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Infection and Immunity, September 2008, p. 4019-4037, Vol. 76, No. 9
0019-9567/08/$08.00+0     doi:10.1128/IAI.00208-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Global Gene Expression as a Function of the Iron Status of the Bacterial Cell: Influence of Differentially Expressed Genes in the Virulence of the Human Pathogen Vibrio vulnificus{triangledown} ,{dagger}

Alejandro F. Alice, Hiroaki Naka, and Jorge H. Crosa*

Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon

Received 14 February 2008/ Returned for modification 6 April 2008/ Accepted 9 June 2008


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ABSTRACT
 
Vibrio vulnificus multiplies rapidly in host tissues under iron-overloaded conditions. To understand the effects of iron in the physiology of this pathogen, we performed a genome-wide transcriptional analysis of V. vulnificus growing at three different iron concentrations, i.e., iron-limiting [Trypticase soy broth with 1.5% NaCl (TSBS) plus ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)], low-iron (1 µg Fe/ml; TSBS), and iron-rich (38 µg Fe/ml; TSBS plus ferric ammonium citrate) concentrations. A few genes were upregulated under the last two conditions, while several genes were expressed differentially under only one of them. A gene upregulated under both conditions encodes the outer membrane porin, OmpH, while others are related to the biosynthesis of amino sugars. An ompH mutant showed sensitivity to sodium dodecyl sulfate (SDS) and polymyxin B and also had a reduced competitive index compared with the wild type in the iron-overloaded mice. Under iron-limiting conditions, two of the TonB systems involved in vulnibactin transport were induced. These genes were essential for virulence in the iron-overloaded mice inoculated subcutaneously, underscoring the importance of active iron transport in infection, even under the high-iron conditions of this animal model. Furthermore, we demonstrated that a RyhB homologue is also essential for virulence in the iron-overloaded mouse. This novel information on the role of genes induced under iron limitation in the iron-overloaded mouse model and the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions shed light on the many strategies used by this pathogen to multiply rapidly in the susceptible host.


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INTRODUCTION
 
Vibrio vulnificus is an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels (17, 43). This estuarine bacterium preferentially affects individuals with underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta thalassemia. In humans, this pathogen frequently causes fatal primary septicemia with a very rapid progress, resulting in a mortality rate of >50% within a few days (17). The common theme in susceptible individuals appears to be high serum iron levels; however, changes in the innate immune response in the host cannot be discarded (17, 19).

Iron is an important element in almost all known organisms. In many bacterial pathogens, the ability to acquire this metal from the host is an important virulence factor (7). Seminal work in Oliver's laboratory showed the importance of iron in V. vulnificus infections (74), as the laboratory identified two siderophores, vulnibactin and a hydroxamate-type siderophore, both involved in iron acquisition (59). Litwin et al. (32-34, 70) studied the role of the siderophore vulnibactin in the uptake of iron from transferrin and hemoglobin and identified genes involved in vulnibactin biosynthesis and transport as well as the heme receptor and Fur, a regulator that represses several genes under iron-limiting conditions (13). More recently, Kim et al. (23) showed the involvement of two nonribosomal peptide synthetases in the biosynthesis of vulnibactin. V. vulnificus can grow in human serum only with transferrin at high iron saturation (6); however, Kim et al. (22) showed that non-transferrin-bound iron is required for the initiation of V. vulnificus growth. It is customary to use iron-overloaded mice in virulence studies with V. vulnificus to mimic the conditions found in susceptible individuals. Mice loaded with iron become highly susceptible to V. vulnificus infection, and the 50% lethal dose (LD50) decreases dramatically, approximately 5 log, when mice are infected intraperitoneally (i.p.) (74) or subcutaneously (s.c.) (63). However, in the latter case, the progress of the disease is slower than in i.p. infections (63). Mice with or without iron overloading died when the bacterial concentration in the blood reached 105 CFU/ml or above, with iron increasing the growth rate of the bacteria, both in the animal and in vitro. Thus, the lethal concentration of bacteria can be reached faster in the iron-overloaded mouse model (19). Starks et al. (62) showed that iron increased the replication of V. vulnificus in skin, but they also observed a reduction in susceptibility of some strains to being killed by animal defenses. There is also a correlation between utilization of iron from transferrin and virulence, in which the siderophore vulnibactin appears to play a role in infections in suckling mice (34, 70) and in normal mice (23), where transferrin saturation levels are expected to be close to the normal range. However, the possible role of vulnibactin in the iron-overloaded mouse model was not evaluated directly, i.e., using specific mutants. Furthermore, the role of Fur and heme transport in the virulence of V. vulnificus in both animal models has not as yet been investigated.

In recent years, a number of virulence factors have been described for this pathogen (10, 24-29, 34, 35, 47-49, 75). Since during infections iron plays an important role in the rapid replication of V. vulnificus, in this work we present the global transcriptional analysis of this bacterium growing under various iron concentrations to elucidate the basis for the increased replication as well as the relevance of several factors in the infection in the progress of the disease.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. The strains and plasmids used in this work are listed in Table 1. Bacteria were grown routinely in Trypticase soy broth with the addition of 1.5% NaCl (TSBS) (V. vulnificus) or in LB broth (Escherichia coli) supplemented with the following antibiotics, as appropriate: ampicillin (500 µg/ml) and chloramphenicol (2 µg/ml) for V. vulnificus and ampicillin (100 µg/ml), chloramphenicol (30 µg/ml), and kanamycin (50 µg/ml) for E. coli. In some experiments, CM9 was used as a minimal medium (11). When needed, 1.5% (wt/vol) agar was added. Thiosulfate-citrate-bile salts-sucrose agar (TCBS) was used as selective medium for V. vulnificus in the conjugations. For iron-limiting conditions, ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA) was added at the indicated concentrations. Iron-rich conditions were obtained by the addition of ferric ammonium citrate (FAC).


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  		TABLE 1. Strains and plasmids used in this work

Bioassays to determine utilization of iron sources. Bacteria were grown overnight in TSBS with the appropriate antibiotics and diluted 1/400 in TSAS (Trypticase soy agar with the addition of 1.5% NaCl) containing 250 µM or 500 µM EDDA. In these experiments, bacterial cells were included in the agar, and upon solidification, plates were spot inoculated with the different iron sources (see Table S2 in the supplemental material). After incubation of the plates at 37°C, halos of bacterial growth surrounding the locations of the spots indicated positive results. In these experiments, FAC, which does not require active transport, was included to confirm that the strain inoculated in the agar was viable. Vulnibactin was purified as described by Simpson and Oliver (59), using supernatants of V. vulnificus cells growing in Chelex 100 (Bio-Rad, Hercules, CA)-treated CM9. The bacterial growth around each spot was monitored for 24 to 48 h after inoculation.

DNA manipulations and sequence analysis. Plasmids were prepared using a Qiaprep miniprep kit (Qiagen, Valencia, CA). Touchdown PCRs were performed with either Taq polymerase (Invitrogen, Carlsbad, CA) or Pfu Taq polymerase (Stratagene, La Jolla, CA), using the following conditions: 95°C for 4 min; 30 cycles of 95°C for 30 s, 63°C for 1 min (the temperature of this step was lowered 0.3°C for each cycle), and 72°C for 1 min; and a final extension step of 72°C for 10 min. Digestions and ligations were performed according to the manufacturer's instructions (New England Biolabs Inc., Ipswich, MA). DNA sequencing reactions were carried out by the Oregon Health and Science University (OHSU) Molecular Microbiology and Immunology Research Core Facility, using a model 377 Applied Biosystems automated fluorescence sequencer. Sequence similarities were analyzed with BLAST (2).

Construction and complementation of V. vulnificus mutants. In-frame deletions of the entire coding sequences of genes were generated using splicing by overlap extension PCR (57). Upstream and downstream regions (approximately 700 bp to 800 bp) flanking each gene were amplified with specific primers, and both fragments were mixed and ligated in a new PCR mixture with primers 1 and 2 for each gene (see Table S1 in the supplemental material). The amplified fragment was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), digested with appropriate restriction enzymes, and subcloned into the suicide vector pDM4, which was previously digested with the same restriction enzymes. E. coli S17-1{lambda}pir transformed with the pDM4 derivatives was conjugated with V. vulnificus, and exconjugants were selected on TCBS agar with chloramphenicol. For the excision of the suicide vector, clones were incubated in the absence of chloramphenicol and plated on TSAS plates containing 15% sucrose. Those colonies that grew on these plates were screened for chloramphenicol sensitivity, and deletions within the genes of interest were confirmed by PCR. For the construction of the fur and ompH disruptant mutants, an internal fragment of each gene was amplified and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), digested with appropriate restriction enzymes, and subcloned into the suicide vector pDM4 as described above. E. coli S17-1{lambda}pir carrying pDM4 derivatives was conjugated into V. vulnificus, and chloramphenicol-resistant exconjugants were selected on TCBS agar with the appropriate antibiotic. When needed, fur::pDM4 and ompH::pDM4 mutants were then grown in the presence of sucrose, and chloramphenicol-sensitive clones were checked for restoration of the wild-type gene by PCR, as described previously (35, 46).

For the complementation experiments, each gene was amplified by PCR with primers containing restriction sites, as indicated in Table S1 in the supplemental material. The fragments were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), sequenced, and then subcloned into pMMB208 (42) under the control of the Ptac promoter, which is under the control of the lacI gene harbored in the vector. The constructs were transferred to V. vulnificus strains by triparental conjugation, using the plasmid helper pRK2013 (14). In order to induce transcription of the cloned genes, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the solid and/or broth medium. For the complementation of the {Delta}ryhB mutant with a wild-type copy of the gene in the lacZ gene locus, the ryhB gene was amplified with primers RyhBSalF and RyhBSalR, possessing SalI restriction sites, and cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA). A deletion of the lacZ gene from V. vulnificus was constructed in the pCR2.1 vector (Invitrogen, Carlsbad, CA) by using splicing by overlap extension as described above. Since primers LacZ3 and LacZ4 have SalI sites, the resulting plasmid was digested with SalI and ligated to a fragment containing the ryhB gene previously digested with the same restriction enzyme. The plasmid thus constructed, pLACRYHB, was digested with SpeI and XbaI, and the fragment of interest was isolated and cloned into pDM4 previously digested with SpeI. The pDMLR plasmid was conjugated into the {Delta}ryhB mutant, and exconjugants were further grown in the absence of chloramphenicol and inoculated on TSAS plates containing 15% sucrose. Colonies obtained on those plates were screened for chloramphenicol sensitivity as described above. Positive clones were then analyzed by PCR to confirm the correct location of the ryhB gene in the lacZ locus.

RNA extractions. Strains were grown to an optical density at 600 nm (OD600) of 0.6 to 0.8 (mid-log phase) in TSBS, TSBS with the addition of 250 µg/ml FAC, and TSBS with the addition of 50 µM EDDA. For the microarray analysis, three independent cultures of the cells grown in each medium were combined and treated as a single sample for the RNA extraction to minimize culture variation. Two samples per condition were used for the microarray analysis. Cells were centrifuged, and the pellets were resuspended in RNAWiz reagent (Ambion, Austin, TX). Total RNA was extracted from each strain according to the manufacturer's instructions. The quality of the RNA was assessed on an Agilent Bioanalyzer 2100 electrophoretic system, and 20 µg was used in the synthesis of double-stranded cDNA. The primer employed for cDNA first-strand synthesis incorporates a 5' T7 promoter sequence that was used to synthesize biotinylated cRNA from the double-stranded cDNA, using a Megascript T7 kit (Ambion, Austin, TX). Following quantification, the cRNA was fragmented with metals and heat and was hybridized to a V. vulnificus CMCP6 NimbleGen array. When RNAs were extracted from cells growing in human serum, an overnight culture was diluted 1/100 in human serum containing 250 µg/ml FAC. Samples were taken after 30 min, 4 h, and 6 h, and RNAs were extracted as described above.

Microarray, hybridization, and analysis. Microarray chips derived from the V. vulnificus CMCP6 genome were prepared by NimbleGen. After hybridization, the array was stained with fluorophore Cy3-streptavidin (Amersham) and scanned with an Axon/API scanner, and the data were extracted with NimbleScan software. By analysis of the genome sequence and annotation, 4,514 targets were identified over the two chromosomes of V. vulnificus CMCP6. Each target is represented on the array by 14 24-mer oligonucleotide pairs/gene. Each pair consists of a sequence perfectly matched to the open reading frame and another adjacent sequence with two mismatched bases, used for background and cross-hybridization determination. Each probe was replicated three times. The oligonucleotide pairs were synthesized on the chip by use of a Maskless array synthesizer developed by NimbleGen. The chips containing the entire V. vulnificus CMCP6 genome were used in the hybridization experiments with the synthesized cRNA. Microarrays were scanned with an Axon Genepix 4000B scanner at 532 nm, with a resolution of 5 µm. Further details about the design of the chips, labeling, hybridization, scanning, and analysis of the samples are available from NimbleGen. The data obtained were further processed using some tools available through the Bioconductor project (http://www.bioconductor.org), using quantile normalization (5) and gene calls generated using the RMA algorithm (20), based on log2 values. We subsequently analyzed the normalized data with GeneSpring 7.0 (Agilent Technologies, Palo Alto, CA) to identify differential gene expression in experimental and control samples. Genes with a >2-fold difference of expression between conditions and a P value of <0.005 were further analyzed and are described here. Adjustment for multiple comparisons was done using the software program q value (65), and after adjustment, the level of statistical significance was set at 0.005, as described previously (66). The Institute for Genomic Research Comprehensive Microbial Resource was consulted for functional classification of each open reading frame (50), as well as the database for annotation, visualization and integrated discovery (DAVID) (12).

RPA. Internal fragments of analyzed genes were amplified by PCR, using the primers described in Table S1 in the supplemental material. In all cases analyzed in this report, probe lengths were between 150 nucleotides (nt) and 200 nt, while the probe for the loading control was 220 nt. Reverse primers were designed to contain the recognition sequence for the T7 polymerase. RNA probes were synthesized and labeled with [{alpha}-32P]UTP by using a MAXIscript SP6/T7 in vitro transcription kit (Ambion, Austin, TX). RNase protection assays (RPAs) were conducted using an RPAIII kit (Ambion, Austin, TX) according to the manufacturer's instructions. Briefly, RNAs were incubated with the indicated probes overnight at 42°C, treated with a mixture of RNase A and RNase T1 for 30 min at 37°C, precipitated, and resuspended in gel loading buffer II (Ambion, Austin, TX). The labeled RNAs were electrophoresed in urea-6% polyacrylamide gels that were then exposed for 4 to 24 h. In all experiments described in this report, the VV13021 gene, encoding a putative protein tyrosine phosphatase, was used as an internal control for loading since the transcription of this gene was similar under all the conditions analyzed.

RT-PCR. RNAs were extracted as described above and treated with DNase Turbo DNA-free (Ambion, Austin, TX) at 37°C for 1 h. cDNA synthesis was performed using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, using the indicated primers. PCRs were performed using 2 µl of each reverse transcription (RT) reaction mix. PCR parameters were as described above, and a control without reverse transcriptase enzyme in the RT reaction mix was used for each PCR. Fragments were resolved by electrophoresis on agarose gels.

RLM-RACE. RNA ligase-mediated rapid amplification of cDNA ends (RML-RACE) can distinguish between primary 5' ends and those generated by processing (3). We used this method as described previously (3), with modifications. RNAs from wild-type CMCP6 were extracted and treated with DNase Turbo DNA-free (Ambion, Austin, TX) as described above. The DNA-free RNA was purified using RNeasy spin columns (Qiagen, Valencia, CA), and 10 µg was used in subsequent reactions. RNAs were treated with tobacco acid phosphatase (TAP), and ligation of 5' RNA adapters to the 5'-terminal ends was carried out using a FirstChoice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer's instructions. The method is based on the fact that primary transcripts in bacteria carry a 5'-triphosphate, which is hydrolyzed by TAP specifically between the {alpha}- and β-phosphate groups. TAP-untreated control reactions were performed, replacing the TAP with nuclease-free water. RT was carried out with primers 42R for VV10842 and RYHBREV-2 for ryhB, using 2 µl of the adapter-ligated RNA and SuperScript II (Invitrogen, Carlsbad, CA) reverse transcriptase in a final volume of 20 µl. The cDNAs thus obtained were subsequently used as a template in a PCR with an adapter-specific 5'-RACE outer primer (Ambion, Austin, TX) and a gene-specific primer (42BAM for VV10842 and RYF for ryhB). PCRs were performed as described above, and the products were analyzed in 2% agarose gels. Fragments obtained exclusively in the TAP-treated samples (5' ends resulting from transcription initiation) compared with the TAP-untreated samples (5' ends resulting from initiation and processing) were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA), cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), and sequenced.

Overexpression and purification of the V. vulnificus Fur(His)6 protein. The DNA fragment coding for Fur was amplified using primers FurVVF and FurVVR and cloned into the expression vector pET200 (Invitrogen, Carlsbad, CA). A recombinant plasmid, pETFUR, with the correct sequence was used for expression of the Fur(His)6 protein in E. coli BL21(DE3) Star. One hundred milliliters of LB supplemented with kanamycin was inoculated with an overnight culture of the strain harboring the pETFUR plasmid, and when the OD600 reached 0.6, 1 mM IPTG was added to induced the expression of the Fur(His)6 protein. The culture was incubated for another 4 h at 37°C, centrifuged, washed, and resuspended in lysis buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cells were sonicated and centrifuged, and the supernatant was passed through a Ni-nitrilotriacetic acid resin (Qiagen, Valencia, CA) according to the manufacturer's instructions. Elution from the column was carried out using lysis buffer with the addition of 250 mM imidazole. After elution, the protein was dialyzed overnight at 4°C against buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 2 mM dithiothreitol, 10% glycerol), and the protein concentration was determined with a bicinchoninic acid assay kit (Pierce, Rockford, IL). The protein was aliquoted and stored at –80°C.

Gel mobility shift assay. The gel mobility shift assay was performed using a second-generation digoxigenin gel shift kit (Roche, Indianapolis, IN). DNA fragments were amplified by PCR and 3' end labeled with digoxigenin-11-ddUTP, using terminal transferase as described by the manufacturer. After the labeling efficiency was determined, each of the labeled probes (4 nM) was incubated with increasing amounts of the Fur(His)6 protein in binding buffer [10 mM bis-Tris-borate, pH 7.5, 100 µg/ml bovine serum albumin, 5% (vol/vol) glycerol, 40 mM KCl, 1 mM MgCl2, 100 µM MnCl2, 1 µg poly(dI-dC)] and incubated for 15 min at room temperature. For competition experiments, 4 nM of the labeled probe was incubated with 100 nM of Fur(His)6 in the presence of increasing amounts of the unlabeled specific probe. Samples were separated in 5% polyacrylamide gels polymerized in the presence of 20 mM bis-Tris-borate, pH 7.5, and 100 µM MnCl2, as described previously (8), and run in the same buffer for 90 min at 110 V. The DNA-protein complex was transferred to a positively charged nylon Hybond N+ membrane (GE Healthcare, Piscataway, NJ), and the chemiluminescent signal was detected according to the manufacturer's instructions (Roche, Indianapolis, IN).

β-Galactosidase assays. The upstream regions of the VV10842 (TonB3 cluster), VV21614 (TonB1 cluster), and VV20364 (TonB2 cluster) genes were amplified with primers 42BAM-42PST, T1SAL-T1XBA, and TB2XBA2-TB2XHO2, respectively, cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), sequenced, and subcloned into the pTL61T vector previously digested with the corresponding restriction enzymes, generating the plasmids p42TL, pT1TL, and pT2TL, respectively. The empty vector and plasmids thus constructed were transferred to the V. vulnificus {Delta}lacZ wild-type strain by triparental conjugation. Bacterial strains harboring the plasmids described above or the empty vector were grown under the conditions shown in Fig. 4. One hundred microliters of each culture was centrifuged, resuspended in the same volume of buffer Z, and used in the assay. β-Galactosidase activities were determined as described previously (39). Due to the turbidity of the human serum used in this work, when samples obtained from this source were analyzed, the CFU/ml counts at each time point were used instead of the OD600 values to normalize the β-galactosidase activities.


Figure 4
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  		FIG. 4. Expression of the TonB1, TonB2, and TonB3 clusters of genes in human serum. The promoter region of each cluster was cloned in front of the promoterless lacZ gene in the pTL61T vector, and plasmids were conjugated into the {Delta}lacZ strain. (A) Cells were grown in human serum (HS), human serum plus FAC at 2 µg/ml (HS + 2), human serum plus FAC at 20 µg/ml (HS + 20), and human serum plus FAC at 200 µg/ml (HS + 200), and samples were taken at different time points to determine β-galactosidase activities. Due to the turbidity of the human serum, OD600 values were not recorded and activities were normalized to 106 CFU/ml. (B) Cells harboring the VV10842-lacZ fusion were grown in TSBS to an OD600 of ~0.6 and inoculated in TSBS or human serum (HS), and samples were taken at different time points (T0, 0 h; T1, 1 h; and T3, 3 h) to determine β-galactosidase activities. Enzymatic activities were determined as described for panel A. The results shown in panels A and B are the means for at least three independent experiments, with standard deviations.

Virulence experiments. Overnight cultures were diluted 1/100 in 10 ml TSBS and incubated for 4 h before 1 ml of the cell suspension was centrifuged, washed, and serially diluted in phosphate-buffered saline (PBS). For LD50 experiments, five 4- to 6-week-old CD-1 mice (Charles River Laboratories, Wilmington, MA) per dilution were injected i.p. or s.c. with 0.1 ml of a dilution of the strain of interest. At least three serial dilutions for each strain were evaluated. For iron overloading of the animals, 900 µg of FAC was injected i.p. 30 min prior to the bacterial challenge (24, 74). For LD50 studies, mortality was monitored at 48 h postinfection. The standard error of the LD50 was calculated by using probit analysis (15, 40, 71). The 95% confidence limits of the LD50 were determined according to the following formula: LD50 ± 1.96 x standard error of the LD50 (71).

For competitive index (CI) experiments, we first constructed a {Delta}lacZ strain and compared its CI with that of the wild type to confirm that both strains behaved similarly in the host. Experiments were performed as follows. Bacterial strains were cultured as described above, and then equal volumes were mixed and serial dilutions performed. A dilution (usually containing ca. 200 to 600 CFU) was injected s.c. into three to six mice. Animals were checked approximately 10 h after inoculation, and when they showed signs of the disease (e.g., lethargy, slow movements, and lack of appetite), they were euthanized with CO2 according to IACUC regulations. Skin samples (one square centimeter around the inoculation site), spleens, and livers were aseptically extracted and homogenized by using a Seward stomacher lab blender in the presence of PBS (1 ml for skin and spleen and 2 ml for liver). Serial dilutions were performed in PBS, and the dilutions were plated on TSAS-5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (TSAS-X-Gal) (0.004%, wt/vol) plates to determine the CFU of the strains under analysis. CI values were obtained as described previously (68). Statistical analysis was performed by using the Student t test with GraphPad Prism 4.0 software.

Determination of total serum iron, total iron-binding capacity, unsaturated iron-binding capacity, and transferrin saturation. Human serum samples from at least two healthy donors were pooled, heat inactivated, centrifuged, divided into aliquots, and stored at –80°C. Serum iron, unsaturated iron-binding capacity, total iron-binding capacity, and transferrin saturation values were obtained by using the bathophenantroline protocol according to the method of Goodwin et al. (16). In some cases, FAC was added at the concentrations indicated, and parameters were measured as described above.

SDS and polymyxin B resistance. For SDS resistance, V. vulnificus cells were grown overnight in TSBS at 37°C and diluted 1/100 in TSBS with the addition of various concentrations of SDS. TSBS without the addition of the detergent was used as a control. After 2 h of growth at 37°C with shaking (200 rpm), the OD600 was determined and the percentage of SDS resistance expressed as follows: (OD600 of sample/OD600 of TSBS sample) x 100. Polymyxin B resistance experiments were conducted as described by Chen et al. (10), with modifications. V. vulnificus cells were grown in TSBS, harvested at the mid-log phase of growth, centrifuged, and resuspended in TSBS, with or without the addition of polymyxin B (20 µg/ml). Cells were incubated and CFU monitored from samples taken at the specified times. The percentage of survival was expressed as follows: (CFU/ml of sample/CFU/ml of TSBS sample) x 100.

Microarray data accession number. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) under GEO Series accession number GSE10633.


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RESULTS
 
Analysis of global gene expression of V. vulnificus growing at various iron concentrations. Since one of the hallmarks of infections by V. vulnificus is the high iron concentration encountered by the bacterium in susceptible patients, we initiated our analysis by comparing the transcriptomes of this bacterium growing at various iron concentrations, i.e., under iron-limiting conditions (TSBS plus 50 µM EDDA), low-iron conditions (1 µg Fe/ml; TSBS), and iron-rich conditions (38 µg Fe/ml; TSBS plus FAC at 250 µg/ml). The iron concentrations of at least two different batches of TSBS were determined by inductively coupled plasma mass spectroscopy. From the results of these experiments, we classified the genes with altered expression into the following three main groups: those induced (i) under iron-limiting conditions, (ii) under both iron-rich and low-iron conditions, and (iii) under either iron-rich or low-iron conditions.

Genes belonging to group i. (i) The tonB systems. One hundred twenty-eight genes were specifically induced under iron-limiting conditions compared with those in the samples obtained from cells growing in TSBS and TSBS plus FAC. Genes grouped in this category belong to some very well-described determinants involved in iron acquisition in this and other pathogens. The cluster of genes that includes those involved in vulnibactin biosynthesis and transport was highly induced, as were the two clusters of genes coding for proteins belonging to the TonB1 (VV21614) and TonB2 (VV20360) complexes (Table 2), which are involved in energy transduction from the inner membrane to the siderophore receptor present in the outer membrane. It is of interest that a third cluster with genes that show similarity to those in another TonB cluster (VV10842 to VV10848; named TonB3 here) did not show any change in transcription at the iron concentrations analyzed. Expression of the three tonB genes was also analyzed by using RPA experiments. We detected increases in the levels of specific mRNAs from the tonB1 and tonB2 genes under iron-limiting conditions, confirming the results obtained in the microarray analysis (Fig. 1A and B, compare lanes TE with lanes T and TF). Furthermore, both the tonB1 and tonB2 clusters are under the control of Fur, as determined by RPA (Fig. 1A and B, compare lanes T and TF for the wild type with lane TF for the fur::pDM4 mutant). For some unknown reason, the expression of the tonB1 gene in the fur::pDM4 mutant in TSBS (low iron) was lower than that in TSBS plus FAC (iron-rich conditions). In agreement with the microarray results, we did not detect tonB3 transcripts under either of those conditions (Fig. 1C). Purified Fur(His)6 can bind to the promoter region of each cluster, as determined by gel shift experiments ( Fig. 2A and B). We then analyzed the specificities of the three TonB systems, using iron source utilization bioassays, as well as their role in the virulence of V. vulnificus, using LD50 and CI experiments. It was also observed in Vibrio cholerae (56) and Vibrio anguillarum (67) that the tonB1 and tonB2 systems have redundant functions in the transport of some iron compounds (heme, vulnibactin, and iron from transferrin and hemoglobin) (see Table S2 in the supplemental material), as detected using iron utilization bioassays. When the virulence of the corresponding mutants was analyzed by determining the LD50 in both animal models, i.e., those with normal iron levels and those overloaded with iron, we found that a double mutation ({Delta}tonB1 {Delta}tonB2) was needed to obtain a significant increase in the LD50 value compared with that of the wild type in the normal mouse model (Table 3). In the case of the iron-overloaded mouse model, there was a smaller change in the LD50s of the double {Delta}tonB1 {Delta}tonB2 and triple {Delta}tonB1 {Delta}tonB2 {Delta}tonB3 mutants, but the death kinetics were significantly different from that of the wild type (see Fig. S3 in the supplemental material), suggesting a role for these systems in infection even under iron-rich conditions in the animal. The experiments described above were conducted using i.p. injections, by which V. vulnificus can reach the bloodstream very quickly. Starks et al. (63) have shown that s.c. injections are not only more sensitive for the analysis of virulence in this bacterium but also resemble more closely the route of infection observed in wounds. Thus, we used the s.c. route with some of the mutants described above, observing an important change in the LD50 values only for the double {Delta}tonB1 {Delta}tonB2 and triple tonB mutants in the iron-overloaded mouse model (Table 3). These results are in agreement with the in vitro bioassays described above, showing the relevance and redundancy of the TonB1 and TonB2 systems in iron transport. We also performed CI experiments between a {Delta}lacZ strain and a triple tonB {Delta}venB mutant. Since the venB gene is involved in vulnibactin biosynthesis (34), we included the venB mutation in this strain to avoid the cross-feeding of the wild type with vulnibactin produced by the mutant, since it has been reported that cross-feeding by a siderophore can occur in vivo inside the host (73). As shown in Fig. 3, the wild-type strain outcompeted the quadruple mutant, even in skin (P < 0.0001 for all organs analyzed), underscoring the relevance of the tonB systems for the growth and/or survival of V. vulnificus in the first steps of the infection.


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  		TABLE 2. Selected genes induced under iron-limiting conditions


Figure 1
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  		FIG. 1. RPA of tonB genes. RNA samples were extracted from the wild-type strain growing under the following conditions: TSBS plus EDDA (TE), TSBS (T), TSBS plus FAC (TF), and human serum with the addition of FAC at 250 µg/ml after 30 min (HS30), 4 h (HS4), and 6 h (HS6) of incubation. RNA samples extracted from fur::pDM4 cells growing in TSBS plus EDDA and TSBS plus FAC are also shown. Arrows indicate the positions of the internal control probe (VV13021) in lanes 1 and the corresponding tonB gene probe without RNase treatment in lanes 2. (A) tonB1; (B) tonB2; (C) tonB3.


Figure 2
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  		FIG. 2. Gel shift of the promoter regions of TonB1, TonB2, and TAD-1 with Fur(His)6. Promoter regions located upstream of the first gene in each cluster were amplified and labeled as described in Materials and Methods and then incubated with increasing concentrations of Fur(His)6 as shown in each panel. (A) TonB1 promoter; (B) TonB2 promoter; (C) TAD-1 promoter. Indicated are free labeled DNA and Fur(His)6-DNA complexes. The rightmost lane in each panel shows the competition between labeled DNA and unlabeled DNA (500 nM).


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  		TABLE 3. Virulence of V. vulnificus strains in various animal models


Figure 3
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  		FIG. 3. CI for iron transport mutant strains. Mixtures of the strains analyzed (0.1 ml) were injected s.c. into iron-overloaded animals. When animals showed signs of disease, organs were removed and homogenized and bacterial cells were plated on TSAS-X-Gal plates. Bars represent the geometric mean CI value for each organ. Experiments were performed at least twice. ***, P < 0.0001.

We then explored the importance of vulnibactin and heme uptake in the iron-overloaded mouse model by using s.c. inoculations, since the virulence of the venB mutant was previously determined intragastrically in mice with normal iron levels (34). In the iron-overloaded mouse, the {Delta}venB mutant showed a similar increase in the LD50 to that observed for the tonB mutant (Table 3); however, the {Delta}hupA (heme receptor) mutant showed an LD50 value similar to that of the wild type (Table 3). It is important that this is the only heme receptor in V. vulnificus (32). These results support the idea that vulnibactin is the major iron chelator during infections, even in the iron-overloaded mouse model. The {Delta}venB mutation did not show any significant change in the LD50 value for i.p. infections with the same animal model (Table 3), underscoring the importance of the route of inoculation when this virulence factor is evaluated.

Although the TonB3 and TonB2 proteins share similarities (49% similarity and 27% identity), the gene arrangements of the two clusters are different. In the TonB3 cluster of genes, the first gene (VV10842) codes for a putative outer membrane receptor that is not present in the TonB2 clusters present in V. vulnificus (VV20360) and V. cholerae (56). Mutants in the VV10842 gene as well as in tonB3 did not show any phenotype in bioassays (see Table S2 in the supplemental material), indicating that under iron-limiting conditions the tonB3 system is not involved in the transport of any iron-related product analyzed, which is in accordance with the absence of transcription of this cluster under those conditions (Fig. 1C). To address the conditions under which tonB3 could be expressed, we constructed a fusion between the putative promoter region of the cluster located upstream of VV10842 and the promoterless lacZ gene present in the pTL61T vector. The plasmid thus obtained was conjugated into the {Delta}lacZ strain, and β-galactosidase activity was analyzed under different growth conditions. When FAC, holo-transferrin, apo-transferrin, ferritin, heme, nickel, and zinc were tested as possible inducers, we did not observe increases in β-galactosidase production, suggesting that none of these compounds was responsible for the activation of this cluster (see Fig. S4A in the supplemental material). Recently, it was described for Neisseria gonorrhoeae that an outer membrane protein that belongs to the TonB-dependent transporters was induced when the bacterium was grown in the presence of host cells or bovine serum (18). Since V. vulnificus can grow in human serum with high levels of transferrin saturation (6) (see Fig. S4B in the supplemental material), we tested the strain carrying the lacZ fusion by growing the cells in TSBS and then transferring them to human serum, with and without the addition of FAC. Interestingly, we found that the TonB3 cluster was specifically induced when V. vulnificus was grown in human serum with the addition of at least 2 µg/ml of FAC. The expression observed was more prominent at higher iron concentrations (Fig. 4A) and when the cells were reaching late log phase or were already at stationary phase. When we performed the same experiment and included the lacZ fusions of the tonB1 and tonB2 promoter regions, we observed that these two clusters were induced when the cells were transferred from TSBS to human serum with the addition of just 2 µg/ml of FAC or without the addition of any iron (approximately 25 to 30% transferrin saturation) (Fig. 4A). These results indicate that at 2 µg/ml FAC, the two TonB systems involved in heme and vulnibactin transport are expressed, while when FAC is added at concentrations of 20 µg/ml and 200 µg/ml, they are repressed (possibly due to the existence of non-transferrin-bound iron).

With respect to the TonB3 system, the microarray results demonstrated that it is not induced at high iron concentrations. However, the gene is indeed induced in human serum when iron is added to allow growth. The question is whether this induction is due to the iron (or iron bound to any protein) or to another serum component. Since we also observed that holo-transferrin was not the inducer (see Fig. S4A in the supplemental material), we grew the cells carrying the VV10842-lacZ fusion in TSBS until an OD600 of ~0.6 and then inoculated them in human serum without the addition of any iron. As shown in Fig. 4B, high β-galactosidase activities were obtained for the cells carrying the VV10842-lacZ fusion even though iron was not added. It is worth noticing that in the latter experiment, the cells grew faster and reached a higher CFU number than in the previous experiment with the same human serum. These results confirm that the induction is not triggered by the added iron but by a component present in human serum that acts when the bacteria reach late log or early stationary phase or a definite number of cells. We confirmed the results obtained with the transcriptional fusion by using RPA experiments with RNAs extracted from bacterial cells growing in human serum with the addition of 250 µg/ml FAC (Fig. 1). Furthermore, RT-PCR experiments established that the entire cluster is transcribed as an operon from a promoter located in front of the VV10842 gene. The transcriptional initiation start point was determined to be located at position –88 relative to the ATG by using RLM-RACE (data not shown).

(ii) The Tad systems. A cluster of genes induced under iron-limiting conditions has similarities to those for the tight adhesion system (Tad) described for Aggregatibacter (Actinobacillus) actinomycetemcomitans and Haemophilus ducreyi (Table 2) (51, 55, 61, 69). Recently, the existence of three Tad systems in V. vulnificus was mentioned (69), and according to our analysis, the other two clusters (named Tad-2 [VV20084 to VV20095] and Tad-3 [VV11745 to VV11758] in this report) did not show any transcriptional change as a function of the iron concentration in the medium. We extracted RNAs from wild-type cells growing under various iron concentrations and performed RPA experiments where we used the tadA gene of each locus as a probe. As can be observed in Fig. 5A and B (compare lanes TE, T, and TF for the wild-type strain), the Tad-1 cluster was the only Tad system under the control of the iron concentration of the medium. Thus, to test if the transcription was under the control of the Fur protein, we analyzed the transcription of the tadA1 gene from the Tad-1 cluster in a fur::pDM4 genetic background and compared it with that in the wild-type strain. Figure 5A shows that the tadA1 gene was expressed under iron-rich conditions in the fur::pDM4 mutant strain, demonstrating that the cluster was under the control of iron in a Fur-dependent manner. When the other two clusters were analyzed in the same genetic background, no changes in transcription were observed, leaving Tad-1 as the only Tad system under the control of Fur (Fig. 5B). We also confirmed that the entire Tad-1 cluster is transcribed as an operon, with the promoter region located upstream of a gene encoding a putative pilin protein that is located upstream of VV12329 (see Fig. S5A and B in the supplemental material). As described recently (69), this gene is not annotated in the CMCP6 genome but is annotated in the genome of V. vulnificus YJ016 (9). We further determined by using gel shift assays that Fur(His)6 can also bind to the promoter region of the Tad-1 cluster of genes, although with an apparently lower affinity than that for the TonB1 and TonB2 promoter regions (Fig. 2C). A putative Fur box binding sequence (13) was identified in this promoter region (data not shown).


Figure 5
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  		FIG. 5. RPA of tadA genes. RNA samples were extracted as described in Materials and Methods from the wild type growing in TSBS plus EDDA (TE), TSBS (T), or TSBS plus FAC (TF) or from the fur::pDM4 mutant growing in TSBS plus FAC (TF). Arrows indicate the positions of the internal control probe without the addition of RNase (VV13021) (lanes C) and the corresponding tadA gene probe (lane A-1, tadA1; lane A-2, tadA2; and lane A-3, tadA3). (A) tadA1; (B) tadA2 (left) and tadA3 (right).

We determined that the Tad-1 cluster does not play any role in the virulence of V. vulnificus by using LD50 and CI experiments with both animal models (Table 3; see Fig. S5C in the supplemental material).

(iii) Fur and RyhB. In previous sections, we reported that several genes are controlled by Fur, and hence, it was important to identify the role of Fur in the virulence of V. vulnificus. The LD50 value for the fur null mutant obtained in the iron-overloaded mice was similar to that for the wild type (Table 3), but using CI tests it was clear that the wild type outcompeted the fur::pDM4 {Delta}lacZ mutant (Fig. 6A) in the organs analyzed (P < 0.0001). A possible explanation for these results is that the fur::pDM4 {Delta}lacZ strain showed slower growth under iron-limiting conditions (Fig. 7A), and as we have shown in the previous section, iron limitation might occur in the first stages of the infection. An alternative explanation might be that this mutant was cross-feeding the wild type with some still uncharacterized compound.


Figure 6
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  		FIG. 6. CI of several mutants. Cells were grown and experiments conducted as described in the legend to Fig. 3. (A) fur::pDM4 {Delta}lacZ mutant versus wild type (n = 5); (B) {Delta}ryhB {Delta}lacZ mutant versus wild type (n = 5); (C) {Delta}ryhB lacZ::ryhB mutant versus wild type (n = 5); (D) {Delta}ryhB fur::pDM4 {Delta}lacZ mutant versus wild type (n = 5). Bars represent the geometric mean CI value for each organ. Experiments were conducted at least twice. ***, P < 0.0001.


Figure 7
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  		FIG. 7. Growth curves under iron-limiting conditions. Cells were grown overnight in TSBS and diluted in TSBS plus 50 µM EDDA, and the OD600 was recorded at various time points. The mean and the standard deviation for each time point for at least two independent experiments are shown.

Another important player in iron regulation under the control of Fur is the small RNA (sRNA) RyhB (36). This gene was previously identified in V. vulnificus by Mey et al. (38), and it is located 331 bp from the 5' end of the VV10901 gene and 264 bp from the 3' end of VV10902. We constructed a {Delta}ryhB mutant to evaluate the role of this gene in the growth of V. vulnificus at various iron concentrations and its role in virulence. It has been described previously for other bacteria that under iron-rich conditions Fur represses the transcription of ryhB, and in consequence, sodB, the target for this sRNA, can be transcribed (36, 38, 44, 72). When iron is limiting, Fur cannot repress ryhB, and as result, this sRNA can be transcribed, increasing the degradation of the sodB mRNA. By using RPAs, we confirmed that ryhB in V. vulnificus is also under the control of Fur (see Fig. S6A in the supplemental material) and determined that ryhB controls the transcription of sodB according to the iron concentration of the medium (see Fig. S6B in the supplemental material). Furthermore, in the absence of ryhB, we detected the sodB transcript under iron-limiting conditions. The {Delta}ryhB mutant was unable to grow under iron-limiting conditions (50 µM EDDA) (Fig. 7B), although we could observe growth when the amount of EDDA was lowered to 30 µM (data not shown), indicating that this sRNA plays an important role in the growth of V. vulnificus under these conditions. To complement the {Delta}ryhB mutant with the wild-type ryhB gene in a low-copy-number plasmid, we determined its transcriptional start point by using RLM-RACE as described in Materials and Methods. As shown in Fig. 7B, the growth defect was relieved in the complemented strain with the pryhB plasmid, where the ryhB gene is under the control of the Ptac promoter.

It was recently reported for E. coli that in a ryhB::cat mutant the intracellular levels of iron are lower than those in the wild type, while in a fur::kan mutant they are higher (21). Since our V. vulnificus {Delta}ryhB mutant was unable to grow under iron-limiting conditions, we tested if a double {Delta}ryhB fur::pDM4 {Delta}lacZ mutant can suppress this defect. As shown in Fig. 7A, this double mutant can grow under iron-limiting conditions at the same level as the wild type, suggesting that any growth defect due to the absence of this sRNA is restored by the absence of Fur.

Next, we determined the role of RyhB in the virulence of V. vulnificus by using s.c. inoculations in the iron-overloaded mouse model. LD50 and CI experiments (Table 3 and Fig. 6B) demonstrated that the {Delta}ryhB {Delta}lacZ mutant is affected in virulence, and we recovered lower numbers of cells than those of the wild-type strain already in the location surrounding the point of inoculation in the skin (P < 0.0001 for each organ analyzed). Figure 6D shows that the double {Delta}ryhB fur::pDM4 {Delta}lacZ mutant had the same phenotype as the single {Delta}ryhB mutant, pointing out that the changes in virulence observed in the {Delta}ryhB mutant cannot be ascribed only to the defect in growth observed for this strain under iron-limiting conditions. To confirm that the finding was due to the lack of expression of the ryhB gene and not to a secondary mutation present in the chromosome of this mutant, we also performed a virulence experiment using a complemented {Delta}ryhB mutant strain. To anticipate the possibility that the pryhB plasmid could be lost during the infection, we cloned the wild-type ryhB gene with its own promoter in the lacZ locus in the chromosome of V. vulnificus as described in Materials and Methods. In this construct, the ryhB gene is expressed ectopically from the chromosome, and as observed for the strain complemented with the ryhB gene expressed from a plasmid, the strain can grow under iron-limiting conditions, although with a slightly different growth rate from that of the wild type (Fig. 7B). This complemented strain showed a lower LD50 value than the mutant, but we still did not observe a full restoration to the wild-type level (Table 3). In fact, the 95% confidence limits of the LD50 values obtained for the complemented strain overlapped with those obtained for the wild-type strain and the mutant. It is worth noticing that from the growth curves for iron-limiting conditions we did not observe a complete restoration of the wild-type phenotype either, indicating that another factor(s) must be missing in the complementation experiments. Figure 6C shows that in CI experiments we recovered similar numbers of cells from the complemented strain to those obtained from the wild type for the different organs, confirming the virulence-enhancing role of ryhB in V. vulnificus infections. We then performed Student t tests contrasting the CI values obtained for the {Delta}ryhB {Delta}lacZ mutant strain with those for the {Delta}ryhB lacZ::ryhB strain, confirming the statistically significant difference between the strains (P < 0.02 for skin and spleen and P < 0.003 for liver). In summary, the attenuated virulence phenotype observed for the {Delta}ryhB mutant of V. vulnificus is possibly due to the growth defect observed in this mutant under iron-limiting conditions, since iron transport is essential in the first stages of infection (see above). However, the facts that the double {Delta}ryhB fur::pDM4 {Delta}lacZ mutant recovered growth under iron-limiting conditions and that this strain still showed a higher LD50 value than the wild type imply that other genes involved in the virulence of V. vulnificus may directly or indirectly be under the control of ryhB.

Genes belonging to group ii (common genes induced in TSBS and TSBS plus FAC). In the group of genes induced in TSBS and TSBS plus FAC, we clustered those that showed at least twofold induction under both conditions compared with the sample coming from cells growing in TSBS plus EDDA. We determined that four genes were found to be consistently upregulated under both conditions (low-iron and iron-rich conditions, in contrast to iron-limiting conditions) (Table 4). Of those genes, we further studied VV12114 and VV10642.


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  		TABLE 4. Genes induced by both TSBS and TSBS plus FAC compared with their expression in TSBS plus EDDA

VV12114 codes for an outer membrane porin that shows similarity to a limited range of outer membrane porins, such as OmpH from Photobacterium profundum (CAA47466) and a porin-like protein H precursor in Vibrio augustum S14 (EAS64080.1). RPA experiments performed to validate our microarray analysis (see Fig. S7 in the supplemental material) clearly showed that this gene is upregulated under low-iron (TSBS) and iron-rich (TSBS plus FAC) conditions. The {Delta}ompH mutant showed an LD50 value similar to that of the wild type (Table 3) when s.c. inoculations were performed; however, when CI experiments were conducted, the mutant was outcompeted by the wild type in all three organs analyzed (P < 0.0001) (Fig. 8), indicating that this protein could be involved in the multiplication of V. vulnificus in the iron-overloaded animals. To confirm that there are no secondary mutations in the mutant analyzed, we constructed a new mutant strain, the ompH::pDM4 mutant, where the gene is interrupted with the pDM4 suicide vector and the wild-type gene is regenerated as described in Materials and Methods, with the suicide vector being excised. As shown in Fig. 8, the restored strain had a similar CI to that of the wild type, confirming that the mutation of this gene rather than a secondary mutation is responsible for the phenotype observed. In addition, a t test performed between the CI values obtained for the ompH::pDM4 mutant and those of the ompH rescued strain confirmed the statistical significance of the values obtained (Fig. 8). Since the downstream gene is not transcribed together with ompH, as determined by RT-PCR (data not shown), the pDM4 suicide vector integrated in this gene does not have any polar effect.


Figure 8
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  		FIG. 8. CI of ompH mutants. Cells were grown and experiments conducted as described in the legend to Fig. 3. (Left) CI between the ompH rescued strain and the {Delta}lacZ mutant (n = 5); (right) CI obtained for the ompH::pDM4 strain versus the {Delta}lacZ mutant (n = 5). Bars represent the geometric mean CI value for each organ. The P values obtained for each comparison are shown. Experiments were conducted at least twice.

The outer membrane of gram-negative bacteria is an effective permeability barrier, allowing only limited diffusion of hydrophobic compounds. It has been described that mutants with a defective outer membrane typically show hypersensitivity toward hydrophobic compounds, anionic or neutral detergents, and cationic peptides. Thus, we assessed whether this outer membrane protein has a role in resistance of V. vulnificus to SDS and polymyxin B. When we challenged the wild type, the ompH::pDM4 mutant, and the original {Delta}ompH mutant with different SDS concentrations, we found that both ompH mutants were more sensitive to this detergent, indicating changes in the properties of the outer membrane in this strain (Fig. 9A). When the sensitivity in the ompH rescued strain was evaluated, similar values to those obtained with the wild type were observed (Fig. 9A).


Figure 9
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  		FIG. 9. Sensitivities of ompH mutants to SDS and polymyxin B. (A) Cells were grown overnight in TSBS and then diluted 1/100 in TSBS with various SDS concentrations, and the OD600 was measured after 2 h of growth. The percentage of SDS resistance is relative to that of the culture without SDS, calculated as described in Materials and Methods. The results shown are the means for three independent experiments, with standard deviations. **, P < 0.01. (B) Cells were grown overnight in TSBS and then diluted 1/100 in TSBS, and when the cells reached mid-log phase, 20 µg/ml polymyxin B was added. A tube with TSBS was used as a control. Samples were taken at determined time points, and serial dilutions were performed in PBS and plated on TSAS plates. The percentage of survival is relative to that of the culture without polymyxin B, as described in Materials and Methods. The results shown are the means for at least three independent experiments, with standard deviations. *, P < 0.05.

Since antimicrobial peptides can also target the outer membrane of V. vulnificus, we evaluated the sensitivities of the wild type and the ompH::pDM4 mutant to polymyxin B. Exposure of ompH mutant bacteria to this peptide resulted in an increase in the sensitivity compared to that of the wild type (P < 0.05), corroborating that this mutant is affected in its outer membrane properties (Fig. 9B). The evaluation of the rescued strain gave similar values to those obtained with the wild type, demonstrating the role of this protein in the resistance to this antimicrobial peptide.

(i) VV10642. VV10642 (glmR) codes for a protein with similarities to the DeoR family of transcriptional regulators, some of which control sugar or amino sugar metabolism, such as GlmR, a transcriptional regulator from Pseudomonas aeruginosa PAO1 (53) (68% identity and 78% similarity). Downstream of VV10642 resides the VV10641 gene (induced in the presence of TSBS plus FAC), whose product has similarities to glucosamine-fructose-6-phosphate aminotransferase, encoded by the glmS gene in many gram-negative and -positive bacteria, which is involved in amino sugar biosynthesis. In addition, both genes are part of the same operon, as demonstrated by RT-PCR (data not shown). Transcriptional analysis performed by RPA with RNAs extracted from the wild-type strain growing at various iron concentrations confirmed the iron regulation observed in the microarray experiments (Fig. 10A, compare lanes TE and TF). We also constructed an in-frame glmR deletion mutant and extracted RNAs from cells growing under iron-rich (TSBS plus FAC) and iron-limiting (TSBS plus EDDA) conditions. As can be observed in Fig. 10A, in the absence of glmR the expression of glmS is deregulated in the mutant in comparison with that in the wild type, suggesting that GlmR controls the expression of glmS. This finding was confirmed by performing a similar analysis with RNAs extracted from cells of the {Delta}glmR mutant complemented with the glmR gene in the pMMB208 vector. As a control, we used RNAs extracted from cells harboring the empty vector. From Fig. 10A, it is clear that the expression in trans of the glmR gene restored the repression of the glmS gene under both growth conditions analyzed (TSBS plus EDDA and TSBS plus FAC). In this case, we did not observe iron regulation of the glmS gene in the presence of glmR, possibly due to the overexpression of the latter gene, which could lead to an imbalance of any substrate that can be sensed by this protein (e.g., glucosamine 6-phosphate or N-acetylglucosamine). The LD50 value for this mutant obtained after s.c. inoculation did not show any difference from that for the wild type in the iron-overloaded mouse model (Table 3). However, we observed a different CI, with a significant increase in the CFU counts of the mutant compared with the wild type, when CI obtained for the spleen were contrasted with those obtained for the skin (P < 0.01) and liver (P < 0.02) (Fig. 10B). We also confirmed that the CI obtained from the spleen is significantly different from 1 (P = 0.0343).


Figure 10
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  		FIG. 10. Analysis of glmR and glmS genes. (A) RPA of the glmS gene. (Right) RNA samples were extracted from the wild type and the {Delta}glmR mutant growing in TSBS plus EDDA (TE) or TSBS plus FAC (TF). (Left) RNA samples were extracted from the {Delta}glmR mutant harboring the pMGlmR vector or the empty vector pMMB208 and growing in TSBS plus EDDA (TE) or TSBS plus FAC (TF). Arrows indicate the positions of the internal control probe without the addition of RNase (VV13021) (lanes 2) and the glmS gene probe (lanes 1). (B) CI of the {Delta}glmR mutant. Cells were grown and experiments conducted as described in the legend to Fig. 3. Bars represent the geometric mean CI value for each organ. Data are representative of three independent experiments. The asterisks indicate the P value for comparison of spleen with skin and spleen with liver, as follows: *, P < 0.01; **, P < 0.02.

Genes belonging to group iii (genes induced either in TSBS or in TSBS plus FAC). We then analyzed the expression of genes under TSBS-plus-EDDA induction and compared it with that of genes under TSBS and TSBS-plus-FAC induction, focusing on those genes that appeared to be induced under only one of the iron-containing conditions. We determined that 62 genes that belong to different categories (Table 5) were specifically induced under the condition of a high iron concentration (i.e., TSBS plus FAC). Interestingly, those genes that code for flagellar proteins, which are virulence factors in V. vulnificus (28), were induced under these growth conditions. Two other genes, VV11712 and VV11713, which code for putative collagenases, were also induced in the presence of a high iron concentration. Since collagenases could play an important role during the invasion of V. vulnificus in the host (60), we determined the involvement of these genes in the virulence of this bacterium by constructing a {Delta}VV11712 {Delta}VV11713 double mutant. LD50 experiments showed that there were no changes between the wild type and the double mutant when s.c. inoculations were analyzed in both animal models (i.e., iron-overloaded mice and mice with normal iron levels) (Table 3). In addition, CI experiments showed similar values to those for the wild type (i.e., ~1), but in this case, we cannot exclude the possibility that collagenases expressed by the wild type cross-fed the mutant, helping it during the invasion. Other genes that were also induced under these conditions included those that encode proteins involved in oxidative phosphorylation and those involved in purine and pyrimidine biosynthesis. The latter could also reflect a rapid synthesis of nucleic acids needed for rapid replication of the cells in the presence of iron, as we observed a shorter doubling time for these samples than for the iron-limited sample (TSBS plus EDDA).


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  		TABLE 5. Genes induced in TSBS plus FAC compared with their expression in TSBS plus EDDA

Under low-iron conditions (i.e., TSBS), we found 171 genes (see Table S8 in the supplemental material) that were exclusively upregulated. These genes belong to different categories, including those that code for ABC transporters, sugar transporters, and ion transporters as well as proteins involved in general metabolism and those with a putative function in peptidoglycan and galactose biosynthesis. An interesting feature of this group is the presence of at least 12 genes that code for putative transcriptional regulators, with 5 genes that encode putative histidine kinases or serine/threonine protein kinases and several methyl-accepting proteins that would be involved in chemotaxis. One of the most remarkable common themes in this list of genes is that they belong to the group of general transcriptional regulators or response regulators, which would indicate that they can sense the environment where V. vulnificus is growing.


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DISCUSSION
 
V. vulnificus can cause severe septicemia in individuals with high levels of iron in serum and in those with liver disease (17, 30). However, at present, there is very scant information on how gene expression influences the physiology and virulence of this bacterium at different iron concentrations. In this work, we investigated the role of genes induced under iron limitation in the iron-overloaded mouse model and report the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions. In this group, except for the genes that code for flagella, we did not find any correlation between the expression of the already well-known virulence factors, such as pili, HlyU, RtxA1, TdkA, and capsule, and the iron content in the medium. This could indicate that a high iron concentration influences the physiology of this bacterium in a very general way, whereas other signals in the host might also contribute to the triggering of virulence.

The iron-overloaded animal model has been used to study V. vulnificus infections due to the similarities observed with susceptible individuals; however, the influence of the vulnibactin system on virulence was examined in the non-iron-treated mouse (34). Moreover, strains unable to take iron from transferrin had been shown to have reduced virulence in the iron-overloaded mouse model; however, those strains also showed differences in other characteristics, such as serum sensitivity and hemolysin production, making it hard to assign the uptake of vulnibactin as the only virulence factor involved (64). Thus, understanding the importance of the siderophore in the virulence of V. vulnificus in the iron-overloaded mouse model has remained elusive. In this work, we evaluated the virulence of a double {Delta}tonB1 {Delta}tonB2 mutant and a {Delta}venB mutant (vulnibactin biosynthesis mutant) in both animal models (i.e., iron-overloaded and nontreated animals) by using both i.p. and s.c. inoculations. Independent of the mutants tested, using the s.c. route and the iron-overloaded model, dramatic changes in LD50 values (~3 log) were observed. In addition, when we performed s.c. infections and quantified them by using CI tests, we were able to establish that iron transport is essential during the first stages of the infection, since we recovered fewer cells from the mutants in the tonB1 and tonB2 systems at the injection site with that animal model. Conversely, we detected only slight differences in LD50 values compared with that of the wild type by using i.p. inoculations in the iron-overloaded animals, suggesting that these mutants still have rapid access to the bloodstream, causing a fulminant septicemia. In a recent study, Adams et al. (1) determined that parenteral iron inoculation in mice increases the iron content in the epidermis and dermis at a high iron dose (5 mg). We did not measure the iron concentration in the skin of the animals used in this study, but our results demonstrate that one of the components involved in the fast replication of V. vulnificus in that organ, previously observed by Starks et al. (62, 63), is the active transport of iron (which may be mainly from transferrin), as demonstrated by the involvement of the tonB genes, which is still crucial for a successful infection in this animal model. It is worth noticing that in iron-overloaded animals the wild-type strain can establish a rapid infection and that the LD50 value is 4 to 5 log smaller than that for non-iron-treated animals. We believe that the paradoxical necessity of an iron transport system in an iron-overloaded animal could be explained in the context of the more natural route of infection provided by s.c. inoculations. Using this route, bacteria need to replicate in the skin and cross many tissue barriers to multiply and cause fatal septicemia.

Expression of the TonB3 cluster occurs only when the bacterium grows in human serum, but we have not been able to characterize the metabolic and/or energetic steps in which TonB3 plays a role which could be involved in other active transport processes (4, 45, 54). Moreover, a TonB-dependent receptor was described for N. gonorrhoeae and is induced when the bacterium is growing in the presence of either eukaryotic cells or bovine serum (18).

Our finding that one of the Tad gene clusters present in V. vulnificus is under the control of iron in a Fur-dependent manner leaves open the possibility that iron controls the adhesion of V. vulnificus to various biotic and abiotic surfaces, such as those that the organism finds in the environment (e.g., oysters or exoskeleton) or those in the host. It was previously described that flagella and type IV pili are important virulence factors in this bacterium and are also important for biofilm formation and adhesion (28, 47, 48). We showed in our study that several genes encoding flagellar proteins were induced under iron-rich conditions, and thus it is tempting to speculate that during the infection process the expression of these genes allows V. vulnificus to move from a low-iron to a high-iron environment.

We have also demonstrated that the sRNA RyhB is an essential component in the virulence repertoire of this bacterium. Previously, Murphy and Payne (44) showed that RyhB controls the transcription of several virulence factors in Shigella dysenteriae, but a direct measurement of virulence in an animal model was not assessed. Our results using the iron-overloaded animal model are, to our knowledge, the first report showing that this type of sRNA is directly involved in the virulence of a pathogen. One of the possible reasons for the attenuated virulence phenotype observed for the {Delta}ryhB mutant of V. vulnificus could be ascribed to the growth defect observed in this mutant under iron-limiting conditions. However, the facts that the double {Delta}ryhB fur::pDM4 {Delta}lacZ mutant recovered growth under iron-limiting conditions and that this strain is still attenuated in virulence imply that other genes involved in the virulence of V. vulnificus may directly or indirectly be under the control of ryhB. An alternative explanation could be that the iron-limiting conditions found for the microorganism inside the animal are different from those analyzed by us in vitro. Experiments to elucidate the role of ryhB in V. vulnificus infections are in progress.

Our results also indicate that in addition to iron limitation, this bacterium senses other iron concentrations for the induction of gene expression. Thus, at the iron concentration found in TSBS (1 µg Fe/ml), several transcriptional regulators were upregulated, as were genes encoding proteins involved in chemotaxis and putative histidine kinase or serine/threonine protein kinases. Genes that were induced only at high iron concentrations (TSBS plus FAC) included those encoding proteins involved in oxidative phosphorylation and purine and pyrimidine biosynthesis as well as those involved in oxidative stress, such as that for superoxide dismutase. There was thus a clear difference from those that were induced or expressed at lower iron concentrations (TSBS) or under iron limitation (TSBS plus EDDA).

In this work, we focused on genes that showed a clear induction in both TSBS and TSBS plus FAC compared with their expression in TSBS plus EDDA (iron limitation), since they represent clear targets for understanding the physiology of V. vulnificus growing in the presence of at least 1 µg Fe/ml. A gene that showed a definite increase in transcription in the presence of iron was VV12114, named ompH here. Mutations in this gene resulted in changes in the biological characteristics of the envelope of the mutant, making this strain sensitive to SDS and polymyxin B. The CI experiments performed using this mutant confirmed that those characteristics are important for the progress of the disease. Our results strongly support the idea that the induction of this gene is needed for V. vulnificus to overcome the response of the host to the infection. The other pair of genes that gave us important clues about the relevance of iron in the physiology of V. vulnificus was glmR and glmS. The enhanced tropism for the spleen observed in the {Delta}glmR mutant would indicate a role for this gene (and the genes under its control) in the virulence of V. vulnificus. It is worth mentioning that GlmS has a key role in the hexosamine pathway by catalyzing the synthesis of glucosamine 6-phosphate from fructose 6-phosphate and glucosamine in gram-negative and gram-positive bacteria (52). This reaction, essential in the absence of external amino sugars, is involved in the synthesis of precursors of both lipopolysaccharide and peptidoglycan.

Lastly, an important outcome of our work is the use of CI as a tool to evaluate the virulence of mutants that do not show significant differences in LD50 values and cannot be cross-fed in vivo by the wild type (or vice versa).


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ACKNOWLEDGMENTS
 
This work was supported by grant AI065981 from the National Institutes of Health to J.H.C.

We thank M. Liu as well as Y. Nakamura and D. Tolle for plasmid constructs. We are grateful to S. McWeeney from the Division of Biostatistics, Department of Public Health and Preventive Medicine, OHSU, for help with the q value. We also thank J. H. Rhee for the CMCP6 strain.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239. Phone: (503) 494-7583. Fax: (503) 494-6862. E-mail: crosajor{at}ohsu.edu Back

{triangledown} Published ahead of print on 23 June 2008. Back

{dagger} Supplemental material for this article may be found at http://iai.asm.org/. Back

Editor: A. Camilli


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REFERENCES
 
    1
  1. Adams, B. D., R. Lazova, N. C. Andrews, and L. M. Milstone. 2005. Iron in skin of mice with three etiologies of systemic iron overload. J. Investig. Dermatol. 125:1200-1205.[CrossRef][Medline]
    2. 2
  2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
    3. 3
  3. Bensing, B. A., B. J. Meyer, and G. M. Dunny. 1996. Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis. Proc. Natl. Acad. Sci. USA 93:7794-7799.[Abstract/Free Full Text]
    4. 4
  4. Blanvillain, S., D. Meyer, A. Boulanger, M. Lautier, C. Guynet, N. Denance, J. Vasse, E. Lauber, and M. Arlat. 2007. Plant carbohydrate scavenging through TonB-dependent receptors: a feature shared by phytopathogenic and aquatic bacteria. PLoS ONE 2:e224.[CrossRef]
    5. 5
  5. Bolstad, B. M., R. A. Irizarry, M. Astrand, and T. P. Speed. 2003. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185-193.[Abstract/Free Full Text]
    6. 6
  6. Brennt, C. E., A. C. Wright, S. K. Dutta, and J. G. Morris, Jr. 1991. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J. Infect. Dis. 164:1030-1032.[Medline]
    7. 7
  7. Bullen, G. E. 1999. Iron and infection, 2nd ed. John Wiley and Sons Ltd., New York, NY.
    8. 8
  8. Chai, S., T. J. Welch, and J. H. Crosa. 1998. Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum. J. Biol. Chem. 273:33841-33847.[Abstract/Free Full Text]
    9. 9
  9. Chen, C. Y., K. M. Wu, Y. C. Chang, C. H. Chang, H. C. Tsai, T. L. Liao, Y. M. Liu, H. J. Chen, A. B. Shen, J. C. Li, T. L. Su, C. P. Shao, C. T. Lee, L. I. Hor, and S. F. Tsai. 2003. Comparative genome analysis of Vibrio vulnificus, a marine pathogen. Genome Res. 13:2577-2587.[Abstract/Free Full Text]
    10. 10
  10. Chen, Y. C., Y. C. Chuang, C. C. Chang, C. L. Jeang, and M. C. Chang. 2004. A K+ uptake protein, TrkA, is required for serum, protamine, and polymyxin B resistance in Vibrio vulnificus. Infect. Immun. 72:629-636.[Abstract/Free Full Text]
    11. 11
  11. Crosa, J. H. 1980. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature 284:566-568.[CrossRef][Medline]
    12. 12
  12. Dennis, G., Jr., B. T. Sherman, D. A. Hosack, J. Yang, W. Gao, H. C. Lane, and R. A. Lempicki. 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4:P3.[CrossRef][Medline]
    13. 13
  13. Escolar, L., J. Perez-Martin, and V. de Lorenzo. 1999. Opening the iron box: transcriptional metalloregulation by the Fur protein. J. Bacteriol. 181:6223-6229.[Free Full Text]
    14. 14
  14. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652.[Abstract/Free Full Text]
    15. 15
  15. Finney, D. J. 1971. Probit analysis: a statistical treatment of the sigmoid response curve. Cambridge University Press, London, United Kingdom.
    16. 16
  16. Goodwin, J. F., B. Murphy, and M. Guillemette. 1966. Direct measurement of serum iron and binding capacity. Clin. Chem. 12:47-57.[Abstract]
    17. 17
  17. Gulig, P. A., K. L. Bourdage, and A. M. Starks. 2005. Molecular pathogenesis of Vibrio vulnificus. J. Microbiol. 43(Spec. No.):118-131.[Medline]
    18. 18
  18. Hagen, T. A., and C. N. Cornelissen. 2006. Neisseria gonorrhoeae requires expression of TonB and the putative transporter TdfF to replicate within cervical epithelial cells. Mol. Microbiol. 62:1144-1157.[CrossRef][Medline]
    19. 19
  19. Hor, L. I., Y. K. Chang, C. C. Chang, H. Y. Lei, and J. T. Ou. 2000. Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection. Microbiol. Immunol. 44:871-878.[Medline]
    20. 20
  20. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U. Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4:249-264.[Abstract]
    21. 21
  21. Jacques, J. F., S. Jang, K. Prevost, G. Desnoyers, M. Desmarais, J. Imlay, and E. Masse. 2006. RyhB small RNA modulates the free intracellular iron pool and is essential for normal growth during iron limitation in Escherichia coli. Mol. Microbiol. 62:1181-1190.[CrossRef][Medline]
    22. 22
  22. Kim, C. M., R. Y. Park, M. H. Choi, H. Y. Sun, and S. H. Shin. 2007. Ferrophilic characteristics of Vibrio vulnificus and potential usefulness of iron chelation therapy. J. Infect. Dis. 195:90-98.[CrossRef][Medline]
    23. 23
  23. Kim, I. H., J. I. Shim, K. E. Lee, W. Hwang, I. J. Kim, S. H. Choi, and K. S. Kim. 2008. Nonribosomal peptide synthetase is responsible for the biosynthesis of siderophore in Vibrio vulnificus MO6-24/O. J. Microbiol. Biotechnol. 18:35-42.[CrossRef][Medline]
    24. 24
  24. Kim, S. Y., S. E. Lee, Y. R. Kim, C. M. Kim, P. Y. Ryu, H. E. Choy, S. S. Chung, and J. H. Rhee. 2003. Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system. Mol. Microbiol. 48:1647-1664.[CrossRef][Medline]
    25. 25
  25. Kim, Y. R., S. Y. Kim, C. M. Kim, S. E. Lee, and J. H. Rhee. 2005. Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence. FEMS Microbiol. Lett. 243:497-503.[CrossRef][Medline]
    26. 26
  26. Kim, Y. R., S. E. Lee, C. M. Kim, S. Y. Kim, E. K. Shin, D. H. Shin, S. S. Chung, H. E. Choy, A. Progulske-Fox, J. D. Hillman, M. Handfield, and J. H. Rhee. 2003. Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect. Immun. 71:5461-5471.[Abstract/Free Full Text]
    27. 27
  27. Lee, J. H., M. W. Kim, B. S. Kim, S. M. Kim, B. C. Lee, T. S. Kim, and S. H. Choi. 2007. Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J. Microbiol. 45:146-152.[Medline]
    28. 28
  28. Lee, J. H., J. B. Rho, K. J. Park, C. B. Kim, Y. S. Han, S. H. Choi, K. H. Lee, and S. J. Park. 2004. Role of flagellum and motility in pathogenesis of Vibrio vulnificus. Infect. Immun. 72:4905-4910.[Abstract/Free Full Text]
    29. 29
  29. Lee, S. E., S. Y. Kim, C. M. Kim, M. K. Kim, Y. R. Kim, K. Jeong, H. J. Ryu, Y. S. Lee, S. S. Chung, H. E. Choy, and J. H. Rhee. 2007. The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor. Infect. Immun. 75:2795-2801.[Abstract/Free Full Text]
    30. 30
  30. Linkous, D. A., and J. D. Oliver. 1999. Pathogenesis of Vibrio vulnificus. FEMS Microbiol. Lett. 174:207-214.[CrossRef][Medline]
    31. 31
  31. Linn, T., and R. St. Pierre. 1990. Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J. Bacteriol. 172:1077-1084.[Abstract/Free Full Text]
    32. 32
  32. Litwin, C. M., and B. L. Byrne. 1998. Cloning and characterization of an outer membrane protein of Vibrio vulnificus required for heme utilization: regulation of expression and determination of the gene sequence. Infect. Immun. 66:3134-3141.[Abstract/Free Full Text]
    33. 33
  33. Litwin, C. M., and S. B. Calderwood. 1993. Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J. Bacteriol. 175:706-715.[Abstract/Free Full Text]
    34. 34
  34. Litwin, C. M., T. W. Rayback, and J. Skinner. 1996. Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect. Immun. 64:2834-2838.[Abstract]
    35. 35
  35. Liu, M., A. F. Alice, H. Naka, and J. H. Crosa. 2007. The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus. Infect. Immun. 75:3282-3289.[Abstract/Free Full Text]
    36. 36
  36. Masse, E., and S. Gottesman. 2002. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc. Natl. Acad. Sci. USA 99:4620-4625.[Abstract/Free Full Text]
    37. 37
  37. Meselson, M., and R. Yuan. 1968. DNA restriction enzyme from E. coli. Nature 217:1110-1114.[CrossRef][Medline]
    38. 38
  38. Mey, A. R., S. A. Craig, and S. M. Payne. 2005. Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect. Immun. 73:5706-5719.[Abstract/Free Full Text]
    39. 39
  39. Miller, J. H. 1992. A short course in bacterial genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    40. 40
  40. Miller, L. C., and M. L. Tainter. 1944. Estimation of the E.D. 50 and its error by means of logarithmic-probit paper. Proc. Soc. Exp. Biol. Med. 57:261-264.[CrossRef]
    41. 41
  41. Milton, D. L., R. O'Toole, P. Horstedt, and H. Wolf-Watz. 1996. Flagellin A is essential for the virulence of Vibrio anguillarum. J. Bacteriol. 178:1310-1319.[Abstract/Free Full Text]
    42. 42
  42. Morales, V. M., A. Backman, and M. Bagdasarian. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:39-47.[CrossRef][Medline]
    43. 43
  43. Morris, J. G., Jr. 1988. Vibrio vulnificus—a new monster of the deep? Ann. Intern. Med. 109:261-263.[Abstract/Free Full Text]
    44. 44
  44. Murphy, E. R., and S. M. Payne. 2007. RyhB, an iron-responsive small RNA molecule, regulates Shigella dysenteriae virulence. Infect. Immun. 75:3470-3477.[Abstract/Free Full Text]
    45. 45
  45. Neugebauer, H., C. Herrmann, W. Kammer, G. Schwarz, A. Nordheim, and V. Braun. 2005. ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus. J. Bacteriol. 187:8300-8311.[Abstract/Free Full Text]
    46. 46
  46. Osorio, C. R., S. Juiz-Rio, and M. L. Lemos. 2006. A siderophore biosynthesis gene cluster from the fish pathogen Photobacterium damselae subsp. piscicida is structurally and functionally related to the Yersinia high-pathogenicity island. Microbiology 152:3327-3341.[Abstract/Free Full Text]
    47. 47
  47. Paranjpye, R. N., J. C. Lara, J. C. Pepe, C. M. Pepe, and M. S. Strom. 1998. The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice. Infect. Immun. 66:5659-5668.[Abstract/Free Full Text]
    48. 48
  48. Paranjpye, R. N., and M. S. Strom. 2005. A Vibrio vulnificus type IV pilin contributes to biofilm formation, adherence to epithelial cells, and virulence. Infect. Immun. 73:1411-1422.[Abstract/Free Full Text]
    49. 49
  49. Park, N. Y., J. H. Lee, M. W. Kim, H. G. Jeong, B. C. Lee, T. S. Kim, and S. H. Choi. 2006. Identification of the Vibrio vulnificus wbpP gene and evaluation of its role in virulence. Infect. Immun. 74:721-728.[Abstract/Free Full Text]
    50. 50
  50. Peterson, J. D., L. A. Umayam, T. Dickinson, E. K. Hickey, and O. White. 2001. The comprehensive microbial resource. Nucleic Acids Res. 29:123-125.[Abstract/Free Full Text]
    51. 51
  51. Planet, P. J., S. C. Kachlany, D. H. Fine, R. DeSalle, and D. H. Figurski. 2003. The widespread colonization island of Actinobacillus actinomycetemcomitans. Nat. Genet. 34:193-198.[CrossRef][Medline]
    52. 52
  52. Plumbridge, J. A., O. Cochet, J. M. Souza, M. M. Altamirano, M. L. Calcagno, and B. Badet. 1993. Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12. J. Bacteriol. 175:4951-4956.[Abstract/Free Full Text]
    53. 53
  53. Ramos-Aires, J., P. Plesiat, L. Kocjancic-Curty, and T. Kohler. 2004. Selection of an antibiotic-hypersusceptible mutant of Pseudomonas aeruginosa: identification of the GlmR transcriptional regulator. Antimicrob. Agents Chemother. 48:843-851.[Abstract/Free Full Text]
    54. 54
  54. Schauer, K., B. Gouget, M. Carriere, A. Labigne, and H. de Reuse. 2007. Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery. Mol. Microbiol. 63:1054-1068.[CrossRef][Medline]
    55. 55
  55. Schreiner, H. C., K. Sinatra, J. B. Kaplan, D. Furgang, S. C. Kachlany, P. J. Planet, B. A. Perez, D. H. Figurski, and D. H. Fine. 2003. Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model. Proc. Natl. Acad. Sci. USA 100:7295-7300.[Abstract/Free Full Text]
    56. 56
  56. Seliger, S. S., A. R. Mey, A. M. Valle, and S. M. Payne. 2001. The two TonB systems of Vibrio cholerae: redundant and specific functions. Mol. Microbiol. 39:801-812.[CrossRef][Medline]
    57. 57
  57. Senanayake, S. D., and D. A. Brian. 1995. Precise large deletions by the PCR-based overlap extension method. Mol. Biotechnol. 4:13-15.[Medline]
    58. 58
  58. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering transposon mutagenesis in gram negative bacteria. Bio/Technology 1:787-796.
    59. 59
  59. Simpson, L. M., and J. D. Oliver. 1983. Siderophore production by Vibrio vulnificus. Infect. Immun. 41:644-649.[Abstract/Free Full Text]
    60. 60
  60. Smith, G. C., and J. R. Merkel. 1982. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect. Immun. 35:1155-1156.[Abstract/Free Full Text]
    61. 61
  61. Spinola, S. M., K. R. Fortney, B. P. Katz, J. L. Latimer, J. R. Mock, M. Vakevainen, and E. J. Hansen. 2003. Haemophilus ducreyi requires an intact flp gene cluster for virulence in humans. Infect. Immun. 71:7178-7182.[Abstract/Free Full Text]
    62. 62
  62. Starks, A. M., K. L. Bourdage, P. C. Thiaville, and P. A. Gulig. 2006. Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice. Mol. Microbiol. 61:310-323.[CrossRef][Medline]
    63. 63
  63. Starks, A. M., T. R. Schoeb, M. L. Tamplin, S. Parveen, T. J. Doyle, P. E. Bomeisl, G. M. Escudero, and P. A. Gulig. 2000. Pathogenesis of infection by clinical and environmental strains of Vibrio vulnificus in iron-dextran-treated mice. Infect. Immun. 68:5785-5793.[Abstract/Free Full Text]
    64. 64
  64. Stelma, G. N., Jr., A. L. Reyes, J. T. Peeler, C. H. Johnson, and P. L. Spaulding. 1992. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl. Environ. Microbiol. 58:2776-2782.[Abstract/Free Full Text]
    65. 65
  65. Storey, J. D. 2002. A direct approach to false discovery rates. J. R. Stat. Soc. B 64:479-498.[CrossRef]
    66. 66
  66. Storey, J. D., and R. Tibshirani. 2003. Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. USA 100:9440-9445.[Abstract/Free Full Text]
    67. 67
  67. Stork, M., M. Di Lorenzo, S. Mourino, C. R. Osorio, M. L. Lemos, and J. H. Crosa. 2004. Two tonB systems function in iron transport in Vibrio anguillarum, but only one is essential for virulence. Infect. Immun. 72:7326-7329.[Abstract/Free Full Text]
    68. 68
  68. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833-2837.[Abstract/Free Full Text]
    69. 69
  69. Tomich, M., P. J. Planet, and D. H. Figurski. 2007. The tad locus: postcards from the widespread colonization island. Nat. Rev. Microbiol. 5:363-375.[CrossRef][Medline]
    70. 70
  70. Webster, A. C., and C. M. Litwin. 2000. Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor. Infect. Immun. 68:526-534.[Abstract/Free Full Text]
    71. 71
  71. Welkos, S., and A. O'Brien. 1994. Determination of median lethal and infectious doses in animal model systems. Methods Enzymol. 235:29-39.[Medline]
    72. 72
  72. Wilderman, P. J., N. A. Sowa, D. J. FitzGerald, P. C. FitzGerald, S. Gottesman, U. A. Ochsner, and M. L. Vasil. 2004. Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc. Natl. Acad. Sci. USA 101:9792-9797.[Abstract/Free Full Text]
    73. 73
  73. zWolf, M. K., and J. H. Crosa. 1986. Evidence for the role of a siderophore in promoting Vibrio anguillarum infections. J. Gen. Microbiol. 132:2949-2952.[Abstract/Free Full Text]
    74. 74
  74. Wright, A. C., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect. Immun. 34:503-507.[Abstract/Free Full Text]
    75. 75
  75. Wright, A. C., L. M. Simpson, J. D. Oliver, and J. G. Morris, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect. Immun. 58:1769-1773.[Abstract/Free Full Text]

Infection and Immunity, September 2008, p. 4019-4037, Vol. 76, No. 9
0019-9567/08/$08.00+0     doi:10.1128/IAI.00208-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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