A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron

Farrell, David H.; Mikesell, Perry; Actis, Luis A.; Crosa, Jorge H.

doi:10.1016/0378-1119(90)90112-5

Cited by 71 publications

(59 citation statements)

References 22 publications

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“…strain M114, and this is discussed below. Significant similarity was also found to a family of enzymes including GrsB (53), TycA (57), SrfA (10), AcvA, PcbA, and PcbB (8,27,40), EntF (37), and AngR (14,52). These enzymes are peptide synthetases, with the last two being involved in siderophore synthesis.…”

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Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.Pseudomonas aeruginosa is an opportunistic pathogen which infects injured, immunodeficient, or otherwise compromised patients. The bacteria secrete a siderophore, pyoverdine, which is likely to play an important role in infection by competing with transferrin for iron in order to overcome the iron-withholding defense mechanism present in mammals (3,11,42). Almost all isolates of P. aeruginosa from infected patients secrete pyoverdine (9,22,29), and it has been shown that pyoverdine is present in the sputa of cystic fibrosis patients infected with P. aeruginosa (21). A mutant of P. aeruginosa which is unable to synthesize pyoverdine showed reduced virulence in an animal model of infection (24).Pyoverdine from P. aeruginosa PAO is a water-soluble, yellow-green fluorescent compound. It consists of a dihydroxyquinoline chromophoric group linked to an eight-residuevia the N-terminal serine, with a small dicarboxylic acid attached to the chromophore. Iron complexation is thought to occur through oxygen atoms present on the dihydroxyquinoline and two hydroxamate units supplied by the L-N 5 -OH-Orn residues. Several Pseudomonas species produce similar compounds, variously called pyoverdines or pseudobactins, all of which have the same dihydroxyquinoline group but differ in the nature of the attached peptide (reviewed in references 1 and 7), and it is likely that all of these are synthesized by similar mechanisms. Synthesis of the chromophoric group begins with condensation of D-tyrosine and L-2,4-diaminobutyric acid (7), with glutamic acid being the precursor of the amide-linked dicarboxylic acid (50). It has been suggested that biosynthesis of the peptide moiety of pyoverdine occurs by a nonribosomal mechanism (30).Little is known about the molecular nature of enzymes involved in the biosynthesis of pyoverdine in P. aeruginosa or any other pseudomonad. Recently, pvdA, which encodes an enzyme (L-ornithine N 5 -oxygenase) responsible for catalyzing the hydroxylation of ornithine, an early step in pyoverdine biosynthesis in P. aeruginosa, has been characterized at the molecular level (55). The pbsC gene, which is involved in synthesis of pseudobactin by Pseudomonas sp. strain M114, has also been seque...

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Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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“…The positive regulation is mediated by the plasmid-encoded 110-kDa AngR protein whose gene lies immediately downstream of fatA and by trans acting factors encoded in a region located noncontiguous to the iron uptake region in the pJM1 plasmid (5,8,9,10). The negative regulation is afforded by the chromosomally encoded Fur protein (11,12) and by an antisense RNA (RNA␣) (13).…”

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Characterization of the Interaction between Fur and the Iron Transport Promoter of the Virulence Plasmid in Vibrio anguillarum

Chai

Welch

Crosa

1998

Journal of Biological Chemistry

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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, RNA␣. 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.

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“…Outer membrane protein pOM2 has been well characterized in vivo as well as in vitro, and all data are consistent with the hypothesis that pOM2 is a surfaceexposed protein that acts as the cell surface receptor for binding of ferric iron-anguibactin complexes (2, 3, 10, 31, 33). The angR region of pJM1 was recently sequenced (16), and pAngR was characterized as an iron-regulated 110-kDa transcriptional activator that is required for iron-regulated expression of pOM2 as well as for iron-regulated expression of anguibactin biosynthetic loci (3,10,16,27,34).Mapping and sequencing studies revealed that the genes encoding p40 and pAngR contain single SalI and PstI recognition sites, respectively (3,16,27), and that the gene encoding pOM2 contains a single StuI site (3) (Fig. 1).…”

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“…Polypeptides p40, pOM2, and pAngR are encoded by contiguous pJM1 DNA regions (Fig. 1), are iron regulated (2,3,10,12,16,27,31,33), and are required for siderophoremediated iron uptake in V. anguillarum 775(pJM1) (9,10). The pJM1 iron transport region encoding these activities has been sequenced, the predicted amino acid sequence of each protein has been determined (3,16), and functional roles in iron uptake have been established for each protein (10).…”

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“…1), are iron regulated (2,3,10,12,16,27,31,33), and are required for siderophoremediated iron uptake in V. anguillarum 775(pJM1) (9,10). The pJM1 iron transport region encoding these activities has been sequenced, the predicted amino acid sequence of each protein has been determined (3,16), and functional roles in iron uptake have been established for each protein (10). The predicted amino acid sequence of p40 suggests that it is a cell membrane-associated polypeptide that, together with outer membrane protein pOM2, is required for transport of ferric iron-anguibactin complexes into the cell (3,10).…”

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Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775

1991

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Insertions were created in three iron uptake genes in plasmid pJMl of Vibrio anguiUarum 775 to assess their in vivo effects on virulence in fish. Insertions that blocked p40, pOM2, and pAngR expression resulted in iron uptake-negative strains and in 4.2 x 105-, 8.8 x 105-, and 2.5 x l05-fold attenuations in virulence, respectively. A strain with an insertion in the pAngR coding region still synthesized significant constitutive levels of the outer membrane protein pOM2 and persisted in fish for at least 14 days postinjection. The results demonstrate a direct relationship between virulence and three pJMl-encoded gene products and also the feasibility of constructing live attenuated strains of V. anguiUarum that might be useful in future vaccines.Vibriosis, caused by the marine bacterium Vibrio anguillarum, is a pervasive killer of marine fishes and has been cited as a worldwide impediment to the successful commercial rearing of salmonid fishes (21). In the case of V. anguillarum 775 and of other V. anguillarum strains isolated worldwide, virulence is strongly correlated with the presence of plasmid pJM1 (8,13,14) or with pJM1-like plasmids (14,30,32,34,37) that encode a low-iron-inducible, highaffinity iron uptake system (1,2,11,12,19,32,34,36,38).Three pJM1-encoded proteins, p40, pOM2, and pAngR, have been shown to be iron regulated and required for iron uptake (3,10,12,29,31), but direct demonstrations that they are also required for virulence of V. anguillarum 775(pJM1) are lacking. This is due in part to the difficulties encountered when attempting to introduce plasmid DNA into V. anguillarum 775(pJM1) in the presence of an apparent pJM1-mediated restriction or exclusion system (6a). Using a restriction-defective recipient strain of V. anguillarum 775(pJM1), we constructed three stable insertion mutations in pJM1 that block expression of p40, pOM2, and pAngR. We assessed the in vivo contribution of each protein to iron uptake and virulence and demonstrated a direct genetic relationship between iron uptake ability, virulence, and the presence of intact p40, pOM2, and pAngR DNA sequences in pJM1.(A preliminary report of these data was presented previously [29a].) Polypeptides p40, pOM2, and pAngR are encoded by contiguous pJM1 DNA regions (Fig. 1), are iron regulated (2,3,10,12,16,27,31,33), and are required for siderophoremediated iron uptake in V. anguillarum 775(pJM1) (9, 10). The pJM1 iron transport region encoding these activities has been sequenced, the predicted amino acid sequence of each protein has been determined (3, 16), and functional roles in iron uptake have been established for each protein (10). The predicted amino acid sequence of p40 suggests that it is a cell membrane-associated polypeptide that, together with outer * Corresponding author. membrane protein pOM2, is required for transport of ferric iron-anguibactin complexes into the cell (3, 10). In vitro expression of p40 DNA results in the biosynthesis of a 40-kDa peptide with an N-terminal hydrophobic region typical of a bacterial signal s...

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A regulatory gene, angR, of the iron uptake system of Vibrio anguillarum: similarity with phage P22 cro and regulation by iron

Cited by 71 publications

References 22 publications

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

Characterization of the Interaction between Fur and the Iron Transport Promoter of the Virulence Plasmid in Vibrio anguillarum

Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775

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