Analysis of Pseudomonas putida KT2440 Gene Expression in the Maize Rhizosphere: In Vitro Expression Technology Capture and Identification of Root-Activated Promoters

Abstract

Pseudomonas putida KT2440, a paradigm organism in biodegradation and a good competitive colonizer of the maize rhizosphere, was the subject of studies undertaken to establish the genetic determinants important for its rhizospheric lifestyle. By using in vivo expression technology (IVET) to positively select single cell survival, we identified 28 rap genes (root-activated promoters) preferentially expressed in the maize rhizosphere. The IVET system had two components: a mutant affected in aspartate-␤-semialdehyde dehydrogenase (asd), which was unable to survive in the rhizosphere, and plasmid pOR1, which carries a promoterless asd gene. pOR1-borne transcriptional fusions of the rap promoters to the essential gene asd, which were integrated into the chromosome at the original position of the corresponding rap gene, were active and allowed growth of the asd strain in the rhizosphere. The fact that five of the rap genes identified in the course of this work had been formerly characterized as being related to root colonization reinforced the IVET approach. Up to nine rap genes encoded proteins either of unknown function or that had been assigned an unspecific role based on conservation of the protein family domains. Rhizosphere-induced fusions included genes with probable functions in the cell envelope, chemotaxis and motility, transport, secretion, DNA metabolism and defense mechanism, regulation, energy metabolism, stress, detoxification, and protein synthesis.The rhizosphere is a densely populated area in which plant roots interact with soilborne microorganisms, including bacteria, fungi, and invertebrates, feeding on an abundant source of organic material (43). Many colonization traits and genes have been identified by random mutagenesis of good competitive root-colonizing bacteria (mainly Pseudomonas fluorescens and Pseudomonas chlororaphis) and through screening for gain or loss of competitive root tip colonization ability (see reference 28 for a recent review). Often these genes have been found to be unimportant for growth in the laboratory, and their role in bacterial fitness has required competition studies involving wild-type and mutant strains.Information on gene expression in the rhizosphere is, however, limited and partial. Previous results obtained by our group have shown that utilization of the imino acid proline by Pseudomonas putida KT2440, which involves uptake and catabolism, was induced by maize root exudates (50). In addition, an aminotransferase involved in the catabolism of lysine was also identified in a screen to select for maize root exudateinduced genes by using a promoter probe transposon (15).The most extensive study so far based on positive selection of bacterial traits as a consequence of their contribution to root colonization is that of Rainey (38). In that work, the in vitro expression technology (IVET) approach was used to select P. fluorescens genes activated during sugar cane root colonization.The study was partial, since only 10% of the genome was analyzed.Our work represents an ...

Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions

“…Lugtenberg and Dekkers (21) have shown that utilization of organic acids by P. fluorescens is the nutritional basis governing the ability of this organism to colonize tomato roots. Genes in pseudomonads encoding proteins with functions in nutrient acquisition and in energy generation are up-regulated in the rhizosphere or when bacteria were exposed to the soil environment as revealed by in vivo expression technology-based approaches (22,23). Differences in expression of these genes in response to the var.…”

“…In vivo expression technology is now established as a method of analyzing bacterial gene expression in the rhizosphere as an approach for the identification of genes involved in microbe-plant interactions (22,23,29,35). One clear advantage of in vivo expression technology is that investigations are carried out in the appropriate environment in the organism of interest; there is no extrapolation from in vitro model systems.…”

Gut and Root Microbiota Commonalities

“…Genes involved in root exudate usage, root attachment, and survival are induced in bacteria colonizing roots (120,121). In vitro expression technology (IVET) (122), proteomic analysis, microarray and RNA Seq transcriptomics, and genetic analysis have revealed rhizobial (120,121,123), Pseudomonas (124,125), Streptomyces (126), and other bacterial genes expressed on roots or rhizospheres. Similarly, bacteria may differentially express genes when in guts.…”

The ColRS Two-Component System Regulates Membrane Functions and Protects Pseudomonas putida against Phenol

“…A publication by Ramos-González et al (37) showed that it can also be true for P. putida, as the promoter of colRS was activated in the maize rhizosphere. In a recent paper, we demonstrated that the P. putida two-component system ColRS is necessary for the accumulation of phenol-utilizing (Phe ϩ ) mutants, which arise due to transposition of Tn4652 (18).…”