Regulatory Mechanisms Of Lfagellar Synthesis And Motility Expression By RpoN And RpoS In Pseudomonas Stutzeri A1501

In order to adapt to the complex environment, prokaryotes need to regulate thetranscription and expression of specific genes by different sigma factors. In the evolution ofmicrobial species，Sigma factors have developed diffirent structure and function. Accordingto the differences of structure and function, Sigma factors can be divided into two families:N70(j and￠7. At present，^o’^and^^aare researched more deeply. Most studies are basedon the level of bacterial transcriptome. Researchers discovered that the regulation oftranscription factors is a fairly complicated process and is the coaction of manyenvironmental factors. In prokaryotes, the multiple regulatory networks are used to regulatethe response to external stimuli. Much evidence indicates that the transcription activationdepends on the interaction of a multitude of transcription factors. In E.coli, we found asynergistic or antagonistic effect on the expression of certain specific genes. Whether thereis the similar regulatory mechanism in E.coli and Pseudomonas is still unclear. The rpoNdeletion mutant was constructed by gene replacement. There is the rpoS deletionmutant in lab. Both of the two mutants were used to study the regulation mechanism ofrpoN gene and rpoS gene on the flagella synthesis and the motility. The main results areas follows:1.The rpoN deletion mutant and the function complementary strain were constructed.2.In LB liquid medium, therpoMnutants of Pseudomonas stutzeri A1501grew veryslowly. The cell density was only50%of the wild type in stable phase. Growth of Comp-rpoiVstrain could recover to80%of the wild-type. The results showed that the deletion ofrpoN affected the growth of strain under normal conditions. On the contrary, the deletion ofrpoS gene had no significant influence on the growth of A1501in rich nutrition. Under theshock of2M NaCl or20mM H2O2, rpoS mutant were more sensitive than WT to the highosmotic pressure and hydrogen peroxide. Interestingly, rpoS mutant was not sensitive tohigh temperature (50*^0), which was different with E. coli.3.Under the condition of nitrogen fixation, rpoN mutant lost the ability of the nitrogen-fixing. And the nitrogenase activity of rpoS mutant decreased by approximately25%compared to the wild type.4.The chemotaxis experiments showed that mutants of rpoN and rpoS exhibited lossand increased motility. The results of transmission electron microscopy indicated that therpoN mutant was the flagellum-defective strain. However, the flagellar of rpoS mutant wasmore slender than WT. 5.The RpoN protein has? alpha helical structures and two beta folding structures analyzed by the phyre2. RpoN contains three functional domains. And the C-terminal exists the RpoN-box conserved sequence:421-lAGLLEAOGIOVARRTVAKYRESLGIAPSSERKRLL-457. The RpoS protein has14alpha helix structures and1beta folded structure. The conserved DNA-binding amino acid sequence of its C terminal is286-EASTLEEVGOEIGLTRERVROIOVEALRRLREILERNGLSSDALFO-332.The Weblogo forecasted that the conserved sequence recognized by RpoN is TGGCACNNNNNTTGC in A1501and the conserved sequence CTAWAWTG is recognized by RpoS.6.The RpoN protein was expressed in E.coli BL21(DE3).The two-intein purificationsystem was developed for the affinity purification of RpoN. We chose fliA which is the keyregulatory gene of flagella synthesis to investigate whether it could bind to the RpoN andRpoS protein. The upstream regions (300bp) of fliA was amplified as the probe.Electrophoresis mobility shitf assay showed that it could bind to RpoS, but it could not bindto RpoN.7.The competition in rhizosphere colonization of rice roots had shown that the absenceof rpoN gene made the colonization ability of bacteria decreased significantly. While thecolonization ability of ISrpoS in rice rhizosphere improved slightly.