Hiroaki Naka

The HlyU Protein Is a Positive Regulator of rtxA1, a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii

ABSTRACT We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2β (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host “addiction” genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.

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.

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.

HlyU acts as an H‐NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6

In Vibrio vulnificus, HlyU upregulates the expression of the large RTX toxin gene. In this work we identified the binding site of HlyU to −417 to −376 bp of the rtxA1 operon transcription start site. lacZ fusions for a series of progressive deletions from the rtxA1 operon promoter showed that transcriptional activity increased independently of HlyU when its binding site was absent. Thus HlyU must regulate the rtxA1 operon expression by antagonizing a negative regulator. Concomitantly we found that an hns mutant resulted in an increase in the expression of the rtxA1 operon genes. Multiple copies of HlyU can increase the promoter activity only in the presence of H‐NS underscoring the hypothesis that HlyU must alleviate the repression by this protein. H‐NS binds to a region that extends upstream and downstream of the rtxA1 operon promoter. In the upstream region it binds to five AT‐rich sites of which two overlap the HlyU binding site. Competitive footprinting and gel shift data demonstrate HlyUs higher affinity as compared with H‐NS resulting in the de‐repression and a corresponding increased expression of the rtxA1 operon.

Biosynthesis of amphi-enterobactin siderophores by Vibrio harveyi BAA-1116: Identification of a bifunctional nonribosomal peptide synthetase condensation domain

The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for enterobactin, yet enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-enterobactins. The amphi-enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980) - a taxonomic opinion

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

Identification and characterization of a novel outer membrane protein receptor FetA for ferric enterobactin transport in Vibrio anguillarum 775 (pJM1).

In this work we demonstrate the existence in Vibrio anguillarum 775 (pJM1) of two chromosomal genes encoding outer membrane proteins that operate in the transport of ferric enterobactin. One of them is a novel receptor that we named FetA and the other is the already characterized FvtA that functions in the uptake of iron complexes of both enterobactin and vanchrobactin. Ferric enterobactin transport proficiency was resumed in double mutants for these two genes when they were complemented with either fetA or fvtA, whereas only the cloned fvtA could complement for ferric vanchrobactin transport. Quantitative RT-PCR assays demonstrated that transcription of the fetA gene is regulated by FetR, that is encoded upstream and in reverse orientation from fetA. This gene as well as fetA, are up-regulated in iron limiting condition in a Fur-dependent manner. The two divergent promoters are located in the intergenic region between fetR and fetA that has a putative Fur binding site and an IrgB binding site in the overlapping promoters of fetR and fetA. FetA and FetR show high homology to V. cholerae IrgA and IrgB respectively and the intergenic regions fetA–fetR and irgA–irgB are also highly related suggesting a vertical transmission of the fetA–fetR cluster from V. cholerae to V. anguillarum.

Genome Sequence of the Marine Bacterium Vibrio campbellii DS40M4, Isolated from Open Ocean Water

Vibrio sp. strain DS40M4 is a marine bacterium that was isolated from open ocean water. In this work, using genomic taxonomy, we were able to classify this bacterium as V. campbellii. Our genomic analysis revealed that V. campbellii DS40M4 harbors genes related to iron transport, virulence, and environmental fitness, such as those encoding anguibactin and vanchrobactin biosynthesis proteins, type II, III, IV, and VI secretion systems, and proteorhodopsin.

The anguibactin biosynthesis and transport genes are encoded in the chromosome of Vibrio harveyi: a possible evolutionary origin for the pJM1 plasmid–encoded system of Vibrio anguillarum?

Many Vibrio anguillarum serotype O1 strains carry 65‐kb pJM1‐type plasmids harboring genes involved in siderophore anguibactin biosynthesis and transport. The anguibactin system is an essential factor for V. anguillarum to survive under iron‐limiting conditions, and as a consequence, it is a very important virulence factor of this bacterium. Our comparative analysis of genomic data identified a cluster harboring homologs of anguibactin biosynthesis and transport genes in the chromosome of Vibrio harveyi. We have purified the putative anguibactin siderophore and demonstrated that it is indeed anguibactin by mass spectrometry and specific bioassays. Furthermore, we characterized two genes, angR and fatA, in this chromosome cluster that, respectively, participate in anguibactin biosynthesis and transport as determined by mutagenesis analysis. Furthermore, we found that the V. harveyi FatA protein is located in the outer membrane fractions as previously demonstrated in V. anguillarum. Based on our data, we propose that the anguibactin biosynthesis and transport cluster in the V. anguillarum pJM1 plasmid have likely evolved from the chromosome cluster of V. harveyi or vice versa.

Fatty Acid Hydrolysis of Acyl Marinobactin Siderophores by Marinobacter Acylases

The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed.