The soil inhabiting rhizobia establish a symbiosis with legume plants through the infection of leguminous roots and the subsequent induction of nitrogen fixing nodules. Biological nitrogen fixation by legumes is particularly important in the nitrogen poor soils of Australia, driving much of Australia's agricultural production. The increased use of legumes is central to the development of sustainable agriculture where it is becoming difficult to use expensive and polluting nitrogenous fertilizers. The rhizobia are divided into the six genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium of the α-subclass of the proteobacteria. The species S. meliloti is the effective microsymbiont for the establishment of Medicago sativa pastures. This plant host is the targeted legume species to overcome the insidious dryland salinity problem in Australian soils. However, the S. meliloti symbiont is particularly sensitive to acidity, an abiotic stress that accompanies salinity in many regions. Stress-responsive genetic circuits have been identified in Sinorhizobium but the sigma factors required for transcriptional activation have not been described. In enteric bacteria the sigma factor RpoS is required and the alternative sigma factor (such as RpoS), are involved in controlling expression of independent subsets of proteins required for acid-tolerance. By mining the established genomic DNA sequence of S. meliloti we have identified that this species does not have the RpoS sigma factor but does have a multitude of other sigma factors. We have identified 1 gene encoding a housekeeping sigma factor, and 13 genes for alternative sigma factors. PCR primers were designed to amplify 500 bp intragenic fragments from each of the genes. PCR conditions were optimised enabling the amplification of 13 intragenic fragments. Each PCR generated fragment is currently being cloned into a plasmid for targeted inactivation in S. meliloti to determine if any of these genes are required for stress response or tolerance
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