Induction of Carbon Starvation-Induced Proteinsin Vibrio vulnificus
DARLA S. MORTON AND JAMES D. OLIVER*
Department of Biology, The University of North Carolina at Charlotte,Charlotte, North Carolina 28223
Received 27 June 1994/Accepted 8 July 1994
By using two-dimensional polyacrylamide gel electrophoresis of pulse-labelled proteins, carbon starvation-induced (Sti) proteins produced by Vrbrio vulnificus were identified. At least 34 proteins were induced over a26-h period of carbon starvation. Although the total rate of protein synthesis over the 26-h starvation periodsuggests that there is a dramatic decrease in total protein synthesis within the first hour of starvation, at least23 of the Sti proteins were induced within the first 20 min of carbon depletion. Six temporal classes of proteinscould be identified, with class A(ii) encompassing the largest (21 proteins) group. All of the proteins in thisgroup could be characterized by one of two patterns of protein synthesis. The addition of chloramphenicol atsequential times throughout starvation revealed that proteins required for starvation survival are made withinthe first 4 h of starvation.
Since most aquatic environments are characterized by lowbioavailability of nutrients, it is understandable that aquaticorganisms, including marine and estuarine bacteria, have de-veloped a starvation response mechanism. Adaptation to con-ditions of low nutrient availability has been shown to allowlong-term survival. Vibrio strain S14 has been reported toremain 100% viable after 2 weeks in unsupplemented artificialseawater, and it is culturable after at least 6 months (13). Wehave observed that Vibrio vulnificus remains viable for over 3years in artificial seawater. V vulnificus is isolated fromestuaries during summer months (19) and can cause bothnecrotic wound infections and fatal septicemias in persons withliver disease or blood disorders that elevate serum iron levels(17). Infections most often occur following ingestion of raw orpartially cooked shellfish, mainly oysters and clams. Unfortu-nately, this organism is responsible for 95% of all seafood-related deaths in the United States, most often affecting menover the age of 40 (17). This study investigated how such a
pathogenic organism can survive long-term nutrient depletionby examining the patterns of proteins produced upon carbonstarvation.The starvation response has been best characterized for
Escherichia coli (9, 22, 23, 30), Salmonella typhimurium (24-27), and the marine Vibrio strain S14 (2, 8, 12-15). Majorbiochemical and morphological changes have been shown tooccur during this response. Miniaturization of cells (7, 10-12,21, 28) occurs as a result of reductive division in which onlyrounds of DNA replication initiated before starvation arecompleted (14). In the case of Vibrio strain S14, formation ofappendages mediating adhesion and aggregation, formation ofouter membrane vesicles, and induced antigenicity due to thepresence of new proteins in the outer membrane all charac-terize the response to starvation (1, 3, 6, 8). Another propertyconferred upon Vibrio strain S14 by starvation is the formationof cross-protection against certain stresses (5, 21). The actualvalue of cross-protection is unclear, but it would appear toallow nonsporulating cells to survive such adverse conditions as
the lethal effects of temperature and exposure to heavy metalsfor a longer period than previously unstressed cells (15). Theonly cross-protection documented for V vulnificus has beenthat against entering the viable but nonculturable state (18).Three phases of starvation have been identified for S.
typhimurium and Vibrio strain S14 (13, 24-26). In Vibrio strainS14, the first or stringent control phase (0 to 30 min ofstarvation) involves a rapid shutdown of RNA, protein, andpeptidoglycan synthesis (13). There is, however, a markedincrease in guanosine 3'-diphosphate 5'-diphosphate levelsand in proteolysis. An apparent relaxation of the stringentresponse characterizes the second phase (1 to 3 h of starva-tion), with decreasing levels of guanosine 3'-diphosphate 5'-diphosphate and increasing macromolecular synthesis (13).Nystrom et al. suggest that this relaxation could be due toproteolysis, which might provide sufficient amino acids tosuppress the stringent response (13). The third reorganiza-tional phase (after 3 h of starvation) involves a gradual declinein macromolecular synthesis and endogenous respiration tolow but detectable levels (8).
Studies classifying groups of proteins induced upon starva-tion in E. coli, S. typhimurium, and Vibrio strain S14 areabundant (12-15, 24-26, 30). Although most of the proteinsare induced at the onset (0 to 3 h) of starvation in these threegenera, groups of proteins are sequentially produced through-out the starvation regimen. These proteins are either tran-siently induced or remain at constant levels throughout star-vation. The identification of temporal classes indicates that theresponse to starvation does not occur all at once. Instead, it isan orderly, programmed sequence of events that equips a cellto survive a stressful change in its environment. We describehere similar induction patterns for proteins of V. vulnificus. Atleast 34 starvation-induced (Sti) proteins were synthesized inV vulnificus over a 26-h period of carbon starvation. Sixtemporal classes of proteins were identified, and most of theproteins were induced within the first 20 min of starvation.Finally, proteins induced at the onset of starvation were shownto be required for short-term starvation survival. Similarresults have been observed with S. typhimurium (24) and withE. coli K-12 (22).
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TABLE 1. Sample spot protein numbers, alphanumeric designations, molecular masses, pls, and temporal classes of Sti proteins
Temporal Sti protein A-N no. Time of starvation (h)' Molecular pI SSP noclass Sipoen AN0.3 0.8 1.3 3.3 6.3 26.3 mass (kDa)
A(i) 1 D 8.7 - 61 4.9 72012 G 9.5 70 6.0 3603
A(ii) 3 H 8.8 * 61 6.25 13034 H 9.0 * 64 6.25 13045 H 9.9 * 74 6.25 16036 G 9.6 * 70 5.9 26057 G 10.1 * 77 5.9 26068 G 10.4 * 81 5.9 27029 G 10.5 * 82 5.9 270310 G 10.9 * 87 6.0 280111 F6.3 * 41 5.6 300412 G 9.0 * 64 5.9 330113 F 9.8 * 73 5.6 450114 F 10.7 85 5.6 480215 D 9.8 73 4.9 650116 D 10.2 78 4.9 660317 D 10.2 78 4.9 661018 D 11.0 * 90 4.9 680219 D 8.8 * 61 4.9 710120 D 10.7 * 85 4.9 780121 C 8.8 * 61 4.6 820122 C 10.1 77 4.6 860323 A 8.9 * 63 4.1 9205
B 24 C/D 9.2 * 66 4.8 8302
C 25 1 8.6 * 60 6.5 020326 G 10.2 * 78 5.9 270127 F 8.5 * 59 5.6 4302
D 28 F 4.6 * 31 5.6 400129 H 9.5 70 6.2 1401
E 30 G 9.3 62 5.9 240431 G 8.8 - 61 5.9 330332 F 8.8 61 5.6 430333 D 9.0 64 4.9 7304
F 34 H 6.0 _ 39 6.25 1002
a Alphanumeric (A-N) designations are coordinates of each spot on a standard gel grid."Times of starvation reported here include the 20-min radiolabelling period.A sample spot protein (SSP) number for each protein was generated with computer software.
MATERIALS AND METHODS
Organism, cultivation, and stress conditions. V vulnificusC7184 (opaque) was grown in heart infusion broth (Difco,Detroit, Mich.) overnight at 22°C on a wrist-action shaker. A1% inoculation into the defined medium, DM, was then made.DM is FMC medium (27) modified to contain 10 g of NaCl perliter, 4 g of glucose per liter, and the following amino acids,which were added according to the method of preparation of acomplete basal medium for Streptococcus equinas (4): alanine(4 mg/liter), aspartic acid (4 mg/liter), glycine (2 mg/liter),histidine (1.24 mg/liter), leucine (2.5 mg/liter), phenylalanine(2 mg/liter), and tryptophan (0.8 mg/liter). Cells were grown tostationary phase with shaking, and a small aliquot was trans-ferred to 30 ml of fresh DM. To initiate carbon limitation,logarithmic-phase cells at an optical density at 610 nm of 0.25were filtered through a 0.2-p.m-pore-size polycarbonate filter(Poretics, Livermore, Calif.), rinsed once with approximately 3ml of DM-salt solution (containing no carbon compounds),
and resuspended into 30 ml of DM-salt solution. Starved cellswere incubated at room temperature (22°C) with shaking.
Labelling and preparation of proteins for electrophoresis.Immediately upon transfer to DM-salt solution, and at 30 min,1 h, 3 h, 6 h, and 26 h after transfer, 1-ml aliquots of thestarved-cell suspensions were pulse labelled for 20 minwith [35S]methionine (Trans35S-Label; ICN Radiochemicals,Costa Mesa, Calif.) at a concentration of 1.1 Ci/ml. Anunstarved control in DM medium was labelled for 20 minwhile cells were in the exponential phase (optical densityat 610 nm of 0.22) during the hour preceding starvation.Labelling reactions were chased for 5 min with 16.7 mMmethionine (29), and the cells were pelleted for 5 min at 13,600x g. Pellets were washed with DM-salt solution and eitherfrozen at -84°C or immediately extracted by a sodium dodecylsulfate (SDS) extraction method (29). Briefly, pellets wereresuspended in SDS-,B-mercaptoethanol solution (0.3% SDS,5% ,B-mercaptoethanol, 0.4% Tris-HCl, 0.26% Trizma base),boiled for 2 min, and incubated for 30 min at 37°C. DNase-
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FIG. 1. Starvation-induced (Sti) proteins in strain C7184. Protein samples were radiolabelled, extracted, and resolved by two-dimensionalpolyacrylamide gel electrophoresis. Densitometry was employed to analyze the autoradiograms. Shown are computer-scanned images ofautoradiograms of samples taken at -1 h (a), 0 h (b), 30 min (c), 1 h (d), 6 h (e), and 26 h (f) of starvation. Arrows indicate the initial inductionof Sti proteins. The 6-h autoradiogram displays proteins that are induced at both 3 and 6 h; larger arrows indicate proteins induced at 3 h. Numberscorrespond to the Sti protein numbers in Table 1.
RNase was added to the cell suspension, the suspension wasincubated on ice for 10 min, and a urea lysis buffer (9.57%urea, 2% Nonidet P-40, 5% ,-mercaptoethanol, 5% Resolyte4-8 [BDH, London, England]) was added to produce proteinextracts.Measurement of relative rates of total protein synthesis. To
determine the rate of protein synthesis, 4 ,ul of each proteinsample was added to 5 ml of ice-cold 5% trichloroacetic acid
and incubated at 0°C for 45 min. Precipitates were collected on0.45-,um-pore-size membrane filters (Gelman Metricel, AnnArbor, Mich.), rinsed twice with 3 ml of ice-cold 5% trichlo-roacetic acid, placed in 10 ml of scintillation cocktail (Scinti-verse BD; Fisher Chemicals), and counted in a Beckmanmodel LS 6000SC liquid scintillation counter. The relative rateof synthesis was calculated by dividing the number of disinte-grations per minute per milliliter of culture by the number of
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CFU per milliliter of culture. All values for rates of synthesisare reported as disintegrations per minute per cell and areaverages of at least two separate experiments.
Resolution of cell proteins by two-dimensional polyacryl-amide gel electrophoresis. Two-dimensional polyacrylamidegel electrophoresis was performed by the method of O'Farrell(16) with modifications (29), by using Bio-Rad electrophoresisunits. The first dimension was a 13-cm isoelectric focusing gelcontaining 2% Resolyte 4-8, and the second dimension was a12% polyacrylamide gel (16 by 20 cm). Equivalent amounts ofradioactivity (approximately 3.5 x 106 dpm) were loaded foreach set of gels. Autoradiograms (RX film; Eastman KodakCo., Rochester, N.Y.) were prepared to permit visualization ofthe 35S-labelled proteins. Densitometry performed by using anImagemaster DTS and Imagemaster software (PharmaciaLKB, Uppsala, Sweden) was employed to detect and quantifyprotein spots. Proteins which increased in quantity twofold ormore compared with the quantity at the previous time pointand the quantity of the control were deemed to be starvationinduced. These proteins were marked and assigned coordi-nates similar to those of an alphanumeric system described byPedersen et al., which consists of letters and numbers desig-nating the relative positions of the protein spots on the gel(20). Relative rates of synthesis of individual proteins werecalculated by dividing the radioactivity value of each proteinspot generated with the densitometer by the total radioactivityvalue from each corresponding gel. The highest percentage foreach spot was then set at a value of 1, and the otherpercentages were converted to values between 0 and 1. Thedata reported are representative of two separate experiments,but they are actual data from one of the two studies.
Requirement of protein synthesis for starvation survival.The temporal requirement of protein synthesis by cells under-going starvation was determined by the addition of chloram-phenicol (CM; 200 ,ug ml-') at various intervals during earlycarbon starvation. Subsamples were allowed to incubate for 4h and were subsequently pelleted and resuspended in DM-saltsolution without CM. Control samples consisted of parallelsamples not exposed to CM. Viability was determined bycolony formation on heart infusion agar.
RESULTS
Growth and starvation of V. vulnificus. V. vulnificus exhibiteda doubling time of ca. 3.25 h in the defined medium DM (datanot shown). In this medium, the cells were motile and had anormal morphology. Cell numbers changed little over the 26-hstarvation period in carbon-depleted DM-salt solution. Asexpected on the basis of starvation experiments with otherbacteria (8, 10-12, 18, 26, 28), cells of V vulnificus decreased insize, and the cells also remained motile for at least 3 days.
Two-dimensional analysis of proteins induced by starvation.V. vulnificus produced at least 34 carbon starvation-induced(Sti) proteins over a 26-h period when placed in the carbon-depleted defined salt medium (Fig. 1). Samples taken at 0 h(Fig. lb), 30 min (Fig. lc), 1 h (Fig. ld), 3 and 6 h (Fig. le), and26 h (Fig. lf) of starvation were selected to study the specificproteins induced during short-term carbon starvation. Themajority of the Sti proteins were induced early in starvation;within the first 20 min, 23 of the 34 total Sti proteins wereinduced (Fig. lb; Table 1).Temporal classes of starvation-induced proteins identified.
Upon examination of the protein induction patterns, synthesisof the various Sti proteins was found to be time dependent, andthey could be grouped according to their initial times ofappearance (Table 1). These proteins were assigned to tem-
a)
b)
.)
V)
(In<u
Time Starvation (h)
10 15
Time Starvation (h)
FIG. 2. Patterns of synthesis of individual proteins in the largesttemporal class, A(ii). Examination of synthesis rates of individualproteins revealed two major induction patterns. (a) Values for threedifferent proteins representative of one induction pattern, SSP1304(U), SSP3301 (@), and SSP1303 (*), are shown. (b) Values for twoproteins representative of the diphasic induction pattern, SSP6501 (-)and SSP6602 (*), are shown. Values for relative rates of synthesiswere calculated as described in Materials and Methods.
poral classes (A through F). Class A included two subgroups,A(i) and A(ii), which were classified according to their times ofdisappearance. A(i) ceased being synthesized after 1.3 h,whereas A(ii) continued to be synthesized after 26.3 h. Overall,the largest group was class A(ii). Two patterns of proteinsynthesis were observed for this subgroup (Fig. 2): (i) a patternof increasing synthesis followed by a constant decrease and (ii)a diphasic pattern. The second pattern was characterized asfollows: after the initial induction, protein synthesis dramati-cally decreased; it subsequently increased until 6 h of starva-tion, and then it decreased constantly.Temporal classes B through F included fewer proteins than
did class A (Table 1). The induction patterns of individualproteins in each of the classes are shown in Fig. 3. Proteininduction patterns of classes B through F were less complexthan those of class A proteins. In each case in which multipleproteins existed within a temporal class, the individual proteinsexhibited similar induction patterns. The expression of thesetemporally induced proteins suggests that the starvation re-sponse in V. vulnificus is complex and is likely to includeproteins whose synthesis is required for survival during theearly periods of carbon starvation.
Effect of CM on starvation survival. To determine whether
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1.25-
0.754 0
ez0.25-
c )
1. 25
A I
, 0.75
4,
t 0.5-
0.25-
b )1.25
a
4,
S 0 5 1 0 1 5 20 25 30
Time Stavation (h)
4)
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1.25
0.75
0 5S
0 25
5 0 5 10 15 20 25 30
Time Starvation (h)
Time Starvation (h)
0 5 10 15 20 25
Time Starvation (h!
e )1.25-
m 1-
0 75 -
0.5-
X 0.25-
-5 0 5 1 0 1 5 20 25 30
Tine Starvation (1)
FIG. 3. Patterns of synthesis of proteins in classes B through F. Relative rates of synthesis were graphed for proteins included in the followingclasses: class B, SSP8302 (a); class C, SSP203 (-), SSP2701 (*), and SSP4302 (0) (b); class D, SSP1401 () and SSP4000 (*) (c); class E, SSP2404(U), SSP3303 (*), SSP4303 (@), and SSP7304 (A) (d); and class F, SSP1002 (e).
protein synthesis was required for survival of carbon starvation,CM was added at different times during starvation, and theeffect on viability was determined. Inhibition of protein syn-thesis initially caused a dramatic decrease in cell viability to2.5% of prestarvation levels. The percent viability increaseduntil about 4 h after CM addition, when the response appearedto level off at 25% of the prestarvation level. Cell viability at 1h of carbon starvation was 0.65%, that at 2 h was 8.6%, that at4 h was 24%, and that at 27 h was 26% of the prestarvationlevel. These data suggest that the first 4 h of protein synthesisare the most critical for starvation survival in the case of V
vulnificus. However, even though important starvation survivalproteins are produced early (within the first 4 h of starvation),continued protein synthesis seems to be required for cells toremain 100% viable.
DISCUSSION
When the total rate of protein synthesis was examined, weobserved a dramatic decrease within the first hour of starva-
tion, and synthesis remained at these lower levels throughoutthe duration of the study. Studies with E. coli and Vibrio strainS14 have resulted in similar rates of total protein synthesis (9,13). It is likely that continuous levels of protein are synthesizedto maintain the cell in a primed state, so that when a carbonsource is again encountered, it can respond quickly andefficiently.The response to carbon limitation in V vulnificus C7184 is
characterized by the sequential expression of Sti proteins. Atleast 34 Sti proteins, defined as those whose synthesis in-creased twofold or more upon starvation, were produced bythis bacterium over a 26-h period of carbon starvation. Sixtemporal classes of proteins (A through F) were identified,with one of the two class A subgroups, A(ii), being the largest,with 21 proteins. These proteins were induced within the first20 min of starvation and continued to be synthesized through-out the 26 h of the study. Two basic patterns of proteininduction were represented equally in the A(ii) class. The firstpattern was characterized by a single peak at around 1 h ofstarvation. A second pattern appeared to be diphasic, involving
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proteins whose synthesis decreased to low levels after an initialinduction, was subsequently increased by a second round ofinduction which peaked between 3 and 6 h, and then decreaseduntil 26 h. It appears that the peak of induction of the firstpattern coincides with the lowest point of induction of thediphasic pattern. If proteases or other molecules responsiblefor the relaxation of the stringent phase were expressed in thegroup characterized by the first pattern, these might eitherdegrade or decrease the synthesis of the proteins in this patternof induction. This could explain the dramatic decrease insynthesis that was observed in the case of this diphasic pattern.The second peak of synthesis in the diphasic pattern wouldthen occur when the proteins characterized by the first patterndecreased in synthesis and the stringent phase was relaxed.
Various patterns of induction have been reported for Stiproteins in Vibiio strain S14 (13) and E. coli (9). The inductionpatterns we observed for class A(ii) in V vulnificus are similarto those observed for some class A proteins in Vibnio strain S14(13), although no diphasic rates of induction were reported.The overall temporal patterns of induction in V vulnificus arestrikingly similar to those in both Vibio strain S14 and E. coli(9, 14). In the case of Vibrio strain S14, 13 of 66 total Stiproteins were induced within the first 10 min of carbonstarvation. Similarly, 23 of 34 total Sti proteins were inducedwithin the first 20 min of carbon starvation in the case of Vvulnificus. The induction patterns of classes B through F arealso similar to patterns of the corresponding classes in Vibriostrain S14 (13). The existence of temporally induced classes ofproteins may imply that the response to starvation in Vvulnificus is as complex as the response in E. coli or in Vibriostrain S14 and that it likely includes proteins whose synthesis isrequired for survival during periods of carbon starvation.The data in this study indicate that protein synthesis during
the first 4 h of carbon starvation is required for starvationsurvival. However, viability of the cells treated with CM neverreached 100%, even after 4 h of starvation. This suggests thateven though a majority of the proteins required for starvationsurvival are produced early, continued protein synthesis isrequired. Similar results have been reported for E. coli andVibrio strain S14 (9, 13). Studies employing CM as an inhibitorof protein synthesis in Vibrio strain S14 have revealed thatprotein synthesis during the first 3 h of starvation is critical forthe survival of starvation (13). Furthermore, CM addition earlyin starvation was found to inhibit synthesis of some of the classC, D, and E Sti proteins of Vibrio strain S14 (13). Similarstudies are being planned to further investigate the inductionproperties of Sti proteins in E vulnificus.
This study indicates that Sti proteins are induced in Vvulnificus in response to carbon starvation and that they arerequired for starvation survival. These experiments are onlythe first step in determining how this organism enters andsurvives the starvation state. Further studies will includecharacterization of individual Sti proteins and examination ofhow the starvation response is regulated.
ACKNOWLEDGMENTS
We thank Thomas Nystrom for consultation concerning the tech-nique of two-dimensional polyacrylamide gel electrophoresis and forinvaluable discussions on the subjects of starvation and data analysis.We thank Staffan Kjelleberg, whose discussions helped initiate thisstudy.
This study was funded in part by grants from National MarineFisheries (NA36FD/0271) and the USDA (NRICGP/USDA 91-37201-6877).
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