γ-Butyrolactone autoregulator-receptor system involved in lankacidin and lankamycin production and morphological differentiation in Streptomyces rochei

Abstract

An afsA homologue (srrX) and three c-butyrolactone receptor gene homologues (srrA, srrB and srrC) are coded on the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two polyketide antibiotics, lankacidin and lankamycin. Construction of gene disruptants and their phenotypic study revealed that srrX and srrA make a c-butyrolactone receptor system in this strain. Addition of a c-butyrolactone fraction to an srrX-deficient mutant restored the production of lankacidin and lankamycin, indicating that the SrrX protein is not necessary for this event. In addition to a positive effect on antibiotic production, srrX showed a negative effect on morphological differentiation. The receptor gene srrA reversed both effects of srrX, while the second receptor gene homologue srrC had only a positive function in spore formation. Furthermore, disruption of the third homologue srrB greatly increased the production of lankacidin and lankamycin. Electron microscopic analysis showed that aerial mycelium formation stopped at a different stage in the srrA and srrC mutants. Overall, these results indicated that srrX, srrA, srrB and srrC constitute a complex regulatory system for antibiotic production and morphological differentiation in S. rochei. INTRODUCTIONThe filamentous Gram-positive soil bacteria of the genus Streptomyces are characterized by three distinct properties: linear chromosome, complex morphological differentiation and ability to produce varieties of secondary metabolites including antibiotics. Antibiotic biosynthetic genes in Streptomyces species usually form a condensed gene cluster on the chromosome. However, several giant linear plasmids encoding an antibiotic gene cluster(s) have been isolated: SCP1 in Streptomyces coelicolor A3(2) carries the biosynthetic gene cluster for methylenomycin (Bentley et al., 2004;Chater & Bruton, 1985;Kinashi et al., 1987;Redenbach et al., 1998), pPZG103 in Streptomyces rimosus has that for oxytetracycline (Gravius et al., 1994; Pandza et al., 1998) and pSLA2-L in Streptomyces rochei has those for lankacidin and lankamycin (Arakawa et al., 2005(Arakawa et al., , 2006Mochizuki et al., 2003;Suwa et al., 2000).S. rochei strain 7434AN4 contains three large linear plasmids (pSLA2-L, -M and -S) (Kinashi et al., 1994) and produces two structurally unrelated polyketide antibiotics, the 17-membered macrocyclic lankacidin and the 14-membered macrolide lankamycin (Fig. 1a). We have been studying the function of the largest linear plasmid pSLA2-L in antibiotic production, and finally determined its 210 614 bp nucleotide sequence (Mochizuki et al., 2003). It was revealed that pSLA2-L contains an unusually condensed gene organization for secondary metabolism (Fig. 1b); two type I PKS gene clusters for lankacidin (lkc, orf4-orf18) and lankamycin (lkm, orf24-orf53), a cryptic type II PKS gene cluster (roc, orf62-orf70) and a carotenoid biosynthetic gene cluster (crt, orf104-orf110). In addition, many regulatory genes were identified on pSLA2-L, including all the homologues in t...

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

“…In contrast, LM is coproduced with LC. Both drugs appear to be coregulated, and thus similar to the streptogramins case, seem to be intended to work together (39,40). In agreement with this hypothesis, our results show their simultaneous binding to the ribosome.…”

“…Furthermore, similar to ERY and other macrolides, desacetyl-LM was reported to be capable of activating expression of inducible macrolide-resistance genes (37). Coregulation of production of LC and LM (39,40) suggests that these drugs have been evolutionary optimized to work together. Nevertheless, although LC and LM are coproduced by the Streptomyces strain, there has been no information about their sites of action, nor any evidence of functional interaction between these two antibiotic compounds.…”

Regulation of Lankamycin Biosynthesis inStreptomyces rocheiby Two SARP Genes,srrYandsrrZ

“…4) It is noteworthy that srrX shows a positive effect on antibiotic production and a negative effect on morphological differentiation. Its receptor gene, srrA, reversed both effects of srrX.…”

“…3) However, recent studies have revealed that the main targets of GB receptors are SARP (Streptomyces antibiotic regulatory protein) family transcriptional activator genes, 4) which act as a master switch for secondary metabolism in streptomycetes. This family of regulatory proteins are characterized by the presence of an OmpRlike DNA-binding domain, 5) and are typified by ActII-ORF4 for actinorhodin biosynthesis in Streptomyces coelicolor, 6) RedD for undecylprodigiosin in S. coelicolor, 7) DnrI for daunorubicin in Streptomyces peucetius, 8) CcaR for cephamycin and clavulanic acid in Streptomyces clavuligerus, 9) and TylS for tylosin in Streptomyces fradiae.…”

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters

“…45 This effect is different from that of afsA in S. griseus, which shows positive effects on both streptomycin production and spore formation. 46 The receptor gene srrA has counteracting functions against both effects of srrX.…”