Analyzing The Function Of Two nodT Genes In Rhizobium Etli Are These Copies Directly Involved In The Nodulation Process

A. Hernández-Mendoza, T. Scheublin, N. Nava, O. Santana, C. Quinto

Departamento de Biología Molecular de Plantas. Instituto de Biotecnología, Universidad Nacional Autónoma de México Apdo. Postal 510-3 C.P. 62251, Cuernavaca, Morelos, México

In Rhizobium etli we have previously described the isolation of two nodT copies, one located on plasmid c (nodTplc) and the other on chromosome (nodTcro) (Hernández-Mendoza 1999)

In order to analyze the nodTcro function we have been making efforts to obtain an insertion in this gene without success. However, if we first complement with the wild type gene in trans, stable insertions were obtained. Since this procedure had been successful for site-directed mutagenesis of the non-essential gene nodTplc in R. etli, we propose that nodTcro is necessary for viability in R. etli under these conditions, or may encode an essential function. We are now isolating double recombinants cured of the complementing plasmid to analyze the nodTcro mutant phenotype. On the other hand, two ORFs were found upstream of nodTcro that have 73% to 63% identity with ameAB from Agrobacterium tumefaciens and mexAB from Pseudomonas aeruginosa, respectively. NodT has 50% and 30% identity with AmeC and OprM, respectively. AmeABC and MexAB-OprM form multidrug efflux pumps.

To gain insight into the possible function of nodTplc gene, this copy has been mutagenized by inserting a spectinomycin cassette. nodTplc mutant does not have a clear nodulation phenotype. However, two ORFs that have 30 to 50% identity with Escherichia coli cpxAR genes were localized upstream of nodTplc. CpxAR form a two-component signal transduction system that in E. coli responds to heat shock and membrane damage. A putative a24 promotor sequence was also found upstream of nodTplc. In order to analyze the role of this gene in response to outer membrane damage stress produced by heat shock and thermotolerance, we analyzed the growth behavior of the mutant with respect to the wild type at different temperatures. However, our results indicate that nodTplc is not involved in bacterial growth at different temperatures neither in basal thermotolerance under the conditions that we have analyzed.

The putative promoter sequence was subcloned into the expression vector pBB53 ::gitóvl in both orientations, upstream of the reporter gene uidA (P-glucoronidase activity). This plasmid was mobilized to wild type R. etli by triparental mating. GUS assays indicated that the promoter in the correct orientation has a 2-4-fold higher expression with respect to that of the promoter in opposite orientation in the presence of ethanol, heat shock conditions or in standard conditions. This suggests that the former promoter orientation has a basal activity but it does not respond to changes in temperature or presence of ethanol in the conditions that we have tested.

Taken together, our results suggest that nodTcro and nodTplc have different, but not complementary roles in the R. etli physiology. However, biochemical, genetical and physiological studies must be still carried out to fully characterize these genes.

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