Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus

FEMS Microbiology Ecology 50 (2004) 133–142 www.fems-microbiology.org

Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus In-Soo Kong a

a,1

, Tonya C. Bates a, Anja H€ ulsmann a, Hosni Hassan b, Ben E. Smith a, James D. Oliver a,*

Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28269, USA b Department of Microbiology, North Carolina State University, Raleigh, NC 27695, USA Received 16 February 2004; received in revised form 19 May 2004; accepted 16 June 2004 First published online 26 June 2004

Abstract Vibrio vulnificus has proven difficult to culture from water or shellfish during winter months, which is attributed to the viable but nonculturable (VBNC) state. Because reactive oxygen species were found to be involved in the low temperature-induced entrance of V. vulnificus into this state, we generated an oxyR mutant which lacks catalase activity. This strain is nonculturable on solid media even at ambient temperature, due to the presence of H2 O2 in such media. Low temperature incubation of the parent resulted in loss of catalase activity, making the cells H2 O2 sensitive, and paralleling the loss of culturability (entry into the VBNC state). Thus, cells of V. vulnificus in the VBNC state are likely exhibiting this response to low in situ temperature and only when the artificial condition of laboratory culture is attempted are the cells nonculturable due to cold-induced loss of catalase activity. To our knowledge, this is the first study providing direct evidence for the metabolic basis of nonculturability and the viable but nonculturable state. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: VBNC; Oxidative stress; Catalase; H2 O2 ; nonculturability

1. Introduction Vibrio vulnificus, an estuarine bacterium, is the cause of several potentially life-threatening human infections [1]. Indeed, V. vulnificus is the number one cause of seafood-borne deaths in the United States, and carries the highest case fatality rate of any food-borne disease in this country [2]. The major source of food-borne infection is the consumption of raw or undercooked oysters, and the primary septicemia which may result carries a 50–60% fatality rate (for recent reviews, see [1,3,4]). Although an anti-phagocytic capsule is clearly required for survival in the host [5], it appears that it is the en-

*

Corresponding author. Tel.: +1-704-687-4049; fax: +1-253-7368431. E-mail address: [email protected] (J.D. Oliver). 1 Present address. Department of Biotechnology and Bioengineering, Pukyong National University, Pusan 608-737, Korea.

dotoxin that causes lethality. Interestingly, 90% of victims are males, and females appear to be protected from the endotoxin by estrogen [6]. V. vulnificus also causes wound infections following contamination of a pre-existing wound with seawater containing the bacterium, or a wound induced by contact with crabs, fish spines, etc. An interesting aspect of the ecology of V. vulnificus is the association of water temperature with decreased recovery from the environment and decreased incidence of human disease. V. vulnificus is readily isolated around the world when the estuarine waters it inhabits are warm. However, when water temperatures are less than
13 °C, it has proven quite difficult to isolate the bacterium from water or shellfish [7,8]. Laboratory studies have shown, however, that this loss of culturability does not reflect loss of viability in these cells. Instead, the cells are said to have entered the viable but nonculturable (VBNC) state [7]. This response, which has now been shown to occur in over 60 bacterial species, results in cells which, while retaining viability, can no longer be

0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.06.004

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cultured on the medium on which the cells are routinely grown [8]. The inducer of the VBNC response varies from species to species, but is low temperature in the case of V. vulnificus [9]. Although it is unlikely that a single underlying mechanism exists for the VBNC phenomenon in all bacteria, the molecular and physiological bases for this response have not been elucidated. Our earlier studies suggested that nutrients may, in some manner, be toxic to V. vulnificus cells incubated at low temperature and that plating such cells to nutrient media may result in their death [10]. Subsequently, Bloomfield et al. [11] speculated that, on exposure to high nutrient levels, growth-arrested cells would likely undergo an imbalance in metabolism which would result in a near instantaneous production of superoxide and other free radicals. In the absence of some pre-adaptation (e.g. starvationinduced stress proteins), they suggested that such cells would not be able to detoxify these toxic radicals and would die. Such a theory could also account for the inability of VBNC cells of V. vulnificus to grow in nutrient media. Recently, several studies have provided evidence for the involvement of reactive oxygen species (ROS) in the VBNC response in V. vulnificus [12,13] and in other bacteria entering this altered physiological state [14–17]. Studies also reported that a significant portion of the VBNC population of V. vulnificus can in fact be cultured if anti-ROS agents (e.g. catalase or sodium pyruvate) are present in the culture medium [12,13]. Such studies led us to hypothesize that catalase might be critical to the ability of V. vulnificus to grow on complex media, and that entry into the VBNC state might reflect loss of catalase activity in these low temperature-stressed cells. Escherichia coli possesses two catalase genes, katG and katE. These code for the periplasmic catalase HPI and the cytoplasmic catalase HPII, respectively. Whereas katG is induced by H2 O2 , katE is induced by entry into the stationary phase [18]. While both genes are regulated by the alternate sigma factor rs , the product of the rpoS gene, the periplasmic catalase is under the control of oxyR [18,19]. Unlike E. coli, however, V. vulnificus has been reported to possess only the katG gene [36]. Interestingly, RT-PCR studies performed on cells of V. vulnificus as they entered the VBNC state indicated the continued production of the oxyR transcript [20]. Thus, we reasoned that, if catalase activity was important in the VBNC response in V. vulnificus, then deletion of OxyR activity should result in a cell which would become nonculturable, similar to that observed in the VBNC state, even at ambient temperature. This would be due to the presence of H2 O2 in routine culture media, a fact which has been shown in a number of studies [21,22], or during metabolism of the cells when exposed to the high nutrient medium, as suggested by Bloomfield et al. [11].

2. Materials and methods 2.1. Bacteria and culture conditions V. vulnificus C7184o is a clinical strain of the encapsulated (‘‘opaque’’) phenotype. It was routinely grown in heart infusion (HI) broth (Difco) under aerobic conditions at
22 °C, with platings to HI agar (1.5% agar, Sigma A7002, added to the HI broth). Studies using culturable cells typically employed cells grown in HI broth to the log phase (OD610 ¼ 0.15–0.2). As required, cells were induced into the VBNC state by inoculation of log phase cells into artificial seawater (ASW; [9]) to give a 1% concentration, with incubation at 5 °C until nonculturable. 2.2. Standard DNA methods Genomic DNA from V. vulnificus was isolated using the Qiagen Genomic-tip System (Qiagen, Valencia, CA). Plasmids were prepared from E. coli using the Qiagen Plasmid Mini Kit. Digestion by endonucleases, ligation reactions and PCR were performed by standard procedures [23]. 2.3. Gene cloning A 600-bp PCR fragment of the oxyR-coding region was generated using V. vulnificus C7184 cells as a template, Taq polymerase (Promega, Madison, WI), and oligonucleotides (Biosynthesis, Lewisville, TX) K3 (50 ATGAAYATHMGNGAYYTNGA) and K2 (50 TGRTCNCKNARRCARTGNCC) derived from the conserved regions of OxyR proteins in other bacteria. More specifically, the 50 terminus of K3 begins with the ATG start codon, and K2 includes the sequence for the conserved cystein C199. Amplification of part of the V. vulnificus oxyR gene was verified by sequencing (Retrogene, San Diego, CA). Protein homology searches were done with BLAST [24]. Multiple alignments were performed using CLUSTAL X [25]. 2.4. Construction of plasmids The amplified fragment was ligated into the cloning vector pCRII Topo (Apr , Kmr ; Invitrogen) using the Topo TA Cloning Kit (Invitrogen, Carlsbad, CA). Cells of E. coli strain Top10 (F mcrA D(mrr-hsdRMSmcrBC)/ 80lacZDM15DlacX74 deoR recA1 araD139 D (araA-leu)7697 galU galK rpsL endA1 nupG; Invitrogen) were transformed with the ligation mixture and ampicillin-resistant transformants were screened for the presence of an oxyR fragment, yielding plasmid pKVR514.

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2.5. Homologous recombination In order to disrupt the oxyR gene of V. vulnificus C7184, plasmid pNQ705 (R6K c ori, oriT of RP4, Cmr ; [26]) was used. Plasmid pKVR514 was digested with the restriction endonucleases XbaI and SstI, and the oxyR fragment was ligated into pNQ705 that had been linearized with the same enzymes and dephosphorylated. E. coli SM10k pir (thi thr tonA lacY supE recA ::RP4-2Te::Mu Kmr ; [27]) cells were transformed with the ligation mixture and chloramphenicol resistant transformants were tested for the presence of the correct fragment by restriction analysis, resulting in plasmid pKNR622. E. coli SM10kpir (donor) was used for mobilizing the suicide construct pKNR622 into V. vulnificus C7184 (recipient). Overnight cultures of donor and recipient were washed in Luria–Bertani (LB) broth. Recipient cells were incubated at 45 °C for 15 min and mixed with donor cells at a 1:3 ratio (v/v). Aliquots (100 ll) were spotted onto a nitrocellulose filter (0.45 lm; Millipore, Bedford, MA) on an LB plate and incubated overnight at 22 °C. The filter was transferred to a microcentrifuge tube containing LB broth. After vortexing, the sample was diluted and aliquots were plated onto LB plates containing 2 lg/ml chloramphenicol (Cm) and 100U polymyxin B. The integral mutation in the transconjugants was subsequently verified by Southern blot analysis, yielding mutant strain V. vulnificus K853. For Southern blot hybridization, the 600-bp oxyR fragment amplified with oligonucleotides K2 and K3 was labeled using a digoxigenin-dUTP (DIG) DNA label and detection kit (Roche Diagnostics GmbH, Mannheim, Germany) and was subsequently used as a probe in Southern hybridization. Hybridization was performed according to the manufacturer’s manual. 2.6. Detection of catalase by native gel electrophoresis Cells of the parent and mutant were grown to both log and stationary phases as described above. A solution of H2 O2 was then added to result in a 10 lM concentration, with incubation for 1 h to induce catalase. Cells were then removed by centrifugation and the cell pellet washed with 50 mM potassium phosphate buffer (pH 7.5). Cell lysates were then prepared by sonication, and native gel electrophoresis performed using 10% polyacrylamide gels. After electrophoresis, the gel was stained for catalase activity by the method of Clare et al. [28].

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room temperature. Cells were plated onto HI solidified with 1.2% agarose (on which the oxyR mutant exhibits unrestricted growth; see text for details) every 10 min for 1 h. 2.8. Effect of anti-ROS agents on culturability of V. vulnificus Cells of the parent and oxyR mutant were grown in HI broth to an OD610 of
0.2 as described above. Cells were then plated onto HI agar with or without catalase (bovine liver, Sigma C9322). The latter was prepared at 20,000 units/ml, filter sterilized and overlayed (100 ll) onto the HI agar. In a separate study on the value of other anti-ROS agents reported to affect culturability, cells were plated onto HI agar supplemented with 100 ll of benzoic acid (100 mM), superoxide dismutase (SOD, 600 units), glutathione (25 mM), histidine (100 mM), or thiourea (25 mM), as well as catalase at 2000 units). In one series of studies, low EEO agarose (Fisher, BP160100) was substituted for the agar in HI agar at a concentration of 1.2%. 2.9. Catalase activity of parent and K853 cells of V. vulnificus Cells were incubated in HI broth or HI broth with 7.5 lg/ml Cm (in the case of K853) overnight with shaking at
22 °C. Fresh broths (2  35 ml) were then inoculated 1:200 with the overnight cultures, and cells were incubated with shaking until early log phase (OD610 of
0.2). Hydrogen peroxide (50 lM) was added to one set of log phase cultures of the parent strain to induce catalase production. Concentrations of 5–50 lM of H2 O2 were added to log phase cells of the mutant. In both cases, the cells were further incubated for 1 h. Cells from
33 ml of log, induced log, and stationary phase cultures were then pelleted and frozen at )80 °C until analyzed for catalase activity. Pellets were resuspended in 1 ml Kpi-EDTA buffer (50 mM potassium phosphate, 0.1 mM EDTA, pH 7.8) and cells were lysed by sonication [29]. After centrifugation (15,000g for 30 min), the supernatants were dialyzed against the Kpi-EDTA buffer for 1 h at 4 °C. Protein concentrations were determined by the Lowry method [30], using bovine serum albumin as a standard. Catalase activities in cell free extracts were determined as described by Beers et al. [31].

2.7. Susceptibility of V. vulnificus to H2 O2

2.10. Catalase activity and culturability of the parent strain incubated at 5 °C

Cells of the parent and oxyR mutant were grown to an optical density of 0.2 as described above. To 98 ll of these cells, 2 ll of 100 mM H2 O2 was added (final concentration of 2 mM H2 O2 ) and the cells incubated at

V. vulnificus was grown to log phase (OD610
0.2). Cells were induced into the VBNC state by 1% inoculation of log phase cells into fresh HI broth with incubation at 5 °C. Samples were periodically removed,

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serially diluted in HI broth and plated onto HI agar. At the same time, samples (50 ml) were removed, centrifuged (5000g for 10 min) and the supernatant decanted. The pellet was frozen in an ethanol and dry ice mixture and stored at )80 °C until assayed for catalase activity [31].

concentrations had previously been found to be the minimum inhibitory dose for V. vulnificus. At intervals, 400 ll of the cells were removed, pelleted and washed with ASW, serially diluted and plated onto HI agar to determine when resuscitation would occur, and if these antibiotics would inhibit resuscitation.

2.11. Presence of H2 O2 in plating media

2.14. Nucleotide sequence

HI agar plates were prepared in the standard way, and maintained at room temperature in the original Petri dish bags. HI agar taken from plates of various ages was analyzed for peroxide content. The concentration of hydrogen peroxide in the media was determined by the phenol red method [32] with the following modifications. About 10–15 g of the solid media were mixed and minced in an equal weight of 10 mM phosphate buffer (pH 6.0); the mixture was kept in the refrigerator for 1 h to allow for the equilibration of H2 O2 between the solid and liquid phases. The mixture was clarified by centrifugation at 15,000g followed by filtering through 0.4 lm Millipore filter. The concentration of H2 O2 in the filtrate was assayed by the Pick and Keisari method, which relies on the oxidation of phenol red, by hydrogen peroxide in the presence of excess Horseradish peroxidase. The oxidation of phenol was measured in a Kontron double-beam spectrophotometer at 600 nm after 1 h incubation at 37 °C. The concentration of H2 O2 in the sample was calculated from a standard curve using known concentrations of H2 O2 . The concentration of H2 O2 in the original solid media was calculated by multiplying the concentration in the extracted sample by the dilution factor (i.e., X2).

The nucleotide sequence of the 600 bp fragment of the V. vulnificus oxyR gene has been deposited to GenBank under Accession No. AY102627.

2.12. Starvation of the oxyR mutant Cells of the parent and mutant were grown as described above, then 100 ll were harvested and washed twice with ASW. ASW (10 ml) was then added to the washed cell pellets and the resuspended cells left at room temperature (
22 °C). Culturable cell concentrations were determined daily, with platings onto HI, HI + catalase, and HI + sodium pyruvate. 2.13. Requirement for protein synthesis for resuscitation of V. vulnificus Following induction of C7184 into the VBNC state, varying concentrations of chloramphenicol were added to 1 ml aliquots of the nonculturable microcosm to provide a final of 1–10 lg/ml of antibiotic. After a temperature upshift to 22 °C for 24 h, cells were plated onto HI agar. In a second study, chloramphenicol (10 lg/ml) or rifampin (10 lg/ml) were added to VBNC microcosms of C7184, which were then subjected to a temperature upshift to 22 °C for 20 h. These antibiotic

3. Results and discussion In a review on the public health significance of viable but nonculturable bacteria [8], it was stated that ‘‘nonculturability may be a consequence of placing cells in/on high nutrient media, due to the production of ROS when cells transport and metabolize organics’’. Such a consequence would be less dramatic in broth media, as any ROS compounds produced would tend to diffuse away from the cell, in contrast to cells which are plated, where the toxic ROS would remain close to the cells. Indeed, our results with V. vulnificus [10] suggested that the nutrient in heart infusion broth is bacteriostatic, and not bactericidal, to those cells. This hypothesis was based on a proposal originally put forward by Boomfield et al. [11] and if correct, then it should be possible to neutralize these ROS substances as they are produced, thus allowing direct culture of the cells. Studies by Mizunoe et al. [14] on the use of ROS-degrading compounds in the restoration of growth by E. coli from the viable but nonculturable state, and similar studies in our lab and by Begosian and Bourneuf [33] and Begosian et al. [12] with V. vulnificus seemed to confirm this. Alternatively, it is also possible that media containing organics might also contain ROS, either naturally or as a consequence of autoclaving [34]. Such a situation would also result in loss of culturability of a population if it was incapable of neutralizing these toxic compounds. To examine these hypotheses, we constructed an oxyR mutant, which would be incapable of neutralizing H2 O2 to a significant degree whether it was present in the medium or as a result of cellular metabolism. Construction of an oxyR mutant. A 600-bp fragment of the oxyR gene in V. vulnificus was amplified and sequenced. The deduced amino acid sequence contained the amino terminal portion of the protein. It also included an amino terminal helix-turn-helix DNA binding motif typical of members of the LysR family. The partial amino acid sequence from our C7184 oxyR gene revealed 85% identity to the corresponding part of the V. cholerae OxyR protein (GenBank Accession No.

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15642631), and was 100% identical with that of the transcriptional regulator sequence (GenBank Accession No. AE009824) from V. vulnificus CMCP6 reported by Lee et al. [35]. This fragment was cloned into plasmid pCRII Topo, then cut out of the vector and ligated into the suicide vector, pNQ705. The oxyR gene in V. vulnificus strain C7184 was inactivated by insertional inactivation during homologous recombination, generating strain K853. A Southern blot analysis was performed to verify the correct recombination event (data not shown).

product in neutralizing H2 O2 , is shown in Fig. 1. Here it can be seen that, while a concentration of 2 mM H2 O2 had no effect on the culturability of wild type cells at room temperature, the oxyR mutant underwent a constant and marked decrease in culturability, ultimately undergoing a
4 log decrease over the 60 min study period. A catalase-deficient mutant of Helicobacter pylori has similarly been reported to be hypersensitive to H2 O2 [37].

3.1. Catalase activity in C7184 and K853

Fig. 2 shows the result when wild type cells of V. vulnificus, which were incubated at 5 °C in ASW, were plated onto HI agar or onto HI agar containing catalase (2000 Units/plate). As shown in Fig. 2(a), the cells fully entered the VBNC state (<0.1 cfu/ml) at day 3 when incubated at 5 °C and plated onto HI agar. However, when the H2 O2 -degrading compound, catalase, was present in the same plating medium, culturability was maintained for 11 days. Catalase and sodium pyruvate are known to be effective and specific scavengers of H2 O2 , and these agents have long been used in bacterial media for the recovery of stressed cells (e.g. see [21,38– 40]). The fact that the parent cells still entered the VBNC state at 5 °C, even in the presence of catalase in the plating medium, suggests that either there remains

Cell-free extracts were prepared from log and stationary phase cells of both the wild type and oxyR mutant, as well as from log phase cells which had been induced by H2 O2 . As shown in Table 1, wild type cells of V. vulnificus exhibited little catalase activity (specific activity of 0.2 U/mg protein) when in the log phase unless induced by H2 O2 . In contrast, log phase cells of the oxyR mutant contained very low catalase activity (0.06 U/mg protein, which is the detection limit) even when exposed to H2 O2 . However, both the wild type and mutant demonstrated catalase activity when grown to the stationary phase, although the mutant exhibited
50% less activity than the parent strain.

3.4. Effect of anti-ROS agents on growth of V. vulnificus

3.2. Detection of cellular catalase by electrophoresis 10 9

3.3. Susceptibility of V. vulnificus to H2 O2 Further evidence for the lack of catalase activity in the mutant strain, and the importance of this oxyR

10 8

CFU/ml

Presence of catalase in the wild type and its absence in the oxyR cells was confirmed by staining for the catalase activity following electrophoresis. Using cell free extracts, native gels revealed the presence of a single catalase band in log cells of the wild type only following induction with H2 O2 . Cells of the oxyR mutant did not exhibit a catalase band, even when H2 O2 -induced (data not shown). This observation is in agreement with Rhee et al. [36], who have reported that, unlike E. coli, V. vulnificus possesses only a single gene (katG) for catalase.

10 7

10 6 10 5 10 4

10 3 0

10

20

30

↓

↓

↓

40

50

60

Time (minutes)

Fig. 1. Culturability of parent (C7184, (--)) and oxyR mutant (K853, (--)) in response to exposure to 2 mM H2 O2 . Following exposure for various periods, cells of the parent and mutant were plated onto HI agar. Whereas peroxide caused a
1 log decrease in culturability of the parent, the mutant underwent greater than a 4 log reduction in culturability within 60 min. Down arrows indicate points below the level of detection.

Table 1 Catalase activity in wild type and oxyR cells of V. vulnificus Catalase (U/mg protein) Log phase C7184 K853 a

0.2 0.06

Log phase (induced)

Stationary phase

5.8a 0.08b ( 0.007)c

4.1 2.1

Wild type induced with 50 lM H2 O2 . Average value for cells of oxyR mutant induced with 5, 10, 20, or 50 lM H2 O2 . c Standard error of the mean of the four studies. b

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I.-S. Kong et al. / FEMS Microbiology Ecology 50 (2004) 133–142 10 9

10 5

10 8 10 7

10 4

CFU/ml

CFU/ml

(a) 10 6

10 3

10 5

10 2

10 4

10 1

10 3 10 2

10 0

0

2

4 6 8 Time (days)

10

K853/HI

12

K853/cat

K853/NaP C7184/HI

Strain/Medium Fig. 3. Effect of anti-ROS agents on culturability of the V. vulnificus oxyR mutant (K853) at room temperature. Cells of the oxyR mutant were plated onto HI agar, or HI agar supplemented with catalase (K853/cat) or pyruvate (K853/NaP), and incubated at room temperature. Also shown is growth of the parent strain (C7184) grown on unsupplemented HI agar. Error bars represent standard error of the means of five independent studies.

(b) 106

105 CFU/ml

10 6

104 103 102

101

the action of catalase or other peroxide destroying agents.

100

0

2

4 6 Time (days)

8

Fig. 2. Effect of catalase and other anti-ROS species on culturability of wild-type V. vulnificus following low temperature incubation. Cells of the parent strain (C7184) were incubated in ASW at 5 °C, then plated onto (a) HI agar (sÞ or HI agar supplemented with catalase (d) after various intervals, or (b) onto HI supplemented with benzoic acid (M), SOD (.), glutathione (
), histidine (), thiourea (), or catalase (d). Error bars in (a) represent standard error of the means of triplicate studies. Down arrows indicate points below the level of detection.

sufficient H2 O2 in the catalase-treated HI agar to cause loss of culturability of V. vulnificus, or H2 O2 sensitivity does not fully account for entry of this pathogen into the VBNC state. At present, we have not differentiated these possibilities. In agreement with a preliminary study from this laboratory [41], the use of ROS inhibitors (SOD, glutathione, thiourea, benzoic acid, histidine) other than catalase or pyruvate in the plating medium did not enhance culturability of this V. vulnificus strain when incubated at 5 °C (Fig. 2(b)), indicating again that it is H2 O2 which is involved in the loss of culturability when V. vulnificus is incubated at low temperature. As seen in Fig. 3, a room temperature-grown population of the parent strain, when inoculated onto HI agar at room temperature, exhibited >108 cfu/ml. In contrast, when an equal number of cells of the oxyR mutant were plated onto HI agar at room temperature, growth was greatly inhibited (
99.999% reduction in culturability) unless the cells were plated onto HI containing catalase or pyruvate. Thus, it would appear that the ability of V. vulnificus to grow on high nutrient, solid media is related to the ability of the cells to detoxify H2 O2 present in, or produced on, these media through

3.5. Presence of H2 O2 in plating media When HI agar was examined for the presence of H2 O2 (Table 2), we found that the medium, whether freshly prepared or stored for up to 2 months, contained an average of 244 ng/ml (7.2 lM), with higher levels of this ROS measured in older media (e.g. 329 ng/ml (9.7 lM) after 5 months). It is clear that this level of peroxide is sufficient to prevent development of V. vulnificus colonies when these cells do not exhibit catalase activity (e.g. when the oxyR mutant is employed). We subsequently found that colony formation was not significantly inhibited on HI made solid by the addition of agarose in place of agar, and direct measurement of H2 O2 in HI/agarose (Table 2) confirmed a low level of H2 O2 (76.5 ng/ml; 2.25 lM). Thus, it appears that the sensitivity of V. vulnificus to organic media is primarily due to the levels of H2 O2 present in agar, and not necessarily to the production of this ROS by cells during metabolism. 3.6. Activity of V. vulnificus catalase at 5 °C If cells of V. vulnificus, grown at room temperature, have adequate levels of catalase to allow growth on solid Table 2 Hydrogen peroxide content of culture media Culture mediuma

H2 O2 (ng/ml)

HI agar HI agarose HI broth

244b 76.5 156.4

a b

See Section 2 for details on media composition and H2 O2 assay. Average of HI agar aged 0–2 months.

I.-S. Kong et al. / FEMS Microbiology Ecology 50 (2004) 133–142

1.5

10 8 10 7 10 6

1.0

10 5 10 4 10 3

0.5

Cfu/ml

Catalase (U/mg protein)

media when incubated at room temperature, why are cold-incubated cells nonculturable when incubated at room temperature on the same solid medium? We theorized that a loss of catalase activity in wild type cells, analogous to that in the oxyR mutant, might be induced by low temperature incubation. Such a result could be due either to (1) low-temperature induced conformational change in the catalase molecule such that it no longer functions to protect the cells against peroxide, or (2) low-temperature induced loss of ability to synthesize catalase de novo, coupled with loss of pre-existent catalase due to normal protein turnover. To examine this question, we prepared cell free extracts of wild type cells which had been incubated at 5 °C for varying periods. Fig. 4 reveals that, as cells underwent cold temperature-induced loss of culturability, there was a concomitant and parallel loss of catalase activity. Thus, these data appear to confirm our hypothesis regarding the effect of low temperature on catalase activity. To investigate which of the above alternatives might account for this finding, we treated cells which were in the nonculturable state with protein and mRNA synthesis inhibitors (chloramphenicol and rifampin, respectively) and examined their ability to return (i.e. resuscitate) to the culturable state following a temperature upshift. In three separate studies, cells resuscitated from a pre-temperature upshift level of <0.005 cfu/ml (achieved by filtering 170–200 ml of the VBNC microcosm and plating the filter to HI agar) to a high level (average of 1.2  106 cfu/ml). Such population levels were very close to those present in the microcosms prior to induction of the VBNC state (average of 2.3  106 cfu/ml). Resuscitation was completely inhibited when protein or mRNA synthesis was inhibited. This suggests that resuscitation of V. vulnificus cells on temperature

10 2 10 1 10 0 10 -1

0.0 0

1

2

3

4

5

6

Incubation Time (days) Fig. 4. Catalase activity and culturability of the parent strain incubated at 5 °C. Cells of the wild-type (C7184) were incubated in HI broth at 5 °C and examined for culturability on HI agar (--) and for catalase activity (--) over time. A concomitant loss of catalase activity was observed as culturability decreased.

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upshift requires de novo synthesis of the catalase necessary for growth on H2 O2 -containing media, as opposed to a low temperature-induced conformational change in the catalase protein. This conclusion is also supported by the observation that resuscitation required between 12 and 24 h, a period greater than that which would be expected to be required for a conformational change to occur in this enzyme. It must be noted, however, that rifampin and chloramphenicol inhibit transcription and translation, respectively, and thus synthesis of proteins in general, and not solely catalase. Thus, their effect on resuscitation of V. vulnificus cannot be assumed to relate exclusively to the inhibition of catalase synthesis. 3.7. Resuscitation of V. vulnificus from the VBNC state We had previously proposed that resuscitation of V. vulnificus from the nonculturable (VBNC) state was likely due to some ‘‘repair’’ events occurring during temperature upshift, or to the appearance of the starvation-induced stress proteins known to be produced by V. vulnificus [10,42]. While such proteins appear to play a role in slowing the rate of entry into the VBNC state [43,44], our present data suggest that the catalase activity is critical to the resuscitation response. Further, we previously reported that resuscitation would only occur in the absence of nutrient [10], and only at permissive (e.g. >13 °C) temperatures [45]. We now believe that entry into the VBNC state is due largely to the loss of catalase activity in these cold-incubated cells, and that resuscitation is likely due to the production of catalase in H2 O2 -free diluents (e.g. ASW) at permissive (e.g. room) temperatures. As seen in Fig. 5, cells of the oxyR mutant incubated in unamended ASW maintained culturability at 105 –106 cfu/ml throughout a 27 day period of starvation when plated onto HI containing either catalase or pyruvate. In contrast, and as previously observed, the mutant was unable to grow (<101 cfu/ml) when the same cells were plated onto unamended HI agar. However, colonies began to develop when cells were plated onto HI agar after 5 days of starvation in ASW, and by
21 days, cells of the oxyR mutant exhibited culturability equal to that of the parent strain. This result suggests that either (1) the production of a second catalase (analogous to the stationary phase-induced HPII of E. coli [46]) during starvation, or that (2) a second form of control (e.g. the stress-related alternate sigma factor, RpoS), in addition to oxyR, exists in V. vulnificus when starved. Because Rhee et al. [36] reported only a single catalase gene for V. vulnificus, and this finding was confirmed in our studies (data not shown), the results shown in Fig. 5 argue for a second form of regulation of the catalase gene. In a study by Mich
an et al. [47] on the function of growth phase and oxidative stress on the oxyR regulon

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I.-S. Kong et al. / FEMS Microbiology Ecology 50 (2004) 133–142 10 7 10

6

Cfu/ml

10 5 10 4 10 3 10 2 10 1 10 0 0

5

15 20 10 Time (days)

25

30

Fig. 5. Development of culturability potential in the oxyR mutant of V. vulnificus following starvation in artificial seawater (ASW). Cells (
106 cfu/ml) were maintained in ASW at room temperature and plated onto HI agar (- -), or HI agar supplemented with catalase (--) or pyruvate (-s-). Whereas cells remained fully culturable when plated on supplemented media, cells exhibited no culturability until after 5 days of starvation. Culturability of the starved cells ultimately reached levels comparable to those plated directly onto HI supplemented with ROSdegrading compounds. Down arrows indicate points below the level of detection.



in E. coli, RpoS was reported to reflect positive transcriptional regulation of the oxyR regulon. We have recently reported [61] that RpoS is important for adaptation to environmental change in V. vulnificus, and that an rpoS mutant is especially sensitive to H2 O2 . Further, we have found that RpoS is required for osmotically induced cross-protection against oxidative (H2 O2 ) stress [62], as observed in Fig. 5 of the present study.

4. Conclusions The VBNC state is likely a survival state, as it allows cells to exist in an essentially dormant state until the inducing factor (low water temperature in the case of V. vulnificus) is again conducive to active metabolism. Indeed, while the majority of cold-shocked cells appear to lose viability, all indications are that a significant number of cells enter a VBNC state where they undergo a myriad of morphological, metabolic, physiologic, and possibly even genetic changes [8,48]. In V. vulnificus, changes include production of starvation and cold shock proteins [49,50] and modifications in cytoplasmic membrane fatty acid composition [51]. Genomic changes have also been suggested in studies reported by Bej et al. [52] and Warner and Oliver [42]. Signoretto et al. [53], in studying the cell wall peptidoglycan of E. coli entering the VBNC state, reported an increase in crosslinking, a threefold increase in unusual DAP–DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the av-

erage length of glycan strands in comparison to exponentially growing cells. VBNC cells were also found to have an autolytic capability far higher than that measured in exponentially growing cells. Similar findings have been reported for Enterococcus faecalis [54]. Yaron and Matthews [55] reported a variety of genes, including those required for 16S rRNA synthesis, continued to be expressed in nonculturable cells of E. coli O157:H7, and Lle o et al. [56] detected message for penicillin binding proteins in several Enterobacter spp. up to 3 months after entering the VBNC state. Similarly, Barrett [20] and Saux et al. [57] have reported continued production of message for various genes after cells of V. vulnificus were in the VBNC state for as long as 4.5 months. ATP levels, generally low in dead and moribund cells, have been found to remain high in VBNC cells [38,58], and continued membrane potential has been reported in some studies [59,60]. We now believe that low temperature inhibits oxyRmediated catalase activity, and that cells in the VBNC state, at least in the case of V. vulnificus, are ‘‘nonculturable’’ because of the loss of catalase activity. While this loss of activity may be of no consequence to cells when in the VBNC state, this low temperatureinduced loss of catalase activity makes the cells sensitive to the H2 O2 naturally present in the routine agar-containing media employed for their culture. That they are ‘‘nonculturable’’ does not necessarily reflect any detriment to the cells in situ; only when the artificial condition of laboratory culture is attempted are the cells nonculturable and may appear dead. Resuscitation of such cells, which in the case of V. vulnificus involves the production of active catalase, reverses this consequence, resulting in metabolically active and fully culturable cells. Our results support the conclusion of Begosian et al. [12] who described cold-shocked cells of V. vulnificus as being ‘‘peroxide sensitive’. However, the argument of these authors that the VBNC state is an artifact, in that only an H2 O2 -sensitive subpopulation can be resuscitated, has been shown not to be correct by other investigators (e.g. [56]). Further, this argument seems to be largely semantic, in that regardless of what subpopulation may be capable of resuscitation, it remains a fact that, as a result of stress, the overall population becomes nonculturable, and at least some portion of that population remains viable and potentially capable of active growth. Whether that viable subpopulation is the ‘‘H2 O2 -sensitive’’ subpopulation described by Begosian et al. [12] for V. vulnificus is unknown. The studies presented here, however, demonstrate that H2 O2 is a critical aspect of nonculturability in V. vulnificus, as is the ability of stressed cells to neutralize this toxic factor with catalase. Thus, resuscitation in V. vulnificus appears to involve the re-synthesis of catalase, allowing cells to again grow on H2 O2 -containing media. However, entry

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into the VBNC state by different bacteria is clearly induced by a large variety of environmental factors [8], and this entry has only been shown to be affected by catalase in a limited number of species. Thus, it would appear that H2 O2 is not a basic aspect of this physiological state, and that different bacteria will likely require different strategies to permit their growth, or resuscitation, from the VBNC state. Acknowledgements This study was supported, in part, by funds from the Sea Grant Program (NOAA) award #NA16RG2251. We thank Maya Dagher for assistance with some of the studies presented here, and Thomas Rosche for helpful discussions. We would like to acknowledge the University of North Carolina at Charlotte Graduate School for supporting publication costs.

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