Pseudomonas putida BIRD-1 is a plant growth-promoting rhizobacterium whose genome size is 5.7 Mbp. It adheres to plant roots and colonizes the rhizosphere to high cell densities even in soils with low moisture. This property is linked to its ability to synthesize trehalose, since a mutant deficient in the synthesis of trehalose exhibited less tolerance to desiccation than the parental strain. The genome of BIRD-1 encodes a wide range of proteins that help it to deal with reactive oxygen stress generated in the plant rhizosphere. BIRD-1 plant growth-promoting rhizobacteria properties derive from its ability to enhance phosphorous and iron solubilization and to produce phytohormones. BIRD-1 is capable of solubilizing insoluble inorganic phosphate forms through acid production. The genome of BIRD-1 encodes at least five phosphatases related to phosphorous solubilization, one of them being a phytase that facilitates the utilization of phytic acid, the main storage form of phosphorous in plants. Pyoverdine is the siderophore produced by this strain, a mutant that in the FvpD siderophore synthase failed to grow on medium without supplementary iron, but the mutant was as competitive as the parental strain in soils because it captures the siderophores produced by other microbes. BIRD-1 overproduces indole-3-acetic acid through convergent pathways.

Maize represents one of the main cultivar for food and energy and crop yields are influenced by soil physicochemical and climatic conditions. To study how maize plants influence soil microbes we have examined microbial communities that colonize maize plants grown in carbonate-rich soil (pH 8.5) using culture-independent, PCR-based methods. We observed a low proportion of unclassified bacteria in this soil whether it was planted or unplanted. Our results indicate that a higher complexity of the bacterial community is present in bulk soil with microbes from nine phyla, while in the rhizosphere microbes from only six phyla were found. The predominant microbes in bulk soil were bacteria of the phyla Acidobacteria, Bacteroidetes and Proteobacteria, while Gammaproteobacteria of the genera Pseudomonas and Lysobacter were the predominant in the rhizosphere. As Gammaproteobacteria respond chemotactically to exudates and are efficient in the utilization of plants exudate products, microbial communities associated to the rhizosphere seem to be plant-driven. It should be noted that Gammaproteobacteria made available inorganic nutrients to the plants favouring plant growth and then the benefit of the interaction is common.

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the “Pseudomonas mandelii subgroup,” within the “Pseudomonas fluorescens group,” Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the “Pseudomonas aeruginosa group,” Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs.

The use of biological control agents (BCAs) is of interest within an integrated management strategy of Verticillium wilt of olive (VWO) caused by the soil-borne fungus Verticillium dahliae Kleb. Previous studies have shown that the root/rhizosphere of healthy olive plants is an important reservoir of microorganisms displaying biocontrol activity against VWO (i.e., Pseudomonas strains PICF7 and PIC141). Moreover, these BCAs are already adapted to the ecological niche where they are deployed. Three novel bacteria (strains PIC28, PIC73 and PIC167) from nursery-produced olive plants were in-depth characterized using a previously implemented approach consisting of in situ isolation, in vitro antagonism tests, in planta bioassays, phenotypic and metabolic characterization, genome analyses and in silico identification of traits involved in plant-bacteria interactions, and multi-locus sequence analyses. All strains displayed in vitro growth inhibition of different olive pathogens and biocontrol effectiveness against Verticillium dahliae, with strain PIC73 being the most effective BCA. Strains PIC73 and PIC167 were identified as Paenibacillus polymyxa (Prazmowski) Ash et al. and Paenibacillus terrae Yoon et al., respectively. Strain PIC28 belongs to the Bacillus genus. Some of these Bacillales members showed in vitro compatibility with previously characterized BCAs (Pseudomonas spp. strains) also originating from the olive rhizosphere, paving the way for the future development of tailored bacterial consortia effective against VWO.

We report the complete sequence of the 5.7-Mbp genome of Pseudomonas putida BIRD-1, a metabolically versatile plant growth-promoting rhizobacterium that is highly tolerant to desiccation and capable of solubilizing inorganic phosphate and iron and of synthesizing phytohormones that stimulate seed germination and plant growth.Bacteria that efficiently colonize the rhizosphere and stimulate plant growth through direct or indirect mechanisms are referred to as plant growth-promoting rhizobacteria (PGPR). Plant growth promotion can be mediated by the synthesis of phytohormones, enhanced uptake of mineral nutrients, antagonizing of soilborne pathogens, induction of systemic resistance, etc. (1). The use of PGPR as inoculants for biofertilization, phytostimulation, and biocontrol avoids the use of chemical fertilizers and pesticides.Pseudomonas putida BIRD-1 is a soil bacterium that is efficient as a PGPR agent through the mobilization of nutrients and stimulation of secondary root proliferation. The complete genome sequence of BIRD-1 was obtained using 454 pyrosequencing technology. The sequences of 499,016 reads were assembled into 106 contigs to give 20ϫ coverage. Fifty-two contigs were longer than 3,000 bp. The complete genome of P. putida BIRD-1 is made up of a 61.74%-GC circular chromosome of 5,731,541 bp with no plasmid. The genome size is similar to that of P. putida W619 (5,774,330 Mb) (4) and smaller than those of P. putida KT2440 (6,181,860 Mb) (2), P. putida F1 (5,959,964 Mb), and P. putida GB-1 (6,078,430 Mb).The genome of BIRD-1 encodes 5,124 proteins, 64 tRNAs, eight 5S rRNAs, seven 16S rRNAs, and 23S rRNA. All essentially conditional genes identified in KT2440 (3) are present in the genome of BIRD-1, and functions were assigned to almost 60% of the total coding genes. The genome of P. putida BIRD-1 encodes a number of root colonization determinants (4) and a number of elements that favor plant growth. The ability of P. putida BIRD-1 to solubilize inorganic phosphate is related to the production of gluconic acid, whereas the ability to solubilize iron is related to the synthesis of pyoverdines. All genes required for these functions have been identified; BIRD-1 produces the indole-3-acetic acid (IAA) plant hormone, a trait associated with stimulation of root initiation and elongation. Two putative tryptophan-dependent IAA biosynthetic pathways have been identified in this strain. The genome of BIRD-1 also contains genes encoding proteins for the synthesis of salicylate, another compound that is relevant in plant responses.Nucleotide sequence accession number. The complete genome sequence of P. putida BIRD-1 is available in GenBank under accession number CP002290.

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SummaryPseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6‐trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification‐related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up‐ and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1‐oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.

Summary Phylogenetic analysis of more than 4000 annotated bacterial acid phosphatases was carried out. Our analysis enabled us to sort these enzymes into the following three types: (1) class B acid phosphatases, which were distantly related to the other types, (2) class C acid phosphatases and (3) generic acid phosphatases (GAP). Although class B phosphatases are found in a limited number of bacterial families, which include known pathogens, class C acid phosphatases and GAP proteins are found in a variety of microbes that inhabit soil, fresh water and marine environments. As part of our analysis, we developed three profiles, named Pfr‐B‐Phos, Pfr‐C‐Phos and Pfr‐GAP, to describe the three groups of acid phosphatases. These sequence‐based profiles were then used to scan genomes and metagenomes to identify a large number of formerly unknown acid phosphatases. A number of proteins in databases annotated as hypothetical proteins were also identified by these profiles as putative acid phosphatases. To validate these in silico results, we cloned genes encoding candidate acid phosphatases from genomic DNA or recovered from metagenomic libraries or genes synthesized in vitro based on protein sequences recovered from metagenomic data. Expression of a number of these genes, followed by enzymatic analysis of the proteins, further confirmed that sequence similarity searches using our profiles could successfully identify previously unknown acid phosphatases.