Functional integration of the Bradyrhizobium diazoefficiens flagellar systems

JIMÉNEZ SÁNCHEZ, C; ITURRALDE, ET; LODEIRO, AR; QUELAS, JI; MONGIARDINI, EJ; ORTEGA CALVO, JJ; ALTHABEGOITI, MJ; MELGAREJO, A; PÉREZ GIMÉNEZ, J

Budapest

Congreso; 12th European Nitrogen Fixation Conference; 2016

Institute of Biochemistry, BRC, Szeged

Biological performance is often characterized by expressing emergent properties that cannot be deduced from the functions of individual parts of the system. Flagella-driven swimming motility is among the most important performance of bacterial cells, in terms of its energy demands and its relevance for colonization of diverse environments. An interesting genus in regard of this performance is Bradyrhizobium, which includes the N2-fixing symbionts of soybean. While B. diazoefficiens and B. japonicum possess dual flagellar systems composed by a subpolar flagellar system and an evolutionary unrelated lateral flagellar system, B. elkanii possesses only the subpolar system, and B. liaoningense lacks functional flagella. However, all these species share the same soil environment and nodulate soybean, which raises some questions about the adaptive value of possessing energy-expensive dual flagellar systems. We approached these questions in B. diazoefficiens, which expresses its subpolar system constitutively, while the expression of the lateral system in liquid medium is conditioned by the carbon source. Thus, we wondered whether their functions might be integrated for the single task of swimming in the water channels of the soil. By comparing the swimming behavior of the wild-type and mutants expressing either the subpolar or the lateral system, we observed that the wild-type swimming behavior does not arise only from additive effects of each flagellar system. Moreover, the subpolar system is the main contributor to propulsion and chemotaxis, while the lateral system contributes to hold swimming near surfaces, in porous media, and in viscous environments. By artificial evolution we obtained B. diazoefficiens and B. japonicum derivatives with higher motility in porous medium, which in field assays were more competitive for soybean nodulation and produced higher grain yield. Therefore, we carried out a deeper study on the B. diazoefficiens derivative. We observed that its subpolar flagellum contributed to higher swimming speed, the lateral flagella were derepressed, and the interaction between both systems was similar to the wild-type. In addition, this derivative had higher swarming motility. All its phenotypes were maintained after passage through nodules and after more than 10 years of use in the laboratory, indicating that they are heritable. Hence, we decided to compare the complete genome sequence of this derivative against the wild-type, and found that none of the known flagellar genes was modified. By contrast, we found only 10 changes, 9 of which were SNP and 1 was a small deletion. The changes occurred in ORFs encoding unknown proteins (3), transcription-related proteins (3), metabolic proteins (2), efflux transport proteins (1), and in an intergenic region (1). We are currently obtaining site-directed mutants in these loci to assess their effects on bacterial motility and lateral flagella regulation.