Detection of Rhizobial Conjugative Transfer by Flow Cytometry: Tools and Applications

TORRES TEJERIZO, G. A.; CERVANTES, L.; ALTHABEGOITI, M. J.; PISTORIO, M.; ROMERO, D.; BROM, S.

Cuatro Ciénagas

Congreso; III Congreso de Bioquímica y Biología Molecular de Bacterias; 2013

Sociedad Mexicana de Bioquímica

One of the major evolutionary forces that have shaped microbial diversity is horizontal gene transfer (HGT), which can be carried out by transformation, transduction or conjugation. Conjugative transfer (CT) has been pointed out as one of the crucial mechanisms for HGT. Rhizobia are Gram-negative bacteria able to grow in the soil in free-living conditions, and in symbiosis associated with the roots of legumes, as nitrogen-fixing organisms. A general feature of rhizobia is the presence of a large amount of plasmid DNA in their genomes. Conjugal transfer of rhizobial plasmids has been well documented since decades ago. Most of the projects evaluating rhizobial CT were performed under laboratory conditions, where plasmid transfer can be easily detected by growing bacteria in selective media (after tagging the plasmids with antibiotic resistance genes). Nevertheless, there are only a few reports that analyze CT in soils, resembling natural conditions. Aiming to detect CT in natural environments and avoiding the need of bacterial growth, we present here a system to specifically detect the transfer of tagged plasmids from known-donor rhizobia. The system was modified from a report originally set up for Pseudomonas (Nancharaiah et al., AEM 69:4846. 2003). The known-donor rhizobial strain was tagged in the chromosome with a Red Fluorescent Protein gene (RFP) and Gentamicin resistance by means of a MiniTn5, and the plasmid was tagged with a Green Fluorescent Protein gene (GFP) and Spectinomycin resistance. To label the plasmids, a set of cassettes with different promoters and antibiotic-resistance genes was constructed, to maximize the versatility of the system. The system allows us to determine conjugal frequencies, after cell separation by Flow Cell Cytometry. The donor rhizobia appeared as bacteria with red and green fluorescence, the receptor bacteria did not show any fluorescence and the transconjugants (after transfer of the plasmid tagged with GFP) showed only green fluorescence. Cell counts by Flow Cell Cytometry and by cultivation of bacteria in selective media were similar, validating the system. This approach will allow us to analyze different enviroments that may affect CT, under laboratory or natural conditions, easily and in a faster way. This system is a valuable tool for the study of the mechanisms involved in the evolution and diversification of rhizobia, by measuring the impact of CT in natural environments, and detecting the factors that influence CT of the plasmids. Acknowledgements: To Ivonne Rosenstein and Erika Melchy for use of the Flow Cytometer, and to CONACYT-CONICET for México-Argentina bilateral cooperation grant Nº 188967