DNA phosphorothioation, conferred by dnd genes, was originally discovered in the soil-dwelling bacterium Streptomyces lividans, and thereafter found to exist in various bacterial genera. However, the physiological significance of this sulfur modification of the DNA backbone remains unknown in S. lividans. Our studies indicate that DNA phosphorothioation has a major role in resistance to oxidative stress in the strain. Although Streptomyces species express multiple catalase/peroxidase and organic hydroperoxide resistance genes to protect them against peroxide damage, a wild type strain of S. lividans exhibited two-fold to 10-fold higher survival, compared to a dnd− mutant, following treatment with peroxides. RNA-seq experiments revealed that, catalase and organic hydroperoxide resistance gene expression were not up-regulated in the wild type strain, suggesting that the resistance to oxidative stress was not due to the up-regulation of these genes by DNA phosphorothioation. Quantitative RT-PCR analysis was conducted to trace the expression of the catalase and the organic hydroperoxide resistance genes after peroxides treatments. A bunch of these genes were activated in the dnd− mutant rather than the wild type strain in response to peroxides. Moreover, the organic hydroperoxide peracetic acid was scavenged more rapidly in the presence than in the absence of phosphorothioate modification, both in vivo and in vitro. The dnd gene cluster can be up-regulated by the disulfide stressor diamide. Overall, our observations suggest that DNA phosphorothioate modification functions as a peroxide resistance system in S. lividans.

The nanchangmycin (NAN) produced by Streptomyces nanchangensis NS3226 is a polyether antibiotic resembling monensin in their gene clusters and the chemical structures. They can inhibit gram-positive bacteria and be a growth promoter for ruminants. Within the nanchangmycin gene cluster (nan), we identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes. Overexpression of NanR1 and NanR2 in wild-type increase NAN yields by at least three folds. Bioinformatic analysis of the immediate upstream DNA sequence of each nan gene and quantitative real-time RT-PCR analysis of the nan operons identified five putative SARP binding sites. Moreover, deletion of an AraC-family repressor gene nanR4 increased expression of NanR1 and R2 and led to a threefold increase in NAN production.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

The peptidyl nucleoside blasticidin S (BS) produced by Streptomyces griseochromogenes was the first non-mercurial fungicide used to prevent rice blast and increasingly used as a selection reagent in transgenic study. Acylation by addition of a leucine residue at the beta amine group of arginine side chain of demethylblasticidin S (DBS) has been proposed as a novel self-resistance to the cytotoxic biosynthetic intermediate. But the resultant product leucyldemethylblasticidin S (LDBS) has not been isolated as a metabolite, and LDBS synthetase activity remained to be demonstrated in S. griseochromogenes. In this study, we isolated LDBS in a BS heterologous producer S. lividans WJ2 upon the deletion of blsL, which encodes a S-Adenosyl methionine-dependent methyltransferase. Purified BlsL efficiently methylated LDBS at the delta N of beta-arginine to generate the ultimate intermediate LBS, but nearly didn’t methylate DBS to final product BS. Above experiments demonstrated that LDBS is indeed an intermediate in BS biosynthetic pathway, and acylation of beta-amino group of arginine side chain is prerequisite for efficient guanidine N-methylation in addition to being a self-resistance mechanism.

The fungicide nucleoside blasticidin S features a β‐arginine, a moiety seldom revealed in the structure of natural products. BlsG, a radical SAM arginine‐2,3‐aminomutase from the blasticidin S biosynthetic pathway, displayed promiscuous activity to three basic amino acids. Here in this study, we demonstrated that BlsG showed high preference toward its natural substrate arginine. The combined structural modeling, steady‐state kinetics, and mutational analyses lead to the detailed understanding of the substrate recognition of BlsG. A single mutation of T340D changed the substrate preference of BlsG leading to a little more preference to lysine than arginine. On the basis of our understanding of the substrate selection of BlsG and bioinformatic analysis, we propose that the D…D motif locationally corresponding to D293 and D330 of KAM is characteristic of lysine 2,3‐aminomutase while the corresponding D…T motif is characteristic of arginine 2,3‐aminomutase. The study may provide a simple way to discern the arginine 2,3‐aminomutase and thus lead to the discovery of new natural compounds with β‐arginine moiety.