Characterization of a purF operon mutation which affects colicin V production

Abstract

A mini-TnlO-kan insertion mutation identified a gene in the chromosome of Escherichia coli required for colicin V production from plasmid pColV-K30. With the complete restriction map of E. coli, the mutation was rapidly mapped to 50.0 min, within the purF operon. Sequence analysis showed that the insertion occurred in a gene with no previously known function which is located directly upstream of purF. We designated this gene cvpA for colicin V production. The mutant requires adenine for growth, probably because of a polar effect on purF expression. However, an adenine auxotroph showed no defect in colicin V production, suggesting that the cvpA mutation is responsible for the effect on colicin V production. Two possible models of cvpAl allele function are discussed.Often, plasmid-borne genes require chromosomally coded molecules for their expression (7). By searching for mutations in the Escherichia coli chromosome that affect the expression of genes from such extrachromosomal elements, specific host-plasmid interactions can be identified and characterized. Therefore, a general search was initiated to identify chromosomal mutations in E. coli that reduce colicin V production from plasmid pColV-K30.Colicin V is a small extracellular protein toxin which kills sensitive cells by disrupting their membrane potential (26). Colicin V is produced from large, low-copy plasmids and requires four plasmid genes for synthesis, export, and immunity (5, 6). The cvaC gene is the structural gene for colicin V, and cvaA and cvaB are required for processing and export of the toxin through the inner and outer membranes; cvi confers immunity to the host cell. There are several stages at which host factors could play a role in colicin V production, and mutations that alter any of these functions should result in lowered levels of extracellular colicin V.In addition to studying the role of host factors in colicin V production, we also wanted to demonstrate the speed and ease with which mutations generated with transposon insertions can be physically and genetically mapped on the E. coli chromosome. Previously, approximate map positions of insertions could be assigned on the basis of interrupted mating experiments and P1 transductions (10, 13), but considerable work was required to map insertion mutations precisely. In this study, we used physical maps of a cloned insertion mutation to quickly locate the cvpA gene within the E. coli chromosome by using the complete physical restriction map of the E. coli chromosome (9). MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used in this study, their relevant characteristics, and their sources are listed in derivative which contains a TnJO inserted into a region nonessential for colicin V production or plasmid replication.Media and culture conditions. Liquid and solid media were prepared as described by Miller (14), and antibiotics were used at the following concentrations: ampicillin, 150 ,ug/ml; kanamycin, 25 ,ug/ml; nalidixic acid, 20 pg/ml; streptomycin, 100 ,u...

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“…5a). Tn5 inserted in a relatively uncharacterized gene required for colicin V production (ewvC) orthologous to cvpA in Escherichia coli 42 . Colicin V is a secreted peptide antibiotic 43 .…”

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

“…Further downstream of mcmI and mcmA, with opposite transcription polarity, the mcmM gene was found, which encodes a protein with 62 % identity to MceF, a protein involved in maturation of microcin E492 (Lagos et al, 2001). McmM also has some similarity (37 % identity) to the protein CvpA, which is encoded on the E. coli K-12 chromosome and is required in an unknown way for colicin V production from plasmid pColV-K30 (Fath et al, 1989). The microcin determinant ends upstream of mcmM, beginning at bp 10 362; the sequence following the microcin determinant is nearly identical to that of the fim operon in E. coli 4787 O155 : v165 : f165 starting at bp 43 (GenBank accession number U098571).…”

Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter

“…The gene organization and map locations (in minutes) are as follows (2,33): purB, 25.2; purHD, 90.3; purEK, 12.2; cvp purF dedF (11,37), 50.0; purMN, 53.5; purC, 53.3; purL, 55.2. In addition, guaBA at min 53.9 is required for the two-step conversion of IMP, to GMP, and purA at min 95.0 and purB are required for conversion of IMP.to AMP.…”