Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases

ABSTRACT A 7.5-kbp fragment of chromosomal DNA downstream of theVibrio cholerae vibriobactin outer membrane receptor,viuA, and the vibriobactin utilization gene,viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli entmutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coliEntF. A mutant with an in-frame deletion of this gene, namedvibF, was created with classical V. choleraestrain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression ofvibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibFpromoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

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Mutagenesis and Molecular Modeling Reveal Three Key Extracellular Loops of the Membrane Receptor HasR That Are Involved in Hemophore HasA Binding

Journal of Bacteriology ◽

10.1128/jb.00251-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5379-5382 ◽

Cited By ~ 10

Author(s):

Clément Barjon ◽

Karine Wecker ◽

Nadia Izadi-Pruneyre ◽

Philippe Delepelaire

Keyword(s):

Molecular Modeling ◽

Outer Membrane ◽

Three Dimensional ◽

Membrane Receptor ◽

Dimensional Model ◽

Outer Membrane Receptor ◽

Three Dimensional Model ◽

Extracellular Loops

ABSTRACT On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.

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Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa

Microbiology ◽

10.1099/mic.0.28552-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 945-954 ◽

Cited By ~ 19

Author(s):

Páraic Ó Cuív ◽

Paul Clarke ◽

Michael O'Connell

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Membrane Receptor ◽

Outer Membrane Receptor ◽

Significant Similarity ◽

Hydroxamate Siderophores ◽

Insertional Inactivation ◽

Titration Assay

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

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Identification of rhtX and fptX, Novel Genes Encoding Proteins That Show Homology and Function in the Utilization of the Siderophores Rhizobactin 1021 by Sinorhizobium meliloti and Pyochelin by Pseudomonas aeruginosa, Respectively

Journal of Bacteriology ◽

10.1128/jb.186.10.2996-3005.2004 ◽

2004 ◽

Vol 186 (10) ◽

pp. 2996-3005 ◽

Cited By ~ 54

Author(s):

Páraic Ó Cuív ◽

Paul Clarke ◽

Damien Lynch ◽

Michael O'Connell

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Membrane Transport ◽

Sinorhizobium Meliloti ◽

Iron Acquisition ◽

Membrane Receptor ◽

Receptor Protein ◽

Outer Membrane Receptor ◽

E Coli ◽

Genes Encoding

ABSTRACT Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium Sinorhizobium meliloti 2011. A regulon comprising rhtA, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon rhbABCDEF; and rhrA, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of rhbA and named rhtX (for “rhizobactin transport”), which is required, in addition to rhtA, to confer the ability to utilize rhizobactin 1021 on a strain of S. meliloti that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in Escherichia coli via the IutA outer membrane receptor and the FhuCDB inner membrane transport system. E. coli expressing iutA and fhuCDB was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in E. coli. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of Pseudomonas aeruginosa, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named fptX (for “ferripyochelin transport”). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.

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Faculty Opinions recommendation of In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1022893.264661 ◽

2004 ◽

Author(s):

Joanna Goldberg

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Membrane Receptor ◽

Outer Membrane Receptor ◽

Expression Host

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Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters

Journal of Bacteriology ◽

10.1128/jb.00569-08 ◽

2008 ◽

Vol 190 (17) ◽

pp. 5953-5962 ◽

Cited By ~ 15

Author(s):

Alexandra R. Mey ◽

Elizabeth E. Wyckoff ◽

Lindsey A. Hoover ◽

Carolyn R. Fisher ◽

Shelley M. Payne

Keyword(s):

Vibrio Cholerae ◽

Transport System ◽

Ferrous Iron ◽

Iron Uptake ◽

Iron Transport ◽

Growth Enhancement ◽

Outer Membrane Receptor ◽

E Coli

ABSTRACT Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.

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