An rplKDelta29-PALG-32 mutation leads to reduced expression of the regulatory genes ccaR and claR and very low transcription of the ceaS2 gene for clavulanic acid biosynthesis in Streptomyces clavuligerus

Abstract

The transcriptional and translational control of the biosynthesis of the beta-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA(trp)-secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S. clavuligerus rplK(DeltaPALG) mutant, with an internal 12 nucleotides in-frame deletion in the rplK gene, encoding the L11 (RplK) ribosomal protein lacking amino acids (29)PALG(32), was constructed by gene replacement. This deletion alters the L11 N-terminal domain that interacts with the RelA and class I releasing factors-mediated translational termination. The mutant grew well, showed threefold higher resistance to thiostrepton, did not form spores and lacked diffusible brown pigments, as compared with the wild-type strain. The wild-type phenotype was recovered by complementation with the native rplK gene. S. clavuligerus rplK(DeltaPALG) produced reduced levels of clavulanic acid (15-26% as compared with the wild type) and cephamycin C (40-50%) in cultures grown in defined SA and complex TSB media. The decreased yields resulted from an impaired transcription of the regulatory genes ccaR and claR and the cefD and ceaS2 genes for cephamycin and clavulanic acid biosynthesis respectively. Expression of ceaS2 encoding carboxyethylarginine synthase (CEAS), the precursor-committing enzyme for clavulanic acid biosynthesis, was particularly affected in this mutant. In the wild-type strain polyphosphorylated nucleotides peaked at 36-48 h of growth in SA cultures whereas expression of the cephamycin and clavulanic acid genes occurred 12-24 h earlier than the increase in ppGpp indicating that there is no strict correlation between the peak of ppGpp and the onset of transcription of the clavulanic acid and cephamycin C biosynthesis. The drastic effect of the rplK(DeltaPALG) mutation on the onset of expression of the ceaS2 and the regulatory ccaR and claR genes and the lack of correlation with ppGpp levels suggest that the onset of transcription of these genes is modulated by the conformational alteration of the N-terminal region of L11 probably by interaction with the nascent peptide releasing factors and with RelA.