A pathway‐specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus

Abstract

SummaryA Streptomyces clavuligerus gene (designated claR ) located downstream from the gene encoding clavaminate synthase in the clavulanic acid biosynthetic gene cluster is involved in regulation of the late steps in clavulanic acid biosynthesis. Nucleotide sequence analysis and database searching of ClaR identified a significant similarity to the helix-turn-helix motif (HTH) region of LysR transcriptional regulators. A gene replacement mutant disrupted in claR was unable to produce clavulanic acid, suggesting that claR is essential for clavulanic acid biosynthesis. Furthermore, the accumulation of clavaminic acid in the claR mutant suggested that ClaR regulates the late steps in the clavulanic acid pathway, i.e. those involved in the conversion of clavaminic acid to clavulanic acid. Transcriptional analysis using RNA isolated from the wild type and the claR mutant showed that the expression of the putative late genes, but not the early genes, was regulated by ClaR. High-resolution S1 nuclease analysis of claR suggested that it is expressed as a monocistronic transcript and also as a bicistronic transcript along with the late gene orf-9. The transcription start site of the monocistronic claR transcript was identified as a C residue 155 nucleotides upstream from the claR start codon.

Construction and analysis of β-lactamase-inhibitory protein (BLIP) non-producer mutants of Streptomyces clavuligerus The GenBank accession number for the sequence determined in this work is M34538.

“…Although BLIP is active against the β-lactamase of S. clavuligerus, it does not seem to affect the innate resistance of S. clavuligerus to penicillin G either in surface-grown or in liquid cultures. This result is in agreement with previous findings that the resistance of S. clavuligerus cultures to β-lactam antibiotics is a result of the production of penicillin-binding proteins having low affinity for β-lactams (Ogawara & Horikawa, 1980 ;Paradkar et al, 1998).…”

“…HPLC analysis was performed as described by Paradkar et al (1998). Clavam-2-carboxylate and clavulanic acid were identified by comparison to authentic standards kindly provided by SmithKline Beecham, Worthing, UK.…”

“…A BLIP non-producer mutant and a clavulanic acid\ BLIP non-producer double mutant were constructed by gene replacement at the bli locus in wild-type S. clavuligerus and in the clavulanic acid production defective mutant, claR (Paradkar et al, 1998), respectively. To construct the bli single mutant, pJOEbli ::tsr was introduced into wild-type S. clavuligerus.…”

“…The claR mutant is defective in clavulanic acid production and was generated by disruption of claR using an apramycin-resistance gene. claR encodes a transcriptional regulator required for the expression of genes involved in the late steps of clavulanic acid biosynthesis (Paradkar et al, 1998). Following sporulation of transformants on non-selective medium, and isolation of Thio R Hyg S colonies, seven putative bli single mutants and one bli claR double mutant were obtained.…”

Transcriptional organization of ThnI-regulated thienamycin biosynthetic genes in Streptomyces cattleya

“…The situation is reminiscent of the one found in the closest ThnI homologue, ClaR, which activates transcription of late clavulanic acid biosynthetic genes in S. clavuligerus. 17,18 ClaR is expressed as a monocistronic transcript from its own promoter, as well as co-transcribed with a biosyntheticregulated gene as a bicistronic transcript, where a similar read through of transcription spanning a stem-loop structure was observed. 17 …”