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Applied and Environmental Microbiology, March 2006, p. 2244-2246, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2244-2246.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

In Situ Gene Expression by Vibrio vulnificus

Ben Smith and James D. Oliver*

Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223

Received 7 October 2005/ Accepted 3 January 2006


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ABSTRACT
 
Strains of Vibrio vulnificus incubated in situ in natural estuarine waters during warm months continued to express katG (periplasmic catalase), rpoS (stress sigma factor), tufA (elongation factor), wza, and wzb (capsule synthesis). vvhA (hemolysin) was differentially expressed between environmental and clinical isolates. These results paralleled our in vitro findings.


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INTRODUCTION
 
Vibrio vulnificus is widely distributed in estuarine environments throughout the world (6, 11, 12, 17, 22). It is an agent of fatal infections in humans (15), causing more seafood-borne fatalities in the United States than any other bacterium (9). Like many bacteria which enter a starvation-survival state following nutrient depletion (5), V. vulnificus produces novel proteins needed for survival and for protection against a variety of environmental stresses (10, 16). At low temperatures, V. vulnificus enters into a metabolically distinct (5, 8, 10, 16), viable but nonculturable (VBNC) state wherein cells retain viability and the ability to cause infection but are nonculturable on routine media (2, 13, 14, 23).

To better understand the starvation response of V. vulnificus, we employed membrane diffusion chambers (7) to monitor the expression of genes encoding putative virulence factors (wza, wzb, and vvhA), a protein synthesis factor (tufA), and stress response factors (rpoS and katG) in strains C7184k/o (clinical isolate), ENV1, and 707o (both environmental isolates) incubated in the natural environment.

Logarithmic-phase cells were inoculated (final density, ca. 106 CFU/ml) into 1/2 ASW (23) microcosms or diffusion chambers as previously described (21). Chambers were suspended at a depth of ca. 1 m in North Carolina estuarine waters in June (27°C, 11{per thousand} salinity) or October (21°C, 19{per thousand} salinity) 2004 as previously described (14, 21). An analysis of estuarine water from the June site indicated an average dissolved organic carbon level of 2.83 mg/liter (4).

At various times, aliquots were removed from the chambers and microcosms and prepared for PCR and reverse transcription-PCR (RT-PCR) analyses as previously described (21). To ensure that our RT-PCR methodology was detecting de novo RNA in viable cells, we determined the approximate half-life of rpoS mRNA in V. vulnificus C7184k/o. Cells were inoculated into 1/2 ASW at room temperature, with aliquots immediately removed and treated with 5 µg/ml rifampin (Sigma) to inhibit de novo RNA synthesis (21). At intervals, culturability was determined, and RNAs were extracted for PCR and RT-PCR analyses. The results indicated a half-life of <5 minutes (data not shown), confirming the continued production of mRNA in the starving cells. Such turnover is typical of bacteria, with reported half-lives of most mRNA species being only a few minutes (3, 20). Indeed, Bernstein et al. (1) found mean half-life values for 16 different functional gene classes in Escherichia coli to vary from only 3.8 to 6.4 min.

All strains remained fully culturable following incubation for 108 h in estuarine waters in June (data not shown). The expression of vvhA, rpoS, and tufA in V. vulnificus C7184k/o and Env1 was continuous throughout this time (Table 1). While strain 707o also expressed rpoS and tufA throughout in situ incubation, the expression of vvhA was never detectable.


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  		TABLE 1. In situ gene expression by clinical and environmental isolates of V. vulnificus during two studies along the North Carolina coast

A short-term (24 h) in situ RT-PCR study was subsequently performed in October to confirm the expression of these genes at a different site as well as to determine the expression of three additional genes (wza, wzb, and katG). As in the June study, cells remained fully culturable during 24 h of incubation in diffusion chambers (data not shown). The in situ expression of rpoS, tufA, wza, wzb, and katG (Fig. 1) was continuous in all three strains (Table 1). As observed in the June study, vvhA was fully expressed in V. vulnificus C7184k/o and Env1 but was not detectable in strain 707o.


Figure 1
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  		FIG. 1. RT-PCR results for katG expression in three strains of V. vulnificus during in situ incubation in estuarine water in October 2004. Lanes: 1, DNA ladder; 2, negative control; 3, positive control; 4, time zero; 5, 1 h; 6, 12 h; 7, 24 h.

We compared our in situ findings to the responses of these strains maintained in laboratory microcosms. The culturabilities of all strains remained high (105 to 106 CFU/ml) during 240 h of in vitro incubation, and the expression of rpoS, tufA, wza, wzb, and katG was observed in all strains (data not shown). vvhA mRNA in strains C7184k/o and Env1 was also observed at all time points, while again, it was never detected in strain 707o (data not shown). Thus, our in vitro gene expression results paralleled our in situ studies for all three V. vulnificus strains. In our previous study involving incubation in cold (<15°C) estuarine waters (21), we observed expression of vvhA in V. vulnificus 707o and Env1 only during the first hour of incubation, suggesting that the production of hemolysin by these strains may be temperature regulated in natural environments.

In a previous study (21), we investigated in situ and in vitro expression of the same six genes in these strains during entry into, persistence within, and resuscitation from the VBNC state. Unlike that study, where the expression of katG (encoding the periplasmic catalase of V. vulnificus [18]) was down-regulated in all three strains during in situ and in vitro experiments in cold (<15°C) waters, in situ and in vitro incubation of these three V. vulnificus strains at permissive temperatures (>20°C) resulted in fully culturable cells and continued expression of katG (Table 1; Fig. 1).

The expression of rpoS both in situ and in vitro at all time points in all three strains paralleled the results observed in our previous study as cells entered the VBNC state (21). This suggests that the alternative sigma factor RpoS is important in both the VBNC and starvation-survival states as V. vulnificus copes with the constant environmental stresses it encounters in estuarine waters.

Since both the starvation and VBNC states of V. vulnificus require de novo production of specific proteins (8, 10), it was not surprising that continued expression of tufA, encoding a protein synthesis elongation factor, was observed during both in situ and in vitro incubation (Table 1) and during the VBNC state (21).

It was recently reported that V. vulnificus possesses genes (wza and wzb) which code for a sugar transferase needed for capsule synthesis (24). We observed continued expression of these two genes under starvation-survival conditions (Table 1), whereas during incubation at low temperatures (21), we found differences in expression between the two newly identified V. vulnificus genotypes (19). Our findings suggest that while capsule synthesis continues in starved cells of V. vulnificus, it is repressed in strains of the clinical genotype during entry into the VBNC state. To our knowledge, this is the first report providing evidence that V. vulnificus transcribes genes essential for capsule production during incubation in warm estuarine waters.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223. Phone: (704) 687-8516. Fax: (704) 687-3457. E-mail: jdoliver{at}uncc.edu. Back


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REFERENCES
 
    1
  1. Bernstein, J. A., A. B. Khodursky, P.-H. Lin, S. Lin-Chao, and S. N. Cohen. 2002. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc. Natl. Acad. Sci. USA 99:9697-9702.[Abstract/Free Full Text]
    2. 2
  2. Colwell, R. R., P. R. Brayton, D. Herrington, S. A. Huq, and M. M. Levine. 1996. Viable but non-culturable Vibrio cholerae O1 reverts to a cultivable state in the human intestine. World J. Microbiol. Biotechnol. 12:28-31.[CrossRef]
    3. 3
  3. Conway, T., and G. K. Schoolnik. 2003. Microarray expression profiling: capturing genome-wide portrait of the transcriptome. Mol. Microbiol. 47:879-889.[CrossRef][Medline]
    4. 4
  4. Dyer-Blackwell, K. 2005. The viable but nonculturable state of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus. M.S. thesis. University of North Carolina at Charlotte, Charlotte.
    5. 5
  5. Heim, S., M. D. M. Lleo, B. Bonato, C. A. Guzman, and P. Canepari. 2002. The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis. J. Bacteriol. 184:6739-6745.[Abstract/Free Full Text]
    6. 6
  6. Kelly, M. T. 1982. Effect of temperature and salinity on Vibrio (Beneckea) vulnificus occurrence in a Gulf Coast environment. Appl. Environ. Microbiol. 44:820-824.[Abstract/Free Full Text]
    7. 7
  7. McFeters, G. A., and D. G. Stuart. 1972. Survival of coliform bacteria in natural waters: field and laboratory studies with membrane diffusion chambers. Appl. Environ. Microbiol. 24:805-811.[Abstract/Free Full Text]
    8. 8
  8. McGovern, V. P., and J. D. Oliver. 1995. Induction of cold-responsive proteins in Vibrio vulnificus. J. Bacteriol. 177:4131-4133.[Abstract/Free Full Text]
    9. 9
  9. Mead, P. S., S. Laurence, V. Dietz, L. F. McCaid, J. S. Breese, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 2000. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:1-38.
    10. 10
  10. Morton, D. S., and J. D. Oliver. 1994. Induction of carbon starvation-induced proteins in Vibrio vulnificus. Appl. Environ. Microbiol. 60:3657-3659.
    11. 11
  11. Motes, M. L., A. DePaola, D. W. Cook, J. E. Veazey, J. C. Hunsucker, W. E. Garthright, R. J. Bladgett, and S. Chirtel. 1998. Influence on water temperature and salinity on Vibrio vulnificus in Northern Gulf Coast and Atlantic Coast oysters (Crassostrea virginica). Appl. Environ. Microbiol. 64:1459-1465.[Abstract/Free Full Text]
    12. 12
  12. Oliver, J. D. 1989. Vibrio vulnificus, p. 596-600. In M. P. Doyle (ed.), Food-borne bacterial pathogens. Marcel Dekker, Inc., New York, N.Y.
    13. 13
  13. Oliver, J. D. 2005. The viable but nonculturable state in bacteria. J. Microbiol. 43:93-100.
    14. 14
  14. Oliver, J. D., F. Hite, D. McDougald, N. L. Andon, and L. M. Simpson. 1995. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl. Environ. Microbiol. 61:2624-2630.[Abstract]
    15. 15
  15. Oliver, J. D., and J. Kaper. 2001. Vibrio species, p. 263-300. In M. P. Doyle, L. R. Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals and frontiers, 2nd ed. ASM Press, Washington, D.C.
    16. 16
  16. Oliver, J. D., L. Nilsson, and S. Kjelleberg. 1991. The formation of nonculturable cells of Vibrio vulnificus and its relationship to the starvation state. Appl. Environ. Microbiol. 57:2640-2644.[Abstract/Free Full Text]
    17. 17
  17. O'Neill, K. R., S. H. Jones, and D. J. Grimes. 1992. Seasonal incidence of Vibrio vulnificus in the Great Bay estuary of New Hampshire and Maine. Appl. Environ. Microbiol. 58:3257-3262.[Abstract/Free Full Text]
    18. 18
  18. Rhee, J. H., C. M. Kim, S. E. Lee, Y. R. Kim, Y. S. Jin, and S. S. Chung. 2000. Cloning and molecular biological characterization of the catalase-peroxidase katG gene of Vibrio vulnificus, abstr. B-309, p. 112. Abstr. 100th Annu. Meet. Am. Soc. Microbiol. American Society for Microbiology, Washington, D.C.
    19. 19
  19. Rosche, T. M., Y. Yano, and J. D. Oliver. 2005. A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation. Microbiol. Immunol. 49:381-389.[Medline]
    20. 20
  20. Sheridan, G. E. C., C. I. Masters, J. A. Shallcross, and B. M. MacKey. 1998. Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli. Appl. Environ. Microbiol. 64:1313-1318.[Abstract/Free Full Text]
    21. 21
  21. Smith, B. E., and J. D. Oliver. 2006. In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state. Appl. Environ. Microbiol., 72:1445-1451.
    22. 22
  22. Tamplin, M., G. E. Rodrick, N. J. Blake, and T. Cuba. 1982. Isolation and characterization of Vibrio vulnificus from two Florida estuaries. Appl. Environ. Microbiol. 44:1466-1470.[Abstract/Free Full Text]
    23. 23
  23. Wolf, P., and J. D. Oliver. 1992. Temperature effects on the viable but nonculturable state of Vibrio vulnificus. FEMS Microbiol. Ecol. 101:33-39.[CrossRef]
    24. 24
  24. Wright, A. C., J. L. Powell, J. B. Kaper, and G. Morris, Jr. 2001. Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus. Infect. Immun. 69:6893-6901.[Abstract/Free Full Text]

Applied and Environmental Microbiology, March 2006, p. 2244-2246, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2244-2246.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jones, M. K., Warner, E., Oliver, J. D. (2008). Survival of and In Situ Gene Expression by Vibrio vulnificus at Varying Salinities in Estuarine Environments. Appl. Environ. Microbiol. 74: 182-187 [Abstract] [Full Text]  
  • Thompson, F. L., Klose, K. E., the AVIB Group, (2006). Vibrio2005: the First International Conference on the Biology of Vibrios. J. Bacteriol. 188: 4592-4596 [Full Text]  

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