Expression regulation of type III secretion system 2 in Vibrio parahaemolyticus by catabolite activator protein.

Vibrio parahaemolyticus has two sets of type III secretion systems that are major pathogenic factors: T3SS1 (cytotoxicity) and T3SS2 (enterotoxicity). V. parahaemolyticus mainly colonizes the distal small intestine after oral infection and may be exposed to carbon-limiting stress due to the lack of readily available carbohydrates in this environment. Catabolite activator protein (CAP), a transcription factor involved in carbon-limiting metabolism in many Gram-negative bacteria, is well known to be involved in the regulation of the expression of many virulence factors. In this study, we determined the effects of CAP on the expression of T3SSs in this bacterium. Based on a lactate dehydrogenase-based cytotoxicity assay, CAP was found to have a greater contribution to the expression of T3SS2-dependent cytotoxicity than to that of T3SS1. Reverse transcription quantitative PCR revealed decreased expression of many T3SS2-related genes, including vpa1348, in the cap gene deletion mutant compared to the parent strain. CAP was demonstrated to bind near the T-rich elements within the vpa1348 promoter region in an electrophoretic mobility shift assay and DNase I footprinting. CAP also enhanced the expression of vpa1348 in a ß-galactosidase reporter assay. Collectively, these results suggest that CAP is involved in T3SS2-mediated virulence by regulating the expression of vpa1348 in V. parahaemolyticus.

Synthesis of Biosynthetic Intermediates of Vibrioferrin and Enzyme Reactions Using Them as Substrates.

Biosynthetic intermediates of siderophore vibrioferrin (VF), O-citryl-L-serine, 2-aminoethyl citrate, and alanine-2-amidoethyl citrate were respectively synthesized as a mixture of stereoisomers. These compounds were used as substrates for enzyme reactions using recombinant PvsA, PvsB, and PvsE proteins as corresponding enzyme equivalents. The results of our study show that each enzyme reacts with a respective substrate and produces VF along the proposed biosynthetic pathway. Furthermore, the results of this study will contribute to the understanding of VF biosynthetic enzymes and may help in the development of antimicrobial drugs by inhibiting siderophore biosynthetic enzymes.

Analysis of the vibrioferrin biosynthetic pathway of Vibrio parahaemolyticus.

Siderophores are small-molecule iron chelators produced by many microorganisms that capture and uptake iron from the natural environment and host. Their biosynthesis in microorganisms is generally performed using non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) enzymes. Vibrio parahaemolyticus secretes its cognate siderophore vibrioferrin under iron-starvation conditions. Vibrioferrin is a dehydrated condensate composed of α-ketoglutarate, L-alanine, aminoethanol, and citrate, and pvsA (the gene encoding the ATP-grasp enzyme), pvsB (the gene encoding the NIS enzyme), pvsD (the gene encoding the NIS enzyme), and pvsE (the gene encoding decarboxylase) are engaged in its biosynthesis. Here, we elucidated the biosynthetic pathway of vibrioferrin through in vitro enzymatic reactions using recombinant PvsA, PvsB, PvsD, and PvsE proteins. We also found that PvsD condenses L-serine and citrate to generate O-citrylserine, and that PvsE decarboxylates O-citrylserine to form O-citrylaminoethanol. In addition, we showed that O-citrylaminoethanol is converted to alanyl-O-citrylaminoethanol by amidification with L-Ala by PvsA and that alanyl-O-citrylaminoethanol is then converted to vibrioferrin by amidification with α-ketoglutarate by PvsB.

Study on the Use of Ozone Water as a Chemical Decontamination Agent for Antineoplastic Drugs in Clinical Settings.

The exposure of healthcare workers to antineoplastic drugs in hospitals has been recognized to be harmful. To minimize the risk of exposure, the removal of these drugs from work environments, such as compounding facilities, has been recommended. In our previous paper, the degradation and inactivation efficacy of ozone water, which is being introduced into Japanese hospitals as a chemical decontamination agent, was reported for its effects on typical antineoplastic drugs (gemcitabine, irinotecan, paclitaxel). This article aims to further investigate the efficacy of ozone water for eight antineoplastic drugs to clarify its application limitations. A small amount (medicinal ingredient: typically ca. 1.5 µmol) of formulation containing 5-fluorouracil, pemetrexed, cisplatin, oxaliplatin, cyclophosphamide, ifosfamide, doxorubicin, or docetaxel was mixed with 50 mL of ozone water (~8 mg/L), and the resulting solutions were analyzed by high-performance liquid chromatography over time to observe the degradation. Consequently, the ozonation was overall effective for the degradation of the drugs, however this varied depending on the chemical structures of the drugs and additives in their formulations. In addition, after the parent drugs were completely degraded by the ozonation, the degradation mixtures were subjected to 1H nuclear magnetic resonance spectroscopy and evaluated for mutagenicity against Salmonella typhimurium strains and cytotoxicity against human cancer cells. The degradation mixtures of cisplatin and ifosfamide were mutagenic while those of the other drugs were non-mutagenic. Further, the ozonation resulted in clear decreases of cytotoxicity for 5-fluorouracil, oxaliplatin, and doxorubicin, but increases of cytotoxicity for pemetrexed, cisplatin, cyclophosphamide, and ifosfamide. These results suggest that the ozone water should be restrictedly used according to the situation of contamination in clinical settings because the ozonation enhances toxicity depending on the drug even if degradation is achieved.

Degradation and inactivation efficacy of ozone water for antineoplastic drugs in hospital settings.

PURPOSE: Occupational exposure to antineoplastic drugs in hospital settings is recognized to be hazardous, and as such environmental decontamination including degradation and inactivation of such drugs is recommended. To data, although various agents such as oxidants have been reported to be useful for decontamination, simpler, safer, and more convenient methods are required. In this study, the degradation and inactivation efficacy of ozone water, which has newly been introduced for decontamination of antineoplastic drugs in spills, was investigated for formulations of gemcitabine, irinotecan, and paclitaxel. METHODS: Antineoplastic formulations (medicinal ingredient: ∼1.5 µmol) were mixed with 50 mL of ozone water (>4 mg/L). The reactions were monitored by high-performance liquid chromatography, and the degradation mixtures were analyzed by 1H nuclear magnetic resonance spectroscopy in order to obtain the structural information of the degradation products. The formulations of gemcitabine and irinotecan and those degradation mixtures were evaluated for their mutagenicity using the Ames test and cytotoxicity against human cancer cells. RESULTS: gemcitabine and irinotecan were found to be readily degraded by the ozone treatment, and their active sites were suggested to be degraded. In contrast, paclitaxel was hard to be decomposed, possibly owing to the consumption of ozone by the polyoxyethylene castor oil added as a pharmaceutical additive of the formulation. No significant mutagenic changes of Salmonella typhimurium strains used for the Ames test were observed for the samples within the concentration ranges examined. The ozone treatment showed obvious increases in cell viability for gemcitabine formulation, and mild increases for irinotecan formulation. CONCLUSIONS: Ozone water was shown to be effective as a decomposition agent for the antineoplastic drug formulations examined, although the efficacy depends on the chemical structures of the drugs and the pharmaceutical additives. It was also suggested that ozone treatment has a tendency to decrease the toxicity of the antineoplastic drug formulations. As such, further studies are required in order to clarify the effects and application limitations of ozone water.

Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Binding of AraC-Type Activator DesR to the Promoter Region of Vibrio vulnificus Ferrioxamine B Receptor Gene.

Vibrio vulnificus can utilize the xenosiderophore desferrioxamine B (DFOB) as an iron source under iron-restricted conditions. We previously identified in V. vulnificus that transcription of the desA gene encoding the outer membrane receptor for ferrioxamine B (FOXB) is activated by the AraC-type transcriptional regulator encoded by desR together with DFOB. In this study, we overexpressed and purified DesR as a glutathione S-transferase-fused protein and examined interaction between the promoter region of desA and DesR. Electrophoretic mobility shift assay (EMSA) revealed that DesR directly binds to the regulatory region of desA, and this binding was enhanced by the presence of DFOB in a concentration-dependent manner, while the presence of FOXB did not affect the potentiation of their binding. Moreover, EMSA identified that DNA fragments lacking a probable DesR binding sequence were unable to form complexes with DesR. Finally, deoxyribonuclease I footprinting assay demonstrated that the DNA binding sequence of DesR is located between -27 and -50 nucleotides upstream of the desA transcription start site. These results strongly indicate that DesR can directly activate the transcription of desA in cooperation with DFOB, which acts as a coactivator for DesR.

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Transcriptional regulation of the ferric aerobactin receptor gene by a GntR-like repressor IutR in Vibrio furnissii.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

Lack of epithelial PPARγ causes cystic adenomatoid malformations in mouse fetal lung.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.

IutB participates in the ferric-vulnibactin utilization system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

Regulation of the Expression of Iron-acquisition System Genes in Pathogenic Vibrio Species.

The genus Vibrio includes >70 species, of which roughly a dozen cause vibriosis such as gastroenteritis, wound infections, and septicemia. Most bacteria, including Vibrio species, require iron for survival and growth. However, the bioavailability of iron is extremely low because it is usually present as an insoluble ferric complex in an aerobic environment or is bound to iron-binding proteins in mammalian hosts. Therefore many bacteria have developed iron acquisition systems, including biosynthesis and secretion of low-molecular-mass iron-chelating compounds called siderophores, and uptake of iron-bound siderophores into bacterial cells through specific active transport systems. Vibrio parahaemolyticus, a major pathogenic Vibrio species, contains multiple iron-acquisition systems mediated by its own siderophore vibrioferrin and several xenosiderophores produced by other microorganisms. In this review, I have focused on the transcriptional and posttranscriptional regulation of genes encoding iron acquisition systems in V. parahaemolyticus. All genes involved in its iron acquisition systems are repressed by Fur, which acts as a ferrous-dependent transcriptional repressor. Furthermore, the stability of polycistronic mRNA involved in vibrioferrin biosynthesis is positively regulated by a small RNA, RyhB, which is repressed by Fur. Expression of PeuA receptor required for utilization of a xenosiderophore, enterobactin, occurs under iron-limiting conditions at alkaline pH. PeuA expression is induced by a two-component regulatory system, PeuRS, which enhances expression of an alternative peuA transcript without an intrinsic translation-inhibitory structure in response to changes in alkaline pH.

The small RNA Spot 42 regulates the expression of the type III secretion system 1 (T3SS1) chaperone protein VP1682 in Vibrio parahaemolyticus.

The cytotoxicity of Vibrio parahaemolyticus has been related to the type III secretion system 1 effector protein VP1680, which is secreted and translocated into host cells with the help of the specific chaperone protein, VP1682. This study sought to confirm the in silico analysis, which predicted that a small regulatory RNA (Spot 42) could base pair with the region encompassing the ribosomal-binding site and initiation codon of the vp1682 mRNA. Electrophoresis mobility shift assays indicated that Spot 42 could bind to the vp1682 mRNA with the help of Hfq. Consistent with these results, the translation of the vp1682 mRNA was inhibited when both Hfq and Spot 42 were added to the in vitro translation reaction. The cytotoxic activity against infected Caco-2 cells was significantly increased in the Spot 42 deletion mutant (Δspf) at 4 h after infection as compared with the parental strain. Additionally, we observed that both VP1682 and VP1680 were more highly expressed in Δspf mutants than in the parental strain. These results indicate that Spot 42 post-transcriptionally regulates the expression of VP1682 in V. parahaemolyticus, which contributes to cytotoxicity in vivo.

Identification and characterization of Aeromonas hydrophila genes encoding the outer membrane receptor of ferrioxamine B and an AraC-type transcriptional regulator.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966(T) could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. ß-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

The RND protein is involved in the vulnibactin export system in Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.

Regulation of the expression of the Vibrio parahaemolyticus peuA gene encoding an alternative ferric enterobactin receptor.

A pvsB-vctA-irgA triple deletion mutant of Vibrio parahaemolyticus can utilize enterobactin under iron-limiting conditions by inducing a previously undescribed receptor, PeuA (VPA0150), in response to extracellular alkaline pH and enterobactin. In silico analyses revealed the existence of a two-component regulatory system operon, peuRS, immediately upstream of peuA, which constitutes an operon with the TonB2 system genes. Both the peuRS and peuA-tonB2 operons were found to be upregulated under iron-limiting conditions in a ferric uptake regulator (Fur)-dependent manner. The involvement of peuA and peuRS in enterobactin utilization was analyzed by complementation experiments using deletion mutants. Primer extension analysis indicated that, under iron-limiting conditions, the transcription of peuA was initiated from the +1 site at pH 7.0 and from both the +1 and +39 sites at pH 8.0 in the presence of enterobactin. The +39 transcript was absent from the peuRS deletion mutant. Secondary structure prediction of their 5'-untranslated regions suggested that translation initiation is blocked in the +1 transcript, but not in the +39 transcript. Consistent with this, in vitro translation analysis demonstrated that production of PeuA was determined only by the +39 transcript. These studies establish a novel gene regulation mechanism in which the two-component regulatory system PeuRS enhances expression of the alternative +39 transcript that possesses non-inhibitory structure, allowing the peuA expression to be regulated at the translation stage.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

The Vibrio parahaemolyticus small RNA RyhB promotes production of the siderophore vibrioferrin by stabilizing the polycistronic mRNA.

High-affinity iron acquisition in Vibrio parahaemolyticus is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon (pvsOp) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938-6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the ryhB and hfq deletion mutants, and a TargetRNA search (http://snowwhite.wellesley.edu/targetRNA/index2.html) revealed that the 5'-untranslated region of pvsOp mRNA (pvsOp 5'-UTR) contains a potential base-pairing region required for the formation of the RyhB-pvsOp 5'-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the pvsOp 5'-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of pvsOp mRNA in the ryhB and hfq mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of pvsOp mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the pvsOp 5'-UTR results in an increase in the stability of pvsOp mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target.

Characterization of a gene encoding the outer membrane receptor for ferric enterobactin in Aeromonas hydrophila ATCC 7966(T).

Aeromonas hydrophila ATCC 7966(T) produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966(T) genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS-PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently with the presence of a predicted Fur box in the promoter region.