Properties of CsnR, the transcriptional repressor of the chitosanase gene, csnA, of Streptomyces lividans

Abstract

A palindromic sequence is present in the intergenic region preceding the chitosanase gene csnA (SSPG_06922) of Streptomyces lividans TK24. This sequence was also found in front of putative chitosanase genes in several other actinomycete genomes and upstream genes encoding putative transcriptional regulators of the ROK family, including csnR (SSPG_04872) in S. lividans. The latter was examined as a possible transcriptional regulator (CsnR) of chitosanase gene expression. In vitro, purified CsnR bound strongly to the palindromic sequences of the csnA and csnR genes (equilibrium dissociation constant [K(D)] = 0.032 and 0.040 nM, respectively). Binding was impaired in the presence of chitosan oligosaccharides and d-glucosamine, and chitosan dimer was found to be the best effector, as determined by an equilibrium competition experiment and 50% inhibitory concentration (IC(50)) determination, while glucose, N-acetyl-glucosamine, and galactosamine had no effect. In vivo, comparison of the S. lividans wild type and ΔCsnR strains using β-lactamase reporter genes showed that CsnR represses the expression of csnA and of its own gene, which was confirmed by quantitative PCR (qPCR). CsnR is localized at the beginning of a gene cluster, possibly an operon, the organization of which is conserved through many actinomycete genomes. The CsnR-mediated chitosanase regulation mechanism seems to be widespread among actinomycetes.

Fig. 1.

Alignment of palindromic sequences found upstream of genes encoding chitosanases or ROK family regulator genes in actinomycetes and LOGO representation of consensus sequence. Complementary bases in palindromes bases are shown in blue. “Pos.” (position) indicates the distance in bp from the central nucleotide of the palindromic sequence to the start codon of the associated gene. K. sp. N106, Kitasatospora sp. N106; S. sp. N174, Streptomyces sp. N174.

Fig. 2.

Purification of CsnR. Protein samples from each stage of the CsnR purification were analyzed by 10% SDS-PAGE and visualized after silver nitrate staining. M, PageRuler prestained molecular mass protein ladder (Fermentas); S, soluble fraction of cell lysate obtained from a culture of E. coli Rosetta-gami 2 (DE3)(pLysS) (pGEX-csnR) induced with 0.1 mM IPTG; (−), purification attempt without previous treatment of the soluble fraction of cell lysate; (+), purification steps with a previous 2 mM ATP and 5 mM MgCl₂ treatment of the soluble fraction of cell lysate; E, eluate collected from the glutathione-Sepharose 4B resin following a 4-h incubation with PreScission protease; F, 20 μl of the size exclusion chromatography fraction with the highest GroEL contamination; P, 20 μl of pooled size exclusion chromatography fractions with purified CsnR.

Fig. 3.

DNase I footprinting analysis of the CsnR binding site to csnA and csnR promoters. (A) A 298-bp labeled probe (csnA-IR) and a 256-bp labeled probe (csnR-IR), both including the entire intergenic regions upstream from csnA and csnR, respectively, were subjected to partial DNase I digestion in the presence (+) or absence (−) of ∼0.5 nmol of purified CsnR. Vertical arrows correspond to the palindromic sequence shown in panel B. (B) Partial intergenic region sequences upstream of csnA and csnR. Boxes correspond to the protected region in panel A. Arrows correspond to the palindromic sequence. Boldface gtg represents the translation initiation codon. **, transcription initiation site as determined by primer extension. The −35 and −10 boxes of the deduced promoter sequence are shown in italic.

Fig. 4.

Effect of saccharides on the interaction between CsnR and the csnA-WT operator. The indicated saccharides were added (500 nM) to binding reaction mixtures containing ∼8.5 ρmol of CsnR and 0.03 nM csnA-WT probe. Free and complexed DNA fragments were separated by 6% polyacrylamide gel electrophoresis and visualized by PhosphorImager.

Fig. 5.

β-Lactamase activities of S. lividans TK24 wild-type or ΔcsnR strains harboring various blaL gene fusions. Solid symbols, 0.5% mannitol medium; open symbols, medium containing 0.125% GlcN and 0.375% chitosan oligomers. Triangles, S. lividans TK24; diamonds, S. lividans ΔcsnR strain. (A to C) BlaL activities obtained from the csnA promoter region (A), the mutated csnA promoter region (B), and the csnR promoter region (C). Means were calculated from two independent experiments. Error bars represent standard errors.

Fig. 6.

RT-PCR expression profiling of putative chitosanase genes belonging to families GH2 (SAV_1223), GH46 (SAV_2015 and SAV_6161), and GH75 (SAV_1288 and SAV_1850) in S. avermitilis grown in the absence (−) or presence (+) of chitosan oligosaccharides. Expression of the SAV_4958 (rps1) gene was used as an internal control. Asterisks indicate chitosanase genes with the CsnR box.