Mar 3, 1994 - Microbiology Section, Department ofEcology and Molecular Biology, The Royal Veterinary and Agricultural University,. DK-1958 Frederiksberg ...
Vol. 60, No. 8
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1994, p. 2944-2948
0099-2240/94/$04.00+0 Copyright X 1994, American Society for Microbiology
Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14 LENE KRAGELUND* AND OLE NYBROE Microbiology Section, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg, Denmark Received 3 March 1994/Accepted 9 June 1994
Changes in culturability and outer membrane protein profiles were investigated in Pseudomonasfluorescens DF57 and Pseudomonas putida DF14 during starvation for carbon, nitrogen, and phosphorus. P. fluorescens DF57 remained fully culturable for 4 days in all starvation regimes. The cell mass increased during starvation for nitrogen and phosphorus, indicating the accumulation of storage compounds, whereas it decreased slightly in carbon-starved cells. P. putida DF14 lost culturability during phosphorus starvation, and the mass of phosphate-starved cells did not increase. Analysis of additional P. fluorescens and P. putida strains, however, showed that the ability to preserve culturability during phosphorus starvation was not species but strain dependent. In DF57, an outer membrane protein of 55 kDa appeared during starvation for phosphorus, while another protein of 63 kDa was seen during all starvation conditions. DF14 induced two outer membrane proteins of 28 and 29 kDa during starvation for carbon and nitrogen, but no phosphorus-specific starvation protein could be detected. Therefore, starvation-induced outer membrane proteins do not seem to be conserved among the fluorescent pseudomonads and a unique starvation response might be found in individual strains. cens DF57 and P. putida DF14, both of which are isolated from the rhizosphere, were studied. The aims were both (i) to identify stable outer membrane components induced by carbon, nitrogen, or phosphorus starvation that could be used as markers for starved cells and (ii) to determine how the starvation conditions affected culturability of the selected strains.
The physiological responses to carbon, nitrogen, or phosphorus starvation have been investigated in detail in Escherichia coli, whereas only limited information is available for Pseudomonas fluorescens and Pseudomonas putida (8). These fluorescent pseudomonads are widely distributed in nature, but special attention has focussed on them because of the plantbeneficial properties of several strains (15, 17, 22). In E. coli, the onset of either starvation regime induces 20 to 40 specific proteins. In addition, some starvation proteins are induced by a variety of different nutritional stresses (10, 21). The expression of starvation proteins is a sequential process; i.e., some are synthesized transiently early during the starvation response, while others occur for extended periods later in the response (10). Some starvation-induced proteins are located in the outer membrane (2, 26, 32). This cellular domain represents the interface between the environment and the interior of the cell and consequently plays a major role in determining how the cell interacts with molecules, surfaces, and other cells (11). Furthermore, protein and lipopolysaccharide (LPS) components of the outer membrane may serve as targets for immunochemically based detection of bacterial cells (4, 23). Starvation is among the stresses encountered in the environment that may induce nonculturable bacterial subpopulations which remain physiologically active (28). Hence, direct methods for detection of bacteria in the environment are an important supplement to studies performed solely by culture techniques (30). However, antibodies ensuring reliable, direct immunochemical detection are directed against stably expressed markers, and therefore information on the physiological state of the detected cells is not obtained. In the present study, the starvation responses of P. fluores-
MATERIALS AND METHODS Bacterial strains and culture media. A panel of fluorescent Pseudomonas strains was used (Table 1). The strains were classified by the API-NE 20 system supplemented by additional tests as described by S0rensen et al. (31). Under nonstarved conditions, the bacteria were grown aerobically in 25 ml of Luria broth (pH 7.2)-0.4% glucose (LB; 1% tryptone, 0.5% yeast extract, 1% NaCl) at room temperature (22 to 25°C) on a rotary shaker at 120 rpm. Starvation experiments. Prior to starvation, the bacteria were grown overnight aerobically in 25 ml of Davis minimal broth [DMB; 0.4% glucose, 30 mM K2HPO4, 14 mM KH2PO4, 0.4 mM MgSO4, 7.6 mM (NH4)2SO4, 1.7 mM sodium citrate supplemented with 1 ml of a trace element solution per liter]. The trace element solution contained 20 mg of CoCl2 6H20, 30 mg of H3B03, 10 mg of ZnSO4 7H20, 1 mg of CuCl22H20, 2 mg of NiCl2 6H20, 3 mg of NaMoO4 * 2H20, 10 mg of FeSO4 7H20, and 2.6 mg of MnSO4 *H20 dissolved in 1,000 ml of reverse osmosis water (Milli-Q-water purification system; Millipore). The overnight cultures were then diluted to an optical density at 600 nm (OD600) of 0.1. When the culture was again growing exponentially (OD600 = 0.6), the cells were harvested by centrifugation at 6,000 x g for 10 min, washed twice with starvation medium (DMB depleted of either carbon, nitrogen, or phosphorus), and resuspended to the original cell density in this medium. Ten-millimolar HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.3, was added to the DMB medium depleted of phosphorus to main-
-
-
* Corresponding author. Mailing address: Microbiology Section, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, Rolighedsvej 21, DK-1958 Frederiksberg, Denmark. Phone: 45 3528 2630. Fax: 45 3528 2624.
2944
OUTER MEMBRANE PROTEINS DURING STARVATION
VOL. 60, 1994
TABLE 1. Fluorescent Pseudomonas spp. isolates and their responses to P starvation
1010
2945
FA
Response to P staivation Isolate
Species
Origin
Induced outer Cultur-
membrane pro-
tein(s) (kDa)a 89 ON24 P. fluorescens Soil 55, 63 DF57 P. fluorescens Cucumber rhizosphere ON13 P. fluorescens Lake sediment 87 Cucumber rhizosphere DF45 P. putida Rhizosphere KT2442 P. putida 47 DF42 P. putida Cucumber rhizosphere DF14 P. putida Cucumber rhizosphere
Cblituy bll
- 8
48
72
108k 107 1010 B
+ + +
109 -g C) .0
tain the pH. Incubation conditions were as already described above. During growth in DMB, the addition of extra nutrient (carbon, nitrogen, or phosphorus) prolonged the logarithmic growth phase for neither DF14 nor DF57 as measured by changes in OD6., indicating that the cultures were not limited by carbon, nitrogen, or phosphorus nutrients during growth. The cultures always resumed exponential growth immediately when transferred back to DMB after harvest, demonstrating that the harvest procedure did not influence the growth potential of the cells. At 5, 24, 48, 72, and 96 h, samples were taken from the cell suspensions undergoing different starvation regimes. The total number of cells was determined by acridine orange direct counts (AODC [14]), and numbers of culturable cells were determined by plating on LB agar. An estimate of cell mass was made by reading the OD600 Preparation of outer membranes. Outer membranes were isolated as described by Filip et al. (5). In brief, the harvested and washed cells were ruptured by sonication with a Branson B12 sonifier at 60 W (10 1-min cycles on ice). Sarkosyl (N-lauroylsarcosine) was added to the suspension of ruptured cells to a final concentration of 2%. After an incubation period of 30 min at 20°C, any intact cells were removed by centrifugation at 3,000 x g at 4°C for 10 min. Outer membranes were centrifuged from the supernatant to form a pellet at 45,000 x g at 4°C for 40 min, washed once with water, and resuspended in water. If the suspension was not used immediately, 200 kallikrein inhibitor units (KIU) of aprotinin (Sigma) ml-' was added to the outer membrane preparations and the samples were stored at -200C. Protein concentration in the outer membrane preparations was determined by the method of Lowry et al. (20) with bovine serum albumin (BSA) as a standard. SDS-PAGE analysis of outer membrane proteins. Onedimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed in 1-mmthick separation gels of 14% (wt/vol) acrylamide and 0.18% (wt/vol) N,N'-methylenebisacrylamide (BIS) overlaid with 5.1% acrylamide-0.26% BIS stacking gels. Samples were solubilized by boiling for 5 min in solubilization-reduction solution (0.75% SDS, 3.3% glycerol, 0.47% dithiothreitol, 0.04% pyronine G [Polysciences Inc., Warrington, Pa.], and 125 mM Tris-HCl [pH 6.8]). Molecular masses were determined from relative mobilities compared with those of standard proteins. The protein molecular mass markers (Bio-Rad) were lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), bovine
#
1-
+ +
a Identified by SDS-PAGE. -, no induced protein identified. bCFU after two weeks of starvation. +, CFU as before starvation; -, decreasing CFU during starvation.
I
CP 'EW 109
108107 3 C
2
[
a
0
I1
0 0
24
96
Time (Hours) FIG. 1. Variations in culture characteristics of P. fluorescens DF57 during starvation for carbon (0), nitrogen (C1), or phosphorus (V). (A) Total number of cells determined as AODC; (B) number of culturable cells; and (C) cell mass determined as OD600. Data from a representative experiment performed in triplicate are shown.
carbonic anhydrase (31 kDa), ovalbumin (45 kDa), BSA (66.2 kDa), and phosphorylase b (97.4 kDa). Electrophoresis was carried out at room temperature for 18 h at 55 V. Gels were silver stained by the method of Blum et al. (3). Six micrograms of protein was loaded on each lane. SDS-PAGE of LPS. Whole-cell extracts for analysis of LPS were prepared as described by S0rensen et al. (31). The gels were silver stained by the method of Fomsgaard et al. (7). RESULTS
Culturability during carbon, nitrogen, or phosphorus star-
vation. P. fluorescens DF57 and P. putida DF14 were harvested in late logarithmic phase in DMB to avoid cells that had experienced starvation conditions and then subjected to starvation by downshift. The effect of carbon, nitrogen, or phosphorus depletion on cell growth and culturability was studied for periods up to 96 h.
The total number of P. fluorescens DF57 cells remained relatively constant during all starvation conditions as measured by AODC (Fig. 1A). The AODC of P. putida DF14 exposed to carbon and nitrogen starvation also remained stable (Fig. 2A), whereas phosphorus starvation for more than 3 days resulted in a twofold decrease due to adhesion of the cells to the tube
APPL. ENVIRON. MICROBIOL.
KRAGELUND AND NYBROE
2946
--El
E
0
C
B
A
1010 FA 97-
109 6645-
108
31 -
107 1010 B
21 -
21
w
-
_
_
21-_
_
_
FIG. 3. Outer membrane proteins of P. fluorescens DF57 exposed U)
to starvation for either carbon, nitrogen, or phosphorus. Lanes A to C, DF57 after 0, 5, and 24 h, respectively, of carbon starvation. Lanes D to G, DF57 after 0, 5, 24, and 96 h, respectively, of nitrogen starvation. Lanes H to K, DF57 after 0, 5, 24, and 48 h, respectively of phosphorus
109
IV
50
starvation. Gels were silver stained, and similar patterns were observed in three separate experiments. Starvation proteins are marked by arrowheads. The migration of molecular-mass-standard proteins is indicated at the margin in kilodaltons.
108 107 3 C 2 ~
a
0
~
~
~
~
-
S v ~~~~
r
e
o-~ ~
~
0
0
24
48
72
96
Time (Hours) FIG. 2. Variations in culture characteristics of P. putida DF14 during starvation for carbon (0), nitrogen (O), or phosphorus (V). (A) Total number of cells determined as AODC; (B) number of culturable cells; and (C) cell mass determined as OD600. Data from a representative experiment performed in triplicate are shown.
wall. Only during phosphorus starvation of DF14 was a decrease in culturability observed (Fig. 1B and 2B). P. fluorescens DF57 exhibited a slight reduction in cell mass (OD600) during starvation for carbon, whereas the cell mass doubled in the cultures starved for nitrogen or phosphorus (Fig. 1C). P. putida DF14 showed similar responses to carbon and nitrogen starvation but showed no increase in cell mass during phosphorus starvation (Fig. 2C). LPS pattern during starvation. The electrophoretic LPS profiles of exponentially growing and starving cells were determined by silver staining and were not affected during carbon, nitrogen, or phosphorus starvation (data not shown). Starvation-induced outer membrane proteins. Outer mem-
brane preparations from the P. fluorescens DF57 and P. putida DF14 strains were analyzed by SDS-PAGE after 5, 24, 48, 72, and 96 h of starvation. The major outer membrane proteins of ca. 21, 36, and 44 kDa observed under standard growth conditions were all maintained, regardless of starvation conditions, in both strains (Fig. 3 and 4).
In P. fluorescens DF57, a 63-kDa protein appeared after 24 h of carbon starvation (Fig. 3, lanes A to C) and a protein of similar mobility was seen after 24 h of starvation for nitrogen (Fig. 3, lanes D to G). During phosphorus starvation, a protein of 55 kDa had already become very abundant after 5 h and a
63-kDa protein was induced later during starvation (Fig. 3, lanes H to K). P. putida DF14 showed an increased abundance of two proteins of 28 and 30 kDa during carbon starvation (Fig. 4, lanes A to C). Two proteins of 30 and 65 kDa were induced during starvation for nitrogen, and the abundances of two other proteins of 70 and 77 kDa increased (Fig. 4, lanes D to F). No new proteins appeared during phosphorus starvation (not shown). Once induced or up-regulated, the above proteins were stably maintained for periods up to 96 h (Fig. 3 and 4). Comparison of the responses of DF57 and DF14 during phosphorus starvation suggested a relation between maintained culturability and expression of new outer membrane proteins. A possible relation was investigated further by comparing the responses of seven Pseudomonas strains. Two strains that did not express phosphate starvation proteins lost culturability. The five remaining strains maintained culturability, and four of five expressed new outer membrane proteins (Table 1).
DISCUSSION P. fluorescens DF57 and P. starvation. during Culturability putida DF14 respond similar to carbon starvation. After down97-
45-l 31-
FIG. 4. Outer membrane proteins of P. putida DF14 exposed to starvation for either carbon, nitrogen, or phosphorus. Lanes A to C, DF14 after 0, 24, and 96 h, respectively, of carbon starvation. Lanes D to G, DF14 after 0, 5, 24, and 96 h, respectively, of nitrogen starvation. Gels were silver stained, and similar patterns were observed in three separate experiments. Starvation proteins are marked by arrowheads. The migration of molecular-mass-standard proteins (in kilodaltons) is indicated in the margin.
VOL. 60, 1994
OUTER MEMBRANE PROTEINS DURING STARVATION
shift, the total number of cells remains constant whereas cell mass declines slightly. Kramer and Singleton (18) found a comparable response of two Vibrio species when starvation was imposed by downshift. However, Nystrom et al. (27) found an increasing number of cells and a rapid decrease in culture OD due to reductive divisions when Vibrio sp. strain S14 was starved for individual nutrients by downshift. During incubation in nitrogen-depleted medium, both strains maintain a constant total number of cells, whereas cell mass increases. Phosphorus limitation results in a comparable response for DF57, but by contrast the cell mass remains constant in DF14. The increase in cell mass is probably due to accumulation of storage compounds such as poly-3-hydroxyalkanoates (16), as has been observed during nitrogen and phosphorus starvation in Pseudomonas aeruginosa and E. coli (12, 33). While neither of the tested starvation conditions influence the culturability of P. fluorescens DF57 in these short-term experiments, P. putida DF14 loses culturability during phosphorus starvation. Comparison of the survival rates of additional P. fluorescens and P. putida strains, however, shows that the ability to remain culturable during starvation for phosphorus is not species but rather strain dependent. Still, both our results and the results of Givskov et al. (8) for P. putida KT2440 indicate that fluorescent pseudomonads are more resistant to carbon, nitrogen, and phosphorus starvation than the extensively characterized Vibrio strain S14 (6, 27). Starvation-induced outer membrane proteins. Starvationinduced cell surface proteins may serve as markers for starved cells provided that they are abundantly and stably expressed during starvation for a specific nutrient. To identify potential starvation markers, outer membrane LPS and protein profiles of starved cells were compared with those of exponentially growing cells. In some bacteria, the LPS profile changes with the growth conditions (1), whereas it was found to remain constant during starvation of Vibrio strain S14 (2). In our test strains, both LPS O side chains and at least three major outer membrane proteins are stably expressed during exponential growth and starvation. Thus, antibodies against these molecules should provide stable recognition of even starved cells. During starvation, we observed no transiently expressed outer membrane proteins, even though this type of starvation protein is frequently found during analysis of whole-cell protein expression (10, 25). A limited number of stable starvation markers were found, including representatives of both specific and general starvation proteins. The outer membrane starvation proteins in P. fluorescens DF57 and P. putida DF14 do not seem closely related in spite of the conservation of major outer membrane proteins within the fluorescent pseudomonads (9, 19, 24). Analogous to our comparison of Pseudomonas strains, Albertson et al. (2) found major differences in the number of starvation-induced outer membrane proteins appearing in two Vibrio strains. Starvation proteins localized in the outer membrane have been identified in Klebsiella spp. and Vibrio spp. (26, 29, 32). In Klebsiella aerogenes NCTC 418, proteins of 36, 46, and 48 kDa appear after starvation for phosphate, ammonia, and glucose, respectively, and comparable proteins of 46 and 48 kDa are seen during starvation of Vibrio strain S14 (26, 32). However, neither of these proteins appears closely related to those observed here for P. fluorescens and P. putida. The function of starvation-induced outer membrane proteins may be to provide the starving cell with the nutrients in demand (32), but for most of the described proteins the exact function remains cryptic.
2947
An exception to this situation is represented by the phosphate-selective porin OprP of P. aeruginosa. This 49-kDa protein is derepressed during starvation for phosphorus, and related proteins have been shown to exist in other fluorescent pseudomonads by immunological and molecular biological methods (13, 29). Conceivably, the 55-kDa protein in P. fluorescens DF57 and the 47-kDa protein in P. putida DF42 may be related to OprP. The failure of several strains to induce phosphorus-starvation-specific outer membrane proteins, however, indicates that this type of responder is not conserved among all fluorescent pseudomonads. Presently, we are preparing antibodies to LPS and to selected outer membrane proteins of DF57. We are hopeful that antibodies for detection of this strain, or of a broader group of fluorescent pseudomonads, can be coupled to an analysis of the distribution of starved subpopulations, e.g., in the rhizosphere environment. ACKNOWLEDGMENTS This work was supported by the Danish Center for Microbial
Ecology. We also gratefully acknowledge the technical assistance of Anita J0rgensen and May-Britt Prahm.
REFERENCES 1. Abeck, D., A. P. Johnson, R. C. Ballard, Y. Dangor, E. A. Fontaine, and D. Taylor-Robinson. 1987. Effect of cultural conditions on the protein and lipopolysaccharide profiles of Haemophilus ducreyi analysed by SDS-PAGE. FEMS Microbiol. Lett. 48:397-399. 2. Albertson, N. H., G. W. Jones, and S. Kjelleberg. 1987. The detection of starvation-specific antigens in two marine bacteria. J. Gen. Microbiol. 133:2225-2231. 3. Blum, H., H. Beier, and H. J. Gross. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93-99. 4. Chester, I. R., and P. M. Meadow. 1973. The relationship between the 0-antigenic lipopolysaccharides and serological specificity in strains of Pseudomonas aeruginosa of different serotypes. J. Gen.
Microbiol. 78:305-318. 5. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauroyl sarcosinate. J. Bacteriol. 115: 717-722. 6. Flardh, K., P. S. Cohen, and S. Kjelleberg. 1992. Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956. J. Bacteriol. 174:6780-6788. 7. Fomsgaard, A., M. A. Freudenberg, and C. Galanos. 1990. Modification of the silver staining technique to detect lipopolysaccharides in polyacrylamide gels. J. Clin. Microbiol. 28:2627-2631. 8. Givskov, M., L. Eberl, S. M0ller, L. K. Poulsen, and S. Molin. 1994. Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content. J. Bacteriol. 176:7-14. 9. Gonzalez, I., R. Martin, T. Garcia, P. Morales, B. Sanz, and P. E. Hernandez. 1993. A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk. J. Appl. Bacteriol. 74:394-401. 10. Groat, R. G., J. E. Schultz, E. Zychlinsky, A. Bockman, and A. Matin. 1986. Starvation proteins in Escherichia coli: kinetics of synthesis and role in starvation survival. J. Bacteriol. 168:486-493. 11. Hancock, R. E. W. 1991. Bacterial outer membranes: evolving concepts. ASM News 57:175-182. 12. Hancock, R. E. W., R. T. Irvin, J. W. Costerton, and A. M. Carey. 1981. Pseudomonas aeruginosa outer membrane: peptidoglycanassociated proteins. J. Bacteriol. 145:628-631. 13. Hancock, R. E. W., R. Siehel, and N. Martin. 1990. Outer membrane of Pseudomonas. Mol. Microbiol. 4:1069-1075. 14. Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. Use of Nuclepore
2948
15. 16.
17.
18. 19. 20. 21. 22.
23.
24.
KRAGELUND AND NYBROE
filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:1225-1228. Hofte, M., J. Boelens, and W. Verstraete. 1991. Seed protection and promoting of seedlings by the plant growth beneficial Pseudomonas strains 7NSK2 and ANP15. Soil Biol. Biochem. 23:407-410. Huisman, G. W., 0. De Leeuw, G. Eggink, and B. Witholt. 1989. Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl. Environ. Microbiol. 55:19491954. Keel, C., P. H. Wirthner, T. H. Oberhansli, C. Voisard, Burger, D. Haas, and G. Defago. 1990. Pseudomonads as antagonists of plant pathogens in the rhizosphere: role of antibiotic 2,4-diacetylphloroglucinol in the suppression of black rot of tobacco. Symbiosis 9:327-341. Kramer, J. G., and F. L. Singleton. 1992. Variations in rRNA content of marine Vibrio spp. during starvation-survival and recovery. Appl. Environ. Microbiol. 58:201-207. Labadie, J. M., and L. Desnier. 1992. Selection of cell wall antigens for the rapid detection of bacteria by immunological methods. J. Appl. Bacteriol. 72:220-226. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Matin, A. 1990. Molecular analysis of the starvation stress in Escherichia coli. FEMS Microbiol. Ecol. 74:185-196. Mazzola, M., and R. J. Coolk 1991. Effects of fungal root pathogens on the population dynamics of biocontrol strains of fluorescent pseudomonads in the wheat rhizosphere. Appl. Environ. Microbiol. 57:2171-2178. Mutharia, L. M., and R. E. W. Hancock. 1985. Monoclonal antibody for an outer membrane lipoprotein of the Pseudomonas fluorescens group of the family Pseudomonadaceae. Int. J. Syst. Bacteriol. 35:530-532. Mutharia, L. M., T. I. Nicas, and R. E. W. Hancoclk 1982. Outer
APPL. ENVIRON. MICROBIOL.
25.
26. 27.
28. 29. 30.
31.
32.
33.
membrane proteins of Pseudomonas aeruginosa serotype strains. J. Infect. Dis. 146:770-779. Nystrom, T., N. H. Albertson, K. Flardh, and S. Kjelleberg. 1990. Physiological and molecular adaption to starvation and recovery from starvation by the marine Vibrio sp. S14. FEMS Microbiol. Ecol. 74:129-140. Nystrom, T., N. H. Albertson, and S. Kjelleberg. 1988. Synthesis of membrane and periplasmic proteins during starvation of a marine librio sp. J. Gen. Microbiol. 134:1645-1651. Nystrom, T., R. M. Olsson, and S. Kjelleberg. 1992. Survival, stress resistance, and alterations in protein expression in the marine Vibrio sp. strain S14 during starvation for different individual nutrients. Appl. Environ. Microbiol. 58:55-65. Oliver, J. D., L. Nilsson, and S. Kjelleberg. 1991. Formation of nonculturable Vibrio vulnificus cells and its relationship to the starvation state. Appl. Environ. Microbiol. 57:2640-2644. Poole, K., T. R. Parr, Jr., and R E. W. Hancoclk 1987. Phosphateselective porins from the outer membrane of fluorescent Pseudomonas sp. Can. J. Microbiol. 33:63-69. Ramos-Gonzaflez, M.-I., F. Ruiz-Cabello, I. Brettar, F. Garrido, and J. L. Ramos. 1992. Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440. J. Bacteriol. 174:2978-2985. S0rensen, J., J. Skouv, A. J0rgensen, and 0. Nybroe. 1992. Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns. FEMS Microbiol. Ecol. 101:41-50. Sterkenberg, A., E. Vlegels, and J. T. M. Wouters. 1984. Influence of nutrient limitation and growth rate on the outer membrane proteins of Kiebsiella aerogenes NCTC 418. J. Gen. Microbiol. 130:2347-2355. Wanner, U., and T. Egli. 1990. Dynamics of microbial growth and cell composition in batch culture. FEMS Microbiol. Ecol. 75:1944.
