Vulnificus CMCP6 Research Articles

Functional conservation of specialized ribosomes bearing genome-encoded variant rRNAs in Vibrio species.

Heterogeneity of ribosomal RNA (rRNA) sequences has recently emerged as a mechanism that can lead to subpopulations of specialized ribosomes. Our previous study showed that ribosomes containing highly divergent rRNAs expressed from the rrnI operon (I-ribosomes) can preferentially translate a subset of mRNAs such as hspA and tpiA in the Vibrio vulnificus CMCP6 strain. Here, we explored the functional conservation of I-ribosomes across Vibrio species. Exogenous expression of the rrnI operon in another V. vulnificus strain, MO6-24/O, and in another Vibrio species, V. fischeri (strain MJ11), decreased heat shock susceptibility by upregulating HspA expression. In addition, we provide direct evidence for the preferential synthesis of HspA by I-ribosomes in the V. vulnificus MO6-24/O strain. Furthermore, exogenous expression of rrnI in V. vulnificus MO6-24/O cells led to higher mortality of infected mice when compared to the wild-type (WT) strain and a strain expressing exogenous rrnG, a redundant rRNA gene in the V. vulnificus CMCP6 strain. Our findings suggest that specialized ribosomes bearing heterogeneous rRNAs play a conserved role in translational regulation among Vibrio species. This study shows the functional importance of rRNA heterogeneity in gene expression control by preferential translation of specific mRNAs, providing another layer of specialized ribosome system.

Transcript-specific selective translation by specialized ribosomes bearing genome-encoded heterogeneous rRNAs in V. vulnificus CMCP6.

Ribosomes composed of genome-encoded heterogeneous rRNAs are implicated in the rapid adaptation of bacterial cells to environmental changes. A previous study showed that ribosomes bearing the most heterogeneous rRNAs expressed from the rrnI operon (I-ribosomes) are implicated in the preferential translation of a subset of mRNAs, including hspA and tpiA, in Vibrio vulnificus CMCP6. In this study, we show that HspA nascent peptides were predominantly bound to I-ribosomes. Specifically, I-ribosomes were enriched more than two-fold in ribosomes that were pulled down by immunoprecipitation of HspA peptides compared with the proportion of I-ribosomes in crude ribosomes and ribosomes pulled down by immunoprecipitation of RNA polymerase subunit β peptides in the wild-type (WT) and rrnI-completed strains. Other methods that utilized the incorporation of an affinity tag in 23S rRNA or chimeric rRNA tethering 16S and 23S rRNAs, which generated specialized functional ribosomes in Escherichia coli, did not result in functional I-ribosomes in V. vulnificus CMCP6. This study provides direct evidence of the preferential translation of hspA mRNA by I-ribosomes.

Sewage Promotes Vibrio vulnificus Growth and Alters Gene Transcription in Vibrio vulnificus CMCP6.

ABSTRACTVibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections.IMPORTANCE Vibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.

RpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19.

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The Km and kcat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 μM and 15.0 s-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and Ki values of 5.2 and 2.5 μM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 μM; Ki: 6.0 μM) and mesotrione (IC50: 4.2 μM; Ki: 1.3 μM) and moderate resistance to tembotrione (IC50: 2.5 μM; Ki: 1.0 μM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.

Structural and functional characterization of a modified legionaminic acid involved in glycosylation of a bacterial lipopolysaccharide

Nonulosonic acids (NulOs) are a diverse family of α-keto acid carbohydrates present across all branches of life. Bacteria biosynthesize NulOs among which are several related prokaryotic-specific isomers and one of which, N-acetylneuraminic acid (sialic acid), is common among all vertebrates. Bacteria display various NulO carbohydrates on lipopolysaccharide (LPS), and the identities of these molecules tune host-pathogen recognition mechanisms. The opportunistic bacterial pathogen Vibrio vulnificus possesses the genes for NulO biosynthesis; however, the structures and functions of the V. vulnificus NulO glycan are unknown. Using genetic and chemical approaches, we show here that the major NulO produced by a clinical V. vulnificus strain CMCP6 is 5-N-acetyl-7-N-acetyl-d-alanyl-legionaminic acid (Leg5Ac7AcAla). The CMCP6 strain could catabolize modified legionaminic acid, whereas V. vulnificus strain YJ016 produced but did not catabolize a NulO without the N-acetyl-d-alanyl modification. In silico analysis suggested that Leg5Ac7AcAla biosynthesis follows a noncanonical pathway but appears to be present in several bacterial species. Leg5Ac7AcAla contributed to bacterial outer-membrane integrity, as mutant strains unable to produce or incorporate Leg5Ac7AcAla into the LPS have increased membrane permeability, sensitivity to bile salts and antimicrobial peptides, and defects in biofilm formation. Using the crustacean model, Artemia franciscana, we demonstrate that Leg5Ac7AcAla-deficient bacteria have decreased virulence potential compared with WT. Our data indicate that different V. vulnificus strains produce multiple NulOs and that the modified legionaminic acid Leg5Ac7AcAla plays a critical role in the physiology, survivability, and pathogenicity of V. vulnificus CMCP6.

Phaeobacter inhibens as biocontrol agent against Vibrio vulnificus in oyster models

Molluscan shellfish can cause food borne diseases and here we investigated if addition of Vibrio-antagonising bacteria could reduce Vibrio vulnificus in model oyster systems and prevent its establishment in live animals. Phaeobacter inhibens, which produces an antibacterial compound, tropodithietic acid (TDA), inhibited V. vulnificus as did pure TDA (MIC of 1–3.9 μM). P. inhibens DSM 17395 (at 106 cfu/ml) eradicated 105 cfu/ml V. vulnificus CMCP6 (a rifampicin resistant variant) from a co-culture oyster model system (oyster juice) whereas the pathogen grew to 107 cfu/ml when co-cultured with a TDA negative Phaeobacter mutant. P. inhibens grew well in oyster juice to 108 CFU/ml and sterile filtered samples from these cultures were inhibitory to Vibrio spp. P. inhibens established itself in live European flat oysters (Ostrea edulis) and remained at 105 cfu/g for five days. However, the presence of P. inhibens could not prevent subsequently added V. vulnificus from entering the live animals, likely because of too low levels of the biocontrol strain. Whilst the oyster model studies provided indication that P. inhibens DSM 17395 could be a good candidate as biocontrol agent against V. vulnificus further optimization is need in the actual animal rearing situation.

All Three TonB Systems Are Required for Vibrio vulnificus CMCP6 Tissue Invasiveness by Controlling Flagellum Expression.

TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis.

인체 병원성 비브리오 균주간 유전자 계통의 불일치성 분석

Lateral gene transfer (LGT) of genes from other bacteria into Vibrio cholerae is expectable because of the pronounced natural competence of the bacterium. In this study, quantitative aspects of LGT among the three species of Vibrio pathogenic to humans were characterized. Genome sequences of V. cholerae N16961, V. parahaemolyticus RIMD2210633, V. vulnificus CMCP6, and Escherichia coli K12 substrain MG1655 were analyzed to determine orthologous quartets of protein coding genes present in all four genomes. Phylogenetic analyses on the quartets were conducted to resolve vertical versus lateral patterns of gene polymorphisms based on congruence versus incongruence of phylogenetic trees. About 70% of the quartets could be resolved as either cohesive topology (75%) or LGT tree topologies (25%). The amount of LGT genes in Vibrio spp. appeared to be abnormally high for a genus and comparable to those of families. Patched distributions of LGT from different donors were observed on a chromosome. In the small chromosome of V. cholerae, physical linkages among LGT loci spanned half the length of the chromosome. Either accumulative selection for the donor alleles in LGT or presence of large-scale LGT events was hypothesized. These findings warrant further studies on the nature of donor-specificity of LGT alleles and its influence on evolution of Vibrio virulence to humans.

Integrative genome‐scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery

Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re-sequenced and fully re-annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome-scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost-effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole-cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.

Role of GacA in virulence of Vibrio vulnificus

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.

Expression of Divergent LSU rRNA Genes in the Vibrio vulnificus CMCP6 Genome During Both Infection and Non-Pathogenic Stages

The Vibrio vulnificus CMCP6 genome harbors nine copies of divergent large subunit (LSU) rRNA genes that may express and constitute four kinds of LSU rRNA molecules in a single cell. Primer extension analyses showed that these heterogeneous LSU rRNA transcripts are all expressed and assembled into ribosomes during both infection and nonpathogenic stages. Phylogenetic analyses of the internal transcribed spacer between SSU and LSU genes indicated that rRNA operons of V. vulnificus CMCP6 can be clustered into three distinct groups in rRNA genes of closely related Vibrio species. These findings imply that divergent rRNA genes in V. vulnificus CMCP6 resulted from interspecies recombination events in V. species, while the consequences of expression of heterogeneous rRNA molecules are not clear.

Direct Interaction between Quorum-sensing Regulator SmcR and RNA Polymerase Is Mediated by Integration Host Factor to Activate vvpE Encoding Elastase in Vibrio vulnificus

It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.

Crystal structure of the transcriptional activator HlyU from Vibrio vulnificus CMCP6

HlyU is a transcription factor of the ArsR/SmtB family and activates the expression of the pathogenic Vibrio vulnificus RTX toxin. In contrast to the other metal-responding ArsR/SmtB proteins, HlyU does not sense metal ions. To provide its structural information, we elucidated the crystal structure of HlyU from V. vulnificus CMCP6 (HlyU_Vv). The monomeric HlyU_Vv architecture of five α-helices and two β-strands, some of which constitute a typical DNA-binding winged helix-turn-helix (wHTH) motif, is very similar to that of other transcription regulators. Nonetheless, the homo-dimeric HlyU_Vv structure shows several different, three-dimensional features in the spatial position and the detailed dimeric interaction, which were not observed in the modeling study based on the same protein family and sequence similarity. Structured summaryMINT-7710072, MINT-7710086: HlyU_Vv (uniprotkb:Q8DES3) and HlyU_Vv (uniprotkb:Q8DES3) bind (MI:0407) by X-ray crystallography (MI:0114)

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

A beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of alpha-d-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Galbeta1 --> 3GlcNAc) and galacto-N-biose (GNB) (Galbeta1 --> 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k(cat)/K(m) for LNB was approximately 60 times higher than the k(cat)/K(m) for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/lacto-N-biose I phosphorylases.

On the origin and function of an insertion element VPaI-1 specific to post-1995 pandemic Vibrio parahaemolyticus strains

Since 1995, new virulent strains of Vibrio parahaemolyticus have emerged and spread throughout the world. These "pandemic" strains have four strain specific genomic islands (GIs), which are considered to be potential factors of the pandemicity. We investigated the origin and function of 24 genes in the so-called VPaI-1, one of the four GIs, by searching homologs in various species in Bacteria and Archaea. Of these 24 genes, two are found only in Vibrio vulnificus CMCP6 and Shewanella sp. MR-7. The genomic segment (- 8 kb) encompassing the two genes shows the synteny among the three species. Since many of the Shewanella species can grow at 4 degrees C, these two genes may be candidates of adaptation to temperature stress. Further, we found a candidate for a swarming gene, which is reported as the V. cholerae virulence gene. Based on these findings, we hypothesized the emergence of pandemicity and discuss the mechanism for how these strains spread throughout the world.

Genomic island identification in Vibrio vulnificus reveals significant genome plasticity in this human pathogen

Genomic islands (GIs) are large chromosomal regions present in a subset of bacterial strains that increase the fitness of the organism under specific conditions. We compared the complete genome sequences of two Vibrio vulnificus strains YJ016 and CMCP6 and identified 14 regions (ranging in size from 14 to 117 kb), which had the characteristics of GIs. Bioinformatic analysis of these 14 GI regions identified the presence of phage-like integrase genes, aberrant GC content and genome signature (dinucleotide frequency) within each GI compared with the core genome indicating that these regions were acquired from an anomalous source. We examined the distribution of the nine GIs from strain YJ016 among 27 V. vulnificus isolates and found that most GIs were absent from the majority of these isolates. The chromosomal insertion sites of three GIs were adjacent to tRNA sites, which contained novel horizontally acquired DNA in all six available sequenced Vibrionaceae genomes. Supplementary data are available at Bioinformatics online.

An acquisition account of genomic islands based on genome signature comparisons.

BackgroundRecent analyses of prokaryotic genome sequences have demonstrated the important force horizontal gene transfer constitutes in genome evolution. Horizontally acquired sequences are detectable by, among others, their dinucleotide composition (genome signature) dissimilarity with the host genome. Genomic islands (GIs) comprise important and interesting horizontally transferred sequences, but information about acquisition events or relatedness between GIs is scarce. In Vibrio vulnificus CMCP6, 10 and 11 GIs have previously been identified in the sequenced chromosomes I and II, respectively. We assessed the compositional similarity and putative acquisition account of these GIs using the genome signature. For this analysis we developed a new algorithm, available as a web application.ResultsOf 21 GIs, VvI-1 and VvI-10 of chromosome I have similar genome signatures, and while artificially divided due to a linear annotation, they are adjacent on the circular chromosome and therefore comprise one GI. Similarly, GIs VvI-3 and VvI-4 of chromosome I together with the region between these two islands are compositionally similar, suggesting that they form one GI (making a total of 19 GIs in chromosome I + chromosome II). Cluster analysis assigned the 19 GIs to 11 different branches above our conservative threshold. This suggests a limited number of compositionally similar donors or intragenomic dispersion of ancestral acquisitions. Furthermore, 2 GIs of chromosome II cluster with chromosome I, while none of the 19 GIs group with chromosome II, suggesting an unidirectional dispersal of large anomalous gene clusters from chromosome I to chromosome II.ConclusionFrom the results, we infer 10 compositionally dissimilar donors for 19 GIs in the V. vulnificus CMCP6 genome, including chromosome I donating to chromosome II. This suggests multiple transfer events from individual donor types or from donors with similar genome signatures. Applied to other prokaryotes, this approach may elucidate the acquisition account in their genome sequences, and facilitate donor identification of GIs.

Identification of an AraC-like regulator gene required for induction of the 78-kDa ferrioxamine B receptor in Vibrio vulnificus

We previously reported that the 78-kDa outer membrane receptor for ferrioxamine B is induced in iron-starved Vibrio vulnificus cells when desferrioxamine B was supplied exogenously. Based on its N-terminal amino acid sequence, a candidate gene for the ferrichrome B receptor was detected in the V. vulnificus CMCP6 genomic database. Here, two contiguous genes, named desR and desA, encoding a member of the AraC family of transcriptional activators and the ferrioxamine B receptor, respectively, were cloned from V. vulnificus M2799 and characterized. Primer extension analysis mapped the iron-regulated transcription initiation sites for desR and desA, and demonstrated involvement of desferrioxamine B in the induction of desA transcription. Insertion mutation of desR resulted in no production of DesA under iron-limiting conditions even in the presence of desferrioxamine B. The DesA production under the same conditions was restored to wild-type levels when the desR mutant was complemented with desR in trans. These results suggest that the desR gene is required for desferrioxamine B-inducible production of DesA in iron-starved cells.

Crystal structure of a conserved hypothetical protein TT1751 from Thermus thermophilus HB8.

Crystal structure of a conserved hypothetical protein TT1751 from Thermus thermophilus HB8.