Enzymatic and genetic characterization of the DasD protein possessingN-acetyl-β-d-glucosaminidase activity inStreptomyces coelicolorA3(2)

Abstract

The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-β-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2) temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin.

Comparative genomics of transport proteins in developmental bacteria: Myxococcus xanthus and Streptomyces coelicolor

“…In the case of the DasABC system, the separately encoded MsiK (multiple sugar import-K) ATPase protein has been shown to serve as the energy-coupling constituent of the system [106]. We infer that the same is true for the AglEFG and MalEFG systems because: (1) each of these sets of proteins are encoded in an operon that lacks a cytoplasmic ATPase, and (2) all three systems belong to the same TC family (CUT1; TC#3.A.1.1) in which interchangeability of ATPases has been documented [106], and (3) an msiK null mutant has been shown to be unable to utilize several disaccharides including maltose [106]. …”

Transition of the Bacterial Community and Culturable Chitinolytic Bacteria in Chitin-treated Upland Soil: From <i>Streptomyces</i> to Methionine-auxotrophic <i>Lysobacter</i> and Other Genera

“…Isolated strains were inoculated into minimal medium (MM) (10 mM K 2 HPO 4 , 10 mM KH 2 PO 4 , 1 mM CaCl 2 , 0.5 mM MgCl 2 supplemented with 0.1% [v/v] trace element solution, and 1.5% [w/v] agar) (Schlochtermeier et al, 1992) containing 0.1% (w/v) colloidal chitin as a carbon source (MMCC medium), as reported previously (Saito et al, 2013) and on MMCC medium supplemented with 0.1% (w/v) yeast extract (YECC medium). Cultures were incubated at 30°C for 10 d. Chitinolytic ability was assessed by the formation of clearing zones around the colonies.…”

Taxonomic and functional diversity ofStreptomycesin a forest soil

“…With the exception of most of the isolates from cluster 9, all the tested isolates showed similar or better growth on xylan plates than on positive mannitol control plates. Thus, all the taxonomic clusters possess hemicellulolytic isolates, suggesting a widespread ability to decompose this compound despite taxonomic distance and confirming that Streptomyces species harbored a large reservoir of hemicellulolytic enzymes (Deesukon et al ., ; Liu et al ., ; Saito et al ., ). CMC utilization assays were carried out at different pH values.…”