Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2).

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor.

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

SCO6992, a Protein with ß-Glucuronidase Activity, Complements a Mutation at the absR Locus and Promotes Antibiotic Biosynthesis in Streptomyces coelicolor.

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong ß-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-ß-D-glucuronide and AS-BI-ß-D-glucuronide. The binding between rSCO6992 and Zn2+ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with ß-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported ß-glucuronidases.

LacI-Family Transcriptional Regulator DagR Acts as a Repressor of the Agarolytic Pathway Genes in Streptomyces coelicolor A3(2).

Actinobacteria utilize various polysaccharides in the soil as carbon source by degrading them via extracellular hydrolytic enzymes. Agarose, a marine algal polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG), is one of the carbon sources used by S. coelicolor A3(2). However, little is known about agar hydrolysis in S. coelicolor A3(2), except that the regulation of agar hydrolysis metabolism is strongly inhibited by glucose as in the catabolic pathways of other polysaccharides. In this study, we elucidated the role of DagR in regulating the expression of three agarase genes (dagA, dagB, and dagC) in S. coelicolor A3(2) by developing a dagR-deletion mutant (Δsco3485). We observed that the Δsco3485 mutant had increased mRNA level of the agarolytic pathway genes and 1.3-folds higher agarase production than the wild type strain, indicating that the dagR gene encodes a cluster-suited repressor. Electrophoretic mobility shift assay revealed that DagR bound to the upstream regions of the three agarase genes. DNase 1 footprinting analysis demonstrated that a palindromic sequence present in the upstream region of the three agarase genes was essential for DagR-binding. Uniquely, the DNA-binding activity of DagR was inhibited by AHG, one of the final degradation products of agarose. AHG-induced agarase production was not observed in the Δsco3485 mutant, as opposed to that in the wild type strain. Therefore, DagR acts as a repressor that binds to the promoter region of the agarase genes, inhibits gene expression at the transcriptional level, and is derepressed by AHG. This is the first report on the regulation of gene expression regarding agar metabolism in S. coelicolor A3(2).