Publications by Year: 1992

Goldberg MB, Boyko SA, Butterton JR, Stoebner JA, Payne SM, Calderwood SB. Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins. Mol Microbiol. 6 (16) :2407-18.Abstract
IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.
Butterton JR, Stoebner JA, Payne SM, Calderwood SB. Cloning, sequencing, and transcriptional regulation of viuA, the gene encoding the ferric vibriobactin receptor of Vibrio cholerae. J Bacteriol. 174 (11) :3729-38.Abstract
A 74-kDa iron-regulated outer membrane protein of Vibrio cholerae acts as the receptor for the V. cholerae iron-siderophore complex, ferric vibriobactin. MBG14, a mutant of V. cholerae 0395 containing a TnphoA insertion in a gene designated viuA, lacks this 74-kDa outer membrane protein and is unable to bind or utilize exogenous ferric vibriobactin. Introduction of a plasmid containing the complete viuA coding sequence and 513 bp of upstream DNA into MBG14 restored ferric vibriobactin utilization to the mutant. The DNA insert in this plasmid was sequenced, revealing a single open reading frame of 2,061 bp, encoding a deduced protein of 687 amino acids with a predicted molecular mass of 76,417 Da and a predicted initial signal sequence of 37 amino acids. ViuA showed only weak homology to two iron-regulated outer membrane proteins in Escherichia coli, IutA and FecA. Construction of viuA::TnphoA gene fusions allowed study of the regulation of viuA expression by iron. This regulation in E. coli was dependent on the fur gene. Northern (RNA) blot analysis of RNA from wild-type V. cholerae grown in high- and low-iron media revealed a monocistronic viuA message that was negatively regulated by iron at the transcriptional level. Primer extension analysis identified a single transcriptional start site, located 243 bp above the translational start site. The promoter region of viuA contained two interrupted dyad symmetric nucleotide sequences, overlapping the -10 and -35 boxes, each similar to the E. coli Fur binding consensus sequence. Another iron-regulated gene in V. cholerae that is negatively regulated by fur, irgA, requires a positive transcriptional activator (irgB) for expression. However, a strain of V. cholerae mutant in irgB was unaffected in viuA expression. These studies suggest that there is conserved, global coordinate iron regulation in V. cholerae by fur; additional regulatory factors, superimposed upon the fur system, may provide more precise control of individual iron-regulated genes.
Stoebner JA, Butterton JR, Calderwood SB, Payne SM. Identification of the vibriobactin receptor of Vibrio cholerae. J Bacteriol. 174 (10) :3270-4.Abstract
Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.