BOCKMAN, AND A. MATIN*Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305

Received 21 Januaryl986/Accepted 14 July 1986

Starvation proteins synthesized by Escherichia coli at the onset of carbon starvation (1R. G. Groat and A.Matin, J. Indust. Microbiol. 1:69-73, 1986) exhibited four temporal classes of synthesis in response to glucoseor succinate starvation, indicating sequential expression of carbon starvation response (cst) genes. A cst mutantof E. coli showed greatly impaired carbon starvation survival. Thus, it appears that E. coli undergoes asignificant molecular realignment in response to starvation, which increases its resistance to this stress. Newpolypeptides were also synthesized by E. coli in response to phosphate or nitrogen starvation. Some of thesepolypeptides were unique to a given starvation regimen, but at least 13 appeared to be synthesized regardlessof the nutrient deprivation causing the starvation.

Starving bacteria are important in both fundamental andapplied contexts. The vicissitudes of nature often subject theresident microflora to starvation, and it has been estimatedthat in some environments-such as the open oceans-thestarvation conditions may last for months at a time (10, 17).Postexponential, starving bacterial cells are important alsoin secontdary metabolite production and as inocula for seed-ing appropriate environments; furthermore, their stabiliza-tion is central to the success of a new, emerging, area offermentation technology, based on the use of immobilizedmicrobial cell bioreactors (9).We have shown that bulk protein synthesis, especially in

the first few hours after the onset of starvation, is essentialfor the longevity of starving Escherichia coli cultures (2, 20),and that, at the onset of starvation for carbon, E. coliinduces about 30 proteins (the starvation proteins), many ofwhich are not synthesized during growth (6). Three ques-tions are addressed in this paper with respect to starvation-protein synthesis. (i) Do these proteins fall into differenttemporal classes in regard to their synthesis after the onsetof starvation? This possibility is suggested by our previousfinding that chloramphenicol has a time-dependent effect onstarvation survival in that the sooner the antibiotic is addedto a cUlture after the onset of starvation, the greater is thesubsequent death rate (20). (ii) Do any of the starvationproteins per se have a role in starvation survival? Towardthis end, we have isolated a number of mutants containingMu dX fusions in carbon-starvation response (cst) genes,and we show here that one of them is markedly compro-mised in its ability to survive carbon starvation. (iii) Arecommon polypeptides induced in response to starvation fordifferent individual nutrients? Wanner (23) and Wanner andMcSharry (24), using E. coli mutants in which lacZY is fusedto pho4phate starvation-inducible (psi gene) promoters, haveshown that expression of some psi genes is also triggered bycarbon or nitrogen starvation. However, this phenomenonhas not yet been investigated in the wild type and at the levelof synthesis of individual proteins.

* Corresponding author.t Dedicated to Professor Sidney Rittenberg on the occasion of his

birthday.

MATERIALS AND METHODS

Organisms and phage. The E. coli K-12 wild type used inthis study was a Stanford strain (21). The phage Mu dX [Mudl B::Tn9 (lac Apr Cmr)] was constructed by Baker et al. (1)and carried as a lysogen in E. coli CAG5050 [F' prolacZ8305::Mu cts62/Mu dX A(pro lac) his met tyr rpsLgyrA]. E. coli MC4100 (F- araD139 AlacU169 rpsL relA)was used as the recipient strain for generating a library of E.coli containing phage insertions. E. coli CAG5050 andMC4100 were kindly provided by Carl Marrs.

Starvation protocol. In all determinations, except one (seebelow), starvation conditions were established by allowingthe culture to grow in M9 medium (21), supplemented witheither glucose (0.025%) or succinate (0.048%), until thecarbon source was exhausted, as determined by direct assayor growth measurements or both. Cultures were incubated at37°C with shaking and attained a density of ca. 3 x 108 cellsper ml at the onset of starvation. The starving culturecontinued to be incubated under these conditions and wassampled at appropriate intervals. The M9 medium wassupplemented with 50 pug of ampicillin per ml for growth andstarvation of Mu dX fusion-bearing strains.A different procedure was used to determine the effect of

starvation for different hutrients on starvation protein syn-thesis (see Fig. 5). This involved rapid harvesting of samplesof a growing culture and suspension of each in appropriatestarvation medium. The medium used in these experimentswas supplemented with a nonphosphate buffer, so as tomaintain buffering capacity during phosphate starvation.The composition of this medium was as follows: 0.05 M Trishydrochloride (pH 7.2)-25 mM KCl-10 mM NaCl-1 mMMgCl2-0.1 mM CaCl2-10 ,M FeCl3-10 mM Na2SO4-0.1 MNa2HPO4 (pH 7.2)-20 mM NH4Cl. An exponential-phaseculture in this medium (plus 0.4% glucose) was divided intosamples and harvested by centrifugation (3,000 x g, 15 min)with a prewarmed rotor. Cells from each sample weresuspended to a density of ca. 3 x 108 cells per ml in one ofthe following starvation media: (i) for carbon starvation,complete medium minus glucose; (ii) for nitrogen starvation,medium minus NH4Cl plus 0.4% glucose; and (iii) for phos-phorus starvation, medium minus Na2HPO4 plus 0.4% glu-cose. To have a control for growing cells under theseconditions, cells harvested from one of the samples were

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E. COLI STARVATION PROTEINS 487

inoculated in complete medium plus 0.4% glucose. Thecontrol subculture resumed exponential growth without a

lag. The absorbancy at 600 nm of carbon- and nitrogen-starving subcultures did not change during a 2-h incubationperiod, whereas that of the phosphorus-starving subcultureincreased with time at a rate of ca. 8% of the growingculture. As discussed previously (6), because the cells expe-

rienced identical treatment before exposure to the abovespecified regimens, it is valid to ascribe their protein synthe-sis pattern to starvation for individual nutrients.Bulk protein synthesis rate and separation of polypeptides.

Samples were removed at selected times during growth andstarvation and were pulse-labeled with L-[35S]methionine(10-8 M, 10 to 25 ,Ci/ml, 1,000 to 1,300 Ci/mmol; AmershamCorp. and New England Nuclear Corp.) for 3 min at 37°C.Labeled proteins were precipitated with 10% (wt/vol) tri-chloroacetic acid at 4°C and washed with 5% trichloroaceticacid and then acetone. Samples were counted on 0.45-,ummembrane filters (Metricel; Gelman Sciences). Protein pre-

cipitates were dissolved in electrophoresis buffer (19) andanalyzed immediately or stored at - 80°C. Conditions fortwo-dimensional electrophoresis were similar to those pre-

viously used (6): in the first dimension, isoelectric focusingto equilibrium (16 h at 350 V, 2 h at 800 V) in 4% acrylamidewith 0.4% ampholines (Bio-Rad Laboratories) (pH 3 to10)-1.6% ampholines (pH 5 to 7); in the second dimension,sodium dodecyl sulfate-polyacrylamide gel electrophoresisin 10% acrylamide. Newly synthesized polypeptides werevisualized by fluorography of dried slab gels (11). Compari-sons of two-dimensional gel maps were made by superim-posing X-ray film (XAR-5; Eastman Kodak Co.)fluorographs of slab gels run in parallel.The pH gradient of the isoelectric focusing gels was

determined after soaking 0.5-in. (ca. 1.25-cm) sections of a

blank gel in deionized water for several hours. To determinemolecular weight distribution, standards (Bio-Rad) were run

with labeled samples on sodium dodecyl sulfate-polyacry-lamide gels, which were stained with Coomassie blue beforefluorography.

Synthesis rates of individual polypeptides. The synthesisrates of polypeptides were determined essentially as de-scribed by Mosteller et al. (18) by measuring [35S]methioninein individual polypeptide spots on two-dimensional gelsprepared from a culture pulse-labeled at different timesduring growth and starvation. Developed films were alignedwith the dried gels, spots of interest were cut out, and gelpieces were solubilized by incubation (2 h at 70°C) in cappedvials containing 30% (vol/vol) H202 plus 1% (vol/vol)NH40H. Catalase (10 ,ug) was added to each vial, andincubation was continued (10 min, room temperature), fol-lowed by counting in a liquid scintillation spectrometer.To assess the recovery of individual 35S-labeled

polypeptides on gels, [3H]leucine-labeled proteins from a

growing culture were mixed with 35S-labeled proteins andserved as an internal standard. Disintegrations per minute ofindividual 3H-labeled polypeptides were relatively constantin the 24 gels analyzed (standard error, + 7 to +12% forindividual gels), indicating that correction for recovery was

not necessary in these experiments. Thus, synthesis rateswere determined by dividing the 35S disintegrations perminute in an individual polypeptide spot by the 35S disinte-grations per minute of the total protein loaded on that gel.Transposon insertion mutagenesis of E. coli MC4100 with

Mu dX (lac Apr Cm,). Mu dX is believed to insert randomlyin the E. coli genome. In the proper orientation, the lacZgene is controlled by the promoter of the gene in which the

phage inserts, making it possible to monitor the expressionof an operon by measuring P-galactosidase synthesis. Phagelysates were prepared as described by Miller (16). E. coliMC4100 was grown to about 109 cells per ml in LB mediumsupplemented with 2.5 mM CaCl2 and 0.5 mM MgSO4. Aphage suspension (1 Mu dX phage per 103 helper phage) wasadded to this culture to give a multiplicity of infection of 0.3for the helper phage; after incubating for 30 min at 30°C, theculture was diluted 10-fold with fresh medium and incubatedat 300C for another 30 min. E. coli::Mu dX lysogens wereselected by plating on LB agar containing ampicillin (50jig/ml) and chloramphenicol (20 jig/ml). Since the probabilityof a given bacterium receiving two Mu dX phages was 5.5 x10-1, each lysogen is likely to contain only a single Mu dXinsertion. Antibiotic-resistant lysogens were screened byreplica plating on M9 medium plus 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside containing either 0.40 or0.02% glucose. During the incubation period (ca. 20 h),starvation conditions for glucose were established only onthe low (0.02%)-glucose plates after formation of visiblecolonies. Of 120 Apr Cmr selectants, 13 turned blue on 0.02%glucose plates but not on the 0.40% glucose plates and wereisolated. These are referred to as carbon starvation response(cst) gene mutants.

Miscellaneous. Viability was determined by spreading se-rial dilutions of the cultures on LB agar plates (21). 3-Galactosidase activity was assayed in duplicate as describedby Clark and Switzer (4) and was corrected for light scatter-ing (16). The activity was expressed as nanomoles of o-nitrophenyl-,5-D-galactoside hydrolyzed per min per unit ofabsorbancy at 660 nm. Glucose was assayed by using Sigmakit 510-A (Sigma Chemical Co.).

RESULTS

Rates of synthesis of individual starvation proteins duringglucose starvation. An E. coli culture was allowed to growuntil glucose (initial concentration, 0.025%) was exhaustedfrom the medium; samples were then removed at varioustimes during starvation and pulse-labeled, and their proteinsynthesis was analyzed by two-dimensional gel electropho-resis (19). The time dependence of synthesis of severalstarvation proteins was readily apparent from the resultinggel maps, as is qualitatively illustrated in Fig. 1 for selectedtime points. The kinetics of synthesis of 20 of the starvationproteins were analyzed in detail as described in Materialsand Methods; these polypeptide spots have been numberedin Fig. 1 for reference.These polypeptides could be grouped in four temporal

categories with respect to their synthesis after glucoseexhaustion from the medium as follows: those synthesizedonly in the first hour of starvation with maximum synthesisrates between 0 to 20 min (Fig. 2A); those maximallysynthesized between 20 and 50 min of starvation withsteadily decreasing synthesis thereafter (Fig. 2B); those witha broad peak of synthesis (Fig. 2C); and those whosemaximal synthesis was primarily between 3 and 4 h ofstarvation (Fig. 2D). The pattern of kinetics shown in Fig. 2Cwas also exhibited by the polypeptides numbered 2, 10, 12,13, 18, and 20. The addition of glucose to a culture sampleafter 50 min of starvation resulted in an immediate decreasein the synthesis of these starvation-induced polypeptides, asis illustrated for a few of them in Fig. 2B and C.

Synthesis of individual polypeptides during succinate star-vation. Glucose is a high-carbon catabolite repression sub-strate, raising the possibility that proteins synthesized by the

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FIG. 1. Two-dimensional gel maps of polypeptides synthesized by E. coli K-12 during exponential growth (A) or at various times afterglucose exhaustion from the medium (30 min in B; 60 min in C; 180 min in D). Culture samples were pulse-labeled with [35S]methionine andprocessed for gel electrophoresis as described previously (6). Circles or ovals indicate the polypeptides whose kinetics of synthesis weremeasured (see below). Empty circles or ovals indicate the position of a spot not present on a given gel map but present on another. The figureis intended to provide primarily a quantitative idea of the appearance and disappearance of individual polypeptide spots during starvation. Thequantitative picture (corrected for total 35S counts loaded on each gel) is presented in Fig. 2.

cells at its exhaustion from the medium were primarilyglucose-repressible proteins (3). If so, protein synthesisduring carbon starvation caused by the depletion of someother carbon substrate should present a different picture.

Accordingly, we studied kinetics of protein synthesis in anE. coli culture entering the postexponential phase due tosuccinate exhaustion from the medium.At the onset of succinate starvation, the bulk protein

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E. COLI STARVATION PROTEINS 489

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STARVATION TIME (MIN)FIG. 2. Rate of synthesis of selected polypeptides inanE. coli K-12 culture entering the postexponential phase due to glucose exhaustion.

Zero time denotes complete exhaustion of glucose from the medium. Polypeptides are grouped in panels A to D according to their temporalpattern of synthesis in response to starvation. Dashed lines show rate of synthesis of some individual polypeptides after glucose readdition(indicated by arrows). The synthesis rate was calculated by dividing 35S disintegrations per minute in a given spot by total 3S disintegrationsper minute loaded on the gel.

synthesis rate (as judged by [35S]methionine incorporation)dropped by about 45% and remained at this rate for the nexthour before declining further to very low values (Fig. 3A).This change was very similar to that observed at the onset ofglucose starvation (Fig. 3B), except that it occurred muchsooner. The kinetics of synthesis of the same 20polypeptides that were investigated in this respect duringglucose starvation (Fig. 1) were determined also duringsuccinate starvation (succinate starvation gel maps notshown). Again, four temporal patterns of synthesis wereobserved, with each polypeptide falling into the same kineticcategory as during glucose starvation (Fig. 2). This is illus-trated by presentation of data for four individualpolypeptides (Fig. 3A), representing each of the four kinetic

categories of glucose starvation proteins shown above (Fig.2). For ease of comparison, the kinetics of synthesis of thesame proteins during glucose starvation are redrawn (Fig.3B), and it is clear that the sequence of synthesis ofindividual starvation polypeptides during succinate or glu-cose starvation was very similar. However, the changeswere accelerated after succinate exhaustion from the me-dium, paralleling the faster change in bulk protein synthesisrate under these conditions (Fig. 3).

Role of a cst gene in starvation survival. The data presentedabove and elsewhere (6) indicate that E. coli possesses a setof genes (cst genes) that are expressed during carbon star-vation. Does this gene expression have a role in starvationsurvival? To address this question, we isolated a number of

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mutants bearing cst::lac fusions as described in Materialsand Methods. Several of these mutants were further testedfor ,B-galactosidase production during growth and starvationand, as expected, were found to exhibit a marked increase inthe production of this enzyme upon glucose exhaustion fromthe medium. One of the mutants, referred to as EZ1, wasselected for starvation-survival studies because it exhibitedstrong cst promoter activity; ,B-galactosidase activity re-mained low in this strain (20 to 40 U) during growth butincreased steeply upon glucose exhaustion and was ca. 280U at 3 h of starvation.

Strain EZ1, when subjected to glucose starvation, lostviability much more rapidly than the parent strain, MC4100(Fig. 4); at the end of a 7-day starvation period, 49% of theparent organisms were still viable as opposed to only 18% ofthe mutant cells. These data are based on the use of twoplates per time point for each strain. In another starvationexperiment, each strain was sampled for plating at the timeof peak viability and after 7 days of starvation, using 10plates per time point. After 7 days of starvation, 50% of theparent cells and 13% of the EZ1 cells were viable. Thestandard deviations for the plate counts were ± 8% or less.

It was possible that the decreased starvation survival ofstrain EZ1 was the result of the presence and expression ofMu dX per se. If so, other insertion strains should also

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FIG. 4. Viability of a glucose-starved lacZ fusion strain, EZ1(0), and its isogenic parent, MC4100 (0). Cultures were grown inM9 medium containing 0.025% glucose and were sampled forviability after growth had ceased. Viability was determined by serialdilution colony counts on LB agar.

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IA',,, * 9 <0 14 -10 exhibit decreased starvation survival. Therefore, another E.bE* 3 Q. coli MC4100 insertion strain, EZ2 (lac Apr Cm'), was tested.

t4g c19 V This strain showed only a weak cst promoter activity in that4\A BULK 8 o P-galactosidase synthesis increased from 0 U during growthA to ca. 20 U at 180 min of starvation. EZ2 exhibited no change

wJ in its starvation survival compared with the isogenic parentr< strain, as determined in four different experiments. SinceEZ1 synthesized a large amount of P-galactosidase during4 Z starvation, whereas the parent strain synthesized none and

2'> EZ2 only a small amount under these conditions, the possi-2 bility remained that the energy expended in enzyme synthe-

.. -xC'2 sis by EZ1 accounted for its impaired starvation survival.tr This possibility was, however, rendered highly unlikely byB ° the finding that E. coli K-12 starved in the presence of 2 mMN>Pn Z isopropyl ,B-D-thiogalactopyranoside (which induces high

w levels of P-galactosidase in this strain) exhibited a survival)A rX-ds-/ 8 z pattern very similar to that when it was starved in the

A-'F'->-<" > ;5 absence of the inducer, i.e., synthesis of ,-galactosidase, 'a¢ / z during starvation made no difference to the starvation-

w survival pattern of this strain. Thus, it is reasonable to*0. conclude that the diminished starvation-survival capacity of/4 strain EZ1 was because of a defective cst gene.S,oZixQ4 ^ Comparison of protein synthesis in E. coli cultures starved

..jP\for carbon, nitrogen, or phosphorus. As noted above, it wasa2 3 of interest to determine whether some of the same starvation

proteins were synthesized by E. coli cultures starved for- different individual nutrients. This was investigated by di-

-40 0 60 120 180 240 viding a log-phase culture into four samples, rapidly harvest-

STAR\/ATION TIME (MIN) ing the cells, and suspending them in appropriate individualRateofsyntARVis oNseletIe (MN)peptides andbulk

media that either permitted logarithmic growth or subjectedRate of synthesis of selected polypeptides and bulk the cells to carbon, phosphorus, or nitrogen starvation underan E. coli K-12 culture entering the stationary phase due otherwise iticalbonditions (e Materialsan ethoundste exhaustion (A). The kinetics of synthesis of the same otherwise identical conditions (see Materials and Methods).es after glucose exhaustion are redrawn from Fig. 2 to Comparisons were made of the protein synthesis pattern ofomparison (B). The synthesis rate was calculated as these subcultures after 30 min of incubation at 37°C (Fig. 5).n the legend to Fig. 2. Note the difference in scale of bulk As was the case during carbon starvation (Fig. 3), nitrogen(nthesis as compared with the scale of individual starvation caused an immediate decrease in bulk proteines. synthesis rate, as judged by [35S]methionine incorporation.

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E. COLI STARVATION PROTEINS 491

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starvation for (B) carbon, (C) phosphate, and (D) nitrogen. The subcultures were sampled at 30 min and pulse-labeled as described inMaterials and Methods. Some of the polypeptides induced by individual starvations are indicated by the following symbols: 0, carbonstarvation; O, nitrogen starvation; A, phosphorus starvation.

In contrast, phosphorus starvation caused only a minimaldecrease in this rate. However, protein synthesis continuedat 20 to 90% of the rate found in growing cells during the firstfew hours of each starvation condition.Under each individual starvation condition, synthesis of

several polypeptides was either newly initiated or increasedrelative to total protein synthesis. As reported before (6),some 26 such polypeptide spots could be easily detected ongel maps of carbon-starving cultures; in addition, 32 and 26such spots were detected on gel maps of cultures starved for

phosphorus and nitrogen, respectively (Fig. 5). Althoughsome of the starvation-induced polypeptides were unique toindividual starvation conditions, several others were com-mon to one or more of these conditions. Thus, as judged bytheir charge-size coordinates on gel maps, 4 starvationpolypeptides were synthesized by cells starved for eithercarbon or phosphorus, 7 were synthesized by those starvedfor carbon or nitrogen, and 13 appeared under all threestarvation conditions. The common spots are identified byappropriate symbols (Fig. 5). These results, along with the

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492 GROAT ET AL.

findings of other workers (23, 24) that some of the psi genesare switched on also by other starvation conditions, stronglysuggest that a common set of starvation proteins is inducedin E. coli by a variety of nutritional stresses. Confirmation ofthis notion awaits further studies, i.e., analysis of the star-vation response for additional nutrients and a more precisebiochemical determination of identity of the 13 commonpolypeptides.Some relatively minor differences can be detected in the

carbon starvation response shown in Fig. 1 and 5. Forexample, proteins labeled as spots 4 and 6 in Fig. 1, whichwe have previously (6) identified as heat shock proteinsdnaK and groEL, although not induced at 30 min of starva-tion in the experiment of Fig. 1, were markedly induced inthat of Fig. 5. (In the experiment of Fig. 1, these proteinswere induced primarily at 3 h of starvation [Fig. 2D].) Thereason for this difference is unknown, although it is probablyrelated to the different protocols used in the two experimentsfor establishing starvation conditions: glucose exhaustionfrom a culture (Fig. 1) versus harvesting and suspension(Fig. 5) (Materials and Methods). Minor differences are alsoevident between the gel maps of growing cells in thesefigures. These are probably related to the fact that cells inexperiment of Fig. 1 were grown at a much lower glucoseconcentration (0.025%) than those of Fig. 5 (0.4%). That thequantitative aspect of the growth environment can influenceprotein synthesis pattern is well established (15).

DISCUSSION

The data presented in this paper show a clear temporalpattern to the synthesis of starvation proteins in E. coli andthus indicate a sequential expression of cst genes. Further-more, at least one cst mutant was markedly compromised instarvation survival. Both of these findings are reminiscent ofthe process of sporulation in Bacillus spp. which, throughtemporally ordered expression of unique genes, leads to thegeneration of resistant structures (14).

Morphologically differentiated resistant structures, likethe Bacillus endospores, are not formed by a large number ofbacterial species. Yet, as might be expected from the factthat recurrent starvation is likely to be the lot of all bacteria,even these are known to possess considerable resistance tostarvation. Thus, an E. coli culture survives carbon starva-tion for several weeks (21), and those of nitrifying bacteria,Pseudomonas cepacia, and the thiobacilli have been re-ported to survive starvation for 5, 14, and 54 years, respec-tively (17). (It is pertinent to note here that several newpolypeptide spots appeared also on gel maps prepared froma starving Pseudomonas aeruginosa culture [R. G. Groatand A. Matin, unpublished data].) Given the very differentphysiological needs of a starving cell as compared with agrowing cell, it is probable that the non-spore-forming bac-teria also undergo a process of molecular realignment at theonset of starvation that may be akin to sporulation. This lineof reasoning suggests that a comparative study of the mo-lecular mechanisms that trigger and regulate the cst (or psi[23]) gene expression in E. coli and those of spo genes inBacillus spp. would be of considerable evolutionary interest.We have previously suggested (20) that not all starvation

proteins are likely to have a role in starvation survival. Someare probably only trivially associated with starvation,whereas many others may be concerned with increasing theprobability of escaping starvation. This is consistent with thefinding reported here that one of the cst- strains, EZ2, wasnot impaired in starvation survival. Work in progress is

aimed at isolating several additional cst mutants that are lessresistant to starvation, with a view of identifying the starva-tion proteins directly involved in starvation survival. Al-though much further work is needed to make any firmpredictions at this stage, it is tempting to speculate that suchproteins would be among the 13 common proteins thatappeared to be synthesized by the starving cells regardless ofthe identity of the starvation nutrient.The proteins that were synthesized transiently very early

in starvation (Fig. 2A) might be proteases or peptidasesspecific to the starvation state. Bacillus subtilis producesproteases during the end of the log phase of growth andduring the early stages of sporulation (5). In addition, anaminopeptidase has been found in E. coli whose levelsincrease fourfold during phosphate starvation (12). Theseproteases may possess altered specificity compared withthose utilized during growth and may be involved in thedegradation of growth-associated proteins at the onset ofstarvation. It is also conceivable that some of these earlyproteins are unique sigma factors involved in the transcrip-tion of the starvation response genes. Unique sigma factorsdirect the transcription of genes activated during the station-ary phase in B. subtilis (13, 22). In addition, the htpR geneproduct of E. coli is believed to be a sigma factor specificallyinvolved in the transcription of the heat shock regulon (7),and a novel form ofRNA polymerase appears to be involvedin the expression of ntr regulon in this bacterium (8). Theseand other possible physiological roles for starvation proteinsare currently under investigation.

ACKNOWLEDGMENTS

This work was supported by a grant from the Center forBiotechnology Research, San Francisco, Calif., and a gift fund fromMonsanto Co., St. Louis, Mo.We thank Carole Reeve Stivers and Madelon Halula for helpful

discussions.

LITERATURE CITED1. Baker, T. A., M. M. Howe, and C. A. Gross. 1983. Mu dX, a

derivative ofMu dl (lac Ap9 which makes stable lacZ fusions athigh temperature. J. Bacteriol. 156:970-974.

2. Bockman, A. T., C. A. Reeve, and A. Matin. 1986. Stabilizationof glucose-starved Escherichia coli K-12 and Salmonellatyphimurium LT2 by peptidase-deficient mutants. J. Gen. Mi-crobiol. 132:231-235.

3. Boucherie, H. 1985. Protein synthesis during transition andstationary phases under glucose limitation in Saccharomycescerevisiae. J. Bacteriol. 161:385-392.

4. Clark, J. M., Jr., and R. L. Switzer. 1977. Experimental bio-chemistry, 2nd ed., p. 97-103. W. H. Freeman & Co., SanFrancisco.

5. Doi, R. H. 1972. Role of proteases in sporulation. Curr. Top.Cell. Regul. 6:1-20.

6. Groat, R. G., and A. Matin. 1986. Synthesis of uniquepolypeptides at the onset of starvation in Escherichia coli. J.Indust. Microbiol. 1:69-73.

7. Grossman, A. D., J. W. Erickson, and C. A. Gross. 1984. ThehtpR gene product of E. coli is a sigma factor for heat-shockpromoters. Cell 38:383-390.

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