Control by A-Factor of a Metalloendopeptidase Gene Involved in Aerial Mycelium Formation in Streptomyces griseus

Abstract

In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position ؊60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position ؊260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.Members of the gram-positive, soil-inhabiting, filamentous bacterial genus Streptomyces produce a wide variety of secondary metabolites and show complex morphological differentiation culminating in sporulation. These characteristics make members of this genus important as industrial microorganisms and as one of the model prokaryotes for studying multicellular differentiation (3, 4). On agar medium, one or more germ tubes formed from a germinating spore grow into substrate hyphae, which branch frequently and grow rapidly by cell wall extension at the hyphal tips. Subsequently, aerial hyphae emerge by reuse of material assimilated into the substrate mycelium, such as DNA, proteins, and storage compounds. Many cells in substrate hyphae thus lyse and die (5,21,31). At this stage of development, therefore, enzymes for the degradation of these substances, such as proteases, nucleases, lipases, and glucanases, are presumably required. In fact, nucleases and serine proteases are associated with the development of aerial hyphae from substrate hyphae (5, 23). When the apical growth of aerial hyphae stops, in contrast to the substrate mycelium, septa are formed at regular intervals along the hyphae to form many unigenomic compartments within a sheath. The sporulation septa consist of two membrane layers separated by a double layer of cell wall material, which permits the eventual separation of adjacent spores (7). Spore chains usua...

DNA microarray analysis of global gene regulation by A-factor in Streptomyces griseus

“…Two DNA fragments containing the AdpA-binding sites of sgmA and sgiA were labelled with digoxigenin (DIG) using the DIG DNA Labelling kit (Roche) and were used as probes. The sgmA probe (sgmA-P) was 300 bp long (nucleotide positions from 2399 to 2100, taking the transcriptional start point as +1) and contained the AdpA-binding B site for sgmA (Kato et al, 2002). The sgiA probe (sgiA-P) was 209 bp (nucleotide positions from 2183 to +26) and contained the AdpAbinding site for sgiA (Hirano et al, 2006).…”

“…The AraC/ XylS family transcriptional regulator AdpA then activates transcription of many genes that are required for secondary metabolism and morphological differentiation, forming an AdpA regulon (Ohnishi et al, 2005). Members of the AdpA regulon identified to date are strR, the pathway-specific transcriptional activator for streptomycin biosynthesis (Ohnishi et al, 1999;Tomono et al, 2005a); an ORF (SGR6071) encoding a probable pathway-specific transcriptional activator for biosynthesis of a polyketide compound (Yamazaki et al, 2004); adsA, encoding an extracytoplasmic function sigma factor of RNA polymerase essential for aerial mycelium formation ; amfR, encoding a transcriptional activator for the amfTSBA operon responsible for production of a surfactant lantibiotic-like peptide SapB (AmfS) (Ueda et al, 2002(Ueda et al, , 2005, which is essential for aerial mycelium formation (Yamazaki et al, 2003b); ssgA, encoding a small acidic protein essential for spore septum formation (Yamazaki et al, 2003a); sgmA, encoding a secreted metalloendopeptidase probably involved in apoptosis of substrate mycelium during aerial mycelium development (Kato et al, 2002); five different genes (sprA, sprB, sprD, sprT and sprU) encoding secreted serine proteases (Kato et al, 2005a;Tomono et al, 2005b); sgiA, encoding a Streptomyces subtilisin inhibitor (SSI) family inhibitor (Hirano et al, 2006); a gene of unknown function (SGR4618, formerly named orfA-AdBS3) (Yamazaki et al, 2004); and an operon of unknown function orf1-2-3-4 of AdBS2 (SGR6559-SGR6556) (Yamazaki et al, 2004). Thus, we have identified 14 AdpA target promoters to date.…”

Streptomyces inside-out: a new perspective on the bacteria that provide us with antibiotics

“…Further, S. griseus produces several proteases that are sensitive to STI-like proteins, and are encoded by AdpA-dependent genes, and at least one of these contributes to aerial development of S. griseus. This provides further evidence that the extracellular protease cascade is controlled by the bldA/AdpA system (Kato et al 2002(Kato et al , 2005a.…”

Anoikis-resistant MDCK cells carrying susceptibilities to TNF-α and verotoxin that are suitable for influenza virus cultivation

“…The cleavage sites are at amide bonds of carbobenzoxyl-Ala-Leu-NH 2 , carbobenzoxyl-Gly-Leu-NH 2 , carbobenzoxyl-Gly-Phe-NH 2 and at p-nitroanilide (pNA) bonds of Pyr-Phe-Leu-pNA, carbobenzoxyl-AlaAla-Leu-pNA and carbobenzoxyl-Gly-Gly-LeupNA. Amino acid sequences of MEP have been reported (Tsuyuki et al 1991;Kojima et al 1998;Kato 2002). Dispase (grade II), a bacterial neutral proteinase, was purchased (Invitrogen, Carlsbad, CA, USA.)…”