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Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden
Correspondence
Debra L. Milton
Debra.Milton{at}molbiol.umu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Center for Research on Intracellular Bacteria (CRIB), Institute for Microbiology, University of Lausanne, Switzerland.
Present address: Guangdong Province, South China Sea Institute of Oceanology, Xinggang Xi Lu, 164, 510301 Guangzhou, China.
The GenBank/EMBL/DDBJ accession numbers for the sequences of rpoS and hfq are EU330190 and EU330191, respectively.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Small diffusible signal molecules, such as N-acylhomoserine lactones, are secreted by the bacteria and accumulate as the population grows. When a signal molecule reaches a threshold concentration at a critical cell density, quorum-sensing systems are activated, inducing or repressing phenotypic expression. Consequently, bacteria coordinate activities as a population, which likely provides a selective advantage in the natural environment. First characterized as a means for regulating light production in the marine bacteria Vibrio fischeri and Vibrio harveyi, quorum sensing is now recognized as a widespread mechanism for global gene regulation in many bacteria.
Vibrio anguillarum is widely distributed in the aquatic environment and is part of the normal microflora of marine fish (Austin & Austin, 1999; Urakawa & Rivera, 2006). When the health or immune system of the fish is compromised, V. anguillarum causes a haemorrhagic septicaemia (vibriosis) (Actis et al., 1999; Austin & Austin, 1999). Production of quorum-sensing signal molecules such as N-acylhomoserine lactones is a common feature of both pathogenic and environmental isolates of V. anguillarum. Thus, quorum sensing may affect the ecology and physiology of this bacterium as well as its pathogenicity (Buch et al., 2003). Moreover, the V. harveyi-like LuxR transcriptional activator in V. anguillarum, VanT, positively regulates extracellular protease activity, pigment production and biofilm formation in response to quorum-sensing signals (Croxatto et al., 2002). Each of these activities may play a role in the survival of V. anguillarum in seawater or in the fish host.
All vibrios analysed so far contain quorum-sensing systems involving phosphorelay systems that are believed to regulate gene expression in response to cell population similarly to the quorum-sensing systems of V. harveyi (Waters & Bassler, 2005; Neiditch et al., 2005; Timmen et al., 2006; Tu & Bassler, 2007). Components of two phosphorelay quorum-sensing systems are known in V. anguillarum and a third is predicted. A model of these signalling systems is given in Fig. 1(a). VanM, an N-acylhomoserine lactone synthase, synthesizes the signal molecules N-hexanoyl-L-homoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL), which are sensed by VanN (Croxatto et al., 2004). VanS likely synthesizes an AI-2 signal molecule, a furanosyl borate diester, which binds the periplasmic protein VanP. The VanP–AI-2 complex then binds to VanQ (Croxatto et al., 2004; Denkin & Nelson, 2004). The CqsA/S signal system has not been characterized but is predicted to be present (Henke & Bassler, 2004). The CAI-1 signal molecule, an (S)-3-hydroxytridecan-4-one (Higgins et al., 2007), is synthesized by CqsA and is believed to bind the receptor CqsS.
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54-dependent activator VanO. When phosphorylated, VanO activates the expression of several small regulatory RNAs (sRNAs) that, together with the RNA chaperone Hfq, destabilize mRNA encoding VanT, the master regulator. At high cell density, the signal molecules accumulate and bind their cognate sensor kinases. Signal binding represses the kinase activity of VanN, VanQ and CqsS, allowing the phosphatase activity to predominate, which leads to dephosphorylation of VanO. Consequently, VanO is inactivated, sRNAs are not transcribed, and VanT expression is induced, activating the quorum-sensing regulon. In this study, the protein profile of VanT expression was analysed during growth of wild-type V. anguillarum. VanT expression could be detected at a population of 3x106 cells ml–1 and the expression peaked as the cells entered late exponential growth. Interestingly, the sigma factor RpoS was shown to play a major role in indirectly inducing VanT expression post-transcriptionally. RpoS stabilizes vanT mRNA by a mechanism involving Hfq. RpoS was not part of the quorum-sensing regulatory cascade since it did not regulate expression or activity of VanO and since RpoS was not regulated by the quorum-sensing systems. Finally, VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production. In summary, RpoS and the quorum-sensing systems work together to modulate expression of VanT, which regulates physiological responses required for survival and stress response.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Plasmid transfers from Escherichia coli to V. anguillarum were done as previously described (Milton et al., 1996). E. coli was routinely grown in Luria broth (per litre: Bacto Tryptone, 10 g; Bacto yeast extract, 5 g; and sodium chloride, 5 g) with the following antibiotic concentrations: 100 µg ml–1 for ampicillin and 25 µg ml–1 for chloramphenicol. For V. anguillarum, Trypticase soy medium from BBL containing 1 % sodium chloride (TSB) was routinely used. For selection against E. coli after conjugation, the Vibrio selective medium TCBS agar (Difco) was used. Antibiotic concentration for V. anguillarum for chloramphenicol was 10 µg ml–1.
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) and sequenced to confirm identity to rpoS. The primers used were VCrpoS-For2 (5'-GGACTAGTCCGTAACCAAAGTAGARGARTT-3') and VCrpoS-Rev2 (5'-GGGGTACCCCAATGGTGCGTGTYTGRTTCAT-3'). The 477 bp PCR fragment was used as a probe to screen a V. anguillarum genomic library in the Lambda Zap II bacteriophage, and the pBluescript plasmids containing a chromosomal insert were excised from positive plaques as previously described (Milton et al., 1992). One plasmid that contained the entire rpoS gene, pBS-rpoS-7, was chosen for sequencing.
To clone the hfq gene, two primers complementary to sequences flanking the hfq gene in Vibrio parahaemolyticus (Makino et al., 2003) were used to amplify a 1519 bp fragment from the V. anguillarum chromosome. Primers used were Hfq-1 (5'-GGACTAGTGCATCAACAACATGTAACAA-3'), which contains a SpeI site at the 5'-end, and Hfq-2 (5'-CTCGAGCTCGGTTACCGACAGATGTGGGA-3'), which contains a SacI site at the 5'-end. This fragment was cloned into pBluescript using SacI and SpeI, creating pBS-hfq, and sequenced.
Mutant and complementation strains.
To create a null mutation in rpoS and hfq, an in-frame deletion was made by allelic exchange as described in detail previously (Milton et al., 1996). Plasmid pDM4-rpoS-AD, which carries an altered allele of rpoS that encodes the first 14 amino acids fused to the last 61 amino acids of RpoS, was used to create strain AC12 (
rpoS). Plasmid pDM4-hfq-AD, carrying an altered allele of hfq encoding the first five amino acids fused to the last five amino acids of Hfq, was used to create strain BW11 (hfq). The in-frame deletions were confirmed by sequencing a PCR-amplified DNA fragment of the deleted chromosomal locus and by Western blot analysis.
For complementation of the rpoS, hfq and vanO mutations, reverse allelic exchange was done. For each, the wild-type gene and flanking DNA were amplified by PCR and cloned into pDM4, resulting in pDM4-rpoS-wt, pDM4-hfq-wt and pDM4-vanO-wt, which were used to exchange the mutant alleles for the wild-type gene, producing strains AC12c, BW11c and AC11c, respectively. Complementation of the mutations was confirmed by Western blot analysis.
PCR conditions, DNA techniques, DNA sequencing and computer analyses.
PCR was performed as previously described (McGee et al., 1996; Croxatto et al., 2002). Unless otherwise stated, all conditions for the various DNA techniques were as described by Sambrook et al. (1989). Reaction conditions for the DNA-modifying enzymes and DNA-restriction enzymes were performed as suggested by the manufacturers. Double-strand DNA sequencing was performed by automated sequencing on an ABI Prism 377 DNA sequencer and by primer walking in two directions from known regions of DNA sequence. DNA sequence editing was done using the Genetics Computer Group Sequence Analysis software (Devereux et al., 1984) of the Genetics Computer Group (University of Wisconsin). Database searches were done using the BLAST program from the National Center for Biotechnology Information. The sequence data have been submitted to the DDBJ/EMBL/GeneBank databases under accession number EU330190 for rpoS and EU330191 for hfq.
Western analysis and preparation of antisera.
VanT, VanO, RpoS and Hfq were purified using the IMPACT T7 system from New England BioLabs. Genes encoding VanT, VanO, RpoS and Hfq were amplified by PCR and cloned into pTYB1 using NdeI and SapI, which fuses the coding region for the protein splicing element intein followed by a chitin-binding tag to the 3'-end of these genes. For optimal self-splicing, an extra glycine codon was added to the 3'-end of the vanT, rpoS and hfq genes. Protein purification was performed as described by the manufacturer except for VanT cleavage from the chitin column, which was done for 48 h. Using the purified proteins, polyclonal rabbit antisera were made by AgriSera AB, Sweden. Before use in Western analyses, the antisera were affinity purified using the MicroLink Protein Coupling kit (Pierce) via the manufacturer's instructions.
For each strain, Western blot analysis was performed on protein samples taken at various time points during growth. Proteins were separated as described by Laemmli (1970) using SDS-12.5 % PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell) using a SemiPhor semidry blotter (Hoefer TE 70 series). Enhanced chemiluminescence (ECL) Western blotting was performed according to the manufacturer's instructions (Amersham Life Sciences). As no suitable loading control was found that could be used to compare all time points during growth, two approaches were taken to determine equal sample loading. A second similarly loaded 12.5 % PAGE was performed and stained with Coomassie blue. In addition, Western analysis was done using protein samples from two time points and two antisera to detect the protein of interest and the outer-membrane protein OmpU, which was used as a loading control. The intensities of the two protein bands were measured using QUANTITY ONE version 4.2.3 software (Bio-Rad) and the protein of interest was equalized to that of OmpU. The mutant to wild-type ratio was then determined.
Construction of transcriptional and translational gfp fusions.
Transcriptional or translational reporter gene fusions with the gene encoding the unstable green fluorescent protein variant Gfp-ASV (Andersen et al., 1998) were made using the suicide vector pDM41. The C-terminal peptide tag ASV renders the stable Gfp protein susceptible to degradation by intracellular tail-specific proteases. The half-life of Gfp-ASV was determined to be 80 min in V. anguillarum (data not shown). To create pDM41, the gfp gene, which lacks a promoter but has a ribosome-binding site (RBS), was removed from pJBA113 by NheI and XbaI digestion. The 850 bp fragment was purified from a 1 % agarose gel using Ultrafree-DA spin columns (Millipore) and cloned into similar restriction sites of the suicide vector pNQ705-1. For transcriptional fusions, the XbaI site, which cleaves upstream of the RBS, was used and for translational fusions, the SphI site, which cleaves at the ATG start codon, was used. For transcriptional fusions of vanT, rpoS, empA, qrr1 and hfq, DNA fragments containing promoters but lacking the possible RBS were amplified by PCR using the following primer sets containing either a SacI or an XbaI site at the 5'-ends: VanT-gfp-S (5'-CTCGAGCTCATTCGTTCCTGAACC-3') and VanT-gfp-X (5'-GCTCTAGAACTGTTGAATTGAGC-3'); RpoS-gfp-S (5'-CTCGAGCTCCCGTTGTCTATTCGG-3') and RpoS-gfp-X (5'-GCTCTAGAGAGCTAGCAAGACAT-3'); EmpA-gfp-1 (5'-CTCGAGCTCATATGCTCAACGAAC-3') and EmpA-gfp-2 (5'-GCTCTAGAGTTATTATTAGCATC-3'); and Rna1-gfp-S (5'-CTCGAGCTCAGCAATATGAGGTCC-3') and Rna1-gfp-X (5'-GCTCTAGATATTGAATAGAGCAC-3'); Hfq-gfp-S (5'-CTCGAGCTCAAGCGTCAGATCACC-3') and Hfq-gfp-X (5'-GCTCTAGAGTTGTAGTTATTTAG-3'). For a translational fusion of vanT, a DNA fragment containing the promoter, the RBS and codons for the first 14 amino acids was amplified by PCR using a primer set containing either a SacI or a SphI site at the 5'-end. The primer pair used was VanT-Sph3 (5'-GGACATGCATGCGTGATAAGCGAGTTC-3') and VanT-gfp-S (listed above). The DNA fragments were gel purified, digested with SacI/XbaI or SacI/SphI and ligated to a similarly digested pDM41, resulting in pDM41-vanT3-TL, pDM41-vanT-TC, pDM41-rpoS-TC, pDM41-empA-TC, pDM41-hfq-TC and pDM41-qrr1-TC. All constructs were sequenced to ensure that the gene fusions were made properly. Each gene fusion was integrated into the promoter region of the respective gene on the chromosome. Since the promoters were duplicated on the chromosomes, these insertions did not disrupt the respective wild-type genes. Chromosomal integrations were checked by PCR analysis.
Green fluorescent protein (Gfp) assays.
V. anguillarum cultures carrying the gfp gene fusions were grown overnight at 24 °C in TSB with aeration. Overnight cultures were diluted to an OD600 of 0.001 in TSB and incubation was continued at 24 °C in TSB with aeration. At various time points during growth (0, 2, 4, 6, 8, 10, 12, 14, 18, 24 h), samples were taken and the OD600 and numbers of c.f.u. were determined. To measure fluorescence at each time point, a cell number equivalent to an OD600 of 0.2 was removed from each sample and, when required, the samples were diluted to an OD600 of 0.2 in a final volume of 2 ml. This was done since the use of too many cells quenched the fluorescence output using a Bio-Rad VersaFluor fluorometer. To minimize any effects on gene expression due to the dilution of the cells, fluorescence was measured immediately following dilution with an excitation wavelength of 490 nm and an emission wavelength at 520 nm, according to the manufacturer's instructions. The fluorescence units were then divided by an OD600 of 0.2 to obtain fluorescence relative to the cell number. The wild-type strain without a gfp gene fusion treated similarly was used as a blank before taking the measurement. All measurements were done in triplicate and averaged.
UV irradiation survival.
Overnight cultures of V. anguillarum grown in TSB at 24 °C with aeration were diluted in the same medium to an OD600 of 0.001 and incubated until an OD600 of 0.5 (5x108 cells ml–1) was reached. Bacteria were diluted in 4 % artificial seawater (Sigma) to a cell density of 1x105 cells ml–1. For the zero time point, c.f.u. were measured from the culture used to inoculate each medium. Bacteria were exposed to shortwave UV light (253.7 nm) for various lengths of time and survival was measured by determining the c.f.u. in each sample. These assays were done at least three times.
RNA isolation and quantification.
Overnight cultures of V. anguillarum grown at 24 °C with aeration were diluted in TSB to an OD600 of 0.001 and incubated until an OD600 of 1.0 was reached. Culture volumes equivalent to 1x108 cells ml–1 were mixed with 2 vols RNAprotect bacteria reagent (Qiagen) to stabilize the RNA transcripts and incubated for 5 min at room temperature. Total RNA was then isolated using the RNeasy minikit (Qiagen). RNA samples were treated with DNA-free DNase (Ambion) and RNA concentrations were then determined using the RiboGreen RNA reagent from Molecular Probes. Both were according to the manufacturers' instructions.
Real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
A one-step real-time qRT-PCR was used to measure the levels of vanT, qrr1 and hpdA mRNA in various V. anguillarum strains. RNA was purified from each strain as described above and 30 ng was used in each RT-PCR reaction. An iCycler iQ (Bio-Rad) and the iScript One-Step RT-PCR kit with SYBR Green (Bio-Rad) were used according to the manufacturer's instructions. To ensure against contaminating chromosomal DNA in the purified RNA, one reaction using purified RNA as template for each sample was done using the iQ SYBR Green Supermix (Bio-Rad) according to the manufacturer's instructions. To ensure that equal amounts of RNA were used, one reaction contained primers for 16S rRNA. RNA samples at each time point were prepared from three separate cultures. Primers used for each gene are as follows: vanT-right (5'-CTTTCGCATGCAAATCAAGA-3') and vanT-left (5'-CCACGCAGATATTGCTGAAA-3'); Qrr1-RT-Fw (5'-AAAGGTCTATTGGCTGTTATTTGTG-3') and Qrr1-RT-Rev (5'-ACCCTTAGGGGTCACCTAG-3'); HpdA-RT-Fw (5'-AGCATTCCTGCGATTTATGG-3') and HpdA-RT-Rev (5'-CGGTCATTCGTTGATTAGCA-3'); 16S-left (5'-CATGCCGCGTGTATGAAGAA-3') and 16S-right (5'-AACAATTATCGTCGTAGTAAACTGC-3'). Calculations for mRNA levels were done according to the standard curve method (Larionov et al., 2005), which normalizes the mRNA levels to that of the 16S mRNA. Each qRT-PCR was done using samples from three independent experiments, the results were averaged, and P-values were determined.
vanT mRNA stability assay.
Overnight cultures of V. anguillarum grown in TSB at 24 °C with aeration were diluted in the same medium to an OD600 of 0.001 and incubated to an OD600 of 0.2 and 1.0. To stop RNA transcription, rifampicin (Sigma-Aldrich) was added to the culture to a final concentration of 200 µg ml–1. For the zero time point, a culture sample (100 µl) was taken before the addition of rifampicin. For mRNA half-life measurements, culture samples (100 µl) were taken at 1, 2, 5 and 10 min after the addition of rifampicin. Total RNA was isolated from each sample and 30 ng RNA was used in a real-time qRT-PCR as described above. For each strain, the zero time point was set to 1.0 and all following time points were normalized to the zero time point. To determine product specificity, standard curves and melting curves were analysed for multiple products. This assay was performed in triplicate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). However, genes regulated by VanT are induced as the cell population enters stationary phase (Croxatto et al., 2002). Thus, we wondered whether VanT protein levels were induced during growth even though it was previously shown that the mRNA levels do not vary greatly. In the wild-type, Western blot analysis (Fig. 2) showed that VanT is detected at a low cell population (OD600 0.007), indicating that the sRNAs do not completely destabilize vanT mRNA. An induction of expression was also seen as the cell population entered stationary phase (OD600 0.4–1.8), after which VanT levels decreased again. To determine if this induction is due to the quorum-sensing systems, VanT expression in a vanO mutant was analysed (Fig. 2). Compared to the wild-type, the vanO mutant showed increased levels of VanT at a low cell population (OD600 0.007–0.4), indicating that the quorum-sensing systems repress VanT expression at low cell densities and the vanO mutation could be complemented with the wild-type gene. However, complete derepression of VanT expression at low cell density did not appear to occur in the vanO mutant and we wondered if an additional regulatory factor may induce the expression of VanT as cell numbers increase.
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; Denkin & Nelson, 2004). Moreover, in E. coli, RpoS is the main regulator of gene expression during stationary phase (for a review see Hengge-Aronis, 2000). The rpoS genetic locus from V. anguillarum was cloned and sequenced. The RpoS amino acid sequence showed 73–86 % identity with RpoS from other Vibrio species in the NCBI database. A mutant (AC12) carrying an in-frame deletion fusing the first 14 amino acids to the last 61 amino acids was made and the mutation was confirmed by Western analysis (data not shown).
To determine if VanT levels are altered in an rpoS mutant, Western analysis of VanT expression was done (Fig. 3a, b). The rpoS mutant did not show the peak of VanT expression during entry into stationary phase that occurred in the wild-type. To establish whether RpoS affects the transcription or translation of vanT, both types of gene fusions were made to a gfp variant that encodes an unstable Gfp protein with a half-life of 80 min in V. anguillarum. Fluorescence was used as a measure of VanT expression throughout growth (Fig. 3c). For the transcriptional fusion, no difference was seen between the wild-type and the rpoS mutant. For the translational fusion, the induction of VanT expression seen during entry into stationary phase in the wild-type was lost in the rpoS mutant. In addition, using real-time qRT-PCR analysis, a decrease in vanT mRNA levels was seen in the rpoS mutant at an OD600 of 1.0 compared to the wild-type (Fig. 3d). For each assay, the loss of VanT expression seen in the rpoS mutant was restored to wild-type levels when the wild-type gene was exchanged for the mutant allele. Interestingly, the increase of VanT expression during late exponential growth correlates with the onset of RpoS expression during growth (Fig. 3a). Together, these data suggest that RpoS indirectly regulates VanT expression via another gene product involved in the post-transcriptional regulation of vanT.
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), VanO is believed to be required for the expression of the sRNAs that destabilize vanT mRNA. However, no difference in VanO levels was seen between the wild-type and the rpoS mutant (Fig. 4a, b). The Western blot analysis does not determine if the activity of VanO is affected. Thus, transcription analysis of an sRNA gene was done to measure the activity of VanO directly. To identify an sRNA gene for these studies, we used the fact that the sRNA Qrr1 (quorum-regulatory RNA) in other Vibrio species is encoded just upstream of luxO; DNA flanking the vanO gene was analysed for qrr1. Similarly in V. anguillarum, a qrr1 homologue with a consensus RpoN (54) binding site in the promoter region was found to be transcribed divergently from vanO (Fig. 1b
). A qrr1 : : gfp transcriptional gene fusion was made and Gfp expression was assayed in the wild-type and in the vanO and rpoN mutants (Fig. 4c). In the wild-type, Gfp was expressed at low cell density and increased in expression during late exponential growth. In the rpoS mutant, Gfp expression from the qrr1 promoter was unchanged compared to that of the wild-type; however, both VanO and RpoN were required for full expression. Real-time qRT-PCR analysis showed that qrr1 encodes a transcript in the wild-type and that the level of qrr1 transcripts decreased in the vanO mutant compared to the wild-type but not in the rpoS mutant (Fig. 4d). Thus, the indirect post-transcriptional regulation of vanT by RpoS is not due to an effect on the expression or activation of VanO and thus expression of Qrr1.
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). Thus, we wondered if RpoS induces expression of VanT by affecting the expression of Hfq. The hfq gene was cloned and the deduced amino acid sequence of Hfq showed 87–90 % identity with Hfq from other Vibrio species in the NCBI database. An hfq : : gfp transcriptional gene fusion was made and Gfp expression was assayed in the wild-type and rpoS mutant (Fig. 5a). In the rpoS mutant, Gfp expression was similar to the wild-type during early growth. At the time point when RpoS is expressed in the wild-type (see Fig. 3), an increase in Gfp expression was seen in the rpoS mutant and Gfp expression remained higher than in the wild-type throughout growth. Western blot analysis showed a similar increase in Hfq levels in the rpoS mutant compared to the wild-type, and exchange of the mutant rpoS gene for the wild-type gene resulted in wild-type Hfq levels (Fig. 5b, c). To determine if an hfq mutation affects VanT expression, an hfq mutant carrying an in-frame deletion fusing the first five amino acids to the last five amino acids was made. In the hfq mutant, VanT expression was derepressed throughout growth when compared to the wild-type (Fig. 5d, e).
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). If an rpoS mutant has increased levels of Hfq, then vanT mRNA would be predicted to be more unstable in this strain than in the wild-type. To test this hypothesis, the stability of vanT mRNA was determined in the wild-type and the rpoS, vanO and hfq mutants at both a low and high cell density. Fig. 6 shows that at both cell densities vanT mRNA is more stable in the hfq mutant than in the wild-type and the vanO mutant and, as expected, in the vanO mutant vanT mRNA is more stable than in the wild-type at low cell densities. However, vanT mRNA in the rpoS mutant, which has increased levels of Hfq, was much less stable than in the wild-type. These data show that Hfq and the Qrr sRNAs destabilize vanT mRNA and that RpoS increases the stability of vanT mRNA by negatively affecting the expression of Hfq, resulting in an increase of VanT expression during late exponential growth.
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). To determine if the vibrio quorum-sensing systems regulate expression of RpoS, Western blot analysis using an antiserum against RpoS was done in the wild-type and the vanO and vanT mutants. In addition, an rpoS : : gfp transcriptional fusion was assayed in the same strains. No difference in RpoS expression was seen in either the vanO or the vanT mutant compared to the wild-type (Fig. 7), indicating that the quorum-sensing systems do not regulate RpoS expression.
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, 2002; Joelsson et al., 2007). RpoS is a major transcriptional regulator essential for adaptation of bacteria to many stress responses during growth (for a review see Hengge-Aronis, 2000). In V. anguillarum, VanT may be part of the RpoS stress response. To test if VanT and RpoS are involved in stress response, the survival of bacterial cells from the wild-type and the rpoS and vanT mutants that were entering stationary phase was measured after exposure to UV irradiation for 45 s. Resistance to UV irradiation, which damages DNA or induces the production of reactive oxygen species, is a likely stress response for many marine vibrios that are exposed to sunlight. Cells from both mutants showed a 100-fold decrease in survival compared to the wild-type and this phenotype could be complemented in the rpoS mutant with the wild-type rpoS gene (Fig. 8a).
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). We asked whether RpoS regulates these genes as well. Using real-time qRT-PCR, a decrease in the hpdA mRNA was seen in the vanT and rpoS mutants as compared to the wild-type (Fig. 8b). Expression of EmpA, which was measured using an empA : : gfp transcriptional fusion, required both RpoS and VanT as no or very little Gfp expression was detected in either of the two mutants (Fig. 8c). Expression of both genes returned to approximately wild-type levels in the rpoS mutant when the mutation was complemented with the wild-type rpoS gene. These data show that RpoS and VanT regulate similar physiological responses. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; McDougald & Kjelleberg, 2006). These quorum-sensing systems tightly repress expression of LuxR-type activators at low cell density. As the bacterial population grows, signal molecules accumulate and repress the phosphorylation relay, resulting in derepressed expression of the LuxR homologues. Previously, we showed that at low cell density, similar quorum-sensing systems in V. anguillarum do not completely repress the mRNA levels of vanT, which encodes a LuxR-type transcriptional activator (Croxatto et al., 2004). This observation led to the suggestion that these regulatory systems limit rather than tightly repress the expression of VanT.
In the present study, we further characterized the expression of VanT in V. anguillarum. As predicted from other vibrio systems, VanO was shown to be required for expression of the sRNA Qrr1 and thus likely activates expression of other qrr genes seen in other Vibrio species but not yet identified in V. anguillarum. Previously, vanT mRNA was shown to be abundant in the wild-type; however, a vanO mutant showed a twofold increase in vanT mRNA, suggesting that repression of VanT expression does occur via the quorum-sensing regulation (Croxatto et al., 2004). Here, vanT mRNA was shown to be more stable in a vanO mutant and significantly more stable in an hfq mutant than in the wild-type. These data suggest that vanT mRNA destabilization occurs via the sRNAs induced by VanO, that sRNAs work with the RNA chaperone Hfq to destabilize vanT mRNA, and that additional unidentified Qrr sRNAs are also likely involved as is the case for V. harveyi and V. cholerae (Lenz et al., 2004; Tu & Bassler, 2007). VanO has less of an effect on destabilizing vanT mRNA than Hfq since it may not be absolutely required for expression of the qrr sRNA genes. A low level of qrr1 expression was seen in the absence of VanO (Fig. 4). Thus, V. anguillarum utilizes the sRNAs induced by VanO to destabilize vanT mRNA; however, repression of VanT expression is not as tight as in other vibrios.
In V. anguillarum, the Qrr sRNAs may be less effective at destabilizing vanT mRNA than Qrr sRNAs in other Vibrio species. One possible reason is that the affinity of the Qrr sRNAs for vanT mRNA is less than that in other vibrios. Moreover, other target mRNAs may exist for the Qrr1 sRNA that are not yet identified and that may be better targets for Qrr1 compared to vanT mRNA. Recently, V. cholerae was shown to have additional target mRNAs other than hapR mRNA that are regulated by the Qrr sRNAs (Hammer & Bassler, 2007). A second explanation may be that the Qrr sRNAs may compete with other sRNAs or RNA-binding proteins for binding to vanT mRNA. One speculation is that since VanU was shown to activate instead of repress VanT expression, VanU may play a pivotal role in VanT expression by creating a balance between repression and activation (Croxatto et al., 2004). To do this, VanU may activate a second response regulator not yet identified that induces the expression of additional sRNAs or an RNA-binding protein that can interfere with Qrr sRNA binding.
This putative balance between repression and activation of VanT expression may easily be influenced by other elements that affect the sRNAs or the response regulator, or possibly by other regulatory elements that act independently of the quorum-sensing phosphorelay mechanism. As shown in this study, RpoS induced post-transcriptionally the expression of VanT at high cell density by negatively affecting Hfq expression. Consequently, vanT mRNA was less stable in an rpoS mutant than in the wild-type (Fig. 6). It is also likely that RpoS may affect vanT expression by other mechanisms not yet characterized. Interestingly, VanO and VanT did not regulate RpoS and RpoS did not regulate VanO, suggesting that RpoS works independently of the quorum-sensing system to regulate VanT. A model for regulation of vanT expression is given in Fig. 9. The role of RpoS in quorum sensing in vibrios differs. In V. harveyi, RpoS is not involved in regulation of luminescence (Lin et al., 2002). In V. vulnificus, RpoS does not affect the expression of SmcR, a LuxR homologue, and vice versa (Jeong et al., 2003). In V. cholerae, RpoS represses LuxO expression during exponential growth, which enhances derepression of HapR expression via the quorum-sensing regulatory systems (Yildiz et al., 2004; Nielsen et al., 2006). In addition, HapR enhances expression of RpoS, suggesting a possible autoregulation loop in this bacterium (Joelsson et al., 2007).
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). A well-studied example of this type of integration is the relationship between RpoS, the global regulator for bacterial adaptation to stress responses (for a review see Hengge-Aronis, 2000), and quorum-sensing regulation in Pseudomonas aeruginosa (Schuster et al., 2004). Transcriptome analyses showed that RpoS regulates, directly or indirectly, more than 40 % of all quorum-sensing-regulated genes. Moreover, the quorum-sensing transcriptional regulators LasR and RhlR weakly induce expression of RpoS and vice versa. Schuster et al. (2004) proposed a model for the interaction between these two global regulatory networks by grouping the regulated genes into classes. Some genes are directly regulated by RpoS but indirectly regulated by quorum sensing, which weakly regulates rpoS. Other genes are directly regulated by quorum sensing and indirectly regulated by RpoS, which weakly regulates lasR and rhlR. However, some genes are directly regulated by both RpoS and quorum sensing. Although not as well studied as in P. aeruginosa, the integration of RpoS regulation with quorum-sensing regulation is seen with other bacteria as well, for instance Burkholderia cepacia (Aguilar et al., 2003), Ralstonia solanacearum (Flavier et al., 1998) and Erwinia carotovora (Mukherjee et al., 2000).
An interesting observation in this study is that Qrr1 expression in V. anguillarum was activated throughout growth and the expression increased during late exponential growth instead of decreasing as was seen for the qrr genes in V. harveyi (Tu & Bassler, 2007). Continual expression of Qrr1 during growth is likely due to VanO since Qrr1 expression required VanO throughout growth. If this is true, it opens up the possibility that VanO is phosphorylated and active at high cell density as well as at low cell density, possibly by a mechanism other than the quorum-sensing systems. Alternatively a more complex regulatory mechanism is likely required for the increase in Qrr1 expression during late exponential growth that involves VanO and possibly another unidentified transcriptional regulator. Further studies are required to determine why Qrr1 expression increases as VanT expression increases and as the bacterial cell population increases.
Taken together these data suggest that VanT expression appears to respond both to the cell population via quorum-sensing regulation and to stress responses via RpoS regulation. In E. coli and P. aeruginosa (Hengge-Aronis, 2000; Schuster et al., 2004) the RpoS induction is suggested to occur during stress responses, such as those to heat, osmotic and oxidative stress, irrespective of the growth phase. VanT and RpoS regulate similar cellular functions, suggesting that VanT is part of the RpoS regulon in V. anguillarum. VanT function can thus be linked to the bacterial stress response and general physiology. Vibrio-specific quorum-sensing systems have previously been suggested to play a role in physiology by regulating starvation adaptation and stress responses (McDougald et al., 2001, 2002). V. anguillarum is widely distributed in the aquatic environment from fresh to deep-sea waters (Urakawa & Rivera, 2006) and forms biofilms on abiotic surfaces in seawater and on fish skin tissue (Tait et al., 2005; Croxatto et al., 2007). Quorum sensing is likely required to coordinate and to perform numerous physiological activities needed for the bacteria to survive as a population in the highly variable aquatic environment.
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ACKNOWLEDGEMENTS |
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Edited by: P. Cornelis
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REFERENCES |
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Received 17 October 2007;
revised 14 December 2007;
accepted 20 December 2007.
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-VanT). As a negative control, the vanT mutant (
