| The two-component system, CbrA-CbrB（catabolic regulation）, was discovered as ansignal-transduction system.It has been described in certain species of Pseudomonads as a globalregulatory system required for the assimilation of carbon and nitrogen sources. CbrA-CbrB formsnetworks with other regulation systems that involve more than one system, and show dependencies andhierarchies, which makes it easily adapt very complex processes and plays an important role in theprocess of microbial adaptation to the environment. CbrA-CbrB in Pseudomonads stutzeri A1501,nitrogen-fixing bacteria, has never been reported. In order to analyze the biological function ofCbrA-CbrB in A1501, a deletion cbrA mutant was constructed and used to detect physiological andbiochemical traits. The major findings of this study were:1ã€Multiple sequence alignment revealed that CbrA ortholog in P. stutzeri A1501had an identity of78%and84%amino acid identity to the well-characterized P. aeruginosa CbrA and CbrB, respectively.The amino-terminal half of CbrA appears to be12possible transmembrane helices, whereas thecarboxy-terminal part shares homology with the histidine of the NtrB family.The CbrB responseregulator shows similarity to the NtrC family members. Which was coincide with the structure ofCbrA-CbrB in P. aeruginosa.2ã€A cbrA deletion mutant of A1501was constructed by gene replacement. The cbrA mutant grew aswell as the wild type parent on LB or the minimal medium containing lactose as sole carbon source. Theutilization of several amino acid（such as L-histidine, L-arginine，L-Alanine and L-Aspartic Acid）,Polyamines（such as putrescine）and carbohydrate（such as-D-glucose and maltose）decreased in cbrAmutant, whereas utilization of D-Fructose and L-asparagine increased. The expression of genes in EMPand ED，both encode enzymes for-D-glucose degradation, were decreased significantly.3ã€In cbrA mutant, the expression of cbrB was1/5as that in A1501under normal conditions, whencrcZ almost no expression. It has been reported CbrB positively regulate crcZ expression inPseudomonas aeruginosa.The experiment result may suggested that the regulation mode of CbrA-CbrBin A1501may be similar to that in P. aeruginosa. Because the expression of cbrB was very low in cbrAdeletion mutant, low-level CbrB cannot start the expression of crcZ.4ã€Here it is demonstrated that CbrA and CbrB play an important role in chemotaxis. The chemotacticring diameter of cbrA deletion mutant was about1/2as that of the wild-type, which showed the deletionof the cbrA affected chemotaxis of bacteria, indicating that the function of CbrA is diversity in A1501,not only affects carbon source metabolism, but also regulate other metabolic pathways.5ã€cbrA affects nitrogenase activity and denitrification capacity in A1501. Nitrogenase activity of thecbrA mutant decreased to approximately40%as that of the wild-type A1501under microaerobicconditions in the free-living state. Real-time PCR revealed that the expression of nifH and nifA geneswere both reduced5-fold in cbrA mutant, consistent with the nitrogenase activity decrease phenotypesof cbrA mutant. The experimental results suggested that CbrA directly or in directly participate in theregulation of nitrogen fixation. What’s more，denitrification capacity of cbrA mutant significantly decreased compared to the wild type.6ã€The recombinant plasmid PBand PAB, used to construct cbrB and cbrAB mutant, have beenobtained. Moreover, we have also got the functional complementary plasmid cbrB-pLAFR3andcbrAB-pLAFR3. Which laid a foundation for cbrB and cbrAB mutant construction and the study of jointmechanism between CbrA-CbrB and NtrB-NtrC.This preliminary work laid experimental and theoretical foundations for the C-N coupling analysisin Pstutzeri A1501. |
