Study Of Bacillus Subtilis Responding To Extracellular Signal Based On Transcriptome

Microorganisms always sense the intracellular and extracellular environmental signals in order to survive. When microorganisms sense environmental signals change, they can quickly adjust their gene expression and metabolic regulation by signal transduction mechanisms. Those mechanisms help microorganisms to survive against the environmental changes and also make their generations to be continued. Discovering and understanding the microorganisms of signal transduction has important significance. For example, culture conditions changes in fermentation processes may lead to significant change in production; nutrition condition and drugs concentration may affect pathogenic bacteria’s drug resistance. These examples may be explained in signal transduction studies. However, the mechanism of signal transduction is complex and hard to study. In this thesis, taking B.subtilis as example, gene microarrays were used to study B. subtilis’responding to some environmental signals. The main research contents are listed below.(1) Dissolved oxygen (DO) is an important parameter in adenosine fermentation. Our previous research results have demonstrated that low oxygen supply in the growth period was good for obtaining high adenosine yield. Herein, for better understanding the relationship between oxygen supply and adenosine productivity in B. subtilis, we sought to systematically explore the effect of DO on genetic regulation and metabolism through transcriptome analysis by using gene microarray. The microarrays representing4,106genes were used to study temporal transcript profiles of B. subtilis fermentation in response to high oxygen supply (agitation700r/min) and low oxygen supply (agitation450r/min). The transcriptome data analysis revealed that low oxygen supply has three major effects on metabolism:Ⅰ) enhance carbon metabolism (glucose metabolism, pyruvate metabolism and carbon overflow); Ⅱ) inhibit degradation of nitrogen sources (glutamate family amino acids and xanthine) and purine synthesis; Ⅲ) enhance respiration, sporulation and protein synthesis. Based on this information, we presented the signal pathway of the DO in B. subtilis and explained the mechanism of oxygen signaled B. subtilis’ metabolism and adenosine production. We also optimized the fermentation conditions according to the proposed mechanism and finally we got30%increased on adenosine production. These results provided new insights into the relationship between oxygen supply and microorganism’s metabolism.(2) Fusaricidins (Fus) are new cyclic lipopeptide antibiotics (CLPs) which have excellent bactericidal activities against Gram-positive bacteria. However, in our study, when B. subtilis was treated with Fus, Fus only inhibited B. subtilis’ growth temporally (with a lot of bacteria lysed at this period) and after couple of hours, B. subtilis’ growth resumed (resuming growth). Microarrays were used to discover the mechanism. Microarray Data analysis indicated that Fus firstly destroyed cell membrane and induced a lot of membrane protective genes (SigW Regulon). The results also indicated that B. subtilis developed drug-resistance to Fus after Fus treatment. During the resuming growth period, genes of cell motility, carbon metabolism and iron transport were most significantly affected by Fus. Further study indicated that the iron metabolism is closely related to drug-resistance. These results provided deeper insights into the mechanism of CLPs.(3) Heavy metal ions are important in microorganism’s metabolism and growth, but they possess cell toxicity. Scientists found heavy metal polluted water would induce microorganism’s cross-resistance to metal ions and antibiotics. Our study showed that high concentration of extracellular Fe3+would specifically increase B. subtilis’ resistance to CLPs. We used microarrays to explore the mechanism underlying this phenomenon. Microarray data analysis showed that high concentration of extracellular Fe3+would affect the bacteria’s membrane proteins, fatty acids composition of cell membrane and kinds of metabolism. Finally, we found that Fe3+can help to maintain PMF that can be disrupted by CLPs. PMF is important for maintaining intact cell in B.subtilis. By keeping the PMF, Fe3+can inhibit cell lysis caused by CLPs, thus leading to higher drug resistance. This study provided new mechanism of antibiotic resistance induced by heavy metal ions.