Improvement of rhizobia by genetic modification with stress response genes

The symbiosis between soil bacteria (rhizobia) and leguminous plants (beans, lentils, chickpeas, etc) provides nitrogen to the plant by biological nitrogen fixation, thus increasing yields and reducing the need for N-fertilisers. Rhizobia have been used as safe seed inoculants in sustainable and ecological agriculture, instead of environmentally damaging chemical N-fertilisers, used in conventional agriculture. Bacterial inoculants should be symbiotically efficient as well as adapted to environmental conditions such as temperature, soil pH or salinity extremes. Knowledge of the molecular bases of symbiotic effectiveness and tolerance to stress is therefore essential for the improvement of rhizobia inoculants, namely by genetic modification. Stress response genes (SRGs) are involved in the response of cells to different types of stress conditions. When subjected to a sudden increase in growth temperature, bacteria show an induction of SRGs, namely genes encoding heat shock proteins (Hsps), such as molecular chaperones GroES/L and DnaK/J, small Hsps and proteases. Knowledge of rhizobia SRGs has improved but stress response mechanisms remain still poorly understood. Our previous studies resulted in the isolation of native chickpea rhizobia strains tolerant to environmental stress, such as acidity, heat and salinity. All isolates belong to the genus Mesorhizobium, specifically M. tianshanense, M.loti and other species, besides the expected chickpea symbionts, M. ciceri and M. mediterraneum. Within the same species we found strains showing tolerance and strains showing sensitivity to stress. The present project is the obvious continuation of the previous one, focusing on the molecular bases of rhizobia response to environmental stress. Different tolerance levels depicted by native Mesorhizobium strains may relay on qualitative or quantitative differences in SRGs. To understand rhizobia response mechanisms to stress, we are investigating global differential gene expression of Mesorhizobium loti under stress (high temperature, low pH and salinity) by transcriptome microarray analysis. M. loti MAFF303099 is the only Mesorhizobium species whose complete genome is published. Hence M. loti DNA microarrays can be used as a comprehensive tool for systematic genome-wide gene expression analysis of the stress response in this bacterium. To investigate the differences between stress response mechanisms in tolerant and sensitive chickpea mesorhizobia, we are also examining specific SRGs regarding sequence, gene organization, expression and functional analysis. These genes include dnaK/J and groeS/L (heat stress), actA and actP (acidity stress), exoN and exoY (osmotic stress), regulatory gene rpoH and novel SRGs identified by microarray analysis from M. loti. Expression analysis of specific SRGs by Northern blot or RT-PCR is showing differential gene induction, thereby clarifying the transcriptional response of tolerant and sensitive strains to stress. Functional analysis of specific SRGs, using rhizobia knockout mutants or gene overexpression, can elucidate their role in the response mechanism to distinct stresses. Additionally, functional analysis of SRGs, particularly chaperon genes, can clarify their contribution to nodulation and nitrogen fixation, by assessment of strain symbiotic performance following gene overexpression.
Identification of genetic determinants of stress tolerance, in M. loti and in native mesorhizobia, is fundamental for strain improvement by genetic modification, in order to obtain chickpea rhizobia inoculants, not only symbiotically efficient but also tolerant to environmental stress conditions. Improved rhizobial inoculants with enhanced stress tolerance are being constructed by genetic transformation of rhizobia with a stable expression vector such as RK2, carrying SRG previously recognized as determinant for stress tolerance. The outcome of overexpressing heterologous SRGs, namely chaperon genes from thermophylic bacteria, is under investigation as well. This project aims to contribute to the comprehension of the mechanisms of response to stress in rhizobia. It comprises the transcriptome analysis of Mesorhizobium loti under stress as well as the analysis of specific SRGs in native chickpea mesorhizobia, tolerant to stress. Finally it intends the improvement of rhizobia by genetic modification with stress response genes. Globally this project can contribute to efficient microbial applications, improving the interaction plant-microbe-environment, in the context of sustainable and ecological agriculture.