Title: Membrane-associated signaling and sensing in Rhizobium etli - Analysisof c-di-GMP-related proteins and the osmoprotective Opp ABC transporter
Other Titles: Membraan-geassocieerde signalisatie en waarneming in Rhizobium etli -Analyse van c-di-GMP-gerelateerde eiwitten en de osmoprotectieve Opp ABCtransporter
Authors: Gao, Shanjun
Issue Date: 6-Mar-2015
Abstract: The Alpha-proteobacterium Rhizobium etli is a soil bacterium able to establish root nodules in a symbiotic interaction with the common bean plant Phaseolus vulgaris. Within the root nodules, R. etli reduces atmospheric nitrogen gas to ammonia which can be assimilated by the plant. In return, the plant provides R. etli with carbon nutrients during the symbiosis. Besides having a symbiotic relationship with the plant, R. etli encounters different environmental stresses when living in the soil. To adapt to the unfavorable conditions either in the symbiotic root nodules or in the free-living state, R. etli has evolved sophisticated strategies. In this dissertation, we investigated two systems for their involvement in symbiosis and free-living stress survival, i.e. cyclic di-GMP mediated signaling pathways and an ATP binding cassette (ABC) transport system in R. etli.
First, we systematically analyzed the presence of c-di-GMP related proteins in different rhizobial species and specifically focused on dissecting the functional and transcriptional profiles of two c-di-GMP related proteins in R. etli. In order to examine the distributions of c-di-GMP metabolizing proteins and their domain arrangement in the genomes of six rhizobial type species, we aligned the c-di-GMP proteins from online databases with the known enzymatically active c-di-GMP proteins and then compiled an inventory of c-di-GMP related proteins in these rhizobial species. We found that c-di-GMP signaling is prevalent in rhizobia, while the degree to which different rhizobial species rely on c-di-GMP signaling might vary. In addition, we identified many c-di-GMP metabolizing proteins that possess degenerate GGDEF or EAL domains in these rhizobial species. Secondly, we selected two c-di-GMP related genes, cdgA and cdgB, located on the symbiotic plasmid of R. etli, for further investigation of the functional roles and expression profiles during symbiosis and free-living conditions. Inactivation of these two genes did not affect R. etli symbiotic phenotypes (e.g. plant root colonization and nitrogen fixation ability) nor exopolysaccharides production, biofilm formation and motility. We confirmed that the proteins encoded both by cdgA and cdgB are bifunctional proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities. Additionally, by using gusA as reporter, we found that cdgB is significantly expressed during plant root colonization as well as in the free-living state, while cdgA expression is low in the free-living state and undetectable on the root surface. Furthermore, we revealed that the stringent response alarmone (p)ppGpp inhibits expression of both cdgA and cdgB.
Secondly, we characterized a novel ABC oligopeptide transporter designated the Opp transporter in R. etli. Firstly, we found that the Opp transporter is essential for establishing effective nitrogen fixation during the R. etli-P. vulgaris symbiosis. Microscopic observation of bacteroids morphology of opp mutants demonstrated that the Opp transporter is crucial for bacteroid development. Secondly, by performing growth tests of opp mutants under high salinity condition, we proved that the Opp transporter is involved in protection of R. etli cells against hyper-osmotic stress in the free-living state. However, expression of opp genes is not induced under hyper-osmotic conditions. Thirdly, we found that R. etli opp mutants displayed increased sensitivity to cell-envelope damaging agents. This effect was reversed in the presence of divalent cations, which are known to stabilize the cell envelope. In agreement with this observation, fatty acid composition differed between opp mutants and wild type. We therefore propose that the Opp transporter is required for maintenance of the structural integrity of the cell envelope of R. etli and fulfills an essential role in bacteroid differentiation.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics

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