streptothricins and beta-lysine

Streptothricin (ST) and its related compounds produced by Streptomyces strains are broad-spectrum antibiotics that consist of carbamoylated d-gulosamine, amino-acid side chain, and streptolidine lactam moieties. BD-12, a streptothricin-related antibiotic, has a glycine-derived side chain and two N-methyl groups, whereas ST-F carrying the l-β-lysine side chain has no methyl group. In our previous studies, we identified and characterized the BD-12 and ST biosynthetic gene clusters. Here we report the functional analysis of two methyltransferase genes (orf 6 and orf 13) in the BD-12 biosynthetic gene cluster. Combinatorial biosynthesis using these two methyltransferase genes and the ST biosynthetic gene cluster resulted in the production of three methylated forms of ST-F. Among them, N,N'-dimethyl-ST-F, a novel compound generated in the present study, showed bacteria-specific antibiotic activities, although ST-F exhibits antibiotic activities against both prokaryotes and eukaryotes. Our findings also demonstrated that the orf 6 and orf 13 genes are responsible for the N-methylations of the amide bonds in the streptolidine lactam and in the amino-acid side chain linkage, respectively, and that N-methyl modification of the streptolidine lactam confers resistance in part against an ST hydrolase, SttH.

Topics: Amino Acids; Anti-Bacterial Agents; Biosynthetic Pathways; Genes, Bacterial; Hydrolases; Lysine; Methylation; Methyltransferases; Multigene Family; Streptomyces; Streptothricins

Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Carrier Proteins; Cloning, Molecular; Lysine; Molecular Sequence Data; Peptide Synthases; Streptomyces; Streptothricins

Racemomycin-B (RM-B), the main component of Streptomyces lavendulae OP-2 which is the basis of 50% of the antibiotics produced, is a streptothricin antibiotic which contains three beta-lysine moieties in the molecule. RM-B had antimicrobial activity against plant-pathogenic microorganisms and growth-inhibitory activity against the root of Brassica rapa L. at the concentration of 50 ppm. It strongly inhibited the growth of Pseudomonas syringae pv. tabaci IFO-3508 (minimum inhibitory concentration (MIC): 0.4 microgram/ml), and also showed antifungal activity against six kinds of Fusarium oxysporum species (MIC: 0.1-2.0 micrograms/ml). The antimicrobial activity of RM-B was much stronger than those of RM-A and -C which contain, respectively, one and two beta-lysine moieties in their molecules. The above activities of RM-A, -C and -B were thus in the order of -B greater than -C greater than -A: namely, the biological activity of racemomycin compounds tended to be stronger with increase in the number of beta-lysine moieties in the molecule.

Topics: Fusarium; Lysine; Pseudomonas; Streptothricins; Structure-Activity Relationship

Nourseothricin (streptothricin) can be inactivated by an acetyl transferase synthesized by E. coli strains containing plasmid pIE 636. Nourseothricin inactivated in the presence of 14C-acetyl-coenzyme A was purified and submitted to partial acidic hydrolysis. By electrophoresis of the hydrolysate a 14C-containing substance moving only slowly towards the cathode could be isolated. This substance after complete hydrolysis yields only unlabelled beta-lysine.

Topics: Acetyltransferases; Anti-Bacterial Agents; Escherichia coli; Isomerism; Lysine; R Factors; Streptothricins

Topics: Anti-Bacterial Agents; Kinetics; Lysine; Mutation; Species Specificity; Streptomyces; Streptothricins