Functional Identification Of Non-coding RNA CrcY In Pseudomonas Stutzeri A1501and Its Regulatory Roles

Bacteria have metabolic regulation systems that allow cells to selectively assimilate a preferred compound among a mixture of several potential carbon sources, at the same time, non-preferred carbon sources are suppressed. This process is known as carbon catabolite repression (CCR). Carbon catabolite regulation system is mainly composed of CbrA/CbrB two-component system, Crc protein and CrcY/CrcZ sRNA. In this system, the levels of free Crc are controlled by CrcZ and CrcY, which are able to bind Crc specifically and regulate the expression of target genes.A analogue of CrcY sRNA is detected through genome and transciptome in Pseudomonas stutzeri A1501.5’-RACE experiment suggests the transcription direction of CrcY is same as upstream gene and downstream gene. Sequence analysis shows that CrcY contain six potential targets for Crc, which form non-paired single strands to combine Crc protein. They have similar initiation site, highly conserved Crc binding sites and similar Rho-dependence termination. Northern blot hybridization proves the existence of CrcY sRNA.We try to explore the effect on the regulation of crcY and CrcZ to strain growth, ability to resist stress and CCR by building crcY mutant and overexpression. In addition, some strains have been used as comparisons, which include crcY mutant, crcYZ double mutant, crc mutant, cbrA mutant and cbrB mutant. We propose that CrcY and CrcZ sRNAs interact and function synergistically in most instances. If one of sRNAs lost, the other will increase substantianlly, which ensure the normal conduct of carbon metabolism. After double mutation, crcYZ double mutant is far away wild type in mixing carbon source, which have prominent carbon catabolite repression. Moreover, the lost of CrcY and crcZ has also weakened bacteria against adversity stress, such as high salt shock, heat shock, H2O2shock, and abilityies in the aspects of chemotaxis and motility.Nitrogenase activity test indicates that deletion of either crcZ or crcY had no effect on nitrogenase activity. However, the simultaneous absence of both sRNAs led to that nitrogenase activity is half of wild type, whose result is same as cbrB mutant and crc mutant. Compared with wild type, overexpression of crcY decreases half level of nitrogenase activity, but overexpression of crcZ has little impact on nitrogenase activity. After a series of nitrogenase activity experiment and RT-PCR analysis, we have found that the amount of CrcY plus CrcZ have something to do with nitrogen fixation within a certain range. But when the volume of CrcY and CrcZ reaches a certain level, a tremendous increase will not significantly enhance the value of nitrogenase activity. And when the total amount is extremely low or not, the value of nitrogenase activity will not disappear, whose efficiency is half of wild type.