Isolation and characterization of plant modulated promoter/genes in the root colonizing bacterium, Pseudomonas aureofaciens.

摘要

Tn5::xylE (promoterless xylE reporter gene) transcriptional fusion mutants of Pseudomonas aureofaciens were screened for the presence of plant root exudate (RE) modulated bacterial promoters/genes. Screening on solid media revealed that 53% of the 2,100 Tn5::xylE mutants tested exhibited constitutive xylE gene expression. Four of the non-constitutive Tn5::xylE mutants strongly expressed the xylE gene while growing in the plant rhizosphere. Three of the four root exudate inducible mutants (2432, 3722, 3735) exhibited xylE gene expression while growing in the rhizosphere of wheat and corn. Two of these mutants, 2432 and 3722, also expressed xylE in the rhizosphere of soybean. XylE expression was absent in all four mutants, when they were grown in the rhizosphere of tomato. The fourth RE-inducible mutant, 3931, exhibited xylE expression only in the rhizosphere of the monocot, corn. XylE expression in two constitutive mutants, 0130 and 0304, was repressed when they were grown in the rhizosphere of wheat, corn, soybean and tomato while a third mutant (3743) showed repressed xylE expression in the rhizosphere of wheat, corn and soybean but expressed xylE in the rhizosphere of tomato. Southern hybridization demonstrated that each of these RE-modulated mutants contained the Tn5::xylE cassette at a unique site in the genome.; DNA sequences flanking the Tn5::xylE insertion site from three of the root exudate inducible mutants (2432, 3722, 3735) have revealed no significant homology to any described DNA/amino acid sequences in the National Center for Biotechnology Information (NCBI) data base. However, the mutant 3931 showed a high degree of homology to nitrite reductase (nirB) in the nasB operon which is involved in nitrate/nitrite assimilation in Bacillus subtilis. Mutant 3931 expressed the xylE gene either when available ammonium was limited or in the presence of ammonium when isolated corn root exudates were present.; In root colonization/competition assays on corn, mutants 3722 and 3735, did not appear to be compromised in their ability to colonize. However, mutants 2432 and 3931 were less able to compete with L11 and the native microbial community in colonizing corn roots growing in non-sterile field soil. These results suggested that the gene(s) insertionally inactivated with the Tn5::xylE cassette in two of the root exudate inducible mutants play a role in root colonization/competition.