Identification and characterization of a Vibrio mimicus gene encoding the heme/hemoglobin receptor

Abstract

The present authors have previously reported that Vibrio mimicus expresses 77-kDa and 80-kDa outer membrane proteins in response to iron-limited conditions, and that the 77-kDa protein serves as the receptor for ferriaerobactin. In this study, it was found that V. mimicus can use heme and hemoglobin as iron sources. FURTA was then applied to V. mimicus 7PT to obtain candidate gene fragments involved in utilization of heme and hemoglobin. One FURTA-positive clone was shown to contain a partial gene, whose predicted amino acid sequence correlated with the N-terminal amino acid sequence determined for the 80-kDa outer membrane protein and also shared homology with heme/hemoglobin receptors of Gram-negative bacteria. Based on this information, the entire gene (named mhuA),andageneupstreamof mhuA (named mhuB) encoding a LysR family of transcriptional activator, were cloned and analyzed. RNA analysis indicated that mhuA and mhuB are each transcribed from individual Fur-regulated promoters. Moreover, RNA analysis of an mhuB deletion mutant and a promoter reporter assay coupled with β-galactosidase suggested that MhuB could function as an activator for mhuA transcription. Finally, the role of MhuA as the heme/hemoglobin receptor was confirmed by construction of an mhuA deletion mutant and its complemented strain followed by growth assay.

Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria

“…If the cloned DNA fragment on the high copy number plasmid contains a Fur-binding site, then Fur proteins will be titrated away from the promoter of fhuF resulting in derepression of the fhuF::lacZ fusion, which can be qualitatively detected during growth on MacConkey agar plates or quantified by a β-galactosidase assay. This assay is called the Fur titration assay (FURTA) and has been used to study Fur regulation for nearly 20 years (Stojiljkovic et al, 1994; Tsolis et al, 1995; Baumler et al, 1996; Fassbinder et al, 2000; Osorio et al, 2004; Haraszthy et al, 2006; Jackson et al, 2010; Tanabe et al, 2010). In toto , these works solidified the role of Fur as a Fe 2+ -dependent transcriptional repressor.…”

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments

“…Heme receptors in other species include HupA (124), HvtA (125), and HupO in V. fluvialis (126); HuvA in V. anguillarum (127,128); and MhuA in V. mimicus (129). HvtA has 68% similarity and 51% identity to V. cholerae HutR, and the operon organization is the same in the two species, indicating a close genetic relationship (120,125).…”

The Vibrio parahaemolyticuspvuA1 gene (formerly termed psuA) encodes a second ferric vibrioferrin receptor that requires tonB2

“…1b), respectively, was ligated into a broad host-range plasmid, pRK415 (Keen et al, 1988). 1b) to construct VPD8/ pRK415-pvuA1 and VPD8/pRK415-pvuA2, respectively, as described previously (Tanabe et al, 2010). 1c), were each mobilized into VPD8 (Fig.…”

“…The primers used to construct the gene-deletion fragments and confirm gene deletions in various mutants are listed in Table S2. PCR amplicons with the respective deletions in the V. parahaemolyticus pvsB, pvuA1, pvuA2, tonB1, tonB2, and tonB3 genes were prepared by PCR-driven overlap extension as described previously (Heckman & Pease, 2007;Tanabe et al, 2010). Briefly, the upstream and downstream regions of the respective genes were amplified in a reaction with corresponding primer pairs #1 and #2, and #3 and #4 shown in Table S2, respectively.…”