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Mutation of rpoS gene decreased resistance to environmental stresses, synthesis of extracellular products and virulence of Vibrio anguillarum

Li Ma, Jixiang Chen, Rui Liu, Xiao-Hua Zhang, Ying-An Jiang
DOI: http://dx.doi.org/10.1111/j.1574-6941.2009.00713.x 286-292 First published online: 8 October 2009

Abstract

Vibrio anguillarum is a gram-negative halophilic bacterium that causes vibriosis in marine fish, freshwater fish and other aquatic animals. Bacteria have developed strategies to survive in harsh environments. The alternative σ factor, RpoS (σS), plays a key role in surviving under stress conditions in some gram-negative bacteria. An rpoS mutant of pathogenic V. anguillarum W-1 was constructed by homologous recombination. The sensitivity of the rpoS mutant to osmotic stress [2.4 M NaCl in artificial seawater (ASW)] did not change obviously, but the sensitivity of the rpoS mutant to high temperature (45 °C in ASW), UV-irradiation and oxidative stress (5 mM H2O2 in ASW) increased 33-fold, sixfold and 10-fold, respectively. The production of extracellular phospholipase, diastase, lipase, caseinase, hemolysin, catalase and protease of the rpoS mutant decreased markedly compared with those of the wild-type strain. Virulence of the rpoS mutant strain was also decreased when it was inoculated intraperitoneally into zebra fish; the lethal dose 50% of the wild type and the mutant was 8.66 × 104 and 2.55 × 106 CFU per fish, respectively. These results indicated that the RpoS of V. anguillarum plays important roles in bacterial adaptation to environmental stresses and its pathogenicity.

Keywords
  • Vibrio anguillarum
  • rpoS mutant
  • environmental stresses
  • virulence

Introduction

Vibrio anguillarum is a halophilic, gram-negative comma-shaped rod bacterium that causes vibriosis, a lethal hemorrhagic septicemia affecting a variety of fish species and other aquatic animals, and causing important economic losses throughout the world (Austin & Austin, 1999). Several virulence-related factors have been identified in V. anguillarum, including an iron-uptake system (Crosa, 1980; Crosa et al., 1980; Stork et al., 2007), extracellular metalloprotease (Norqvist et al., 1990; Milton et al., 1992; Yang et al., 2007), hemolysin (Munn, 1980; Hirono et al., 1996; Rock & Nelson, 2006), dermatotoxin, hemagglutinin and cytotoxin (Toranzo et al., 1983; Toranzo & Barja, 1993), genes affecting chemotaxis and motility (McGee et al., 1996; Milton et al., 1996; O'Toole et al., 1996). Recently, a repeat-in-toxin (rtxA) is also found to be a major virulence factor for V. anguillarum (Li et al., 2008). However, the mechanisms of the pathogenicity are not completely understood in V. anguillarum and the causes of disease are still elusive.

Bacteria encounter various stresses, including high temperatures, periods of nutrient starvation, osmotic changes and oxidative stress in their natural environment and in the systemic environments of host fish. The alternative σ factor, RpoS (σS), plays a key role in the survival of bacteria during starvation or exposure to stress conditions and is required for the expression of many genes in the stationary phase of growth. The rpoS of Escherichia coli is involved in survival under conditions of famine, oxidative stress, osmotic shock and low pH (Lange & Hengge-Aronis, 1991; Hengge-Aronis, 2002; Frey et al., 2007). More than 100 genes of E. coli were regulated by the RpoS regulon under starvation (Ishihama, 2000; Patten et al., 2004). The rpoS is also reported to control expression of virulence genes in some pathogenic bacteria. RpoS mutant of Salmonella enterica serovar Typhimurium showed significant attenuation in virulence (Fang et al., 1992). The mRNA levels of Pseudomonas aeruginosa rpoS were increased in chronically infected cystic fibrosis patients, suggesting an upregulation of rpoS upon entry into the human host and therefore a role during infection (Suh et al., 1999). The rpoS of V. anguillarum was reported to induce expression of quorum-sensing regulator VanT and was needed for survival following UV-irradiation and for pigment and metalloprotease production (Weber et al., 2008). In this study, we inactivated the rpoS of V. anguillarum by homologous recombination to investigate the roles of the rpoS in survival under environmental stresses such as hydrogen peroxide (H2O2), osmotic stress and high temperature, as well as the production of the extracellular products and pathogenesis.

Materials and methods

Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Vibrio anguillarum W-1 was cultured on Zobell's 2216E medium at 28 °C for 24 h. Escherichia coli strains were grown in Luria–Bertani (LB) medium at 37 °C. The complete rpoS fragment was ligated with the pUCm-T vector and introduced into JM109. After transformation, colonies were screened on LB medium supplemented with ampicillin at 100 μg mL−1. For cultivation and selection of the rpoS mutant of V. anguillarum, chloramphenicol was added to the growth medium at 25 μg mL−1.

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1

Bacteria and plasmids used in this study

Strains or plasmidsRelevant characteristic(s)Source
Vibrio anguillarum
W-1Wild typeLaboratory collection
MUT W-1Cmr, rpoS insertion mutation from W-1This study
Escherichia coli
JM109K-12 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiΔ(lac-proAB) [F′traD36 proAB lacIqlacZΔM15]Laboratory collection
SY327 (λpir)Δ (lac pro) argE(Am) recA56rpoBλpir, host for π-requiring plasmidsUmeå University
S17-1 (λpir)Tpr SmrrecAthipro rK mKRP4:2-Tc:MuKm Tn7λpirUmeå University
SY327/pR3SY327 containing pR3This study
S17-1/pR3S17-1 containing pR3This study
Plasmids
pUCm-TAmpr, 2.7 kb, high-copy-number cloning vectorSangon, China
pNQ705Cmr, 4.5 kb, R6Kγori, oriT of RP4, suicide vectorUmeå University
pR1Ampr, pUCm-T with 1002-bp fragment containing rpoSThis study
pR2Ampr, pUCm-T with a 486-bp fragment of rpoSThis study
pR3Cmr, pNQ705 with a 472-bp SacI–SalI fragment of rpoSThis study

Cloning of the rpoS gene from V. anguillarum

Chromosomal and plasmid DNA preparation, DNA ligation, bacterial transformation and agarose gel electrophoresis were carried out using standard protocols described previously (Sambrook et al., 1989). Forward primer P11 (5′-ATGAGTATCAGCAATGCAGTAACG-3′) and reverse primer P12 (5′-TTAGTCGTATTCAACATTAAACAGC-3′) were designed according to the sequence of the rpoS genes of V. anguillarum and other Vibrio species. PCR was performed at 94 °C for 5 min, 30 cycles at 94 °C for 1 min, 1 min at 53 °C and 1 min at 72 °C, and the reaction was completed by incubation at 72 °C for 10 min. The PCR product was cloned into pUCm-T and sequenced by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China). Sequence analysis was performed using the National Centre for Biotechnology Information blast network service.

Construction of the rpoS mutant

An rpoS mutant of V. anguillarum was constructed by inserting a suicide vector into the chromosome DNA of V. anguillarum W-1 as described before (Miller & Mekalanos, 1988). Briefly, a 486-bp fragment of rpoS gene was amplified from the plasmid containing the rpoS gene of V. anguillarum with primers P21 5′-TCGAGCTCTAGTGATGAAGAACTCAC-3′ and P22 5′-CTCGTCGACAAGGTAGATATTCAACT-3′, containing SacI and SalI sites. This amplicon was subcloned into the SacI–SalI sites of the suicide vector pNQ705. The resulting suicide plasmid was transferred into E. coli SY327 and then to E. coli S17-1. An rpoS mutant was constructed by integration of the suicide plasmid containing the 486-bp fragment of the rpoS gene into the chromosomal rpoS gene of V. anguillarum W-1 as described previously (Ramos-González & Molin, 1998; Yildiz & Schoolnik, 1998). The rpoS mutant was selected on thiosulfate–citrate–bile salts–sucrose (TCBS) media containing chloramphenicol (25 μg mL−1). The rpoS gene disruption was finally identified by sequence analysis of a PCR-amplified fragment with primer P31 (5′-CCAGTGGCTTCTGTTTCTATCA-3′, complementary to the plasmid pNQ705 just outside the linker region) and P11 (5′-ATGAGTATCAGCAATGCAGTAACG-3′, complementary to the rpoS gene just outside of the 486-bp region).

Exoenzyme expression assays

To determine the production of extracellular enzymes, the rpoS mutant and wild-type cells of V. anguillarum were spotted on 2216E agar plates containing the following substrates: 1% yolk for phospholipase, 0.2% amylum for diastase, 1% Tween 80 for lipase, 1% casein for caseinase and 5% fish red blood cells for hemolysin, respectively. The plates were then incubated at 25 °C for 48 h and the clearing zones were measured. All experiments were performed in duplicate and repeated at least once.

Catalase assays

The wild-type and the mutant cells were cultured in 2216E broth at 28 °C for 24 h, and the culture supernatant was collected by centrifuging at 10 000 g for 10 min at 4 °C. The catalase assay was performed using the method described previously (Beers & Sizer, 1952). The reduction in the amount of H2O2 was monitored by measuring the OD240 nm of the reaction mixture using a spectrophotometer. Phosphate buffer was used as control. One unit of enzyme was defined as the amount that degraded 1 μmol H2O2 min−1 under the standard assay conditions.

Protease activity assays

The wild-type and the mutant cells were cultured and the culture supernatant was collected as described above. Protease activity was determined using azocasein (Sigma Chemical Co., St. Louis, MO) as the substrate by a modified method of Inamura et al. (1985). Briefly, 0.25 mL of the culture supernatant was added to 0.25 mL of azocasein (5 mg mL−1) in 50 mM Tris-HCl buffer (pH 8.0). The mixture was incubated at 25 °C for 20 min. The reaction was terminated by adding 1.75 mL of 5% trichloroacetic acid and keeping on ice for 10 min. The mixture was centrifuged at 380 g for 5 min, 1.75 mL of the supernatant was mixed with an equal amount of 0.5 N NaOH, and the OD440 nm was determined. One unit of protease activity was defined as the amount of enzyme producing an increase of 0.01 OD440 nm under the specified conditions.

Survival assays

The rpoS mutant and the wild-type cells of V. anguillarum were grown in 2216E at 28 °C for 24 h to reach the stationary phase. The bacterial cells were harvested by centrifugation at 2350 g at 4 °C for 10 min and washed three times with artificial seawater (ASW). The washed cells were resuspended in ASW at a density of c. 108 cells mL−1. For survival assays under different stress conditions, we used osmotic challenge (2.4 M NaCl in ASW), heat challenge (45 °C in ASW), UV-irradiation (wavelength 254 nm), H2O2 (5 mM H2O2 in ASW) and starvation challenge (4 °C in ASW). In each experiment, 0.1-mL aliquots of the bacterial cultures were removed and diluted to determine the viable counts at indicated time points by spreading on 2216E plate in duplicate. For starvation challenge assays, the cells were incubated in ASW at 4 °C for 40 days, and the viable counts were determined as described above. All experiments were performed in duplicate and repeated at least once.

Virulence assays

To investigate virulence retention of the rpoS mutant, 180 zebra fish were bought from a fish market in Qingdao in China and randomly divided into nine groups, each comprising 20 fish. The fish were kept in 2-L glass tanks containing 1 L aerated water at 20–25 °C and fed with commercial pellets. The groups were injected intraperitoneally with 20 μL of the wild-type cells of V. anguillarum (103, 104, 105 and 106 CFU mL−1), the mutant cells (104, 105, 106 and 107 CFU mL−1) and autoclaved saline, respectively. The inoculated fish were then kept at 20–25 °C for 30 days and the mortality was recorded. Lethal dose 50% (LD50) was calculated by the method of Reed & Munench (1938).

Statistical analysis

Data were expressed as mean ± SD. The χ2 test was used to assess the differences in survival analysis under environmental stresses and productions of extracellular enzymes between the rpoS mutant and wild-type strains of V. anguillarum. A P-value of <0.01 or <0.05 was taken to indicate statistically distinct significance or significance, respectively.

Results

Construction of the rpoS mutant in V. anguillarum

A 1002-bp fragment was amplified from the genomic DNA of V. anguillarum W-1 by PCR and was cloned into pUCm-T. The fragment contained a 999-bp ORF, coding for a predicted 333-amino acid protein (accession number FJ599688). The encoded protein showed 93%, 86%, 85%, 85%, 82% and 73% identity with the corresponding amino acid sequence of rpoS in Vibrio parahaemolyticus (ZP01991880), Vibrio cholerae (NP230185), Vibrio harveyi (ZP01988417), Vibrio alginolyticus (ZP01262007), Vibrio vulnificus (NP760484) and E. coli (YP542094), respectively.

A 486-bp fragment (designated ΔrpoS gene) was amplified from the plasmid (pR1) containing the rpoS gene of V. anguillarum and cloned into plasmid pUCm-T (yielding pR2). It was then subcloned into the corresponding sites of the suicide vector pNQ705 (yielding pR3). The plasmid pR3 was transferred into E. coli S17-1. An rpoS mutant was constructed by integration of the suicide plasmid containing the 486-bp fragment of rpoS gene into the chromosomal rpoS gene of V. anguillarum W-1. The mutant strain was selected by growth on TCBS agar plate containing chloromycetin and identified by sequence analysis of the PCR-amplified fragment, which containing three sections: partial sequence of the rpoS gene, the ΔrpoS gene and the partial sequence of the pNQ705.

Survival of the rpoS mutant under environmental stresses

The response of the rpoS mutant to environmental stresses was examined by determining the viable plate counts after the samples of rpoS mutant were exposed to various stresses. The sensitivity of the rpoS mutant to osmotic stress (2.4 M NaCl) did not change obviously (P>0.05) (Fig. 1a). The rpoS mutant was more sensitive to oxidative stress than the wild-type counterparts, and the survival of the rpoS mutant was determined to be 10-fold less than that of the wild type when they were incubated in nutrient-free ASW containing 5 mM of H2O2 for 60 min (P<0.05) (Fig. 1b). The sensitivity of the rpoS mutant to heat shock was also increased when the stationary-phase cells were incubated at 45 °C for 45 min; the survival of the rpoS mutant was c. 33-fold less than that of the wild type (P<0.05) (Fig. 1c). The resistance of the rpoS mutant to UV-irradiation for 60 min was estimated to be almost sixfold less than that of the wild-type counterparts (P<0.05) (Fig. 1d). The response to prolonged starvation at low temperature did not show an obvious change after a 40-day period of starvation at 4 °C in ASW; the direct viable counts of the rpoS mutant declined from 5.73 × 106 to 2.20 × 104 cells mL−1, whereas the wild-type strain declined from 5.00 × 107 to 5.30 × 104 cells mL−1 (P>0.05) (Fig. 1e).

1

Survival of the rpoS mutant and wild-type cells of Vibrio anguillarum to different stresses. The survival rates were determined as the percentage of viable plate counts at the indicated time points. Bar limits represent SD. *Significant differences (P<0.05) between the values of the rpoS mutant and wild-type cells of V. anguillarum. ▲, the wild-type strain; ▪, the mutant strain. (a) 2.4 M NaCl, (b) 5 mM of H2O2, (c) 45°C in ASW, (d) UV-irradiation, (e) starvation (4°C in ASW).

Influence of rpoS mutation on the production of extracellular enzymes

The rpoS mutant and the wild-type cells of V. anguillarum were spotted on 2216E agar plates containing various substrates to determine the production of extracellular enzymes. Clearing zones were observed on the agar plates for phospholipase, lipase, diastase, caseinase and hemolytic activity, but the enzyme activities of the rpoS mutant decreased to 35.71% (P<0.01), 40% (P<0.05), 66.70% (P<0.05), 38.03% (P<0.05) and 16% (P<0.05) when compared with those of the wild-type strain, respectively (Table 2).

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The extracellular enzyme activities of the mutant and wild-type cells of Vibrio anguillarum

Extracellular enzymesWild-type cellsMutant cellsRemaining activities (%)
Phospholipase (width of zero−width of colony) (mm)7.0 ± 1.22.5 ± 0.7**35.71
Lipase (width of zero−width of colony) (mm)10.0 ± 0.74.0 ± 1.0*40.00
Diastase (width of zero−width of colony) (mm)12.0 ± 1.68.0 ± 1.1*66.70
Caseinase (width of zero−width of colony) (mm)7.1 ± 0.92.7 ± 0.3*38.03
Hemolysin (width of zero−width of colony) (mm)12.5 ± 3.22.0 ± 1.0*16.00
Catalase (U mL−1)112 ± 9.636 ± 3.7**32.14
Protease (U mL−1)309 ± 31.9232 ± 26.1*75.15
  • Remaining activity was represented as the percent of enzyme activity of the mutant cells compared with that of the wild-type cells.

  • The χ2 test was used to assess the differences in productions of extracellular enzymes between the rpoS mutant and wild-type strains of Vibrio anguillarum.

  • ** P<0.01 was taken to indicate statistically distinct significance.

  • * P<0.05 was taken to indicate statistical significance.

The cell culture supernatant of wild-type and mutant cells was also collected to determine the production of extracellular catalase and protease. The catalase activity of the rpoS mutant decreased to 32.14% compared with that of the wild-type strain (P<0.01). The protease activity of the mutant cells decreased to 75.15% when determined with azocasein as the substrate (P<0.01) (Table 2).

Virulence of the mutant

To investigate virulence retention of the rpoS mutant, zebra fish were intraperitoneally injected with different amounts of the wild-type and the rpoS mutant cells of V. anguillarum. Some of the inoculated fish died within 30 days. LD50 of the wild type and the rpoS mutant were 8.66 × 104 and 2.55 × 106 CFU per fish, respectively (Table 3). Culturable cells of V. anguillarum could be isolated from the ascites fluid. The bacterium was identified by PCR amplification and analysis of the 16S rRNA gene. The fish inoculated with autoclaved saline remained alive during the experimental time, and no V. anguillarum cells were isolated from these animals.

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Virulence to zebra fish of the mutant and the wild-type cells of Vibrio anguillarum

GroupsDose (CFU per fish)No. of fishNo. of dead fishMortality (%)LD50 (CFU per fish)
Wild-type cells3.70 × 1062020100.008.66 × 104
3.70 × 105201575.00
3.70 × 10420735.00
3.70 × 1032000.00
Mutant cells3.88 × 1072020100.002.55 × 106
3.88 × 106201155.00
3.88 × 10520315.00
3.88 × 1042000.00
Sterile saline20 μL2000.00

Discussion

RpoS has been identified as a central regulator during the stationary phase in response to environmental stresses, but its roles in regulating expression of genes varied in bacterial species. The rpoS mutation of V. anguillarum led to decreased survival rates, responding to a variety of stresses, including hyperosmotic stress, H2O2, heat and UV-irradiation, in agreement with that reported in E. coli and V. cholerae (Yildiz & Schoolnik, 1998). The rpoS of E. coli was reported to be responsible for coordinating the expression of more than 200 genes during the stationary phase, which are involved in survival during conditions of famine, oxidative stress, osmotic shock and low pH (Lange & Hengge-Aronis, 1991; Patten et al., 2004; Vijayakumar et al., 2004). The V. cholerae rpoS was found to positively regulate expression of at least 25 different genes for survival under a variety of stressful situations upon entry into the stationary phase (Yildiz & Schoolnik, 1998). Pseudomonas aeruginosa rpoS mutant was more sensitive to carbon starvation, H2O2, heat, hyperosmotic stress, acid pH and ethanol (Jørgensen et al., 1999; Suh et al., 1999). The rpoS in V. harveyi did not appear to have a role in survival following oxidative or hyperosmotic challenge, but it did function in survival after ethanol stress, persisting throughout the stationary phase of growth (Lin et al., 2002).

However, rpoS mutation of V. anguillarum did not seem to affect survival in prolonged carbon starvation. The direct viable counts of the rpoS mutant strain declined to the same extent as that of the wild-type strain after a 40-day period of starvation in sterilized nutrition-free ASW microcosms at 4 °C. Both strains were able to survive carbon starvation at 4 °C for a long time. This result is in contrast to results reported in E. coli, V. cholerae and other species. The viable counts of Pseudomonas putida KT2440 did not change significantly after 3–4 weeks, but the viable count of an rpoS-deficient strain was reduced about 100-fold after 2 weeks of carbon starvation (Ramos-González & Molin, 1998). Most bacterial genomes encode multiple σ factors. Escherichia coli contained several alternative factors such as σ70, σ32, σ54N), σE, σF, σfecl and σS; by binding to RNA polymerase, they regulate expression of housekeeping genes including heat shock, starvation, stationary-phase adaptive response and nitrogen-regulated genes. Carbon starvation protection in P. putida was regulated by both rpoS and rpoN (Kim et al., 1995; Ramos-González & Molin, 1998); the latter is a σ factor regulating nitrogen metabolism in several bacteria. Our result in V. anguillarum suggested that alternative systems may be available to protect against carbon starvation in V. anguillarum.

RpoS was reported to control the expression of various virulence genes. RpoS of V. vulnificus controlled the expression of elastase, exoproteases (Jeong et al., 2001; Hülsmann et al., 2003) and fur gene of virulence-associated iron-uptake systems (Litwin & Quackenbush, 2001). In V. alginolyticus, RpoS regulated the expression of albuminase, collagenase, gelatinase and caseinase activities (Tian et al., 2008). The rpoS mutant of V. anguillarum exhibited decreased production of catalase, phospholipase, diastase, lipase and hemolysis activity compared with the wild-type cells. The catalase activity decreased threefold in the rpoS mutant. The rpoS mutation decreased the pathogenicity of V. anguillarum to fish, and the LD50 of the rpoS mutant was almost 29-fold higher than that of the wild-type strain, which is in agreement with reports that the rpoS mutant in S. typhimurium resulted in a 1000-fold increase in oral LD (Fang et al., 1992). In contrast, there was no significant difference of LD50 between the mutant strain and the wild-type strain in Yersinia enterocolitica (Iriarte et al., 1995). Vibrio cholerae rpoS mutants had no role in promoting virulence in mice (Yildiz & Schoolnik, 1998). It is likely that the role of rpoS in pathogenicity varies in different bacterial species.

In conclusion, RpoS has been shown to regulate expression of some environmental stress genes and virulence-related genes in V. anguillarum. The rpoS mutant decreased the ability to survive diverse environmental stresses, including exposure to H2O2, osmotic stress and high temperature. The rpoS mutation led to reduced production of hemolysin, catalase and phospholipase. The virulence of the rpoS mutant was decreased 29-fold compared with that of the wild-type strain, which indicated that the rpoS plays important roles in environmental survival and pathogenicity in V. anguillarum.

Acknowledgements

We thank Dr Debra L. Milton (Umeå University, Sweden) for providing E. coli S17-1 (λpir), SY327 (λpir) and plasmid pNQ705. This work was supported by grants of National High-Tech R&D Program (2007AA09Z416), Major State Basic Research Development Program of China (2006CB101803) and Special Non-Profit Research Projects from Ministry of Agriculture of China (nyhyzx 07-046).

Footnotes

  • Editor: Jizheng He

References

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