Aims: To isolate the rhizosphere competence-defective transposon Tn5 mutant of Pantoea agglomerans NBRISRM (SRM) and to identify the gene causing defect in its root colonization ability.
Methods and results: From over 5000 clones containing Tn5, one mutant P. agglomerans NBRISRMT (SRMT) showing 6 log units less colonization when compared with SRM, after 30 days in sand-nonsterilized soil assay system was selected for further work to determine the effects of the mutation on rhizosphere competence. Southern hybridization analysis of restricted genomic DNA of SRMT demonstrated that the mutant had a single Tn5 insert. SRM increased in titre to about 2 x 10(8) CFU g(-1) root, compared with the indigenous bacterial population of heterotrophs of about 5 x 10(7) CFU g(-1) root. In contrast, 30 days later, the titre value of SRMT was almost undetectable at 1 x 10(2) CFU g(-1) root, demonstrating its inability to survive and colonize the rhizosphere. Sequencing of the flanking region of the Tn5 mutant revealed that Tn5 disrupted the purB gene.
Conclusions: A defect in the colonization phenotype of the SRMT was attributed to the disruption in adenylosuccinate lyase (EC 4.3.2.2) which is encoded by the pur B gene and is required for rhizosphere colonization in P. agglomerans. Significantly less exopolysaccharide and biofilm was formed by SRMT when compared to SRM, because of the disruption of the purB gene.
Significance and impact of the study: This work provides the first evidence for a functional role of purB gene in rhizosphere competence and root colonization by any rhizobacteria.