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Applied and Environmental Microbiology, June 2006, p. 4356-4359, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02937-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evidence for an Intermediate Colony Morphology of Vibrio vulnificus

Thomas M. Rosche, Ben Smith, and James D. Oliver^*

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

Received 13 December 2005/ Accepted 26 March 2006

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Vibrio vulnificus causes both food-borne disease and wound infections. Most V. vulnificus strains express capsular polysaccharide (CPS), which is required for the virulence of this organism. Under standard growth conditions, CPS expression is lost at a relatively high frequency (10^–3 to 10^–4), resulting in a switch from an opaque (Op, CPS⁺) colony morphology to a translucent (Tr, CPS^–) colony morphology. The wzb gene, which encodes a phosphatase required for CPS expression, has been proposed to be involved in this switch through a site-specific deletion of the entire gene. In an examination of five strains, we found that the frequency of wzb deletion in Tr colonies varies by strain and therefore does not account for all the Tr colonies that are seen. In addition, we have identified a third, intermediate (Int) colony morphotype, in which the colonies appear less opaque but are not fully translucent. PCR studies have demonstrated that Int colonies still contain the wzb gene, while reverse transcriptase PCR studies have shown that although Int strains retain expression of wzb, in some cases the transcription of wzb is reduced. Int strains switch to a true Tr (wzb negative) morphotype at a very high frequency (nearly 100%) under certain conditions. Finally, Int colonies, which in some cases can easily be mistaken for Tr colonies, have been observed to occasionally revert to Op, while Tr colonies containing a wzb deletion presumably are unable to revert to the encapsulated form.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Of all concerns which face the shellfish industry, none are currently as critical as the presence of Vibrio vulnificus in oysters. In fact, 95% of all deaths resulting from seafood consumption are caused by this bacterium (8). V. vulnificus is part of the normal bacterial flora of estuarine waters and occurs in high numbers in molluscan shellfish. Fatality rates of over 60%, with median incubation times to onset of symptoms being as low as 7 h, have been reported (8). Between 1989 and 2000, 274 cases involving oyster ingestion occurred in the United States, of which 142 (52%) were fatal. Most (95%) cases occur in individuals who are immunocompromised, have diabetes, or have underlying diseases/syndromes which result in elevated serum iron levels, primarily liver cirrhosis secondary to alcohol abuse/alcoholism (7). Additionally, most (95%) cases occur in males, since females are protected from the V. vulnificus endotoxin by estrogen (5). Recent evidence also suggests that only certain strains are able to cause infection (6, 9).

One of the few experimentally proven virulence factors for V. vulnificus is the capsular polysaccharide (CPS) expressed on the surface of the bacterial cell. It has been suggested that the CPS aids the bacterium in immune evasion, and mutant strains unable to produce or express surface CPS are essentially avirulent in mice (10, 13, 16). CPS expression is visible in the colony morphology of V. vulnificus, with colonies of cells expressing high levels of CPS appearing opaque (Op), while those of cells lacking CPS, or expressing it in low levels, are translucent (Tr). Recently, an additional rugose colony morphotype (3) which produces a more extensive biofilm than either the Op or the Tr morphotype was identified. Under standard laboratory growth conditions, Op strains have been observed to produce Tr colonies at a relatively high frequency (10^–3 to 10^–4), which has led to the suggestion that CPS expression is a phase-variable trait (11, 15). Although studies of mutants have identified many genes involved in CPS expression, the mechanism of loss of CPS expression is not known (11, 14). An analysis of one of the published V. vulnificus genomes (CMCP6) showed that one gene required for CPS expression, wzb, is flanked on either side by a series of direct repeats (14). Because the sequence of the repeats is exactly the same on both sides of the wzb gene, it seems possible that wzb could be deleted by homologous recombination. The frequency of deletion could be determined by the length of the repeats, which in turn could increase or decrease due to the slipped-strand DNA replication errors common in phase-variable systems (1). Chatzidaki and Wright (2) proposed such a mechanism and showed preliminary data indicating that some Tr cells have lost wzb (4). The second V. vulnificus genome to be sequenced, YJ016, does not contain repeats flanking wzb, nor is the flanking sequence homologous, suggesting that for some V. vulnificus strains the mechanisms for wzb deletion may differ. In the current study, we examined Tr isolates of several different V. vulnificus strains and identified a third colony morphotype, which we term intermediate (Int). Int colonies have a stable phenotype that appears to express lower levels of CPS than the Op phenotype but higher levels than the Tr phenotype.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains and culture preparations.
V. vulnificus strains C7184 and YJ016 (clinical isolates) and Env1, JY1305, and JY1701 (environmental isolates) were used in this study. Cultures were routinely grown in heart infusion (HI) broth or agar (Difco, Detroit, MI) at 22°C. All dilutions were performed in artificial seawater (12).

Determination of wzb genotype.
V. vulnificus colonies to be tested were grown overnight in HI broth. To isolate DNA, the cultures were boiled for 5 min and stored at 4°C until used as templates in PCRs. PCR was performed with 50-µl reaction mixtures containing 5 µl of 10x buffer, 4 µl deoxyribonucleotide triphosphate (dNTP) mix, 1 µl wzb up (5'-GGTAAGCCAGCCGATGC-3'), 1 µl wzb dn (5' CTTTGCCCAGGCTTGTG-3'), 37.75 µl distilled water, 0.25 µl TaKaRa Ex-taq (Panvera, Madison, WI), and 1 µl template. These primers amplify a 296-bp region within the 439-bp open reading frame; this region does not include the direct repeats, which occur outside the open reading frame. The reaction conditions were 94°C for 1 min; 30 cycles of 94°C for 20 s, 58°C for 20 s, and 72°C for 20 s; and a final extension of 72°C for 1 min. The products were then run on a 2% agarose gel. As a template control in wzb-negative samples, PCR for the hemolysin gene (vvhA) was performed under the same conditions but substituting primers vvhA up (5'-CGCCGCTCACTGGGGCAGTGGCTG-3') and vvhA dn (5'-CCAGCCGTTAACCGAACCACCCGC-3').

Colony switch assay.
In order to generate higher-than-normal loss of capsule expression, we used the medium we previously described for inducing capsule switching (10). Briefly, stationary-phase cultures were diluted 1:10 (final volume, 5 ml) into modified maintenance medium (10 g proteose peptone no. 3, 10 g NaCl, 20 mg/liter cysteine, pH 7.0), grown without shaking at 37°C for 72 h, and then diluted and plated onto HI agar. After 24 to 48 h at 22°C, Op, Int, and Tr colonies could easily be distinguished, and the percentage of non-Op colonies was determined. These conditions are similar to those reported by Jones et al. (4) in their studies designed to enhance capsule switching. The genotypes of Int and Tr colonies were confirmed using PCR with the wzb primers.

RNA extraction.
Op and Int isolates of C7184k, YJ016, Env1, JY1305, and JY1701 were grown overnight at 22°C. A 500-µl aliquot from each strain was immediately treated with 1 ml RNAprotect bacterial reagent (QIAGEN, Valencia, CA) to stabilize the RNA, vortexed for 5 seconds, and incubated at 22°C, undisturbed, for 5 min. Each sample was then centrifuged at 14,000 x g at 4°C for 15 min, the supernatants were decanted, and the pellets were stored at –20°C. Total RNA was extracted using the TRIzol Max bacterial RNA isolation kit (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. In some experiments with Env1, single Int and Op colonies were individually resuspended in 50 µl of water and treated with 100 µl of RNAprotect bacterial reagent; then RNA was extracted as described above. Following RNA extraction, total RNA was quantified by Gene Spec analysis (MiraiBio, Inc., Alameda, CA). For all samples, 1 µg RNA was used for DNase I treatment according to the manufacturer's instructions (Invitrogen). After DNase I treatment, each sample was adjusted to 20 ng/µl.

RT-PCR.
For PCR analysis of DNase I-treated RNA, a master mix containing 5 µl 10x buffer, 2 µl (25 mM) MgCl₂, 1 µl (10 mM) dNTP, 2.5 µl (6 µM) wzb up, 2.5 µl (6 µM) wzb dn, 0.25 µl (5 units/µl) HotStarTaq DNA polymerase (QIAGEN), and 31.75 µl water was used per sample for a total volume of 45 µl/sample. DNase I-treated RNA (5 µl) added to 45 µl of master mix was used for PCR analysis to detect DNA contamination; any samples with positive amplification were not used. Positive controls for PCR consisted of 45 µl master mix, 1 µl DNA extracted from stationary-phase V. vulnificus Env1, and 4 µl water. Negative controls for PCR consisted of 45 µl master mix and 5 µl water (no-DNA template). PCR amplification consisted of an initial heating step of 15 min at 95°C to activate the HostStarTaq DNA polymerase, followed by 30 cycles of denaturation (94°C for 30 s), annealing (57°C for 1 min), and extension (72°C for 30 s). After cycling was complete, a final extension (72°C for 10 min) was performed. A reverse transcriptase PCR (RT-PCR) master mix for each sample consisted of 10 µl 5x buffer containing 12.5 mM MgCl₂, 2 µl (10 mM) dNTP, 5 µl (6 µM) wzb up, 5 µl (6 µM) wzb dn, 2 µl One Step enzyme mix, and 21 µl RNase-free water for a volume of 45 µl per sample. DNase I-treated RNA (5 µl) and 45 µl master mix were used for RT-PCR (QIAGEN RT-PCR one-step kit) as described by the manufacturer. Positive controls for RT-PCR consisted of 5 µl DNase-free RNA and 45 µl master mix, and negative controls consisted of 5 µl RNase-free water and 45 µl master mix. RT-PCR amplifications consisted of a reverse transcription step for 30 min at 50°C followed by an initial PCR step of 15 min at 95°C to activate the HotStarTaq DNA polymerase. Next, 30 cycles of denaturation (94°C for 30 s), annealing (57°C for 1 min), and extension (72°C for 1 min) were performed, followed by a final extension at 72°C for 10 min. PCR and RT-PCR products were visualized on a 2% agarose gel. As a control for total RNA levels, the primers tufA sense (5'-TCTCAATCCAAGGTCGTGGT-3') and tufA antisense (5-ACCAGGCATTACCATTTCT-3') were used to detect this elongation factor. For tufA reactions, an annealing temperature of 50°C was used.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Role of wzb deletion in Tr morphotype.
The frequency of wzb deletion in Tr colonies (as determined by PCR) of five strains (two clinical and three environmental) is shown in Table 1. In three of the strains, no wzb deletions were seen, indicating that the deletions are relatively uncommon in those isolates, while in the other two strains, the wzb deletion was observed. All Op colonies from all strains tested were wzb⁺. All wzb-negative templates were found to be vvhA positive, indicating the presence of genomic DNA in these samples. In all cases, at least some colonies appearing translucent still contained the wzb gene, indicating that deletion of this gene is not required for the Tr morphotype to develop.

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TABLE 1. Presence of wzb in Tr colony isolates of V. vulnificus

It seems possible that the exact series of direct repeats occurring on both sides of the wzb in CMCP6, for which the entire genome has been published, argues for deletion to be mediated by homologous recombination at these sites. However, a second published V. vulnificus strain, YJ016, lacks any flanking homologous sequences. We examined the five strains included in the present study and found that two (C7184 and JY1701) had the repeats, while the remaining three strains lacked them. Thus, it is clear that while homologous recombination of the direct repeats may provide such a means for deletion of wzb, more than one mechanism must exist for its deletion.

Intermediate colony morphology.
In the course of these studies, we observed that some apparently Tr colonies were more opaque in appearance than others. We termed intermediate any colony that appeared to have an opacity between that of the Op and Tr morphotypes (Fig. 1). In some cases these colonies were closer to Op in appearance, while in other cases, the Int colonies more closely resembled the Tr morphotype. This intermediate phenotype could have been caused by a colony that contained both Op (CPS⁺) and Tr (CPS^–) cells. If this were the case, a dilution and plating of the cells in the Int colony would produce only Op and Tr colonies, with no (or very few) Int colonies. In order to test whether the Int colony phenotype was due to such a mixture, we picked and diluted several Int colonies and plated them onto HI agar. We found the resulting colonies to be uniform in morphotype (Int), indicating that the Int morphotype is a true and stable phenotype, not simply a mixture of Op and Tr cells. Furthermore, when Int colonies (regardless of strain) were picked and grown overnight in HI broth and then replated, they retained the Int phenotype. Finally, all Int colonies tested (n = 10) contained the wzb gene.

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FIG. 1. Tr, Int, and Op colony morphotypes of V. vulnificus strain JY1305. The strains were grown from freezer stocks on HI agar at 22°C. (A) Single colonies of Tr, Int, and Op morphotypes after 48 h growth; (B) growth of Tr, Int, and Op strains after 24 h.

Switching of Int to Tr.
Since the Int and Tr colonies could most easily be visually differentiated in strain JY1305, we used this strain to study switching from Int to Tr. We observed that non-Op (both Int and Tr) colonies arose from Op strains at a low to moderate rate (ranging from 2.2% to 26.2%) but that Int strains yielded Tr colonies at a much higher rate (ranging from 75.1% to 98.8%). Representative colonies of the Int and Tr morphotypes were confirmed using PCR, with Int colonies always found to be wzb⁺, while all Tr colonies were wzb negative. We have also observed Int strains reverting to Op at a low frequency (<1/1,000) under a variety of conditions (e.g., various growth media, incubation temperatures of 23 to 40°C, and with and without aeration). However, we have not observed, nor would we expect, a Tr strain to revert to Op (or Int), as the Tr strains lack wzb. The data suggest that our culture conditions favor cells that express low levels of CPS.

The Int morphology is obviously a result of decreased CPS at the cell surface, and it is possibly due to down-regulation of one or more of the capsule genes. Using RT-PCR, we examined the expression of wzb from five strains of V. vulnificus with the Int morphotype grown from freezer stocks. Figure 2 shows that all the Int strains, like their Op parents, continued to express wzb. In experiments with Env1, in which RNA was extracted directly from Int and Op colonies which had been isolated immediately after exposure to the switch conditions, wzb expression appeared somewhat lower in the Int colonies than in the Op colony (Fig. 3A). In contrast, expression of the elongation factor tufA in Int colonies was similar to that in the Op colony (Fig. 3B). While RT-PCR is considered by some researchers not to be quantitative, this finding for every Int colony examined suggests that wzb expression is transiently lowered while in the elevated switch conditions (maintenance medium, 37°C, without shaking) but, upon exposure to routine lab conditions (HI agar, 22°C, vigorous shaking), returns to high levels. Alternatively, other genes involved in CPS expression, including regulatory genes, could be down-regulated under these conditions.

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FIG. 2. Expression of wzb in Op and Int strains of V. vulnificus. Lanes: 1, negative control; 2, positive control; 3, Env1 Op; 4, Env1 Int; 5, YJ106 Op; 6, YJ016 Int; 7, C7184o Op; 8, C7184o Int; 9, JY1701 Op; 10, JY1701 Int; 11, JY1305 Op; 12, JY1305 Int.

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FIG. 3. Expression of (A) wzb and (B) tufA in Op and Int isolates of V. vulnificus strain Env1. Total RNA was extracted directly from the colonies. Lanes: 1, negative control; 2, positive control; 3, Env1 Op; 4 to 8, Env1 Int colonies 1, 2, 3, 4, and 5, respectively.

We suggest that there are at least two mechanisms by which V. vulnificus can lower surface CPS expression. Since wzb is required for CPS expression (4), it is self-evident that the loss of wzb causes irreversible loss of CPS. Alternatively, in agreement with the observations of Wright et al. (15), CPS genes could be down-regulated under certain conditions, leading to lower CPS expression and colonies which appear less Op. We have observed that the Int morphotype is stable; CPS expression does not return immediately upon transfer from elevated switch conditions to growth in aerated HI agar. This implies either that CPS expression must be reinduced by a specific factor (rather than being repressed only) or that cells exhibiting high levels of CPS arise randomly. It is also important to note that the identification of the Int morphotype likely explains reports of Tr strains reverting to Op. For many strains of V. vulnificus, the majority of non-Op colonies are actually Int (Table 1). We believe that non-Op strains that revert to Op actually have the Int morphotype which undergoes reinduction of CPS expression. "True" Tr strains contain deletions in the wzb locus and are presumably unable to revert to encapsulated forms.

 
These studies were funded by a grant (2002-1240-42) from the North Carolina Sea Grant Program. We gratefully acknowledge the UNC Charlotte Graduate School for supporting page charges.

 
* 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.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, June 2006, p. 4356-4359, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02937-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Brown, R. N., Gulig, P. A. (2009). Roles of RseB, {sigma}E, and DegP in Virulence and Phase Variation of Colony Morphotype of Vibrio vulnificus. Infect. Immun. 77: 3768-3781 [Abstract] [Full Text]  
• Jones, M. K., Oliver, J. D. (2009). Vibrio vulnificus: Disease and Pathogenesis. Infect. Immun. 77: 1723-1733 [Full Text]  

This Article Abstract Full Text (PDF) An author's correction has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rosche, T. M. Articles by Oliver, J. D. Search for Related Content PubMed PubMed Citation Articles by Rosche, T. M. Articles by Oliver, J. D. Agricola Articles by Rosche, T. M. Articles by Oliver, J. D.