Viability was determined by plating aliquots of serial dilutions on PSY agar and colony‐forming unit counting. Cells were challenged by incubation at 48☌ before (open symbols) or after (closed symbols) being subjected to overnight carbon starvation. Cultures were grown at 30☌ in PSY medium to an OD 600 of 1. Thermal and dessication resistance of B. japonicum wild type, Δ phyR mutant 8402 and Δ ecfG mutant 8404. A. This shows that carbon starvation induces stress resistance, resulting in a cross protection against heat shock and desiccation, which is partially PhyR‐ and σ EcfG‐dependent. However, when cells were subjected to carbon starvation in minimal medium before exposure to stress, we could show that the phyR mutant 8402 and ecfG mutant 8404 were more sensitive than the wild type to subsequent heat exposure and desiccation whereas no difference was found with respect to salt, H 2O 2 and UV tolerance. The finding that carbon starvation induced protection against a second stress, i.e. Subsequently, PhyR was found to be involved in resistance to heat shock and desiccation, as well as oxidative, UV, ethanol and osmotic stresses. PhyR was discovered as a regulator induced upon phyllosphere colonization of the epiphytic bacterium and shown to be essential for plant colonization ( Gourion et al., 2006). Recently, insights into the regulatory path resulting in general stress response in the Alphaproteobacterium Methylobacterium extorquens AM1 were provided. However, the alpha subdivision of the Proteobacteria lacks orthologues of rpoS and sigB, and different regulators have to be assumed to regulate the general stress response. The general stress response has been extensively studied in Escherichia coli and Bacillus subtilis, in which σ S (RpoS) and σ B (SigB), respectively, were identified as master regulators controlling this response (for recent reviews, Hecker et al., 2007 Klauck et al., 2007).
These include desiccation, pH shifts, osmotic stress, carbon starvation in bulk soil and presence of reactive oxygen species during the infection process ( Gibson et al., 2008). Under free‐living conditions in the bulk soil/rhizosphere as well as during invasion, rhizobia are exposed to distinct and fluctuating stresses.
The latter symbiotic state represents a well‐balanced physiological interaction: the microsymbiont converts atmospheric nitrogen into ammonium that is provided to the plant host in exchange for carbon and energy generated by photosynthesis ( Prell and Poole, 2006). Rhizobia have different lifestyles either as free‐living bacteria in the soil and as bacteroids in nodules after infection and differentiation. Phylogenetically, they belong to the Proteobacteria, in which most of the currently described rhizobial strains fall into several dispersed lineages within the class of Alphaproteobacteria, and include genera such as Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium and Methylobacterium ( Sawada et al., 2003). This finding was further substantiated by in vitro protein–protein interaction studies which are in line with a partner‐switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. Microarray analysis revealed that PhyR and σ EcfG control congruent regulons suggesting both regulators are part of the same signalling cascade. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF σ factor σ EcfG are involved in heat shock and desiccation resistance upon carbon starvation.
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