Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440

A major challenge in microbiology is the elucidation of the genetic and ecophysiological basis of habitat specificity of microbes. Pseudomonas putida is a paradigm of a ubiquitous metabolically versatile soil bacterium. Strain KT2440, a safety strain that has become a laboratory workhorse worldwide, has been recently sequenced and its genome annotated. By drawing on both published information and on original in silico analysis of its genome, we address here the question of what genomic features of KT2440 could explain or are consistent with its ubiquity, metabolic versatility and adaptability. The genome of KT2440 exhibits combinations of features characteristic of terrestrial, rhizosphere and aquatic bacteria, which thrive in either copiotrophic or oligotrophic habitats, and suggests that P. putida has evolved and acquired functions that equip it to thrive in diverse, often inhospitable environments, either free-living, or in close association with plants. The high diversity of protein families encoded by its genome, the large number and variety of small aralogous families, insertion elements, repetitive extragenic palindromic sequences, as well as the mosaic structure of the genome (with many regions of 'atypical' composition) and the multiplicity of mobile elements, reflect a high functional diversity in P. putida and are indicative of its evolutionary trajectory and adaptation to the diverse habitats in which it thrives. The unusual wealth of determinants for high affinity nutrient acquisition systems, mono- and di-oxygenases, oxido-reductases, ferredoxins and cytochromes, dehydrogenases, sulfur metabolism proteins, for efflux pumps and glutathione-S-transfereases, and for the extensive array of extracytoplasmatic function sigma factors, regulators, and stress response systems, constitute the genomic basis for the exceptional nutritional versatility and opportunism of P. putida , its ubiquity in diverse soil, rhizosphere and aquatic systems, and its renowned tolerance of natural and anthropogenic stresses. This metabolic diversity is also the basis of the impressive evolutionary potential of KT2440, and its utility for the experimental design of novel pathways for the catabolism of organic, particularly aromatic, pollutants, and its potential for bioremediation of soils contaminated with such compounds as well as for its application in the production of high-added value compounds.     

Articles selected by Faculty of 1000: proteomics of macromolecular complexes; verification of microarray data; chromosome 22 transcript map; arraying maize tissue-specific genes; profiling plant defense responses

A selection of evaluations from Faculty of 1000 covering the Pseudomonas putida genome, SAGE and microarray analysis of cell death, a screen for phosphopeptide binding domains and an engineered bacterium that uses a non-natural amino acid.     

Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440

The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates - perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms.     

Degradation of lawsone by Pseudomonas putida L2

From humus obtained from Stuttgart, a bacterium was isolated with lawsone (2-hydroxy-1,4-naphthoquinone) as selective source of carbon. This bacterium is capable of utilizing lawsone as sole source of carbon and energy. Morphological and physiological characteristics of the bacterium were examined and it was identified as a strain of Pseudomonas putida. The organism is referred to as Pseudomonas putida L2. The degradation of lawsone by Pseudomonas putida L2 was investigated. Salicylic acid and catechol were isolated and identified as metabolites. In lawsone-induced cells of Pseudomonas putida L2, salicylic acid is converted to catechol by salicylate 1-monooxygenase. Catechol 1,2-dioxygenase catalyses ortho-fission of catechol which is then metabolized via the beta-ketoadipate pathway. Formation of cis,cis-muconate and beta-ketoadipate was demonstrated by enzyme assays. Salicylate 1-monooxygenase and catechol 1,2-dioxygenase are induced sequentially. The enzymes of the beta-ketoadipate pathway are also inducible. Naphthoquinone hydroxylase, however, was demonstrated in induced and non-induced cells. This constitutive enzyme enables Pseudomonas putida L2 to degrade various 1,4-naphthoquinones in experiments with resting cells.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila

Pseudomonas entomophila is an entomopathogenic bacterium that, upon ingestion, kills Drosophila melanogaster as well as insects from different orders. The complete sequence of the 5.9-Mb genome was determined and compared to the sequenced genomes of four Pseudomonas species. P. entomophila possesses most of the catabolic genes of the closely related strain P. putida KT2440, revealing its metabolically versatile properties and its soil lifestyle. Several features that probably contribute to its entomopathogenic properties were disclosed. Unexpectedly for an animal pathogen, P. entomophila is devoid of a type III secretion system and associated toxins but rather relies on a number of potential virulence factors such as insecticidal toxins, proteases, putative hemolysins, hydrogen cyanide and novel secondary metabolites to infect and kill insects. Genome-wide random mutagenesis revealed the major role of the two-component system GacS/GacA that regulates most of the potential virulence factors identified.     

Complete Genome Sequence of the Naphthalene-Degrading Pseudomonas putida Strain ND6

Pseudomonas putida strain ND6 is an efficient naphthalene-degrading bacterium. The complete genome of strain ND6 was sequenced and annotated. The genes encoding the enzymes involved in catechol degradation by the ortho-cleavage pathway were found in the chromosomal sequence, which indicated that strain ND6 is able to metabolize naphthalene by the catechol meta- and ortho-cleavage pathways.     

Separation of d-(+)-Nicotine from a Racemic Mixture by Stereospecific Degradation of the l-(-) Isomer with Pseudomonas putida

Incubation of racemic mixtures of dl-(+/-)-nicotine with Pseudomonas putida resulted in a complete stereoselective degradation of the l-(-) isomer. Unnatural d-(+)-nicotine, which is of pharmacological interest for stereochemical studies of various nicotine-responsive systems, was not affected by the bacterium and was recovered by extraction.     

Biodesulfurization in biphasic systems containing organic solvents

Biphasic systems can overcome the problem of low productivity in conventional media and have been exploited for biocatalysis. Solvent-tolerant microorganisms are useful in biotransformation with whole cells in biphasic reactions. A solvent-tolerant desulfurizing bacterium, Pseudomonas putida A4, was constructed by introducing the biodesulfurizing gene cluster dszABCD, which was from Rhodococcus erythropolis XP, into the solvent-tolerant strain P. putida Idaho. Biphasic reactions were performed to investigate the desulfurization of various sulfur-containing heterocyclic compounds in the presence of various organic solvents. P. putida A4 had the same substrate range as R. erythropolis XP and could degrade dibenzothiophene at a specific rate of 1.29 mM g (dry weight) of cells(-1) h(-1) for the first 2 h in the presence of 10% (vol/vol) p-xylene. P. putida A4 was also able to degrade dibenzothiophene in the presence of many other organic solvents at a concentration of 10% (vol/vol). This study is a significant step in the exploration of the biotechnological potential of novel biocatalysts for developing an efficient biodesulfurization process in biphasic reaction mixtures containing toxic organic solvents.     

Selective Enhancement of the Fluorescent Pseudomonad Population After Amending the Recirculating Nutrient Solution of Hydroponically Grown Plants with a Nitrogen Stabilizer

Fluorescent pseudomonads have been associated, via diverse mechanisms, with suppression of root disease caused by numerous fungal and fungal-like pathogens. However, inconsistent performance in disease abatement, after their employment, has been a problem. This has been attributed, in part, to the inability of the biocontrol bacterium to maintain a critical threshold population necessary for sustained biocontrol activity. Our results indicate that a nitrogen stabilizer (N-Serve(R), Dow Agrosciences) selectively and significantly enhanced, by two to three orders of magnitude, the resident population of fluorescent pseudomonads in the amended (i.e., 25 mug ml(-1) nitrapyrin, the active ingredient) and recycled nutrient solution used in the cultivation of hydroponically grown gerbera and pepper plants. Pseudomonas putida was confirmed as the predominant bacterium selectively enhanced. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA suggested that N-Serve(R) selectively increased P. putida and reduced bacterial diversity 72 h after application. In vitro tests revealed that the observed population increases of fluorescent pseudomonads were preceded by an early growth suppression of indigenous aerobic heterotrophic bacteria (AHB) population. Interestingly, the fluorescent pseudomonad population did not undergo this decrease, as shown in competition assays. Xylene and 1,2,4-trimethylbenzene (i.e., the inert ingredients in N-Serve(R)) were responsible for a significant percentage of the fluorescent pseudomonad population increase. Furthermore, those increases were significantly higher when the active ingredient (i.e., nitrapyrin) and the inert ingredients were combined, which suggests a synergistic response. P. putida strains were screened for the ability to produce antifungal compounds and for the antifungal activity against Pythium aphanidermatum and Phytophthora capsici. The results of this study suggest the presence of diverse mechanisms with disease-suppressing potential. This study demonstrates the possibility of using a specific substrate to selectively enhance and maintain desired populations of a natural-occurring bacterium such as P. putida, a trait considered to have great potential in biocontrol applications for plant protection.     

Determination of the toxicity of several aromatic carbonylic compounds and their reduced derivatives on Phanerochaete chrysosporium using a Pseudomonas putida test system

We tested four aromatic carbonylic compounds and their corresponding reduced derivatives, possible substrates, and products of a biotransformation for toxicity against the white-rot fungus Phanerochaete chrysosporium. The bacterium Pseudomonas putida, which has been proven to be a good test organism for investigating toxic effects, was used as a primary screen. For both P. chrysosporium and P. putida, all ketones showed a higher toxicity than their corresponding alcohol derivatives. Within one chemical group a direct correlation between the hydrophobicity (logP values) of the compounds and their toxicity could be observed. Furthermore, all tested compounds also caused an isomerization of cis to trans unsaturated fatty acids in P. putida, a mechanism of this bacterium to adapt its membrane to toxic environmental influences. Toxicity of aromatic carbonylic compounds in an established biotransformation system with P. chrysosporium can be estimated by calculating the corresponding logP values of the substrates and potential products. P. putida can be used to test the toxicity of aromatic ketones to the basic diomycete P. chrysosporium.     

A Tn7-based broad-range bacterial cloning and expression system

For many bacteria, cloning and expression systems are either scarce or nonexistent. We constructed several mini-Tn7 vectors and evaluated their potential as broad-range cloning and expression systems. In bacteria with a single chromosome, including Pseudomonas aeruginosa, Pseudomonas putida and Yersinia pestis, and in the presence of a helper plasmid encoding the site-specific transposition pathway, site- and orientation-specific Tn7 insertions occurred at a single attTn7 site downstream of the glmS gene. Burkholderia thailandensis contains two chromosomes, each containing a glmS gene and an attTn7 site. The Tn7 system allows engineering of diverse genetic traits into bacteria, as demonstrated by complementing a biofilm-growth defect of P. aeruginosa, establishing expression systems in P. aeruginosa and P. putida, and 'GFP-tagging' Y. pestis. This system will thus have widespread biomedical and environmental applications, especially in environments where plasmids and antibiotic selection are not feasible, namely in plant and animal models or biofilms.     

Isolation and characterization of a unicellular manganese-oxidizing bacterium from a freshwater lake in northwestern Russia

A unicellular manganese-oxidizing bacterium (strain L7), isolated from Lake Ladoga, is identified as "Siderocapsa" sp. according to its morphology. However, this bacterium belongs to the phylogenetic cluster of Pseudomonas putida. The physiological characteristics (utilization of sugars, polyatomic alcohols, organic acids, and phenolic substrates as carbon and energy sources) also indicate the similarity of strain L7 to representatives of the genus Pseudomonas. The growing culture oxidizes Mn(II); the rate of oxidation depends on the type of added organic substrate. Carbonate requirement for this process indicates mixotrophic metabolism. The relatedness of the isolated bacterium to the representatives of the genus Pseudonomas and their phenotypic similarity provide a basis for considering strain L7 not as "Siderocapsa" sp., but as a new species, Pseudomonas siderocapsa sp. nov., of the P. putida cluster.     

Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida

Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.     

多功能降解菌Pseudomonas putida KT2440-DOP的构建与降解特性研究

三唑磷水解酶基因为研究发现的一个新的广谱有机磷水解酶基因,通过PCR从有机磷降解菌株Ochrobactrumsp.mp-4总DNA扩增了tpd,将tpd定向克隆到pBBRMCS-5载体上,构建重组质粒pTPD,在辅助质粒pRK2013的帮助下,通过三亲接合将pTPD转移到模式菌株Pseudomonas putidaKT2440中,获得的工程菌PseudomonasputidaKT2440-DOP可以降解多种有机磷农药及芳香烃化合物;KT2440-DOP的有机磷水解酶活较出发菌株MP-4提高了一倍左右,且遗传性状稳定。     

Experimental validation of in silico estimated biomass yields of Pseudomonas putida KT2440

Pseudomonas putida is rapidly becoming a microbial cell platform for biotechnological applications. In order to understand genotype‐phenotype relationships genome scale models represent helpful tools. However, the validation of in silico predictions of genome scale models is a task that is rarely performed. In this study the theoretical biomass yields of Pseudomonas putida KT2440 were estimated for 57 different carbon sources based on a genome scale stoichiometric model applying flux balance analysis. The batch growth of P. putida KT2440 with six individual carbon sources covering the range of maximal to minimal in silico biomass yields (acetate, glycerol, citrate, succinate, malate and methanol, respectively) was studied in a defined mineral medium in a fully controlled stirred‐tank bioreactor on a 3 L scale. The highest growth rate of P. putida KT2440 was measured with succinate as carbon source (0.51 h^?1). Among the 57 carbon sources tested, glycerol resulted in the highest estimated biomass yield (0.61 molC_Biomass molC^?1_Glycerol) which was experimentally confirmed. The comparison of experimental determined biomass yields with a modified version of the model iJP815 showed deviations of only up to 10%. The experimental data generated in this study can also be used in future studies to further improve the genome scale models of P. putida KT2440. Improved models will then help to gain deeper insights in genotype‐phenotype relationships.     

Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis

The genome of Pseudomonas putida KT2440 encodes an unexpected capacity to tolerate heavy metals and metalloids. The availability of the complete chromosomal sequence allowed the categorization of 61 open reading frames likely to be involved in metal tolerance or homeostasis, plus seven more possibly involved in metal resistance mechanisms. Some systems appeared to be duplicated. These might perform redundant functions or be involved in tolerance to different metals. In total, P. putida was found to bear two systems for arsenic (arsRBCH), one for chromate (chrA), four to six systems for divalent cations (two cadA and two to four czc chemiosmotic antiporters), two systems for monovalent cations: pacS, cusCBA (plus one cryptic silP gene containing a frameshift mutation), two operons for Cu chelation (copAB), one metallothionein for metal(loid) binding, one system for Te/Se methylation (tpmT) and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (one nikABCDE, two znuACB and one mobABC). Some of the metal-related clusters are located in gene islands with atypical genome signatures. The predicted capacity of P. putida to endure exposure to heavy metals is discussed from an evolutionary perspective.     

Transformation of bacteria with plasmid DNA by electroporation

The possibility of electric field-mediated transformation ("electroporation") of a gram-positive bacterium (Enterococcus faecalis) and two gram-negative bacteria (Escherichia coli and Pseudomonas putida) with plasmid DNA was investigated. E. faecalis protoplasts could be transformed by electroporation with a transformation frequency of 10(4) to 10(5) transformants/micrograms plasmid. Untreated--i.e., washed--cells of E. coli could be transformed with rates of 1 X 10(5) transformants/micrograms plasmid DNA. Transformation rates for P. putida cells were up to 3 X 10(4) if the method developed for E. coli was used. Detailed protocols for these systems, including the results of various optimization experiments, are given.