Pleiotrophic Alterations in the Cellular Protein Synthesis of Tn5-Induced Rhizobium Mutants as Revealed by Two-Dimensional Gel Electrophoresis

Abstract

Two-dimensional gel electrophoresis (2-DE) is currently unequalled in its capacity to separate individual components of complex protein mixtures, making it the method of choice for monitoring global changes in gene expression in response to defined conditions (Guerreiro et al, 1997). Using 2-DE we have examined the global changes in Rhizobium gene expression which occur in response to specific Tn5-induced mutations that lead to defective symbiotic phenotypes. Exopolysaccharides (EPS) of Rhizobium leguminosarum have been implicated to be involved in a complex series of interactions which lead to the establishment of an effective Rhizobium -legume symbiosis, but the precise role of EPS in this interaction is not well defined. The mutant ANU437, a derivative of R. l. bv. trifolli strain ANU794, contains a Tn5 insertion in pssA (also called pss4), produces very low levels of acidic exopolysaccharide and induces delayed formation of small non-nitrogen-fixing nodules on various clover species (Rolfe et al, 1996). Using 2-DE we have shown ANU437 to exhibit multiple differences in gene expression when compared with its wild-type parent. Such pleiotrophies would have been difficult to show via other methodologies. Twenty-one proteins in ANU437 were either newly-induced or up-regulated in their level of synthesis. Six of these proteins have been characterised by N-terminal sequencing, all were novel proteins except for one which showed a high similarity to a phosphogluconate dehydrogenase. These results have shown the PSSA protein, in addition to being a glycosyltransferase, may be directly or indirectly involved in influencing the expression of a complex series of genes whose levels of expression may be the actual contributing factor to the ineffective nodulation observed with exo− mutants.KeywordsBiological Nitrogen FixationComplex SeriesRhizobium LeguminosarumPhosphogluconate DehydrogenaseMultiple DifferenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.