Analysis of Pseudomonas putida KT2440 gene expression in the maize rhizosphere: in vivo [corrected] 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-beta-semialdehyde dehydrogenase (asd), which was unable to survive in the rhizosphere, and plasmid pOR1, which carries a promoter-less 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.

FIG. 1.

Map of pOR1 plasmid (IVET vector) used to identify P. putida root-activated promoters. Modifications to the original plasmid pIVPRO (20) are described in the text. This plasmid has the replication origin oriR6K and thus requires protein Π for stable maintenance. The plasmid is therefore a suicide in Pseudomonas and is mobilizable as tra functions are supplied in trans.

FIG. 2.

Effect of rap promoter activity on colonization ability. (A) Survival of chromosomal IVET cointegrates pool in the rhizosphere. ○, KT2440R; •, Δasd; ▵, a pool of approximately 500 clones of P. putida IVET cointegrates. (B) Survival of rap fusion strains. ○, KT2440R; •, Δasd; ▵, rap_1-2 fusion; and ▴, rap_1-4 fusion. Maize seeds were inoculated and plants grown as described in Materials and Methods. Bacterial cells were recovered on plates at the indicated times. At time zero, CFU was per g of seed; afterwards, CFU is plotted per g of rhizosphere including root. Means and standard deviations for three independent experiments are shown.

FIG. 3.

β-Galactosidase activity (Miller units) of rap fusions during seedling colonization in hydroponic culture. Dotted bars show values from cells in suspension in the presence of maize seedling. Open bars are values in 2.5 mM citrate-supplied M9 minimal medium with all requirements to support the growth of the rap fusions, as described in Materials and Methods. Inoculum size was 2.5 × 10⁶ to 5 × 10⁶ CFU/ml. Means and standard deviations from three independent experiments are shown.

FIG. 4.

β-Galactosidase activity of rap fusions in the rhizosphere. Hatched bars show values from cells recovered from the maize rhizosphere supplied with PNS. Open bars are values from control cells in sand supplied with PNS, citrate, and all requirements to support the growth of the rap fusions as described in Materials and Methods. Inoculum size for controls was 1 × 10⁴ to 2.5 × 10⁴, similar to the number of cells attached to the seeds. Means and standard deviations from three independent experiments are shown. Units are given as the concentration of 4-methyl-umbelliferone (femtomolar) per CFU.