Cloning, Regulation And Functional Analysis Of NoeAB From Sinorhizobium Meliloti 042BM

The Tn5-1063 containing promoterless luxAB genes was used to mutagenize S. meliloti 042BM Three mutants involved in nodulation were obtained, named as 042BMR5, 042BMR11 and 042BM29, respectively .All of them showed some changes of ability in symbiosis. Further, their luciferase activities were determined in TY medium, and showed that Tn5-1063 was inserted directly into loci downstream of promoters in 042BM genome. DNA sequences flanking Tn5-1063 of 042BMR5 were amplified using inverse PCR , it was found that Tn5-1063 was inserted into noeB gene. The noeB of 042BM is identical to that of S. meliloti 1021 at 98% level, and similarity of amino acid sequences of their NoeB is 95%. Hydrophobicity analysis of the NoeB showed that it is a transmembrane protein and includes four transmembrane regions at N-terminal., consisting of three primary helixs and one secondary helix.042BM noeA was obtained by PCR, it is identical to that of strain 1021 at 99% level, and similarity of their NoeA is 97%. In addition, it was also found that this protein shares limited but significant homology with the SAM-dependent methyltransferase of Mesorhizobium sp.BNC1(32% similarity) , and the similarity of its 303-362 region to the 160-220 domains of L11 methyltransferases of E.coli (PrmA) is 41%. It is suggested that methylation of L11 resulted in effects of noeA on nodulation of 042BM.Compared to 042BM, the noeA deletion mutant 042BMA-Km showed different degree of increase in nodule number, weight of nodule nodule and plant top dry weight on cultivars of Putong Zihua, Baoding, Ningxia, Baifa and Aohan, but decrease on Milu. However, this mutant has no significant change in ability to nodulate cultivars of Huanghou and Zahua. Hence, noeA is involved in cultivar-specific nodulation.Trigonelline could not elevate the level of noeAB expression, which indicated that these genes aren't regulated by nodD2. Since association of nodD3 and syrM can't change the level of the genes expression, they aren't also controlled by nodDS-syrM system. However, Induction of luteolin resulted in 16 times increase of noeAB expression, which indicated that noeAB was regulated by nodD1. Most interestingly, more than 30 times increase in its expression was observed on TY medium withoutany flavonoid. Thus, it was suggested that noeAB may be controlled by another unknown mechanism.Proteomic analysis of 042BMA-Km was carried out using two-dimensional gel electrophoresis. Deletion of noeA had a pleiotropic effect on protein synthesis levels. A minimum of 44 proeitn differences were observed for the noeA mutant. Among them, at least 15 proteins were up-regulated, over 28proteins were newly synthesized, and one protein was down-regulated.