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Antimicrobial Agents and Chemotherapy, March 2001, p. 969-972, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.969-972.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

María F. Azpiroz, Eliana Rodríguez, and Magela Laviña^*

Sección de Fisiología y Genética Bacterianas, Facultad de Ciencias, Montevideo 11.400, Uruguay

Received 22 September 2000/Returned for modification 31 October 2000/Accepted 1 December 2000

	ABSTRACT

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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.

	TEXT

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For gram-negative bacteria, several three-component ATP-binding 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).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   (A) DNA region containing the MccH47 genetic system. The physical map and the mch genes, with their extension and direction of transcription, are shown. The genes mchABCD are involved in MccH47 synthesis, mchI codes for the immunity peptide, and mchX appears to be related to regulatory functions. mchE and mchF are microcin secretion genes. (B) Enlargement of the 4,197-bp sequenced DNA region containing mchEF. The physical map, as well as the extension and direction of transcription of the mch genes, is shown. B, BamHI; E, EcoRI; H, HindIII.

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) (5). Protein homology analysis of the deduced amino acid sequences for MchE and MchF revealed 98 and 89% identity with CvaA and CvaB, respectively, indicating that MchF is an ABC protein and MchE is an MFP (Fig. 2). These results are consistent with the identification of a double glycine leader peptide located in the 15 N-terminal residues of the MccH47 precursor, which would be processed concomitantly with export (9, 17). The alignment of MchE and CvaA included the methionine where a second in-frame shorter protein, CvaA*, and a putative MchE* homologue begin (Fig. 1B and 2A) (6). In fact, two proteins had previously been detected in polyacrylamide gel electrophoresis systems as products of mchE (5).

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence alignments using the program LALIGN (10). The numbers on the right refer to amino acid positions; nonconserved residues are shaded. (A) Alignment of the entire MchE and CvaA sequences. The first residue of MchE* and CvaA* is boxed. (B) Alignment of the entire MchF and CvaB sequences.

The mchE and mchF genes are expressed in the same direction and present a small overlap, identical to that found between cvaA and cvaB, indicating that they are arranged in an operon (Fig. 1B) (6). A previously isolated insertion on mchE, Tnlac 7.1, conferring -galactosidase activity and thus presumed to generate a gene fusion, led us to infer that mchEF were expressed from the right to the left (Table 1) (5). Since the opposite direction was now confirmed, the Tnlac 7.1 insertion site was sequenced, showing that no fusion existed: Tnlac mapped in mchE, with lacZ oriented from right to left.

View this table: [in this window] [in a new window]   TABLE 1.   Reporter enzymatic activities in strains with TnphoA or Tnlac insertions in mchE

New mutagenesis experiments with TnphoA and Tnlac were performed to analyze mchE expression. Strains harboring pMVD14, a pACYC184 derivative plasmid carrying the EcoRI-EcoRI fragment that contains mchE, were mutagenized as described previously (7, 12). Clones bearing mchE-phoA and mchE-lacZ active gene fusions were isolated, and their respective enzymatic activities when grown in Luria-Bertani medium were measured (Table 1) (2, 13). These activities increased from logarithmic to stationary phase, which could be indicative of a growth phase regulation of mchE expression. The junction sites in the mchE-phoA fusions were distributed between codons 54 and 343, a result that indicates a periplasmic MchE segment, in agreement with previously reported data on cvaA-phoA fusions. In both cases, no active fusions with PhoA were isolated at the C-terminal portion of MchE or CvaA (19). On the contrary, two active mchE-lacZ fusions were located near the end of mchE, indicating that their products would reside in the cytoplasm. We propose that these fusions encode two types of hybrid proteins, MchE-LacZ and MchE*-LacZ, and that the latter is responsible for the detected enzymatic activity. MchE* lacks predictable transmembrane segments and thus would be a cytoplasmic protein.

In view of the strong similarities between MchEF and CvaAB, DNA homologies were searched for using the program CLUSTAL W (20). A 92.1% identity was found along the entire coding sequences of mchEF and cvaAB and ceased abruptly in noncoding DNA. Upstream of mchE no counterpart of the Fur box, which is responsible for cvaAB iron regulation, was found (1). When analyzed with the program FASTA (14), sequences downstream of mchF, i.e., beyond the limits of the MccH47 genetic system, exhibited 53.4% identity with cvi and cvaC, the ColV immunity and activity genes, respectively. In fact, two small open reading frames reminiscent of these ColV genes were found (data not shown). However, no antibacterial or immunity function is encoded by these sequences, as saturation mutageneses of the MccH47 system and its surroundings revealed (5, 11). This finding suggests the ancestral occurrence of a ColV genetic system that integrated next to the mch sequences; the determinants for its export apparatus became dedicated to MccH47 secretion, while the ColV activity and immunity genes lost their function.

A heterologous complementation analysis for MccH47 secretion by the ColV exporter was performed. For this purpose, a plasmid carrying the ColV genetic system, pUY270, was constructed by cloning a 7-kb HindIII-BglII fragment from pColV-K270 (15) into HindIII/BamHI-digested pUC13. Six RYC1000 derivative strains, each bearing plasmid pEX100 (containing the MccH47 system) with a different insertion mutation in mchE or mchF, were used. These strains produce MccH47 but are unable to secrete it; therefore, they do not give rise to growth inhibition halos (5). pUY270 was used to transform these strains. In a halo assay performed on minimal M63 glucose medium (13, 17), the transformant clones were found to efficiently produce extracellular MccH47 (Fig. 3). In parallel, they were assayed on a lawn of an MccH47-immune and ColV-sensitive strain, confirming that they also produced ColV (data not shown). In sum, all the experimental clones produced and secreted both antibiotics, clearly revealing that the ColV exporter is competent for recognizing and exporting MccH47 to the extracellular medium in substantial amounts.

View larger version (144K): [in this window] [in a new window]   FIG. 3.   Heterologous complementation analysis for MccH47 secretion. MccH47 production was assayed on a lawn of the MccH47-sensitive ColV-immune indicator strain PAP222 (15). Six different RYC1000 derivative strains were stabbed, all carrying pUY270 and the following pEX100 derivative plasmids: pEX100 mchF::MudI1681 30.9 (stab 1), pEX100 mchF::MudI1681 40.1 (stab 2), pEX100 mchE::MudI1681 30.4 (stab 3), pEX100 mchE::MudI1681 30.5 (stab 4), pEX100 mchE::MudI1681 80.9 (stab 5), and pEX100 mchE::Tnlac 7.1 (stab 6). RYC1000(pUY270) (stab 7) and RYC1000(pEX100) (stab 8), ColV- and MccH47-producing strains, respectively, were also stabbed onto the lawn. The plates were incubated at 37°C for 16 h to detect inhibition halos.

We can conclude that MccH47 is secreted by an ABC export apparatus. The ABC and the second component proteins are encoded by genes belonging to the MccH47 genetic system, while the outer membrane protein would be encoded by the unlinked tolC gene. The MccH47 and ColV exporters were found to be strongly related from the structural and functional points of view. Moreover, DNA sequence homologies revealed that the MccH47 exporter genes most probably derived from those of ColV.

Nucleotide sequence accession number. The sequence of the 4,197-bp EcoRI-BamHI DNA segment containing the mchE and mchF genes has been deposited in the EMBL database under accession number AJ278866.

	ACKNOWLEDGMENTS

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas "Fondo Clemente Estable" grant 4059 and by Programa de Desarrollo de las Ciencias Básicas, Uruguay.

We thank Enrique P. Lessa for helpful discussion. We are also indebted to María Parente for excellent technical assistance.

	FOOTNOTES

^* Corresponding author. Mailing address: Sección de Fisiología y Genética Bacterianas, Facultad de Ciencias, Iguá 4225, Malvín Norte, Montevideo 11.400, Uruguay. Phone: (5982) 525 86 18, ext. 143. Fax: (5982) 525 86 29. E-mail: magela{at}fcien.edu.uy.

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Antimicrobial Agents and Chemotherapy, March 2001, p. 969-972, Vol. 45, No. 3
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.3.969-972.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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