The regulatory effect of CcaR activator on the cephamycin C gene cluster of streptomyces clavuligerus

Kurt, Aslıhan
Streptomyces clavuligerus produces industrially important secondary metabolites such as cephamycin C (a β-lactam antibiotic) and clavulanic acid (a potent β-lactamase inhibitor). Cephamycin C is active against penicillin-resistant bacteria due to presence of methoxyl group in C-7 position of cephalosporin nucleus. Clavulanic acid is prescribed in combination with β-lactams for treatment of various bacterial infections. Cephamycin C and clavulanic acid gene clusters form β-lactam supercluster in S. clavuligerus genome. CcaR (Cephamycin C-Clavulanic Acid Regulator), encoded by ccaR, located in cephamycin C gene cluster, is a positive regulator of β-lactam supercluster. Previous studies on cephamycin C gene cluster have used different techniques, such as S1 nuclease (Paradkar et al., 1994), Northern blot (Perez-Llarena et al., 1997), and Western blot (Alexander and Jensen, 1998) to determine expression of cephamycin C genes at mRNA level and to identify their functions at protein level, and they have studied on different parts of the cluster. Hence, a comprehensive study is needed to understand molecular mechanisms of pathway-specific regulation of cephamycin C production by S. clavuligerus. In this study, time-dependent expression levels of cephamycin C gene cluster in a ccaR-disrupted mutant and ccaR-overexpressed recombinant strain of S. clavuligerus as compared to those in the wild strain were analysed by RT-PCR and qRT-PCR. In addition, DNA-binding sequences of CcaR on cephamycin C gene cluster were examined by EMSA. The effect of ccaR disruption and overexpression on cephamycin C and clavulanic acid yields were determined by bioassay and HPLC. Three polycistronic and two monocistronic transcripts were obtained by RT-PCR. CcaR regulation showed its effect on mostly ccaR, lat, cmcI, cefD, blp and cefF expression levels. qRT-PCR data was supported by EMSA showing CcaR binding to lat, cefD–cmcI and ccaR promoters. ccaR overexpression from multi-copy recombinant plasmid resulted in significant increase in cephamycin C and clavulanic acid yields, making the respective recombinant strain as an attractive industrial strain. qRT-PCR data presented herein constitute the first that reveal the effect of CcaR activator on the expression of cephamycin C genes in a time-dependent manner.


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Citation Formats
A. Kurt, “The regulatory effect of CcaR activator on the cephamycin C gene cluster of streptomyces clavuligerus,” Ph.D. - Doctoral Program, Middle East Technical University, 2011.