The Structure, Function, and Origin of the Microcin H47 ATP-Binding Cassette Exporter Indicate Its Relatedness to That of Colicin V

Abstract

Microcin H47, a gene-encoded peptide antibiotic produced by a natural Escherichia coli strain, was shown to be secreted by a three-component ATP-binding cassette exporter which was revealed to be strongly related to that of colicin V. The results of sequence and gene fusion analyses, as well as heterologous complementation assays, are presented.For gram-negative bacteria, several three-component ATPbinding cassette (ABC) exporters dedicated to protein secretion have been described. They are composed of an ABC transporter protein, a second component of the membrane fusion protein (MFP) family, and an outer membrane protein (3,4,21). There is a single peptide described to be secreted by a three-component ABC apparatus: Escherichia coli colicin V (ColV), an antibiotic of the microcin family. Its exporter comprises the ABC protein CvaB, the MFP CvaA, and the outer membrane protein TolC (6). The ColV ABC transporter contains a proteolytic domain, and consistent with this, the ColV precursor bears a double glycine leader peptide which is processed during export (8, 9).In this work, results are presented on the mode of secretion of microcin H47 (MccH47), an E. coli antibiotic peptide. Genes for its synthesis, immunity, and secretion are clustered in a 10-kb DNA segment (Fig. 1A) (5,11,16,17). The secretion function was assigned to the products of two genes, mchE and mchF. In addition, tolC mutants were shown to produce reduced amounts of microcin. It has been proposed that MccH47 is secreted by an ABC exporter, constituted by MchF, MchE, and TolC (5). A DNA segment containing the mchE and mchF genes was sequenced, partly in our laboratory (18) and partly in the DNA Sequencing Core Laboratory Service of the University of Florida. Two open reading frames were found in the positions expected for these genes (Fig. 1B)

The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN

“…sequenced genes, mchE and mchF, encoding the exporter for microcin H47 (Azpiroz et al, 2001), are 98 and 99 % identical to the cvaA and cvaB genes, respectively, from strain DSM 6601. We therefore designate the genes mchE and mchF.…”

“…Partial sequencing of plasmid pEX4 (Azpiroz et al, 2001), which encodes microcin H47, indicated that the high similarity observed for the sequence encoding MchXIBCDEF extended beyond the region deposited in the database. A comparison of the mcmI and mcmA genes on pEX4 to the sequence of strain DSM 6601 revealed several mutations in pEX4 that do not allow the expression of an active microcin M and its immunity protein.…”

Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins

“…One of these microcins (microcin H47) had already been described before for another E. coli strain, whereas the second microcin was unknown so far. The latter has been termed `microcin M´ (M from Mutaflor ® ) by Klaus Hanke and colleagues (Azpiroz et al 2001, Gaggero et al 1993, Patzer et al 2003, Rodriguez et al 1999 (Fig. 1).…”

Involvement of Enterobactin Synthesis Pathway in Production of Microcin H47

“…3 kb where insertion mutations did not significantly affect the antibiosis phenotypes of production or immunity. This DNA segment was thus provisionally called the silent region (2,8,19,20).…”