Transcriptional and translational analysis of the ccaR gene from Streptomyces clavuligerus

Abstract

CcaR is a positive-acting transcriptional regulator involved in cephamycin C and clavulanic acid biosynthesis in Streptomyces clavuligerus. Previous sequence analyses of the ccaR 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, ccaR was expressed, either from the GTG or ATG codon, in Escherichia coli. A protein product was only obtained from the ATG construct. Similarly, ccaR constructs originating from ATG or GTG and designed for expression from a glycerol-regulated promoter in Streptomyces species were prepared and used to complement a S. clavuligerus ccaR mutant. Bioassays showed that only the ATG construct could complement the ccaR 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 ccaR from its native promoter also initiated at the ATG rather than GTG, a conservative point mutation was introduced into ccaR, converting the GTG to GTC. The GTC construct still fully complemented a ccaR mutant, confirming that ATG is the true start codon. Inspection of the region upstream of ccaR 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.

pcd Mutants of Streptomyces clavuligerus Still Produce Cephamycin C

“…Oligonucleotide primers Pcd-fwd (5Ј-CGC CGC ACA TCC AAG GCA TAG AGA GCA CAC ACC GTC ATG ATT CCG GGG ATC CGT CGA CC) and Pcd-rev (5Ј-CAC GGG ACG GCG CCC GGC GCC GCG GGC CGG GGC GGA CTA TGT AGG CTG GAG CTG CTT C) were used to amplify a disruption cassette carrying oriT and the aac(3)IV gene flanked by sequences homologous to the 5Ј and 3Ј ends of the pcd gene by use of plasmid pIJ773 as the template. The pcd gene was then exchanged for the disruption cassette within cosmid 2B8 by means of the RED recombination functions carried within E. coli BW25113, and the recombinant cosmid lacking pcd was conjugated into S. clavuligerus as described previously (25), where the entire native pcd gene was replaced by the disruption cassette by homologous recombination.…”

“…The resulting vector, pSET-Neo, was digested with EcoRV, and the 3.2-kb pcd-containing BamHI fragment from pDA109 was inserted after it had been made blunt by treatment with the Klenow fragment of DNA polymerase I. The resulting construct, pSET-Neo-pcd, was then introduced into the pcd::apr mutant by conjugation using established techniques (25).…”

Expression ofccaR, Encoding the Positive Activator of Cephamycin C and Clavulanic Acid Production inStreptomyces clavuligerus, Is Dependent onbldG

“…The BldG primary antibody (9) was used at a dilution of 1 in 10,000, while the CcaR-specific antibody (1) was used at a dilution of 1 in 5,000. Extracts isolated from wild-type S. coelicolor M145 and from the S. coelicolor bldG mutant ⌬bldG 1DB (Table 1) were used as positive and negative controls for BldG detection, respectively, while purified CcaR (56) and extract isolated from a ccaR mutant strain (Table 1) …”

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

“…The ccaR gene product from the cephamycin C biosynthetic gene cluster, which activates the expression of genes involved in cephamycin C biosynthesis (16,19), is similar to strR in streptomycin biosynthesis. We, therefore, examined cephamycin C productivity in S. avermitilis by introducing conjugation of an extra copy of ccaR expressed from the alternative rpsJ promoter.…”