SACE_0012, a TetR-Family Transcriptional Regulator, Affects the Morphogenesis of Saccharopolyspora erythraea

Xiaojuan Yin 0 Xinqiang Xu 0 Hang Wu 0 Li Yuan 0 Xunduan Huang 0 Buchang Zhang 0 0 X. Yin X. Xu H. Wu (&) L. Yuan X. Huang B. Zhang (&) Institute of Health Sciences, School of Life Sciences, Anhui University , Jiu Long Road No. 111, Hefei 230601, China Saccharopolyspora erythraea, a myceliumforming actinomycete, produces a clinically important antibiotic erythromycin. Extensive investigations have provided insights into erythromycin biosynthesis in S. erythraea, but knowledge of its morphogenesis remains limited. By gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012 was identified to be a negative regulator of mycelium formation of S. erythraea A226. Detected by quantitative real-time PCR, the relative transcription of SACE_7115, the amfC homolog for an aerial mycelium formation protein, was dramatically increased in SACE_0012 mutant, whereas erythromycin biosynthetic gene eryA, a pleiotropic regulatory gene bldD, and the genes SACE_2141, SACE_6464, SACE_6040, that are the homologs to the sporulation regulators WhiA, WhiB, WhiG, were not differentially expressed. SACE_0012 disruption could not restore its defect of aerial development in bldD mutant, and also did not further accelerate the mycelium formation in the mutant of SACE_7040 gene, that was previously identified to be a morphogenesis repressor. Furthermore, the transcriptional level of SACE_0012 had not markedly changed in bldD and SACE_7040 mutant over A226. Taken together, these results suggest that SACE_0012 is a negative regulator of S. erythraea morphogenesis by mainly increasing the transcription of amfC gene, independently of the BldD regulatory system. - During its life cycle, the soil-inhabiting Actinomycetes undergoes a complex morphological differentiation to adapt to adverse environments [1]. Growth of Actinomycetes begins with spore germination and hyphal outgrowth, leading to the formation of a vegetative, or substrate mycelium. Sensing of nutrient deprivation stimulates reproductive growth resulting in the development of aerial hyphae and spore chains [2]. Saccharopolyspora erythraea could form the aerial hyphae, and produce erythromycin, which is a macrolide antibiotic with broad-spectrum antimicrobial activity. Extensive genetic and biochemical studies have provided detailed insights into the genes involved in erythromycin biosynthesis in S. erythraea [3, 4], yet its morphological differentiation remains poorly understood. In recent years, the availability of the complete genome sequence of S. erythraea allowed a deeper exploration of the molecular processes controlling its morphogenesis [5]. Guided by in vitro and in vivo investigations, BldD (SACE_2077) was discovered to be a key developmental regulator in actinomycetes [1], controlling erythromycin biosynthesis and morphological differentiation in S. erythraea [6]. Furthermore, we identified a TetR-family transcriptional regulator (SACE_7040) involving in S. erythraea mycelium formation, and established genetic evidence for the crosstalk between SACE_7040 and BldD [7]. In this study, we have used gene inactivation, complementation, and transcriptional analysis to delineate the role of a new TetR-family regulator (SACE_0012) in the development differentiation of S. erythraea. Deletion of SACE_0012 principally influences the transcription of a putative aerial mycelium formation gene SACE_7115, that is homologous to amfC of Streptomyces. Materials and Methods Strains and Growth Conditions Saccharopolyspora erythraea A226 and its derivatives were incubated in TSB medium at 30 C for DNA extraction, protoplast preparation, and in liquid fermentation medium R5 for analysis of erythromycin production. R3M agar medium was used for protoplast regeneration, phenotypic observations, and RNA extraction [7]. Escherichia coli DH5a was the host for plasmid construction [8]. Bacillus subtilis PUB110 was used for an inhibition test of erythromycin production in S. erythraea. Plasmid, DNA Isolation, and Manipulation Plasmid pUCTSR [9] was a pUC18 derivative containing a 1.36 kb fragment of a thiostrepton resistance cassette (tsr) cloned into the BamHI/SmaI sites. The E. coli-S. erythraea integrative shuttle expression vector pZMW [4, 10] was used for the gene complementation. DNA isolation and manipulation in E. coli and S. erythraea were carried out according to the standard methods [8, 11]. Disruption of SACE_0012 in S. erythraea A226, bldD, and SACE_7040 Mutant Two 1.5 kb fragments flanking the SACE_0012 gene were amplified from genomic DNA of S. erythraea A226 by PCR using the primer pairs P1/P2 (50-TGC GAA TTC CTC CTC Fig. 1 Inactivation of the TetR-family regulatory gene SACE_0012. a Schematic deletion of SACE_0012 in S. erythraea A226. b Confirmation of SACE_0012 deletion mutant by PCR analysis with the primer pair P1/P4. The size of 3.69 kb for the PCR-amplified bands was observed in wild-type A226, while a band of the size 4.36 kb was observed in mutant DSACE_0012, suggesting that the SACE_0012 gene was completely deleted GGC CGG TGA GCA GC-30; 50-GAT GGT ACC ATA CGA GCG GCC CCA ACC CGA AAG CCC-30) and P3/P4 (50-ATT TCT AGA ACA CGC CCG CCA CCG GCT TCG C-30; 50-ACC AAG CTT AAG GGC TCG ATC GAC TCC TGG CGG-30). Then, the two DNA PCR products were inserted into the EcoRI/KpnI and XbaI/HindIII sites of pUCTSR, respectively, yielding pUCTSRD0012. By linearized fragment homologous recombination [7], the SACE_0012 gene was replaced with the thiostrepton resistance gene in the S. erythraea A226 chromosome, and the selected mutants were verified by PCR using the primers P1/ P4 (Fig. 1a, b). Similarly, SACE_0012 disruption was formed in the bldD and SACE_7040 mutant strains. Genetic Complementation of the SACE_0012 Mutant For complementation, the SACE_0012 gene was amplified by the primers P5 (50-TAA CAT ATG TTG AAA ACG GCG TCA ATC CTC ATC CCG-30) and P6 (50-CGC GAT ATC TCA GCG ATC GGC GGT AGT CG-30) from genomic DNA of S. erythraea A226, and was ligated into the NdeI/EcoRV sites of pZMW [9] to generate pZMW0012. Then, pZMW-0012 was introduced into SACE_0012 mutant by PEG-mediated protoplast transformation, generating the complemented strain DSACE_0012/pZMW0012. Quantitative Real-Time PCR (qRT-PCR) The transcriptional levels of eryA, bldD, SACE_0012, and homologous genes of whiA, whiB, whiG, and amfC associated with morphogenesis in Streptomyces (Table S1) [12], were determined by qRT-PCR. Specific primers were designed as listed in Table S2. Total RNA was isolated from S. erythraea A226 and the mutants of SACE_0012, bldD, and SACE_7040 after 2 or 4 days growth on R3M agar medium. Then, extracted RNA was treated with DNase I (Fermentas), and reverse transcription was accomplished using a cDNA synthesis kit (Fermentas). qRT-PCR reactions were performed on the Applied Biosystems StepOnePlus system with MaximaTM SYBR Green/ROX qPCR Master Mix (Fermentas). The hrdB gene encoding the major sigma factor in S. (...truncated)