References

Galibert F et al. (2001) Science 293, 668-672 Gôttfert M et al. (2001) J. Bacteriol. 183, 1405-1412 Gouzy J et al. (1999) Computers and Chemistry 23, 333-340 Kaneko T et al. (2000) DNA Res. 7, 331-338

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) (1).

The 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 nodTplc. CpxAR form a two-component signal transduction system that in E. coli responds to heat shock and membrane damage. A putative cr24 promotor sequence was also found upstream nodTplc. We are analyzing now the role of this copy in heat resistance.

In order to analyze the nodT cro 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, suggesting that this gene is essential. On the other hand, two ORFs were found upstream 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.

Our results suggest that in R. etli, nodT genes are not directly involved in the nodulation process.

References

Hernández-Mendoza A (1999) M.S. Thesis, IBT-UNAM Acknowledgement

A. H.-M. was supported by a CONACyT Student Fellowship No. 90288.

A SIMPLE SYSTEM FOR CLONING RHIZOBIUM PLASMIDS

I. Hernández-Lucas1, T.M. Finan2, P. Mavingui1, E. Martinez1

dentro de Investigación sobre Fijación de Nitrógeno, UNAM, Apdo. Postal 565-A

Cuernavaca, Morelos, México department of Biology, McMaster University, Hamilton, Ontario, Canada

In this work, we developed a simple system to clone Rhizobium plasmids in E. coli. This method can be used to clone plasmids potentially from many gram-negative bacteria. The requirements for this method are a Tn5 inserted in the replicón to be cloned, the integrative vector pTH509 (Chain 2000), which contains 300 bp of the IS50, the oriT (RK2), a QSp cassette and the F origin of replication. The system consists of a conjugation between E. coli harboring pTH509 and Rhizobium-bewc'mg plasmid with a Tn5 insertion. A single cross-over occurs between the IS50 of the integrative vector and that of target Rhizobium plasmid. Rhizobium transconjugants are selected and mated with E. coli. The resultant E. coli strain carries the Rhizobium plasmid under the control of the F origin of replication. We have applied this simple system (based on two matings) to clone plasmids of Rhizobium etli, Rhizobium tropici and Sinorhizobium meliloti. The average size of the replicons that has been obtained is 200 kb. In contrast, plasmids of higher molecular weight (600 kb) were recalcitrant to cloning as a whole molecule, although fragments of 200 kb of such replicons were obtained. In the case of R. tropici pSym we obtain three different regions that cover almost the complete symbiotic plasmid of 600 kb, one of these fragments carried all the genes necessary to nodulate and fix nitrogen. Our results showed that this technique can be used to isolate different parts of high molecular replicons or to clone directly plasmids of moderate size, from Rhizobium species. Currently some of the replicons that we isolated are being sequenced.

References

Chain P et al. (2000) J. Bacteriol. 182, 5486-5494 Acknowledgements

We thank José Augusto, Ramírez -Trujillo, Miguel Angel Gaitan, José A Gama, Victor González for technical help and discussion, and Susana Brom, who kindly supplied strains.

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