Transcriptional Characteristics Of Non-coding RNA Genes CrcZ And CrcY And Their RNA Stability In Nitrogen-fixing Pseudomonas Stutzeri

Carbon Catabolite Repression(CCR)allows bacteria to acclimate rapidly to preferred carbon source and inhibits the assimilation of non-preferred carbon source thereby maximizing growth rate in complicated nutritional environment.The resent study indicates that non-coding RNA(ncRNA)interacts with RNA-binding proteins(Crc and Hfq),down-regulating the utility of non-preferred carbon sources at post-transcriptional level.The numerical relationship between ncRNA and inhibitor protein effects the hierarchy of carbon source utility.When preferred carbon source is present,RNA-binding protein binds the mRNA of non-preferred carbon source assimilation genes,causing the loss of the ability to utilize non-preferred carbon source;when preferred carbon source is depleted,ncRNA binds RNA-binding protein,leading to the target mRNA translation therefore the initiation of non-preferred carbon source consumption.The ncRNA mediating CCR exists in almost all Pseudomonas species,but the number and type are different.There exist two ncRNAs in a root-associated bacterium Pseudomonas stutzeri A1501,namely CrcZ and CrcY,however,the function and mechanism of CrcZ and CrcY are still unclear.In this thesis we study the transcriptional characteristic of ncRNA genes crcZ and crcY and their RNA stability in Pseudomonas stutzeri A1501.The main results are described as follows:1.The transcriptional characteristics of crcZ and crcY are studied by using PcrcZ-lacZ and PcrcY-lacZ fusion vectors,?-galactosidase activity results show that the transcriptional activity of crcZ in WT cultured in restrictive(glucose and lactate as the sole carbon source respectively)medium are similar and are 4.9 folds as much as 85 U,which is the transcriptional activity of crcZ in WT cultured in LB medium;transcriptional activities of crcY in the above three culture condition are significantly different,the transcriptional activity of crcY in WT cultured in LB medium is 209 U,the transcriptional activity of crcY in restrictive(lactate as sole carbon source)cultured WT is 0.57 folds as much as that in restrictive(glucose as sole carbon source)cultured WT and is 2.03 folds as much as that in WT cultured in LB medium.Compared with the nitrogen-restricting condition,the transcriptional activity of crcZ and crcY in nitrogen-fixing condition decreases 0.1 and 0.16 folds respectively.Additionally,we find that both transcriptional activities of crcZ and crcY in WT cultured in carbon-scanty conditions are higher than these in carbon-abundant condition.In the culture condition mentioned above the transcriptional activities of crcZ are all obviously lower than those of crcY in WT,however,ddPCR results show that the copies of CrcZ is 36.8 folds as much as CrcY in nitrogen-fixing cultured WT,which indicate that the stability regulatory mechanisms of the two ncRNAs are different.2.qRT-PCR and transcription activity results show that the level of CrcZ and the transcription activity of crcZ in ?crc cultured in restrictive(benzoate as sole carbon source)medium decrease dramatically compared with these of WT(0.016 and 0.3 folds respectively);the level of CrcY and the transcription activity of crcY in ?crc also decrease compared with these of WT(0.21 and 0.18 folds respectively),implying that Crc may affect not only the transcription of crcZ and crcY but also their stability.Compared with WT the expressional level of cbrB and rpoN in ?crc decrease1 fold under the cultural condition with benzoate as the sole carbon source,indicating that Crc affects the expression of cbrB and rpoN thereby up-regulating crcZ and crcY transcription.RNA stability measurement result shows that after 50 min CrcZ and CrcY in WT still retain 90% and 60% respectively,but after 17 min both CrcZ and CrcY in ?crc retain only 50% respectively.Moreover,we check the CrcZ and CrcY mRNA stability in RNA chaperon hfq mutant and find that after 50 min the CrcZ and CrcY level in ?hfq retained 79% and 64% respectively and that after 5 min both CrcZ and CrcY level retain only about 60% respectively.These results indicate that both Crc and Hfq have differentiated effect on stability of CrcZ and CrcY transcript.Bioinformatics analysis shows that the stem-loops of the predicted second structure of CrcZ and CrcY have several “CA motifs” and in the vicinity of “CA motifs” RNase E splice site(single strand AU-rich RNA)is found,suggesting that RNase E may be a specific degradation enzyme in the regulatory network of CrcZ and CrcY activity.In this study we initially discuss the transcriptional characteristic of crcZ and crcY of A1501 in different nutritional conditions and regulatory nodes and their stability,which provide a theoretical foundation for further investigating the CCR and nitrogen fixation network regulatory mechanism.