The chromosomal region encoding the secY gene of Streptomyces griseus N2-3-11 was cloned and analyzed. The secY gene encodes a polypeptide of 438 aa with a molecular mass of 47.5 kDa. The transcriptional start point of the secY gene was determined. Northern blot analysis revealed a growth phase-dependent secY expression supporting our previous findings for secA gene expression in S. griseus. Overproduction of the SecY protein was obtained when using Streptomyces lividans TK23 as host. The interaction of the SecY proteins of S. griseus, S. lividans, and Escherichia coli, respectively, with the purified SecA protein of S. griseus was demonstrated for the first time by using ligand affinity blot assays.

Galbonolides A and B are antifungal compounds, which are produced by Streptomyces galbus. A multimodular polyketide synthase (PKS) was predicted to catalyze their biosynthesis, and a methoxymalonyl-acyl carrier protein (methoxymalonyl-ACP) was expected to be involved in the biosynthesis of galbonolide A. Cloning of a methoxymalonyl-ACP biosynthesis locus (galGHIJK) and the flanking regions has revealed that the locus is colocalized with beta-ketoacyl synthase (KAS)-related genes (orf3, 4, and 5), but separated from any multimodular PKS gene cluster in S. galbus. A galI-disruption mutant (SK-galI-5) is unable to produce galbonolide A, but can synthesize galbonolide B, indicating that galGHIJK is involved in the biosynthesis of galbonolide A. A disruption mutant of orf4 is severely impaired in the production of both galbonolides A and B. These results indicate that galGHIJK and the KAS genes are involved in the biosynthesis of galbonolides, although they are not colocalized with a multimodular PKS gene cluster. We further propose that a single galbonolide PKS generates two discrete structures, galbonolides A and B, by alternatively incorporating methoxymalonate and methylmalonate, respectively.

We present evidence for the coexistence and coevolution of antibiotic resistance and biosynthesis genes in soil bacteria. The distribution of the streptomycin (strA) and viomycin (vph) resistance genes was examined in Streptomyces isolates. strA and vph were found either within a biosynthetic gene cluster or independently. Streptomyces griseus strains possessing the streptomycin cluster formed part of a clonal complex. All S. griseus strains possessing solely strA belonged to two clades; both were closely related to the streptomycin producers. Other more distantly related S. griseus strains did not contain strA. S. griseus strains with only vph also formed two clades, but they were more distantly related to the producers and to one another. The expression of the strA gene was constitutive in a resistance-only strain whereas streptomycin producers showed peak strA expression in late log phase that correlates with the switch on of streptomycin biosynthesis. While there is evidence that antibiotics have diverse roles in nature, our data clearly support the coevolution of resistance in the presence of antibiotic biosynthetic capability within closely related soil dwelling bacteria. This reinforces the view that, for some antibiotics at least, the primary role is one of antibiosis during competition in soil for resources.

A combined approach, comprising PCR screening and genome mining, was used to unravel the diversity and phylogeny of genes encoding 5-aminolevulinic acid synthases (ALASs, hemA gene products) in streptomycetes-related strains. In actinomycetes, these genes were believed to be directly connected with the production of secondary metabolites carrying the C5N unit, 2-amino-3-hydroxycyclopent-2-enone, with biological activities making them attractive for future use in medicine and agriculture. Unlike "classical" primary metabolism ALAS, the C5N unit-forming cyclizing ALAS (cALAS) catalyses intramolecular cyclization of nascent 5-aminolevulinate. Specific amino acid sequence changes can be traced by comparison of "classical" ALASs against cALASs. PCR screening revealed 226 hemA gene-carrying strains from 1,500 tested, with 87% putatively encoding cALAS. Phylogenetic analysis of the hemA homologs revealed strain clustering according to putative type of metabolic product, which could be used to select producers of specific C5N compound classes. Supporting information was acquired through analysis of actinomycete genomic sequence data available in GenBank and further genetic or metabolic characterization of selected strains. Comparison of 16S rRNA taxonomic identification and BOX-PCR profiles provided evidence for numerous horizontal gene transfers of biosynthetic genes or gene clusters within actinomycete populations and even from non-actinomycete organisms. Our results underline the importance of environmental and evolutionary data in the design of efficient techniques for identification of novel producers.

Three novel proteinaceous inhibitors, which had been identified as "Streptomyces subtilisin inhibitor-like (SIL) proteins" and exhibited trypsin inhibition in addition to strong inhibition toward subtilisin BPN', were purified from the culture broth of three Streptomyces strains: SIL10 from S. thermotolerans, SIL13 from S. galbus, and SIL14 from S. azureus. Their primary structures were determined by sequence analysis of intact SIL inhibitors and peptides obtained by enzymatic digestions of S-pyridylethylated SIL inhibitors. These inhibitors were composed of about 110 amino acids and existed as dimer proteins. The reactive site was identified as Lys-Gln for all three inhibitors by sequence analysis of their modified forms in which the reactive-site peptide bond was specifically cleaved by subtilisin BPN' under acidic conditions. Thus, their inhibition toward trypsin and subtilisin BPN' was due to the presence of a Lys residue at the P1 site. Inhibitor constants toward subtilisin BPN' and trypsin were also determined. These inhibitors showed relatively high sequence homology to other SSI-family inhibitors possessing a Lys residue at the P1 site, with amino acid replacements on their molecular surface.