Lázaro Molina

Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions.

Pseudomonas putida KT2440 is a root colonizer of potential interest for the rhizoremediation of pollutants and the biological control of pests. The short- and long-term survival of this strain, as well as the possible eAects of its introduction on diAerent populations of indigenous soil bacteria, were tested in soil under greenhouse and field conditions. The greenhouse studies showed that inoculated P. putida KT2440 was able to establish itself after 3 d in nonvegetated soils at a density of 822 10 3 CFU g ˇ1 soil. The introduction of this strain had no significant eAect on the number of several soil bacteria including those that were resistant to tetracycline; those that utilized p-hydroxyphenylacetic acid as the sole C-source, and total fluorescent pseudomonads. In four independent field assays in nonplanted soils, the numbers of P. putida KT2440 decreased during 50 d from an initial density of 1 10 6 CFU g ˇ1 soil to approximately 221 10 2 CFU g ˇ1 soil. Thereafter, the number of cells was below detection limits (i.e. <10 2 CFU g ˇ1 soil), although they were still present because they could be recovered using selective enrichment from the soil for up to 200 d after the beginning of the experiment. This suggested that P. putida was maintained at a low cell density long after inoculation. In contrast, when P. putida KT2440 was introduced in the soil as a coating of corn (Zea mays) or broad bean (Vicia faba) seeds, the bacteria established at high cell densities in the rhizosphere (10 4 ‐10 5 CFU g ˇ1 soil in corn; 10 6 ‐10 7 CFU g ˇ1 soil in broad beans) during the growth of the crops over 12 to 16 weeks. The numbers of P. putida in the bulk soil after 2 weeks were 1 to 2 orders of magnitude below those in the rhizosphere. During the field assays, the population of p-hydroxyphenylacetic acid users was also monitored in the rhizosphere and the bulk soil. No significant seasonal variations were found. # 2000 Elsevier Science Ltd. All rights reserved.

Solvent tolerance in Gram-negative bacteria

Bacteria have been found in all niches explored on Earth, their ubiquity derives from their enormous metabolic diversity and their capacity to adapt to changes in the environment. Some bacterial strains are able to thrive in the presence of high concentrations of toxic organic chemicals, such as aromatic compounds, aliphatic alcohols and solvents. The extrusion of these toxic compounds from the cell to the external medium represents the most relevant aspect in the solvent tolerance of bacteria, however, solvent tolerance is a multifactorial process that involves a wide range of genetic and physiological changes to overcome solvent damage. These additional elements include reduced membrane permeabilization, implementation of a stress response programme, and in some cases degradation of the toxic compound. We discuss the recent advances in our understanding of the mechanisms involved in solvent tolerance.

Analysis of the plant growth‐promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD‐1

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.

Complete Genome of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida BIRD-1

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.

The pGRT1 plasmid of Pseudomonas putida DOT‐T1E encodes functions relevant for survival under harsh conditions in the environment

Pseudomonas putida DOT-T1E has the capacity to grow in the presence of high concentrations of toluene. This ability is mainly conferred by an efflux pump encoded in a self-transmissible 133 kb plasmid named pGRT1. Sequence analysis of the pGRT1 plasmid revealed several key features. Most of the genes related to the plasmid maintenance functions show similarity with those encoded on pBVIE04 from Burkholderia vietnamensis G4, and knock-out mutants in several of these genes confirmed their roles. Two additional plasmid DNA fragments were incorporated into the plasmid backbone by recombination and/or transposition; in these DNA regions, apart from multiple recombinases and transposases, several stress-related and environmentally relevant functions are encoded. We report that plasmid pGRT1 not only confers the cells with tolerance to toluene but also resistance to ultraviolet light. We show here the implication of a new protein in solvent tolerance which controls the level of expression of the TtgGHI efflux pump, as well as the implication of a protein with homology to the universal stress protein in solvent tolerance and ultraviolet light resistance. Furthermore, this plasmid encodes functions that allow the cells to chemotactically respond to toluene and participate in iron scavenging.

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.

Analysis of the core genome and pangenome of Pseudomonas putida

Pseudomonas putida are strict aerobes that proliferate in a range of temperate niches and are of interest for environmental applications due to their capacity to degrade pollutants and ability to promote plant growth. Furthermore solvent-tolerant strains are useful for biosynthesis of added-value chemicals. We present a comprehensive comparative analysis of nine strains and the first characterization of the Pseudomonas putida pangenome. The core genome of P. putida comprises approximately 3386 genes. The most abundant genes within the core genome are those that encode nutrient transporters. Other conserved genes include those for central carbon metabolism through the Entner-Doudoroff pathway, the pentose phosphate cycle, arginine and proline metabolism, and pathways for degradation of aromatic chemicals. Genes that encode transporters, enzymes and regulators for amino acid metabolism (synthesis and degradation) are all part of the core genome, as well as various electron transporters, which enable aerobic metabolism under different oxygen regimes. Within the core genome are 30 genes for flagella biosynthesis and 12 key genes for biofilm formation. Pseudomonas putida strains share 85% of the coding regions with Pseudomonas aeruginosa; however, in P. putida, virulence factors such as exotoxins and type III secretion systems are absent.

Analysis of solvent tolerance in Pseudomonas putida DOT‐T1E based on its genome sequence and a collection of mutants

Pseudomonas putida strains are prevalent in a variety of pristine and polluted environments. The genome of the solvent‐tolerant P. putida strain DOT‐T1E which thrives in the presence of high concentrations of monoaromatic hydrocarbons, contains a circular 6.3 Mbp chromosome and a 133 kbp plasmid. Omics information has been used to identify the genes and proteins involved in solvent tolerance in this bacterium. This strain uses a multifactorial response that involves fine‐tuning of lipid fluidity, activation of a general stress‐response system, enhanced energy generation, and induction of specific efflux pumps that extrude solvents to the medium. Local and global transcriptional regulators participate in a complex network of metabolic functions, acting as the decision makers in the response to solvents.

Metabolic potential of the organic-solvent tolerant Pseudomonas putida DOT-T1E deduced from its annotated genome

Pseudomonas putida DOT‐T1E is an organic solvent tolerant strain capable of degrading aromatic hydrocarbons. Here we report the DOT‐T1E genomic sequence (6 394 153 bp) and its metabolic atlas based on the classification of enzyme activities. The genome encodes for at least 1751 enzymatic reactions that account for the known pattern of C, N, P and S utilization by this strain. Based on the potential of this strain to thrive in the presence of organic solvents and the subclasses of enzymes encoded in the genome, its metabolic map can be drawn and a number of potential biotransformation reactions can be deduced. This information may prove useful for adapting desired reactions to create value‐added products. This bioengineering potential may be realized via direct transformation of substrates, or may require genetic engineering to block an existing pathway, or to re‐organize operons and genes, as well as possibly requiring the recruitment of enzymes from other sources to achieve the desired transformation.

Complete Genome Sequence of a Pseudomonas putida Clinical Isolate, Strain H8234

ABSTRACT We report the complete genome sequence of Pseudomonas putida strain H8234, which was isolated from a hospital patient presenting with bacteremia. This strain has a single chromosome (6,870,827 bp) that contains 6,305 open reading frames. The strain is not a pathogen but exhibits multidrug resistance associated with 40 genomic islands.