The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus

Abstract

Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the other clavam metabolites in Streptomyces clavuligerus. The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene (egfp) as a reporter. ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starchasparagine (SA) media. The transcriptional start points of the two genes were also mapped to a C residue 98 bp upstream of ceaS1 and a G residue 51 bp upstream of the ceaS2 start codon by S1 nuclease protection and primer extension analyses. Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase (bls1) and proclavaminate amidinohydrolase (pah1) isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of ϳ4.9 kb includes ceaS1, bls1, and pah1. The expression of ceaS1 and ceaS2 in a mutant strain defective in the regulatory protein CcaR was also examined. ceaS1 transcription was not affected in the ccaR mutant, whereas that of ceaS2 was greatly reduced compared to the wild-type strain. Overall, our results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and presumably also of other paralogous genes that encode proteins involved in the early stages of clavulanic acid and clavam metabolite biosynthesis.

Molecular Regulation of Antibiotic Biosynthesis in Streptomyces

“…Other sites contain only two obvious heptamers, such as sanO-sanN in S. ansochromogenes (221), polB and polC in Streptomyces cacaoi subsp. asoensis (222), cmcI and ceaS2-II in S. clavuligerus (223), and fdmR2 in S. griseus (219). Degeneracy of the repeats may make the structure of the targets difficult to characterize, perhaps explaining why the spacers appear to vary from 4 to 15 nt in different targets (216,217,219).…”

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

“…Streptomyces clavuligerus is a gram-positive bacterium that, like most other Streptomyces spp., produces a wide variety of natural products, including penicillin, clavulanic acid, and holomycin (8,9). The genome of S. clavuligerus ATCC 27604 encodes 20 putative terpene synthases (10).…”

Carboxyethylarginine Synthase Genes Show Complex Cross-Regulation in Streptomyces clavuligerus

“…1B) (16), as the template, along with primers Mutate ceaS2-For and Mutate ceaS2-Rev to amplify a 580-bp fragment from the 5= end and upstream of ceaS2. The amplified fragment encompassed naturally occurring NcoI and SacII sites and introduced a GA-to-TG mutation to change the glutamate 57 residue of CeaS2 to tryptophan.…”

“…pSET-ceaS1 (16), which carries the promoter region of ceaS1 coupled to a promoterless egfp gene, was conjugated into wild-type and ⌬ceaS2-IF mutant strains of S. clavuligerus with selection for apr. Confocal microscopy was carried out using a Leica DM IRB inverted microscope to detect enhanced green fluorescent protein (EGFP) production under conditions described previously (16), except samples were harvested after 48 h of growth in soy medium. An argon laser with 50 to 52% attenuation was used to provide excitation at 488 nm, and EGFP excitation was detected between 500 nm and 520 nm.…”

“…The genes of the clavam and paralogue clusters are associated with the production of the 5S clavam metabolites, while the clavulanic acid cluster is involved with the production of clavulanic acid. The two processes are regulated independently of one another (11)(12)(13)(14)(15)(16), although the shared early steps in the pathway mean that there is some cross talk between clavulanic acid and 5S clavam biosynthesis. The ceaS1 gene is highly similar to ceaS2 (66% identity and 80% similarity at the amino acid level), but deletion of the ceaS1 gene caused only a minor decrease in clavulanic acid production (10), which left open the question of why ceaS2 mutants typically do not produce any clavam metabolites.…”