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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).

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). [ABSTRACT FROM AUTHOR]

Homologs of PtxS are ubiquitous transcriptional regulators controlling the expression of the glucose dehydrogenase and kgu operon to globally regulate the 2-ketogluconic acid (2KGA) metabolism in Pseudomonas. In the present study, a PtxS from a 2KGA industrial producer Pseudomonas plecoglossicida JUIM01 (PpPtxS) was heterologously expressed in E. coli BL21(DE3), then structurally and functionally characterized. The obtained results showed that PpPtxS was a 36.65-kDa LacI-family transcriptional regulator. 2KGA was the sole effector of PpPtxS. Glucose negatively affected the molecular binding of PpPtxS and 2KGA, and gluconic acid inhibited the PpPtxS-2KGA binding reaction. PpPtxS in water solution mainly existed as a dimer and bound to two molecules of 2KGA. The effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS by interacting with the 299th to the 301st amino acids (Ala, Gln, Pro, Thr, Glu and Arg). PpPtxS specifically recognized and bound to a 14-bp palindrome sequence (5′-TGAAACCGGTTTCA-3′) due to its conserved HTH motif at the N-terminal. The characterization of PpPtxS in this study would provide a theoretical guidance for the industrial production of 2KGA. • PtxS from a 2KGA industrial producer P. plecoglossicida was heterologously expressed. • PpPtxS was a 36.65-kDa LacI-family transcriptional regulator. • PpPtxS had a sole effector of 2KGA. • PpPtxS was mainly dimeric in water solution and bound to two molecules of 2KGA. • PpPtxS specifically recognized and bound to a 14-bp palindrome sequence. [ABSTRACT FROM AUTHOR]