Showing papers on "Pseudomonas putida published in 2015"

Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms.

Abstract: Bacteria secrete signalling molecules (AHLs) to coordinate actions such as biofilm formation and the release of public goods, in a process called quorum sensing. Here, the authors show that AHLs are stochastically produced and control asocial (self-directed) traits in young biofilms of P. putida.

Pseudomonas putida—a versatile host for the production of natural products

Abstract: The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out.

Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida.

Abstract: The soil bacterium Pseudomonas putida is increasingly attracting considerable interest as a platform for advanced metabolic engineering through synthetic biology approaches. However, genomic context, gene copy number, and transcription/translation interplay often introduce considerable uncertainty to the design of reliable genetic constructs. In this work, we have established a standardized heterologous expression device in which the promoter strength is the only variable; the remaining parameters of the flow have stable default values. To this end, we tailored a mini-Tn7 delivery transposon vector that inserts the constructs in a single genomic locus of P. putida's chromosome. This was then merged with a promoter insertion site, an unvarying translational coupler, and a downstream location for placing the gene(s) of interest under fixed assembly rules. This arrangement was exploited to benchmark a collection of synthetic promoters with low transcriptional noise in this bacterial host. Growth experiments and flow cytometry with single-copy promoter-GFP constructs revealed a robust, constitutive behavior of these promoters, whose strengths and properties could be faithfully compared. This standardized expression device significantly extends the repertoire of tools available for reliable metabolic engineering and other genetic enhancements of P. putida.

Mechanisms of solvent resistance mediated by interplay of cellular factors in Pseudomonas putida

Abstract: A number of microorganisms have the ability to thrive in the presence of a range of toxic solvents. Tolerance to these chemicals is a multifactorial process, meaning that bacterial cells use a set of physiological and gene expression changes to overcome the damage imparted by these chemicals. This review focuses mainly on issues related to tolerance to aromatic hydrocarbons and butanol in Pseudomonas, although other microorganisms are also discussed. Pseudomonas putida strains contain a circular chromosome of approximately 6 Mbp which encodes about 5300 genes. A combination of physiological and biochemical assays, a genome-wide collection of mutants and several omics approaches have provided useful information to help identify functions involved in solvent tolerance in P. putida. The solvent response involves fine-tuning of lipid fluidity to adjust membrane functions including impermeabilization, activation of a general stress-response system, increased energy generation and induction of specific efflux pumps that extrude solvents to the medium. These responses are modulated at the transcriptional level by local and global regulators as well as by a number of sRNAs whose levels fluctuate with the presence of solvents in the environment. Taken as a whole these regulatory inputs orchestrate the complex network of metabolic responses observed after solvent addition.

Genome reduction boosts heterologous gene expression in Pseudomonas putida.

Abstract: The implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome). In this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440. The two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

The production of exopolysaccharide by Pseudomonas putida GAP-P45 under various abiotic stress conditions and its role in soil aggregation

Abstract: Exopolysaccharides (EPS) production is modified in response to environmental changes and is believed to protect bacteria. In the present study Pseudomonas putida strain GAP-P45 identified by 16S rDNA sequence analysis was exposed to stresses such as drought, temperature and salt to test the ability to tolerate and produce EPS. Strain GAP-P45 could tolerate matric stress up to −0.73 MPa, Temperature of 50°C and 1.4 M salt and produced EPS, which increased with increase in stress levels. Among all stress conditions, the production was high under drought stress. HPLC analysis revealed that monosaccharide composition and ratio of sugars in EPS increased under stress that might induce osmotic and thermal tolerance in GAP-P45. Rhamnose was reported as major sugar under all stress conditions. Among the different carbon sources tested, glycerol was found to be best for EPS production under stressed as well as non-stressed conditions. Inoculation with GAP-P45 resulted in better soil aggregation and aggregate stability under different stress conditions. However inoculation effect was more under drought-stress. The significance of these findings shows that abiotic stresses influence the EPS composition and ratios of sugars, which influence the tolerance of the microorganisms which can be employed for improvement in water holding capacity under unfavorable environmental conditions.

Engineering mediator-based electroactivity in the obligate aerobic bacterium Pseudomonas putida KT2440

Abstract: Pseudomonas putida strains are being developed as microbial production hosts for production of a range of amphiphilic and hydrophobic biochemicals P putida’s obligate aerobic growth thereby can be an economical and technical challenge because it requires constant rigorous aeration and often causes reactor foaming Here, we engineered a strain of P putida KT2440 that can produce phenazine redox-mediators from Pseudomonas aeruginosa to allow partial redox balancing with an electrode under oxygen-limited conditions P aeruginosa is known to employ its phenazine-type redox mediators for electron exchange with an anode in bioelectrochemical systems We transferred the seven core phenazine biosynthesis genes phzA-G and the two specific genes phzM and phzS required for pyocyanin synthesis from P aeruginosa on two inducible plasmids into P putida KT2440 The best clone, P putida pPhz, produced 45 mg/ L pyocyanin over 25 h of growth, which was visible as blue color formation and is comparable to the pyocyanin production of P aeruginosa This new strain was then characterized under different oxygen-limited conditions with electrochemical redox control and changes in central energy metabolism were evaluated in comparison to the unmodified P putida KT2440 In the new strain, phenazine synthesis with supernatant concentrations up to 33 µg/ mL correlated linearly with the ability to discharge electrons to an anode, whereby phenazine-1-carboxylic acid served as the dominating redox mediator P putida pPhz sustained strongly oxygen-limited metabolism for up to 2 weeks at up to 12 µA/ cm² anodic current density Together, this work lays a foundation for future oxygen-limited biocatalysis with P putida strains

Effect of plant growth-promoting rhizobacteria inoculation on cadmium (Cd) uptake by Eruca sativa.

Abstract: Microbe-assisted phyto-remediation approach is widely applied and appropriate choice to reduce the environmental risk of heavy metals originated from contaminated soils. The present study was designed to screen out the nested belongings of Eruca sativa plants and Pseudomonas putida (ATCC 39213) at varying cadmium (Cd) levels and their potential to deal with Cd uptake from soils. We carried out pot trial experiment by examining the soil containing E. sativa seedlings either treated with P. putida and/or untreated plants subjected to three different levels (ppm) of Cd (i.e., 150, 250, and 500). In all studied cases, we observed an increase in Cd uptake for E. sativa plants inoculated with P. putida than those of un-inoculated plants. Cd toxicity was assessed by recording different parameters including stunted shoot growth, poor rooting, and Cd residual levels in the plants that were not inoculated with P. putida. Significant difference (p < 0.05) of different growth parameters for inoculated vs non-inoculated plants was observed at all given treatments. However, among the different treatments, E. sativa exhibited increased values for different growth parameters (except proline contents) at lower Cd levels than those of their corresponding higher levels, shoot length (up to 27 %), root length (up to 32 %), whole fresh plant (up to 40 %), dry weight (up to 22 %), and chlorophyll contents (up to 26 %). Despite the hyperaccumulation of Cd in whole plant of E. sativa, P. putida improved the plant growth at varying levels of Cd supply than those of associated non-inoculated plants. Present results indicated that inoculation with P. putida enhanced the Cd uptake potential of E. sativa and favors the healthy growth under Cd stress.

Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains

Abstract: Pseudomonas putida strains are ubiquitous in soil and water but have also been reported as opportunistic human pathogens capable of causing nosocomial infections. In this study we describe the multilocus sequence typing of four P. putida strains (HB13667, HB8234, HB4184, and HB3267) isolated from in-patients at the Besancon Hospital (France). The four isolates (in particular HB3267) were resistant to a number of antibiotics. The pathogenicity and virulence potential of the strains was tested ex vivo and in vivo using different biological models: human tissue culture, mammalian tissues, and insect larvae. Our results showed a significant variability in the ability of the four strains to damage the host; HB13667 did not exhibit any pathogenic traits, HB4184 caused damage only ex vivo in human tissue cultures, and HB8234 had a deleterious effect in tissue culture and in vivo on rat skin, but not in insect larvae. Interestingly, strain HB3267 caused damage in all the model systems studied. The putative evolution of these strains in medical environments is discussed.

Inhibition of Cell Differentiation in Bacillus subtilis by Pseudomonas protegens

Abstract: Interspecies interactions have been described for numerous bacterial systems, leading to the identification of chemical compounds that impact bacterial physiology and differentiation for processes such as biofilm formation. Here, we identified soil microbes that inhibit biofilm formation and sporulation in the common soil bacterium Bacillus subtilis. We did so by creating a reporter strain that fluoresces when the transcription of a biofilm-specific gene is repressed. Using this reporter in a coculture screen, we identified Pseudomonas putida and Pseudomonas protegens as bacteria that secrete compounds that inhibit biofilm gene expression in B. subtilis. The active compound produced by P. protegens was identified as the antibiotic and antifungal molecule 2,4-diacetylphloroglucinol (DAPG). Colonies of B. subtilis grown adjacent to a DAPG-producing P. protegens strain had altered colony morphologies relative to B. subtilis colonies grown next to a DAPG-null P. protegens strain ( phlD strain). Using a subinhibitory concentration of purified DAPG in a pellicle assay, we saw that biofilm-specific gene transcription was delayed relative to transcription in untreated samples. These transcriptional changes also corresponded to phenotypic alterations: both biofilm biomass and spore formation were reduced in B. subtilis liquid cultures treated with subinhibitory concentrations of DAPG. Our results add DAPG to the growing list of antibiotics that impact bacterial development and physiology at subinhibitory concentrations. These findings also demonstrate the utility of using coculture as a means to uncover chemically mediated interspecies interactions between bacteria. IMPORTANCE Biofilms are communities of bacteria adhered to surfaces by an extracellular matrix; such biofilms can have important effects in both clinical and agricultural settings. To identify chemical compounds that inhibited biofilm formation, we used a fluorescent reporter to screen for bacteria that inhibited biofilm gene expression in Bacillus subtilis. We identified Pseudomonas protegens as one such bacterium and found that the biofilm-inhibiting compound it produces was the antibiotic 2,4-diacetylphloroglucinol (DAPG). We showed that even at subinhibitory concentrations, DAPG inhibits biofilm formation and sporulation in B. subtilis. These findings have potential implications for understanding the interactions between these two microbes in the natural world and support the idea that many compounds considered antibiotics can impact bacterial development at subinhibitory concentrations.

The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

Abstract: The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy andflow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inac- tive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional (glpD-gfp) fusion (a proxy of the glycerol-3- phosphate (G3P) dehydrogenase activity) linked the macroscopic phenotype to the expression of the glpgenes. Either deleting glpR(encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRDgene cluster) or alter- ing G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpDexpression. These manipula- tions eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpRintransrestored the phenotypes lost in the glpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regu- latory device controlling the transcription of the glpgenes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional re- sponse, a layout fostering the observed time-dependent shift between two alternative physiological states. IMPORTANCE Phenotypic variation is a widespread attribute of prokaryotes that leads, inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mecha- nisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of met- abolic genes when cells face specific nutrients (e.g., glycerol). Pseudomonas putida may have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. De- feating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.

Efficient recombinant production of prodigiosin in Pseudomonas putida

Abstract: Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20 °C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

Molecular mechanism of nicotine degradation by a newly isolated strain, Ochrobactrum sp. strain SJY1.

Abstract: A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.

Isolation and characterization of bacterial endophytes from the roots of Cassia tora L.

Abstract: In the present investigation, five endophytic bacterial strains isolated from the roots of Cassia tora L were identified as Bacillus subtilis, Agrobacterium tumefaciens, Bacillus sp, Pseudomonas putida, and Pseudomonas sp based on biochemical characteristics as well as 16S rRNA gene sequencing Isolates were screened for plant growth promoting traits, antibacterial activity, antifungal activity, antibiotic sensitivity, and salinity tolerance The majority of the endophytic strains produced phytohormone indole acetic acid (IAA), ammonia, and also solubilized phosphate Siderophore and HCN production were observed in A tumefaciens, P putida, and Pseudomonas sp The antibiotic sensitivity profile indicated that the isolates were resistant to chloramphenicol, while highly susceptible to neomycin and streptomycin Bacterial endophytes gave a definite stamp on their antibacterial activity against Escherichia coli and Klebsiella pneumoniae and antifungal activity against Pythium ultimum, Aureobasidium pullulans, and Alternaria alternata Pseudomonas sp showed maximum salt tolerance (6 % NaCl) contrary to A tumefaciens that was least tolerant

Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

Abstract: Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a “sulfoglycolytic” pathway, in addition to the classical glycolytic (Embden–Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner–Doudoroff pathway for glucose-6-phosphate: It involves an NAD+-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)+-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

Antimicrobial resistance of Pseudomonas spp. isolated from wastewater and wastewater-impacted marine coastal zone

Abstract: In this study, species distribution and antimicrobial susceptibility of cultivated Pseudomonas spp. were studied in influent (INF), effluent (EFF), and marine outfall (MOut) of wastewater treatment plant (WWTP). The susceptibility was tested against 8 antimicrobial classes, active against Pseudomonas spp.: aminoglycosides, carbapenems, broad-spectrum cephalosporins from the 3rd and 4th generation, extended-spectrum penicillins, as well as their combination with the β-lactamase inhibitors, monobactams, fluoroquinolones, and polymyxins. Among identified species, resistance to all antimicrobials but colistin was shown by Pseudomonas putida, the predominant species in all sampling points. In other species, resistance was observed mainly against ceftazidime, ticarcillin, ticarcillin-clavulanate, and aztreonam, although some isolates of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas pseudoalcaligenes, and Pseudomonas protegens showed multidrug-resistance (MDR) phenotype. Among P. putida, resistance to β-lactams and to fluoroquinolones as well as multidrug resistance become more prevalent after wastewater treatment, but the resistance rate decreased in marine water samples. Obtained data, however, suggests that Pseudomonas spp. are equipped or are able to acquire a wide range of antibiotic resistance mechanisms, and thus should be monitored as possible source of resistance genes.

Enzymatic production of 5-aminovalerate from L-lysine using L-lysine monooxygenase and 5-aminovaleramide amidohydrolase.

Abstract: 5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of L-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from L-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L L-lysine in 12 h. Because L-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate.

Plant growth-promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study.

Abstract: Plant growth-promoting rhizobacteria (PGPR) affect growth of host plants through various direct and indirect mechanisms. Three native PGPR (Pseudomonas putida) strains isolated from rhizospheric soil of a Mentha piperita (peppermint) crop field near Cordoba, Argentina, were characterised and screened in vitro for plant growth-promoting characteristics, such as indole-3-acetic acid (IAA) production, phosphate solubilisation and siderophore production, effects of direct inoculation on plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) and accumulation and composition of essential oils. Each of the three native strains was capable of phosphate solubilisation and IAA production. Only strain SJ04 produced siderophores. Plants directly inoculated with the native PGPR strains showed increased shoot fresh weight, glandular trichome number, ramification number and root dry weight in comparison with controls. The inoculated plants had increased essential oil yield (without alteration of essential oil composition) and biosynthesis of major essential oil components. Native strains of P. putida and other PGPR have clear potential as bio-inoculants for improving productivity of aromatic crop plants. There have been no comparative studies on the role of inoculation with native strains on plant growth and secondary metabolite production (specially monoterpenes). Native bacterial isolates are generally preferable for inoculation of crop plants because they are already adapted to the environment and have a competitive advantage over non-native strains.

Functional coexistence of twin arsenic resistance systems in Pseudomonas putida KT2440.

Abstract: Summary The genome of the soil bacterium Pseudomonas putida KT2440 bears two virtually identical arsRBCH operons putatively encoding resistance to inorganic arsenic species. Single and double chromosomal deletions in each of these ars clusters of this bacte- rium were tested for arsenic sensitivity and found that the contribution of each operon to the resistance to the metalloid was not additive, as either cluster suf- ficed to endow cells with high-level resistance. However, otherwise identical traits linked to each of the ars sites diverged when temperature was decreased. Growth of the various mutants at 15°C (instead of the standard 30°C for P. putida) uncovered that ars2 affords a much higher resistance to As (III) than the ars1 counterpart. Reverse transcription poly- merase chain reaction of arsB1 and arsB2 genes as well as lacZ fusions to the Pars1 and Pars2 promoters traced the difference to variations in transcription of the corresponding gene sets at each temperature. Functional redundancy may thus be selected as a stable condition - rather than just as transient state - if it affords one key activity to be expressed under a wider range of physicochemical settings. This seems to provide a straightforward solution to regulatory problems in environmental bacteria that thrive under changing scenarios.

High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply

Abstract: High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L−1 h−1) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L−1 was achieved in 33 h with a biomass productivity of 3.1 g L−1 h−1 which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L−1 h−1) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA−1. Biotechnol. Bioeng. 2015;112: 725–733. © 2014 Wiley Periodicals, Inc.

Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of Chlorella vulgaris and Pseudomonas putida

Abstract: The co-culture system of photosynthetic microalgae Chlorella vulgaris and aerobic heterotrophic bacteria Pseudomonas putida was investigated as a possible combination of symbiotic mixed culture for the simultaneous removal of nutrients (ammonium and phosphate) and organic contaminants. Using synthetic municipal wastewater, the co-culture system exhibited symbiotic enhancement in the removal of nutrients and organic carbon compared to each of axenic cultures. The co-culture system performed successfully in removing both of ammonium and chemical oxygen demand (COD), showing around 80% removal for 4 days. Strategies of nitrogen and phosphorous starvation in C. vulgaris for two days prior to main treatment did not increase the performance of nutrients removal, indicating that the nutrient starvation as a pretreatment is unnecessary. Without alkalinity (as bicarbonate), nutrients and COD were not removed significantly, implying that the existence of alkalinity is essential for symbiotic treatment of both nutrients and organics. Results demonstrated that coculture system composed of C. vulgaris and P. putida can be a potential candidate of mixed culture system for the simultaneous removal of nutrients and organic carbon in wastewater treatment using a single reactor.

Different Ancestries of R Tailocins in Rhizospheric Pseudomonas Isolates.

Abstract: Bacterial genomes accommodate a variety of mobile genetic elements, including bacteriophage-related clusters that encode phage tail-like protein complexes playing a role in interactions with eukaryotic or prokaryotic cells. Such tailocins are unable to replicate inside target cells due to the lack of a phage head with associated DNA. A subset of tailocins mediate antagonistic activities with bacteriocin-like specificity. Functional characterization of bactericidal tailocins of two Pseudomonas putida rhizosphere isolates revealed not only extensive similarity with the tail assembly module of the Pseudomonas aeruginosa R-type pyocins but also differences in genomic integration site, regulatory genes, and lytic release modules. Conversely, these three features are quite similar between strains of the P. putida and Pseudomonas fluorescens clades, although phylogenetic analysis of tail genes suggests them to have evolved separately. Unlike P. aeruginosa R pyocin elements, the tailocin gene clusters of other pseudomonads frequently carry cargo genes, including bacteriocins. Compared with P. aeruginosa, the tailocin tail fiber sequences that act as specificity determinants have diverged much more extensively among the other pseudomonad species, mostly isolates from soil and plant environments. Activity of the P. putida antibacterial particles requires a functional lipopolysaccharide layer on target cells, but contrary to R pyocins from P. aeruginosa, strain susceptibilities surpass species boundaries.

Integration of chemotaxis, transport and catabolism in Pseudomonas putida and identification of the aromatic acid chemoreceptor PcaY

Abstract: Summary Aromatic and hydroaromatic compounds that are metabolized through the β-ketoadipate catabolic pathway serve as chemoattractants for Pseu- domonas putida F1. A screen of P. putida F1 mutants, each lacking one of the genes encoding the 18 puta- tive methyl-accepting chemotaxis proteins (MCPs), revealed that pcaY encodes the MCP required for metabolism-independent chemotaxis to vanillate, vanillin, 4-hydroxybenzoate, benzoate, protocat- echuate, quinate, shikimate, as well as 10 substituted benzoates that do not serve as growth substrates for P. putida F1. Chemotaxis was induced during growth on aromatic compounds, and an analysis of a pcaY- lacZ fusion revealed that pcaY is expressed in the presence of β-ketoadipate, a common intermediate in the pathway. pcaY expression also required the tran- scriptional activator PcaR, indicating that pcaY is a member of the pca regulon, which includes three unlinked gene clusters that encode five enzymes required for the conversion of 4-hydroxybenzoate to tricarboxylic acid cycle intermediates as well as the major facilitator superfamily transport protein PcaK. The 4-hydroxybenzoate permease PcaK was shown to modulate the chemotactic response by facilitating the uptake of 4-hydroxybenzoate, which leads to the accumulation of β-ketoadipate, thereby increasing pcaY expression. The results show that chemotaxis, transport and metabolism of aromatic compounds are intimately linked in P. putida.

Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts

Abstract: Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes. In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen. Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells. Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.