| Carboxyethylarginine synthase (CeaS) catalyzes the first reaction in the partially shared biosynthetic pathway leading to clavulanic acid and the 5S clavams in Streptomyces clavuligerus . Using Southern analysis, a second gene (ceaS1) encoding CeaS was isolated, which is 73% identical at the nucleotide level to the original ceaS2 gene from the clavulanic acid gene cluster. Along with c7p and skn, which encode a putative transcriptional regulator and sensor kinase, respectively, ceaS1 is located next to other paralogous genes, which are also involved in the early stages of clavulanic acid and 5S clavam metabolite biosynthesis. This group of genes comprising ceaS1 was designated as the paralogue gene cluster to distinguish it from the clavulanic acid and clavam gene clusters, which are also involved in the biosynthesis of the respective metabolites. Chromosome walking and Southern analysis of macro-restriction fragments of genomic DNA separated by pulsed field gel electrophoresis indicated that the above three genes clusters are not physically linked.; Single mutants defective in ceaS1 and ceaS2 were prepared, and were found to be affected to varying degrees in their ability to produce clavulanic acid and the 5S clavams, whereas the ceaS1/ceaS2 double mutant was completely blocked in the biosynthesis of the respective metabolites. In addition, preparation and analysis of mutant strains defective in c7p and skn revealed that they did not produce any detectable levels of the 5S clavams, but were unaffected in clavulanic acid production.; The nutritional regulation of ceaS1 and cea S2 expression was analyzed and it was found that ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starch asparagine media. As well, ceaS1 transcription was not affected in a mutant defective in the regulatory protein CcaR, whereas that of ceaS2 was greatly reduced in the same mutant when compared to the wild type strain.; Overall, results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and despite the structural and biosynthetic relatedness of clavulanic acid and the 5 S clavams, the genes involved in their production reside in three unlinked gene clusters. |
