SCO3129, a TetR family regulator, is responsible for osmotic stress in Streptomyces coelicolor

Abstract

Streptomyces are the soil-dwelling bacteria with a complex lifecycle and a considerable ability to produce a variety of secondary metabolites. Osmoregulation is important for their lifecycle in nature. In the genome of Streptomyces coelicolor M145, SCO3128 (encodes a putative fatty acid desaturase), SCO3129 (encodes a putative TetR family regulator) and SCO3130 (encodes a putative l-carnitine dehydratase) constitute a transcriptional unit, and its transcript was found to be in response to osmotic stress. Disruption of SCO3130 led to a bald phenotype on MMG medium and the mycelia lysis on the edge of the colony when KCl/NaCl was added to the medium. These results indicated that SCO3130 is important for the osmotic stress resistance in S. coelicolor. Transcriptional analysis and electrophoretic mobility shift assays (EMSA) demonstrated that SCO3129 repressed the transcription of SCO3128-3130 operon through directly binding to the promoter region of SCO3128, indicating that SCO3129 regulates the transcription of SCO3128-3130 in response to osmotic stress.

Keywords: Osmotic stress; Regulation; Streptomyces coelicolor.

Fig. 1

Gene organization of SCO3128-3130 and its transcription profile with or without KCl. (A) Gene organization of SCO3128-3130 and RT-PCR analysis of the transcription of SCO3128-3130 in S. coelicolor M145. The PCR templates were RNA (1, as negative control), genomic DNA (2, as positive control) and cDNA (3), respectively. (B) The transcription of SCO3128 was induced by KCl. Strains were grown in YEME containing 10.3% sucrose for 15 h and treated with or without 1 M of KCl. RT-PCR analysis was done with the samples taken at 1 h, 3 h and 12 h after treated by KCl.

Fig. 2

Phenotype of strainsSCO3128DM, SCO3129DM, SCO3130DMand M145 on MM agar. (A) Schematic representation of gene disruption. SCO3128DM, the SCO3128 disruption mutant; SCO3129DM, the SCO3129 disruption mutant; SCO3130DM, the SCO3130 disruption mutant. (B) Phenotype of strains on MMM (mannitol as the sole carbon source), MS (soya flour with mannitol) and MMG (glucose as the sole carbon source). a, S. coelicolor M145; b, SCO3128DM; c, SCO3129DM; d, SCO3130DM. Scanning electron microscopy of SCO3128DM, SCO3129DM and SCO3130DM grown on MMG for 4 days.

Fig. 3

Salt sensitivity analysis of each mutant. Salt sensitivity of SCO3128DM, SCO3129DM and SCO3130DM was examined by streaking cells on MM plates containing 1 M of KCl. A, Colony morphology of each mutant. Strains were grown at 28 °C for 5 days on MMM or MMG. B, Analysis of cell weight of SCO3130DM and M145 on MM agar with or without KCl. 1 × 10⁷ pre-germinated spores of SCO3130DM and M145 were spread on cellophane membrane on MMM or MMG (containing 1 M of KCl) respectively. Mycelia were harvested after 5 days growth, and then the dry cell weight was measured. The data was obtained as average of three independent experiments.

Fig. 4

Phenotypes ofSCO3130DMand its complemented strain. SCO3130DM-C1 and SCO3130DM-C2 were random chosen as the complemented strains of SCO3130DM; Wild-type, the S. coelicolor M145. Phenotypes of these four strains were observed on MMG for 2–7 days.

Fig. 5

Comparison of the deduced SCO3129 with other TetR family regulators. The boxes showed N-terminal conserved helix-turn-helix (HTH) DNA binding domain, a typical feature of TetR family regulators. Helix-turn-helix (HTH) designated as α1, TTT and α2. Dashes represent gaps introduced into the sequence to obtain the best consensus. Homologous regions of the SCO3129 polypeptides and the other five polypeptides are shaded in black or grey, respectively. The following sequences were used for comparison: AcrR (Accession no. NP752516.1); QacR (Accession no. NP115320.1); TtgR (Accession no. 743546.1); JadR2 (Accession no. AAB36583.1); EnvR (Accession no. NP627346.1); MtrR (Accession no. ZP05107326.1).

Fig. 6

Transcriptional analysis of SCO3128 in M145 and SCO3129DM. A, The enhanced level of SCO3128 and SCO3127 transcripts in SCO3129DM. S. coelicolor M145 and SCO3129DM were cultured in YEME liquid medium containing 10.3% sucrose for 16 h. Total RNA was isolated and analyzed by realtime RT-PCR. B, Transcription level of SCO3128 in M145 and SCO3129DM after treated with or without KCl for a serial times. Strains were grown in YEME containing 10.3% sucrose for 15 h and treated with or without 1 M of KCl. Sample harvested just before treated with KCl designated 0 h, then other three samples were taken at 1, 3 and 12 h, respectively, after treated with KCl.

Fig. 7

Purification of His₆-SCO3129 and analysis of its binding to the promoter region of SCO3128-3130. A, Expression and purification of SCO3129-His₆. M, molecular size markers; 1, cellular lysate of E. coli BL21(DE3) carrying pET28a vector as negative control; 2, cellular lysate of E. coli BL21(DE3) carrying pET28a::SCO3129, but not induced by IPTG; 3, whole cellular proteins of E. coli BL21(DE3) with pET28a::SCO3129 induced by IPTG; 4, soluble proteins of E. coli BL21(DE3) with pET28a::SCO3129 induced by IPTG; 5–8, purified His₆-SCO3129 fractions from Ni-NTA affinity chromatography. B, EMSA analysis of SCO3129-His₆ binding ability to the promoter region of SCO3128-3130. The probe PE28 containing the upstream regions of SCO3128 were incubated with the increasing amounts of SCO3129-His₆ (lanes 1–6 contain 0, 1, 2, 4, 10 and 50 ng proteins). EMSAs of 50 ng SCO3129-His₆ with 200 fold excess of unlabelled specific probe (cold probe) are shown in lane 7 and 200 fold excess of non-specific competitor PET27 is shown in lane 8. The arrows indicate the free probes and the braces show SCO3129 -DNA complexes.