| Stable and high efficiency micro-plant interaction is essential to the optimal nitrogenase activity of root-associated nitrogen fixation bacteria. There are several factors that participate in this process including the recognition of outside signal, the chemotaxis, colonization on the roots and the invasion into the superficial layers of the root cortex. Diazotrophic bacteria can be induced by some certain signals from environment and move to the surface of roots directionally. During this process, the flagellum plays an important role. Pseudomonas stutzeri A1501, isolated from rice paddy soils in south China, exhibits good nitrogen-fixing activity under the condition of low oxygen tension and ammonia concentration. At present, the main research regions of A1501 are focused on the regulation of nitrogen-fixation (nif) genes and the uptake system of carbon source, which have been made great progress. However, it is still unclear on how the associative micro-plant interaction was established. Analysis of the recently published A1501 genome sequence, at least 100 genes related to the flagella biosynthesis and chemotaxis were identified. We further select the putative regulator gene fleQ as the target gene for mutation and function identification.Sequence analysis of P. stutzeri A1501 genome showed that the fleQ and the two downstream genes, fleS and fleR encoded regulator are closely related to each other. The RT-PCR results showed that fleQ belonged to a transcribed unit, while fleS and fleR were shared in an operon. Amino acid sequences were compared using the NCBI-BLAST search system. A1501 FleQ was found to share protein sequence homology with known FleQ regulator proteins bacterial species, which showed highest homology to P. aeruginosa FleQ (identity = 83%). To analyze the function of A1501 regulator gene fleQ, we constructed the suicide plasmid vector pfleQ, containing the fleQ gene fragment. Then, pfleQ was transfered into the wild type strain A1501 by tri-parental mating assay and the mutation strainâ–³FleQ was obtained by homologous recombination selection. The complementary strain (FleQComp) carrying the expressing fleQ gene plasmid was also constructed. The mutant of fleQ leads to the disappearing of flagellum, but it can be recovered by the complementation strain FleQComp. This result was observed and confirmed by the transmission electron microscopy (TEM). Disruption of fleQ doesn't affect the growth and nitrogenase ability of the mutant, but influences the ability to move and affect the formation of the biofilms. Real-time RT-PCR assays show that the expressions of some key structure genes encoding of the flagellum biosynthesis, such as flgB, flgE, fliC and fliG, were descend down-regulated dramatically by RT-PCR in the fleQ mutant. The results showed indicates that FleQ can be a positive regulateor on positively the expression of the flagella gene, and the mutant will stop the the synthesis of flagellum, which will influence the assembly of flagellum. The results from experiment of rice-breeding experiment with by inoculate the mutant and WT A1501 strains found that the roots and height of rice were significantly weaker in mutant than in WT A1501. This suggests that the growth of crops was influenced, because the system of the demolishment of the associative nitrogen fixation system owning to the incapacity to produce functional flagella.From all the results above, we make a hypothesis that fleQ may be an important regulation gene for the synthesis of flagellum and may be function at the upper regulation lever compared to the traditional regulator genes in each transcriptional operon. However, the exact function of fleQ in flagellum biosynthesis system needs further investigations.
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