Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces’ arsenal of signaling and antagonistic compounds.

No abstract

Microbial biofilms are considerably more resistant to antibiotics than planktonic cells. It has been reported that chitosan coupling with the aminoglycoside antibiotic streptomycin dramatically disrupted biofilms of several Gram-positive bacteria. This finding suggested the application of the covalent conjugate of antimicrobial natural polysaccharides and antibiotics on anti-infection therapy. However, the underlying molecular mechanism of the chitosan-streptomycin conjugate (CS-Strep) remains unclear and the poor water-solubility of the conjugate might restrict its applications for anti-infection therapy. In this study, we conjugated streptomycin with water-soluble chitosan oligosaccharides (COS). Unlike CS-Strep, the COS-streptomycin conjugate (COS-Strep) barely affected biofilms of tested Gram-positive bacteria. However, COS-Strep efficiently eradicated established biofilms of the Gram-negative pathogen Pseudomonas aeruginosa. This activity of COS-Strep was influenced by the degree of polymerization of chitosan oligosaccharide. The increased susceptibility of P. aeruginosa biofilms to antibiotics after conjugating might be related to the following: Suppression of the activation of MexX-MexY drug efflux pump system induced by streptomycin treatment; and down-regulation of the biosynthesis of biofilm exopolysaccharides. Thus, this work indicated that covalently linking antibiotics to chitosan oligosaccharides was a possible approach for the development of antimicrobial drugs against biofilm-related infections.

Specialized metabolic clusters in Streptomyces are the source of many clinically prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2.

1Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-2 producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins or 3 LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 4 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. 5 Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic 6 nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 7 activity in model species and uncharacterized isolates resulted in the production of new metabolites not 8 seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by 9Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and 10 provide global control over Streptomyces' arsenal of signalling and antagonistic compounds. 11 12 Interestingly, however, most clusters are poorly expressed under normal laboratory conditions, and in 54 many cases their associated metabolites remain uncharacterized. This is also the case for the 55 filamentous fungi, many of whom have a broad, untapped specialized metabolic repertoire, courtesy of 56 transcriptional silencing by histones (Pfannenstiel and Keller, 2019). Significant efforts are being made to 57 stimulate the production of these 'cryptic' metabolites in both bacteria and fungi, as they are widely 58 regarded as productive sources of new natural products (Craney et al., 2013;Ochi and Hosaka, 2013; 59 Scharf and Brakhage, 2013;Yoon and Nodwell, 2014; Daniel-Ivad et al., 2017;Onaka, 2017). 60We sought to investigate the role of the nucleoid-associated proteins Lsr2 and LsrL in gene 61 regulation in Streptomyces. We found that deleting lsr2 from the chromosome of Streptomyces 62 venezuelae had minor effects on S. venezuelae growth and development and major effects on 63 metabolism. In contrast, deleting lsrL had no detectable impact on development, and only a minor effect 64 on metabolism. Focussing on Lsr2, we determined that it bound AT-rich regions, generally repressed the 65 expression of prophage genes and other genes unique to S. venezuelae (presumably acquired by lateral 66 gene transfer), and suppressed antisense gene expression. The most profound effect of lsr2 deletion, 67 however, was the large-scale activation of specialized metabolic cluster gene expression. Lsr2 directly 68 repressed the transcription of many cryptic clusters in a way that is analogous to Lsr2-and H-NS-69 mediated repression of pathogenicity islands in other bacteria, and histone-mediated cluster silencing in 70 fungi. Unexpectedly, Lsr2 also controlled the expression of well-characterized and highly-conserved 71 clusters, suggesting that Lsr2 control has been broadly integrated into the regulatory cascades governing 72 specialized metabolism. Our results suggest that Lsr2 functions as a metabolic gatekeeper in the 73 strepto...