Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution.

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.

Two ABC transporter systems participate in siderophore transport in the marine pathogen Vibrio anguillarum 775 (pJM1).

ORF40 (named fatE) in the Vibrio anguillarum pJM1 plasmid-encoding anguibactin iron transport systems is a homolog of ATPase genes involved in ferric-siderophore transport. Mutation of fatE did not affect ferric-anguibactin transport, indicating that there must be other ATPase gene(s) in addition to fatE. By searching the genomic sequence of V. anguillarum 775(pJM1), we identified a homolog of fatE named fvtE on chromosome 2. It is of interest that in this locus, we also identified homologs of fatB, fatC, and fatD that we named fvtB, fvtC and fvtD, respectively. The fvtE mutant still showed ferric-anguibactin transport, while the double fatE and fvtE mutation completely abolished the ferric-anguibactin transport indicating that fatE and fvtE are functional ATPase homologs for ferric-anguibactin transport. Furthermore, we demonstrate that fvtB, fvtC, fvtD, and fvtE are essential for ferric-vanchrobactin and ferric-enterobactin transport.

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.

The regulator HlyU, the repeat-in-toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections.

HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

The ttpC gene is contained in two of three TonB systems in the human pathogen Vibrio vulnificus, but only one is active in iron transport and virulence.

The TonB system of proteins is required for the energy-dependent active transport of iron-bound substrates across the outer membrane of gram-negative bacteria. We have identified three TonB systems within the human pathogen Vibrio vulnificus. The TonB1 system contains the TonB1, ExbD1, and ExbB1 proteins, whereas both the TtpC2-TonB2 and TtpC3-TonB3 systems contain an additional fourth protein, TtpC. Here we report that TtpC3, although highly related to TtpC2, is inactive in iron transport, whereas TtpC2 is essential for the function of the TtpC2-TonB2 system in V. vulnificus. This protein, together with TonB2, is absolutely required for both the uptake of endogenously produced iron-bound siderophores as well as siderophores produced from other organisms. Through complementation we show that V. vulnificus is capable of using different TtpC2 proteins from other Vibrio species to drive the uptake of multiple siderophores. We have also determined that aerobactin, a common bacterial siderophore involved in virulence of enteric bacteria, can only be brought into the cell using the TtpC2-TonB2 system, indicating an important evolutionary adaptation of TtpC2 and TonB2. Furthermore, in the absence of TonB1, TtpC2 is essential for a fully virulent phenotype as demonstrated using 50% lethal dose (LD(50)) experiments in mice.

Secondary structure of antisense RNAß, an internal transcriptional terminator of the plasmid-encoded iron transport-biosynthesis operon of Vibrio anguillarum.

RNAß affects the transcription process of the iron transport-biosynthesis operon encoded in the pJM1 plasmid of Vibrio anguillarum at a stem-loop structure located in the intergenic region between the fatA and angR genes. The net result is a higher level of the fatD, fatC, fatB, and fatA moiety as compared with the longer transcript encoding those genes as well as the angR and angT genes. In this work we report the secondary structure of RNAß determined by treatment with single and double strand specific ribonucleases as well as lead acetate followed by sequencing. The generated in vitro structural data indicated that three of the four previously described loops are in agreement with the original model, however, the alteration of loop IV as well as several other structural differences in the overall shape of the molecule led to the necessity of creating a new in silico model. Using the sites of mutations in the various loops we modeled the change in the RNAß secondary structure induced by those mutations. Mutations of loops III and IV to their complementary bases alter the overall structure of the RNAß significantly and increase its function while mutations in loops I and II have the opposite effect, the structure is unchanged but the activity of RNAß decreases. This indicates that loops I and II are necessary for interaction with the target mRNA. It is possible that the structural rearrangement introduced by mutations in loops III and IV promote activity and binding in loops I and II through reducing steric hindrance or increased binding to the target. This result also indicates that the exact relative positions of the critical loops are unimportant for activity.

The TonB3 system in the human pathogen Vibrio vulnificus is under the control of the global regulators Lrp and cyclic AMP receptor protein.

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

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.

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum.

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.

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.

Identification and characterization of a novel outer membrane protein receptor required for hemin utilization in Vibrio vulnificus.

Vibrio vulnificus, the cause of septicemia and serious wound infection in humans and fishes, require iron for its pathogenesis. Hemin uptake through the outer membrane receptor, HupA, is one of its many mechanisms by which it acquires iron. We report here the identification of an additional TonB-dependent hemin receptor HvtA, that is needed in conjunction with the HupA protein for optimal hemin utilization. The HvtA protein is significantly homologous to other outer membrane hemin receptors and its expression in trans restored the uptake of hemin and hemoglobin, the latter to a weaker extent, in a mutant strain that was defective in both receptors. Quantitative RT-PCR suggested that transcription of the hvtA gene was iron regulated. The operon containing the hvtA gene is homologous to the operon in V. cholerae containing the hemin receptor gene hutR suggesting a vertical transmission of the hvtA cluster from V. cholerae to V. vulnificus.

Homodimerization and binding of specific domains to the target DNA are essential requirements for HlyU to regulate expression of the virulence gene rtxA1, encoding the repeat-in-toxin protein in the human pathogen Vibrio vulnificus.

The virulence gene rtxA1, encoding the repeat-in-toxin protein, plays an essential role in the pathogenesis of Vibrio vulnificus infections. Expression of this gene is controlled by the HlyU regulator by direct contact of the DNA upstream of the rtxA1 toxin operon acting as a derepressor of the H-NS protein. The crystal structure suggests that HlyU forms a homodimer in vitro. However, knowledge of the biological implications of these findings in vivo is limited. In this work, we endeavored to dissect, using genetic and biochemical approaches, the domains of this protein that are essential for homodimer formation and the interaction of HlyU with the target DNA. We identified that residues L18, N22, R25, S54, Q55, L57, W59, R61, K70, and Y77 are essential for the HlyU protein binding to the DNA and that amino acids L17 and L91 are important for HlyU dimerization. We also determined that HlyU homodimer formation is an essential requirement for binding to the upstream region of the rtxA1 operon and is the key feature in relieving the H-NS repression of rtxA1 transcription.

Genetic Determinants of Virulence in the Marine Fish Pathogen Vibrio anguillarum.

One of the most studied fish pathogens is Vibrio anguillarum. Development of the genetics and biochemistry of the mechanisms of virulence in this fish pathogen together with clinical and ecologic studies has permitted the intensive development of microbiology in fish diseases. It is the intention of this review to compile the exhaustive knowledge accumulated on this bacterium and its interaction with the host fish by reporting a complete analysis of the V. anguillarum virulence factors and the genetics of their complexity.

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.

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 O2ß (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.

Power plays: iron transport and energy transduction in pathogenic vibrios.

The Vibrios are a unique group of bacteria inhabiting a vast array of aquatic environments. Many Vibrio species are capable of infecting a wide assortment of hosts. Some of these species include V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. anguillarum, and V. cholerae. The ability of these organisms to utilize iron is essential in establishing both an infection in their hosts as well as surviving in the environment. Bacteria are able to sequester iron through the secretion of low molecular weight iron chelators termed siderophores. The iron-siderophore complexes are bound by specific outer membrane receptors and are brought through both the outer and inner membranes of the cell. The energy needed to drive this active transport is achieved through the TonB energy transduction system. When first elucidated in E. coli, the TonB system was shown to be a three protein complex consisting of TonB, ExbB and ExbD. Most Vibrio species carry two TonB systems. The second TonB system includes a fourth protein; TtpC, which is essential for TonB2 mediated iron transport. Some Vibrio species have been shown to carry a third TonB system that also includes a TtpC protein.

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.

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775.

Anguibactin, the siderophore produced by Vibrio anguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

The TonB energy transduction systems in Vibrio species.

Studying the organization and conservation of the TonB systems across the genus Vibrio, we can tease out trends in gene arrangement and function that lead to clues about the evolution and necessity of the proteins in multiple TonB systems. The TonB2 systems, with additional TtpC proteins, are in general more promiscuous regarding their interactions with many different TonB-dependent transporters in the outer membrane. Studies show that the TtpC protein spans the periplasmic space, suggesting that it can be the connection between the energy from the proton motive force and the outer membrane protein receptors, which the shorter TonB2 cannot provide. As an earlier system, the combination of the TtpC protein and a TonB2 system must have been necessary for the function of the smaller TonB2 protein and to transduce energy in a medium that can have osmotic challenges.

Role of the pJM1 plasmid-encoded transport proteins FatB, C and D in ferric anguibactin uptake in the fish pathogen Vibrio anguillarum.

Vibrio anguillarum serotype O1 is part of the natural flora in the aquatic habitat, but under certain circumstances it can cause terminal haemorrhagic septicemia in marine and fresh water fish due to the action of the anguibactin iron uptake system encoded by the virulence plasmid pJM1. This plasmid harbours the genes for the biosynthesis of the siderophore anguibactin and the ferric anguibactin transport proteins FatD, C, B and A encoded in the iron transport operon. The FatA protein is the outer membrane receptor for the ferric siderophore complex and the FatB lipoprotein provides the periplasmic domain for its internalization, whereas the FatC and D proteins are located in the cytoplasmic membrane and might play a role as part of the ABC transporter for internalization of the ferric siderophore. In this work we demonstrate the essential role of these two inner membrane proteins in ferric anguibactin transport and that the lipo-protein nature of FatB is not necessary for ferric anguibactin transport.

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 HlyU's higher affinity as compared with H-NS resulting in the de-repression and a corresponding increased expression of the rtxA1 operon.