1887

Abstract

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in . Previous sequence analyses of the gene revealed two possible start codons, an ATG, and a GTG located in-frame 18 bp downstream of the ATG. To determine the true start codon, was expressed, either from the GTG or ATG codon, in . A protein product was only obtained from the ATG construct. Similarly, constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in species were prepared and used to complement a mutant. Bioassays showed that only the ATG construct could complement the mutant to restore cephamycin C production, and Western analysis confirmed the presence of CcaR in the mutant complemented with the ATG construct only. To ensure that expression of from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into , converting the GTG to GTC. The GTC construct still fully complemented a mutant, confirming that ATG is the true start codon. Inspection of the region upstream of by S1 nuclease protection and primer extension analyses indicated the presence of two transcript start points that mapped to residues located 74 and 173 bp upstream of the ATG codon.

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2004-12-01
2019-10-21
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