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Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.

A ptsH homologue of Streptomyces coelicolor A3(2) was identified in the emerging genome sequence, cloned in Escherichia coli and the S. coelicolor HPr over-produced and purified. The protein was phosphorylated in vitro in a phosphoenolpyruvate (PEP)-dependent manner by purified enzyme I (EI) from Bacillus subtilis, and much less efficiently in an ATP-dependent manner by purified HPr kinase, also from B. subtilis. There was no indication of ATP-dependent phosphorylation of the purified protein by cell extracts of either S. coelicolor or Streptomyces lividans. Deletion of the ptsH homologue from the S. coelicolor and S. lividans chromosomes had no effect on growth when fructose was supplied as sole carbon source, and in S. coelicolor it had no effect on glucose repression of agarase and galactokinase synthesis, suggesting that the HPr encoded by this gene does not play an essential role in fructose transport nor a general role in carbon catabolite repression.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.

A skimmed-milk clearing assay was used to identify, in a multicopy Streptomyces lividans 66 genomic library, DNA fragments that lead to increased expression of protease activity in S. lividans 66. Three independent loci were identified. The majority class (slpA, which represented 68 of 71 clones) produced large zones of clearing. Two other classes (designated slpB and slpC) showed smaller zones than slpA. Subcloning and deletion analysis of the slpA locus delineated the relevant DNA to within a 2.5 kilobase pair fragment. DNA sequence analysis revealed a structural gene associated with the appearance of an extracellular protein in the culture medium. The derived amino acid sequence indicated the presence of a zinc-binding motif, which was previously noted to be characteristic of metalloprotease enzymes. However, the relatively small size of the protein (apparent molecular weight 20,000-24,000) suggests that it represents a novel class of neutral proteases distinct from the thermolysin-type enzymes. An adjacent divergent open reading frame was identified and shown to cause a significant increase in protease activity when present together with the protease structural gene on a multicopy plasmid in S. lividans 66. The derived amino acid sequence of this open reading frame showed homology with previously characterized regulatory proteins of the LysR family of transcriptional regulator proteins.