Two Non-coding RNAs CrcZ And CrcY Modulate The Carbon Catabolite Repression In Nitrogen-fixing Pseudomonas Stutzeri

Carbon Catabolite Regulation (CCR) is an important and complex regulatory response that allows the bacteria to selectively assimilate a preferred compound as a source of carbon and energy in competitive environments. The regulatory factors or systems known to be involved in CCR of Pseudomonas are CbrAB, Crc and ncRNAs (CrcZ, CrcY and CrcX), ncRNAs play a central role in CCR, howerver wheather the nitrogenase genes were regulated by the CCR systems have not reported. P. stutzeri A1501is an associative nitrogen-fixing bacterium which colonizes the rhizosphere of rice. The multiple sequence alignment revealed that the CCR regulatory systems as described in other Pseudomonas were in the genome of P. stutzeri A1501, but the regulatory mechanism of the two ncRNAs is not cleared. In current study, we investigated the roles of CrcZ and CrcY in modulating the carbon metabolism in P. stutzeri A1501, meanwhile we went through the role of the ncRNAs in nitrogenase regulatory. The major findings were:1. P. stutzeri A1501grown in medium K containing4mM succinate plus15mM glucose, which exhibited diauxic growth. That was due to A1501utilized succinate exclusively until it was exhausted and then shifted to catabolism of glucose.The results showed that sodium lactate and succinate are preferred carbon source for A1501, benzoate and glucose are less-preferred carbon source. We observed a typical catabolic repression phenotype in A1501.2. Transcriptome analysis of A1501under nitrogen-fixation conditions identified two ncRNAs CrcZ and CrcY in P. stutzeri A1501. The CrcZ of P. stutzeri A1501and which in P. aeruginosa are rather similar, sharing a70%nucleotide identity. CrcY of P. stutzeri A1501and which in P. putida are sharing a65%nucleotide identity. The homology of CrcZ and CrcY in P. stutzeri A1501is60%. The results of Northern blot and Real-time PCR showed that the expression of crcZ and crcY was reduced sharply in the cbrB and rpoN deletion mutant. The orientation and transcription start site of CrcZ and CrcY were determined by5’RACE. Well-conserved a54(RpoN) and CbrB recognition sequence occurred upstream of these ncRNAs. The EMSA revealed that CbrB binds to the promoter regions of crcZ and crcY. Therefore expression of crcZ and crcY is dependent upon RpoN together with the two-component system CbrAB with the alternative sigma factor RpoN. The CbrAB two-component system, CrcZ and CrcY were expressed at a low level in preferred carbon source and a high level in less-preferred carbon source. It means that expression of this system was influenced by the carbon source.3. In order to investigate the regulatory role of CrcZ and CrcY in carbon catabolite regulation in P. stutzeri A1501, we have constructed the crcZ and crcY individual and double mutants (△crcZ,△crcY, andAcrcZ/Y). Deletion of either crcZ or crcY has no effect on catabolite repression of P. stutzeri A1501. However crcZ/Y double mutant showed a growth defect in less-preferred carbon source, also the diauxic growth that exhibited in wild type and crcZ or crcY mutant was vanished. The results of Real-time PCR suggested that lack of CrcZ or CrcY leads to a compensatory increase in the levels of the remaining one. Thus, we propose that CrcZ and CrcY are functionally redundant in P. stutzeri A1501.4. The nitrogenase activity of the crcZ/Y mutant was reduced1/3compared with the wild-type using sodium lactate as the sole carbon source. The previous reasearch showed the nitrogenase activity of cbrA, cbrB, and crc mutant was approximately60%that of A1501. The expression of nitrogen fixation related genes was all reduced sharply in crcZ/Y mutant. Thus, we propose that the consequences of CCR system are not limited to regulate carbon catabolite, it exerts an important influence in nitrogenase activity of A1501.This study revealed the ncRNAs CrcZ and CrcY played a central role in CCR of P. stutzeri A1501. They could ensure A1501assimilate preferentially one specific compound that provides the most efficient growth and high efficiency nitrogen fixation. We established a model of CrcZ and CrcY in modulating the carbon metabolism and nitrogen fixation regulatory in P. stutzeri A1501, and laid a theoretical foundation of global regulation of carbon and nitrogen catabolite in P. stutzeri A1501.