SACE₀012, A TetR Family Transcriptional Regulator,Affects The Morphogenesis Of Saccharopolyspora Erythraea

Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces erythromycin, a clinically important antibiotic. Extensive investigations have provided insights into erythromycin biosynthesis in Sac. erythraea, but knowledge of its morphogenesis remains limited so far. The TetR family regulates a wide range of cellular activities, such as multidrug resistance, antibiotic biosynthesis, and morphological differentiation, which is widely distributed among bacteria. It was reported that Sac. erythraea contains101TetR-like transcriptional regulators. TetR-family transcriptional regulator SACE₀012had been disrupted by Xinqiang Xu in our lab previously, and was identified to be a negative regulator of mycelium formation of Sac. erythraea. The aim of this study was to further explore the molecular mechanism of SACE₀012in mycelium formation of Sac. erythraea.To verify the function of SACE₀012, ASACE₀012was inoculated into the flat of R3M at30℃. The results showed that ASACE0012mutant formed aerial mycelium24h earlier than the original strain A226. But after a longer cultivation to the sixth day, no significant phenotypic difference was observed between the wild-type strain A226and mutant ΔSACE₀012, revealing that SACE₀012was responsible for the early aerial hypha formation of Sac. erythraea. And by HPLC analysis, erythromycin A production was not apparently changed between ΔSACE₀012and Sac. erythraea A226. The results suggested that SACE₀012did not affect the synthesis of erythromycin. The ASACE₀012/pZMW-0012strain had restored the timing of aerial mycelium with a single copy of SACE₀012in pZMW to illustrate that it was the SACE₀012deletion that caused the aerial mycelium to form earlier in Sac. erythraea. By using gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE₀012was identified to be a negative regulator of early aerial hypha formation of Sac. erythraea, and not involved in erythromycin biosynthesis.In order to explore the molecular mechanism of SACE₀012in mycelium formation, we compared A226and ΔSACE₀012for the transcriptional change to sporulation genes （whi, bldD, amfC） and the erythromycin structure gene eryA. ASACE₀012and Sac. erythraea A226were inoculated into the flat of R3M at30℃, the total RNA were extracted after the cells were finely ground in liguid nitrogen. Then, DNase1was used for degradation of genomic DNA from RNA preparations. RNA was reversely transcribed into cDNA, which was the template for quantitative real time PCR. The RT-PCR results showed that erythromycin structure gene eryA and global regulator gene bldD were not differentially expressed, the homologous genes of whi A, whiB, whiG involved in the regulation of sporulation in Streptomyces （SACE₂141, SACE₆464, SACE₆040, respectively） were also not differentially expressed, while the transcriptional level of SACE₇115（homologous to amfC） was2.0-fold higher in the mutant ΔSACE₀012than in the parent strain A226. The transcription of SACE₇115in the ΔSACE₀012/pZMW₀012complement mutant was back to the original level. The results showed that SACE₀012was not possiblly involved in the regulation of erythromycin biosynthesis, while affected the early aerial hypha formation of Sac.erythraea through AmfC pathway.ΔbldD/ASACE₀012and ASACE₇040/ASACE₀012had constructed by Xinqiang Xu in our lab previously. The results showed that SACE₀012disruption failed to restore the defect in the mycelium formation of the bldD Mutant, and did not further accelerate the mycelium formation of the ASACE₇040mutant. To futher examine the relationship of SACE₀012and BldD or SACE₇040regulatory system, the transcriptional levels of SACE₀012in the AbldD and ΔSACE₇040strains and bldD in ASACE₀012strain were analysised by qRT-PCR. The results showed that SACE₀012transcriptions were slightly decreased but not obviously different in the AbldD and ASACE?₇040, and the transcriptional level of bldD in ΔSACE₀012were not differentially expressed compared to original strain A226, indicating that SACE₀012, although influencing morphological differentiation, was likely independent of the BldD regulatory system and the possible cause of the early aerial hyphae formation in ASACE₀012was the higher transcriptional level of SACE₇115.The SACE₇115mutant was constructed to address the relationship between SACE₀012and AmfC. The method was as follows:two1.5kb DNA fragments that upstream and downstream fragments of SACE₇115of the adjacent regions were amplified from the genome of Sac. erythraea A226. Then the amplified products were digested and sequentially inserted into the corresponding sites of pUCTSR, yielding pUCTSRΔ7115. Secondly, the fragments, which carrying the thiostrepton resistance gene and the flanking fragments of SACE₇115, were introduced into protoplasts of Sac. erythraea A226through PEG-mediated transformation. SACE₇115disruption mutant were selected by growing on R3M agar medium flooded with thiostrepton （30μg/mL） selection. It was found that aerial hyphae mycelium of ASACE₇115was delayed compared to the original strain A226, and that was consistent with the results in SACE₀012deletion mutant, and the increased expression of SACE₇115in the ΔSACE₀012mutant. Those futher confirmed our conclusion that the early aerial hyphae formation in ΔSACE₀012is caused by the higher transcriptional level of amfC.In summary, through gene inactivation and complementation with further transcriptional analysis, SACE₀012was shown not to be related to the BldD regulatory cascade and not to be related to erythromycin biosynthesis, and it regulated early aerial hypha formation of Sac.erythraea by AmfC. This conclusion was useful for comprehensive understanding regulatory mechanism of morphological differentiation in Sac. erythraea.