Genomic analysis reveals the major driving forces of bacterial life in the rhizosphere

Abstract

Background: Mutualistic interactions less well known than those between rhizobia and legumes are commonly found between plants and bacteria, frequently pseudomonads, which colonize roots and adjacent soil areas (the rhizosphere).

Results: A global analysis of Pseudomonas putida genes expressed during their interaction with maize roots revealed how a bacterial population adjusts its genetic program to this lifestyle. Differentially expressed genes were identified by comparing rhizosphere-colonizing populations with three distinct controls covering a variety of nutrients, growth phases and life styles (planktonic and sessile). Ninety rhizosphere up-regulated (rup) genes, which were induced relative to all three controls, were identified, whereas there was no repressed gene in common between the experiments. Genes involved in amino acid uptake and metabolism of aromatic compounds were preferentially expressed in the rhizosphere, which reflects the availability of particular nutrients in root exudates. The induction of efflux pumps and enzymes for glutathione metabolism indicates that adaptation to adverse conditions and stress (oxidative) response are crucial for bacterial life in this environment. The finding of a GGDEF/EAL domain response regulator among the induced genes suggests a role for the turnover of the secondary messenger c-diGMP in root colonization. Several mutants in rup genes showed reduced fitness in competitive root colonization.

Conclusion: Our results show the importance of two selective forces of different nature to colonize the rhizosphere: stress adaptation and availability of particular nutrients. We also identify new traits conferring bacterial survival in this niche and open a way to the characterization of specific signalling and regulatory processes governing the plant-Pseudomonas association.

Figure 1

Microarray profiling of bacterial gene expression in the rhizosphere. (a-c) Global gene expression of P. putida KT2440 was analyzed in the rhizosphere versus that of LB log bacteria (OD₆₆₀ = 0.7) (a, a'), LB stationary phase cells (OD₆₆₀ = 3.3) (b, b'), and sessile bacterial cells incubated in sand microcosms (c, c'). Experimental set up and cDNA preparation are described in detail in Materials and methods. Genes induced (red) and repressed (green), with a P value < 0.05 and A >64 were clustered according to their fold change (>2 to >10) and (≤2 to ≤10) and the number is plotted.

Figure 2

Venn diagrams showing the overlap between differentially expressed genes in the rhizosphere. (a) Down-regulated and (b) up-regulated genes resulting from the individual analysis of each experiment: rhizosphere versus LB exponentially growing cells (Rhi/Log), rhizosphere versus LB stationary phase cells (Rhi/St), and rhizosphere versus sessile cells in microcosm without plant (Rhi/Microcosm). Common genes were clustered automatically with a freely available informatics tool [48]. (c) The result of the combined analysis of the three experiments before and after applying adjustments in the P value. Out of the 57 Bonferroni genes, 54 are included in the 90 overlapping rup genes.

Figure 3

Rhizosphere fitness of mutant strains in competition with KT2440RTn7-Sm. The knocked-out open reading frame in the mutant strains is indicated by the locus name. Proportion of mutant (grey) and wild type (white), which was 50% ± 2% in the initial inocula, is plotted after 12 days of colonization. Data are the averages and standard error for six plants. KT2440RTn7-Sm, a streptomycin resistant derivative of KT2440R (see Materials and methods), was used as the wild-type strain in the experiments. KT2440RTn7-Sm and KT2440R are equally competitive in root colonization (not shown). Sm resistance marker of the wild-type bacteria allowed their specific selection against the mutants, which were kanamycin resistant derivatives of KT2440R. Statistical analysis was carried out using SPSS software (version 12.0.1 for Windows, SPSS Inc., Chicago, IL, USA). The linear model univariate analysis of variance rendered significant differences for the mutants shown in the figure (P value < 0.05) in comparison with the wild type. Seed adhesion rate was similar for mutants and KT2440R (0.5%), with the exception of PP0110 and PP1477 (0.1%). The growth of the mutants under laboratory conditions (rich and defined medium) was indistinguishable from that of KT2440RTn7-Sm. The transcriptional organization of mutated genes is shown in the bottom. The space between the 3' end of PP1476 and the 5' end of PP1477 is 7 bp. Translational coupling between PP3281 and PP3280 (8 bp) was observed. PP3279 is probably in an independent operon; however, PP3279 and PP3281 code for enzymes in the same degradative pathway [28]. Translational coupling between PP4987 and PP4988 was also observed (8 bp). Arrows indicate direction of transcription. Transposon insertion is indicated by inverted triangles.

Figure 4

Integrated scheme showing relevant bacterial functions induced in the rhizosphere-Pseudomonas interaction. Functions related to genes included in Additional data file 3 have also been included in this figure. See text for details.