Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Abstract

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of pHUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.

Isolation and characterization of the haeminbinding proteins from Neisseria meningitidis

“…The identification of two meningococcal surface-exposed iron-regulated haemin-binding proteins in this study is consistent with the supposition that haem-iron uptake may proceed via such a receptormediated process in N. meningitidis. The recent genetic demonstrations that haemin uptake in Yersinia enterocolitica (Stojiljkovic & Hantke, 1992) and in Vibrio cholerae (Henderson & Payne, 1993) requires a haemin-specific iron-regulated outer membrane receptor support this contention. Using a solid-phase dot enzyme binding assay, in which biotinylated human haemoglobin was used to probe iron-limited meningococcal whole-cells and total membranes for haemoglobin-binding activity, a previous investigation reported the presence of an outer membrane haemoglobin-binding protein(s) in N metzingitidis (Lee & Hill, 1772).…”

Vibrio cholerae iron transport: haem transport genes are linked to one of two sets of tonB, exbB, exbD genes

“…In V. cholerae, mutations in the hut genes had less effect on haem transport; haemin utilization was observed in strains that contained chromosomal mutations in hutB or hutC (data not shown) and in DHH11 containing plasmids with mutations in hutB, hutC, or hutD. Only a polar insertion mutation (strain DHH8), that presumably eliminated expression of all three hut genes, eliminated haem transport (Henderson and Payne, 1993). This suggests that there is significant cross-talk among the V. cholerae permeases and that proteins of other transport systems can substitute for components of the haem transport system in part but not in its entirety.…”

“…The diameter of the zone of bacterial growth in the medium around the spot was measured after 15-18 h of incubation. Henderson and Payne (1993) pHUT10::Tn5f Cm R , pHUT10 with a Tn5 insertion in a non-essential region of DNA with respect to TonB-dependent iron transport Henderson and Payne (1993) pHUT7 Cm R , 6.7 kb Sal I-Hin dIII fragment of pHUT10::Tn5 f cloned into the Sal I/Hin dIII sites of pACYC184 For genes within the TonB1 region, the Xho/Sal I fragments of plasmids containing the aphA-3 disruptions were inserted into the Sal I site of pACSac to form pTONB1::aphA-3, pEXBB1:: aphA-3, pEXBD1::aphA-3, pHUTB::aphA-3, pHUTC::aphA-3 and pHUTD::aphA-3. Each of these plasmids was transferred to CA401 by electroporation, and allelic exchange mutants were isolated by selecting for colonies that were Km R Cm S and sucrose resistant (5% w/v sucrose) as described previously (Wyckoff et al, 1997).…”

“…HutA, the haem receptor (Henderson and Payne, 1994a), and ViuA, the vibriobactin receptor Stoebner et al, 1992), are expressed in response to iron limitation. As is the case for bacterial iron transport systems in general, the transcription of the genes encoding these receptors is repressed by the Fur protein in the presence of iron (Bagg and Neilands, 1987;Butterton et al, 1992;Henderson and Payne, 1993).…”

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

“…This bacterium can also use iron contained in heme or hemoglobin and also produces an iron-regulated hemolysin which may intervene in iron acquisition in vivo (51,80,81,(137)(138)(139). V. cholerae can use ferri-ferrichrome as well (95).…”