Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing.

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.

Genetic organization of the region encoding regulation, biosynthesis, and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti.

Eight genes have been identified that function in the regulation, biosynthesis, and transport of rhizobactin 1021, a hydroxamate siderophore produced under iron stress by Sinorhizobium meliloti. The genes were sequenced, and transposon insertion mutants were constructed for phenotypic analysis. Six of the genes, named rhbABCDEF, function in the biosynthesis of the siderophore and were shown to constitute an operon that is repressed under iron-replete conditions. Another gene in the cluster, named rhtA, encodes the outer membrane receptor protein for rhizobactin 1021. It was shown to be regulated by iron and to encode a product having 61% similarity to IutA, the outer membrane receptor for aerobactin. Transcription of both the rhbABCDEF operon and the rhtA gene was found to be positively regulated by the product of the eighth gene in the cluster, named rhrA, which has characteristics of an AraC-type transcriptional activator. The six genes in the rhbABCDEF operon have interesting gene junctions with short base overlaps existing between the genes. Similarities between the protein products of the biosynthesis genes and other proteins suggest that rhizobactin 1021 is synthesized by the formation of a novel siderophore precursor, 1,3-diaminopropane, which is then modified and attached to citrate in steps resembling those of the aerobactin biosynthetic pathway. The cluster of genes is located on the pSyma megaplasmid of S. meliloti 2011. Reverse transcription-PCR with RNA isolated from mature alfalfa nodules yielded no products for rhbF or rhtA at a time when the nifH gene was strongly expressed, indicating that siderophore biosynthesis and transport genes are not strongly expressed when nitrogenase is being formed in root nodules. Mutants having transposon insertions in the biosynthesis or transport genes induced effective nitrogen-fixing nodules on alfalfa plants.

The overlapping angB and angG genes are encoded within the trans-acting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis.

Products encoded in the trans-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of Vibrio anguillarum. Here we identify angB, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by angB and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2, 3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of various angB mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designated angG, also has an essential biosynthetic function. The angG gene directs the expression of three polypeptides when overexpressed in Escherichia coli, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the angB and angG genes.

Characterization of the DNA- and metal-binding properties of Vibrio anguillarum fur reveals conservation of a structural Zn(2+) ion.

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn(2+), Co(2+), Cd(2+), and to a lesser extent Ni(2+) but, unlike E. coli Fur, not in the presence of Zn(2+). We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.

Characterization of the angR gene of Vibrio anguillarum: essential role in virulence.

The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum. Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system. AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin. Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen. In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics. In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives. Both virulence and anguibactin production were dramatically affected by each one of the angR modifications. In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases. The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V. anguillarum. We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression.

Fur and iron transport proteins in the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius.

The Brazilian purpuric fever (BPF) clone of Haemophilus influenzae biogroup aegyptius causes a fatal septicaemic disease, resembling fulminant meningococcal sepsis, in children. When isolate F3031 was grown under iron-limiting conditions, the presence of several iron-regulated proteins of 38-110 kDa was revealed by electrophoretic analysis and a Fur homologue was shown by immunoblotting. Dot-blot assays and immunoblotting indicated that BPF cells bound human transferrin and contained transferrin-binding proteins in the outer membrane. However, the binding activity and the biosynthesis of these proteins were detected even under iron-rich conditions. Immunoblot analysis demonstrated the presence of a periplasmic protein related to the ferric iron-binding protein A (FbpA), the major iron-binding protein described in Neisseria spp. However, the FbpA homologue in strain F3031 was constitutively expressed and was smaller than the periplasmic protein detected in H. influenzae type b strain Eagan. The periplasm of strain F3031 also contained a protein related to the Streptococcus parasanguis FimA protein which recently has been shown to be involved in iron acquisition in Yersinia pestis. Although the Eagan and F3031 FimA homologues had a similar mol. wt, of 31 kDa, the expression of the BPF fimA-like gene was not regulated by the iron concentration of the culture medium.

Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds.

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

Characterization of the interaction between Fur and the iron transport promoter of the virulence plasmid in Vibrio anguillarum.

The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNAalpha. Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V. anguillarum Fur protein and this promoter. The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain. Gel retardation and DNase I footprint analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hypersensitivity to the nuclease in the nontemplate strand. The periodicity of the hypersensitive sites suggested that the promoter DNA undergoes a structural change upon binding to Fur, which might play a role in the repression of gene expression.

Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.

Iron is an essential element for nearly all living cells. Thus, the ability of bacteria to utilize iron is a crucial survival mechanism independent of the ecological niche in which the microorganism lives, because iron is scarce both in potential biological hosts, where it is bound by high-affinity iron-binding proteins, and in the environment, where it is present as part of insoluble complex hydroxides. Therefore, pathogens attempting to establish an infection and environmental microorganisms must all be able to utilize the otherwise unavailable iron. One of the strategies to perform this task is the possession of siderophore-mediated iron uptake systems that are capable of scavenging the hoarded iron. This metal is, however, a double-edged sword for the cell because it can catalyze the production of deadly free hydroxyl radicals, which are harmful to the cells. It is therefore imperative for the cell to control the concentration of iron at levels that permit key metabolic steps to occur without becoming a messenger of cell death. Early work identified a repressor, Fur, which as a complex with iron repressed the expression of most iron uptake systems as well as other iron-regulated genes when the iron concentration reached a certain level. However, later work demonstrated that this regulation by Fur was not the only answer under low-iron conditions, there was a need for activation of iron uptake genes as well as siderophore biosynthetic genes. Furthermore, it was also realized that in some instances the actual ferric iron-siderophore complex induced the transcription of the cognate receptor and transport genes. It became evident that control of the expression of iron-regulated genes was more complex than originally envisioned. In this review, I analyze the processes of signal transduction, transcriptional control, and posttranscriptional control of iron-regulated genes as reported for the ferric dicitrate system in Escherichia coli; the pyochelin, pyoverdin, and enterobactin systems in Pseudomonas species; the irgB system in Vibrio cholerae; and the plasmid-mediated anguibactin system in Vibrio anguillarum. I hope that by using these diverse paradigms, I will be able to convey a unifying picture of these mechanism and their importance in the maintenance and prosperity of bacteria within their ecological niches.

The AngR protein and the siderophore anguibactin positively regulate the expression of iron-transport genes in Vibrio anguillarum.

Vibrio anguillarum virulence is associated with the presence of a plasmid-mediated iron-uptake system expressed under iron-limiting conditions, which consists of the siderophore anguibactin and specific iron-transport proteins. This system is maximally expressed under iron-limiting conditions and requires the AngR protein that acts as a positive regulator of anguibactin biosynthesis and also possess an EntE-like enzymatic function that may play a role in anguibactin biosynthesis. In this work, we demonstrate that in addition to possessing these functions related to anguibactin production, AngR also positively regulated transcription of the iron-transport genes fatA and fatB. We also show that transcription of angR is repressed by Fur under iron-rich conditions. In addition, we present evidence that anguibactin itself enhanced transcription of the iron-transport genes fatA and fatB, independently of AngR and the trans-acting factor (TAF) product(s). The presence of either AngR (together with the TAF product(s)) or anguibactin alone led to a partial level of expression of the iron-transport genes fatA and fatB, while full expression is achieved when AngR, the TAF products and anguibactin are all present.

Antisense RNA, fur, iron, and the regulation of iron transport genes in Vibrio anguillarum.

The negative regulation of the expression of iron transport genes fatA and fatB in Vibrio anguillarum is mediated by a chromosome-encoded Fur protein and a plasmid pJM1-derived antisense RNA (RNAalpha), which is preferentially expressed under iron-rich conditions. In this work, we characterized the RNAalpha promoter region, and by using promoter fusion and rifampicin experiments we were able to demonstrate that iron regulates RNAalpha synthesis posttranscriptionally by stabilizing RNAalpha half-life rather than enhancing transcription initiation. The Fur protein is also essential for RNAalpha synthesis at the transcription initiation level, independently of the iron status of the cell. From experiments assessing the relative contribution of Fur and RNAalpha, we were able to show that RNAalpha may indeed play an important role on the negative regulation of the expression of the iron transport genes under physiological conditions.

Transcription of the region encoding the ferric dicitrate-transport system in Escherichia coli: similarity between promoters for fecA and for extracytoplasmic function sigma factors.

We have analyzed the molecular mechanism of regulation of the ferric dicitrate transport system in Escherichia coli (Ec), by studying the transcription of the regulatory and structural genes under various environmental conditions, and by determining the location of their transcriptional start points and promoter regions. We report here that the main species observed in Northern hybridization analyses were a 2.5-kb mRNA, encoded by the outer membrane protein receptor gene fecA, and a 1.5-kb mRNA encoded by a region including the fecIR genes. The synthesis of the 2.5-kb fecA mRNA is regulated by both citrate and iron. Furthermore, transcription of fecA is dependent on the presence of FecI. The promoter region for the fecA mRNA, a likely site of action for FecI, is not related to the consensus promoter region for sigma 70 RNA polymerase in Ec K-12. However, it shows greatest similarity with promoters of genes regulated by a new sub-family of sigma factors, i.e., the extracytoplasmic function (ECF) sigma factors, which are associated with the expression of genes involved in extracytoplasmic functions, suggesting that FecI may act as a specialized sigma factor. We also show that the fecB,C,D,E transport genes are linked in operon fashion to fecA. Since the levels of the fecB,C,D,E RNAs are extremely low, as compared to the level of fecA mRNA, it is likely that processing from the 3' end must occur and stop near the end of fecA where a hairpin structure is located.

Antisense RNA regulation of the fatB iron transport protein gene in Vibrio anguillarum.

We have recently identified an antisense RNA (RNA alpha) that regulates the expression of the fatA iron transport gene encoding the outer membrane receptor for the iron-anguibactin complex. In this work, we demonstrate that RNA alpha also inhibits the expression of fatB, which encodes a 35 kDa iron transport protein and has domains homologous to other periplasmic transport proteins. The expression of fatA and fatB is repressed under iron-rich conditions, in which RNA alpha is induced. RNA alpha is homologous to two-thirds of the coding region of fatB. By cloning RNA alpha coding sequences immediately downstream of a tet promoter, we were able to obtain constitutive expression of the antisense RNA. The cloned region contains approximately 83% of the 650 nucleotide RNA alpha and is complementary to only 51% of the fatB mRNA but is still capable of causing a repression of the expression of the fatB gene. Our results in this work demonstrate that RNA alpha probably affects the stability of the fatB-specific mRNA.

Regulation of angR, a gene with regulatory and biosynthetic functions in the pJM1 plasmid-mediated iron uptake system of Vibrio anguillarum.

AngR and the product(s) encoded in the trans-acting factor (TAF) region are necessary for the full expression of the pJM1 plasmid-mediated anguibactin iron-uptake system in Vibrio anguillarum (Va). In this report, we analyzed the factors that affect the expression of the angR gene. Northern blot analysis showed that angR encodes a 3.1-kb transcript which is expressed only under iron-limiting conditions. Measurement of steady-state RNA levels show that, under iron-limiting conditions, angR is positively regulated at the transcriptional level by product(s) of the Va TAF region. However, this enhancement of angR expression by TAF does not occur at high levels of the AngR protein, as assessed by using an angR::lacZ fusion in the presence of a construct containing angR under the control of ptac. We also report that repression of angR by iron could possibly be mediated by an endogenous Va antisense RNA beta, which contains a stem-loop structure complementary to the stem-loop structure located at the 5' end of angR.

Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid.

The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin. Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore. In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport. By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations. Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region. FatB, the product of the fatB gene, is isolated with the membrane fraction. In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence. The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein.

Iron transport genes of the pJM1-mediated iron uptake system of Vibrio anguillarum are included in a transposonlike structure.

The pJM1 genes encoding the proteins involved in iron transport in the anguibactin iron uptake system were found to be flanked by insertion sequences in a composite transposonlike structure. These Vibrio anguillarum insertion sequences, ISV-A1 and ISV-A2, are related to IS903, IS102, and the ISVs found in Vibrio parahaemoliticus, Vibrio mimicus, and non-O1 Vibrio cholerae flanking various tdh (thermostable direct hemolysin) genes. The inverted repeats at the ends of ISV-A1 and ISV-A2 have no more than three mismatches when compared to the inverted repeats of the other ISVs or IS903 and IS102. ISV-A1 and ISV-A2 are flanked by 9-bp direct repeats, which is the number of bases that are duplicated upon IS903 or IS102 transposition. The similarities found between the V. anguillarum ISVs and the other ISVs as well as IS903 and IS102 suggest that they derive from a common ancestral insertion sequence. At the end of ISV-A1 there is a -35 sequence region followed by a -10 sequence found in the pJM1 sequence immediately outside the ISV. This promoter region is followed by an open reading frame with the potential to encode a polypeptide of 26,985 Da whose function is still unknown. The functionality of this promoter has been demonstrated and expression analysis showed that the promoter is regulated by the iron concentration of the media.

A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1 is essential for virulence: histamine is a precursor in the biosynthesis of anguibactin.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJM1 which encodes a histidine decarboxylase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44,259.69 Da. The amino acid sequence has extensive homology with the pyridoxal-P-dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3-HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJM1 iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A. V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron-limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional analyses of mutant Fur proteins with impaired regulatory function.

Vibrio anguillarum Fur mutants, 775met9 and 775met11, were characterized. V. anguillarum 775met9 had a change of D to G at position 104 located in the carboxy terminus resulting in impaired Fur activity. Computer analysis predicts perturbation of an alpha-helix in the carboxy terminus which may interfere with Fur protein conformation. Strain 775met11 had a change in the start codon resulting in no protein synthesis. The mutants are unstable, and reversion to the wild type occurs frequently.

Chromosome-mediated 2,3-dihydroxybenzoic acid is a precursor in the biosynthesis of the plasmid-mediated siderophore anguibactin in Vibrio anguillarum.

We have isolated a recombinant clone harboring the chromosomal aroC gene, encoding chorismate synthase, from Vibrio anguillarum 775 by complementation of the Escherichia coli aroC mutant AB2849 which was transfected with a cosmid gene bank of the plasmidless V. anguillarum H775-3. The nucleotide sequence was determined, and an open reading frame that corresponds to a protein of 372 amino acids was found. The calculated mass of 40,417 Da was correlated with the size of the V. anguillarum aroC product detected in vitro. The homology of the V. anguillarum aroC gene to the aroC genes of E. coli and Salmonella typhi is 68% at the nucleotide level and 78% at the protein level. The expression of the aroC transcript is not regulated by iron, as determined by Northern (RNA) blot hybridization analysis. After insertion of an antibiotic resistance gene cassette within the cloned aroC gene, an aroC mutant of V. anguillarum was generated by allelic exchange. This mutant is deficient in the production of 2,3-dihydroxybenzoic acid (2,3-DHBA). Our bioassay and complementation experiments with this mutant demonstrate that the chromosome-mediated 2,3-DHBA is a precursor of the pJM1 plasmid-mediated siderophore anguibactin.

Localization of the replication region of the pMJ101 plasmid from Vibrio ordalii.

The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far. The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants. Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions. However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein. Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein. The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E. coli strain. Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M. Couturier et al. (Microbiol. Rev. 52, 375-395, 1988).