Impact of Pseudomonas putida RRF3 on the root transcriptome of rice plants: Insights into defense response, secondary metabolism and root exudation

The parasitic nematode Haemonchus contortus is one of the world’s most important parasites of small ruminants that causessignificant economic losses to the livestock sector. The population structure and selection in its various strains are poorlyunderstood. No study so far compared its different populations using genome-wide data. Here, we focused on differentgeographic populations of H. contours from China (Tibet, TB; Hubei, HB; Inner Mongolia, IM; Sichuan, SC), UK andAustralia (AS), using genome-wide population-genomic approaches, to explore genetic diversity, population structure andselection. We first performed next-generation high-throughput 2b RAD pool sequencing using Illumina technology, andidentified single-nucleotide polymorphisms (SNPs) in all the strains. We identified 75,187 SNPs for TB, 82,271 for HB,82,420 for IM, 79,803 for SC, 83,504 for AS and 78,747 for UK strain. The SNPs revealed low-nucleotide diversity (p =0.0092–0.0133) within each strain, and a significant differentiation level (average Fst = 0.34264) among them. Chinesepopulations TB and SC, along with the UK strain, were more divergent populations. Chinese populations IM and HBshowed affinities to the Australian strain. We then analysed signature of selection and detected 44 (UK) and 03 (AS) privateselective sweeps containing 49 and 05 genes, respectively. Finally, we performed the functional annotation of selectivesweeps and proposed biological significance to signature of selection. Our data suggest that 2b-RAD pool sequencing canbe used to assess the signature of selection in H. contortus.

Isolation and characterization of diethyl phthalate degrading bacteria from crude oil contaminated soil

Aim: The aim of this study was to isolate, identify and characterize diethyl phthalate degrading bacterial strains isolated from a crude oil contaminated soil from a landfill dump site of a petroleum refinery in Mersin, Turkey. Methodology: The bacteria was isolated from a crude oil contaminated soil and characterized by 16S rRNA analysis. Bacterial genomic DNA was identified by 16S rRNA analyses. Biodegredation experiments were conducted for 5 days and plasmid curing experiment was performed. Catechol test was carried out to determine phthalate degradation pathways. Results: The isolated bacteria from soil were identified as Pseudomonas putida based on 16S rRNA sequences. The size of the plasmid was estimated to be about 15.9 kb. Results of biodegradation experiments indicated that the diethyl phthalate concentrations were reduced by 85.5% after 5 days of incubation at pH 7.0 and 30°C. The ability of P. putida degrading diethyl phthalate was found to be plasmid-mediated through curing experiments. Interpretation: The study suggested that plasmid of Pseudomonas putida PAG5 could be involved in effective degradation of diethyl phthalate

Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

Transcriptomic profiling of substrate-dependent autoaggregation in Pseudomonas putida CP1

Aims@#Pseudomonas putida CP1 is an interesting environmental isolate which exhibits substrate-dependent autoaggregation when the organism was grown on 0.5% (w/v) fructose. Autoaggregation is a process of a single bacterial species to develop clumps of cells during a substrate stress. This study was carried out to investigate the genetic changes in the bacterium during aggregate formation. @*Methodology and results@#P. putida CP1 was grown on 0.5% (w/v) fructose in batch culture at 30 °C and 150 rpm. The removal of fructose from the medium corresponded with aggregation of the cells which started after 8 h incubation. Microarray gene expression profiling using a P. putida KT2440 Genome Oligonucleotide Array (Progenika, Spain) showed that 838 genes involved in metabolism and adaptation were differentially expressed in P. putida CP1. Global transcriptomic profiling studies showed that P. putida CP1 growing on fructose resulted in the induction of genes encoding for proteins mainly involved in protein translation, ABC transporters, oxidative phosphorylation and two-component systems (TCS). Novel genes, associated with autoaggregation, were identified using transcriptomic analysis involved in ABC transporter, TCS, flagella assembly and lipopolysaccharide biosynthesis. It was also associated with the up-regulation of genes involved in the flagellar assembly including the fliE gene which encodes for the flagellar hook-basal body protein. @*Conclusion, significance and impact of study@#The identification of new genes involved in autoaggregation formation is important to understand the molecular basis of strain variation and the mechanisms implicated in cell-cell communication.

Epidemiological characteristics and antimicrobial resistance of 91 strains of Pseudomonas putida / 中国感染控制杂志

Objective To analyze the epidemiological characteristics and antimicrobial resistance of clinically isolated Pseudomonas putida (P.putida),and provide basis for rational prevention and treatment of P.putida infection.Methods P.putida isolated between January 2010 and December 2015,as well as clinical data of patients infected with P.putida were collected,antimicrobial susceptibility of isolates was determined by Kirby-Bauer disk diffusion susceptibility testing of Clinical and Laboratory Standards Institute (CLSI) of America,susceptibility testing results of isolated strains were analyzed by WHONET 5.5 software.Results A total of 91 strains of P.putida were isolated from clinical specimens,most were from elderly patients aged ＞60 years(70.33％);the major underlying disease was community-acquired pneumonia (23.08％),followed by chronic pulmonary heart disease (15.38％);the main specimen was sputum(57.14％),followed by urine(27.47％);P.putida mainly distributed in department of respiratory medicine (28.57 ％),followed by department of cardiovascular medicine (13.19 ％).P.putida had high resistance rate to aztreonam (52.75 ％),while resistance rates to gentamicin,imipenem,levofloxacin,ceftazidime,meropenem,and ciprofloxacin were 7.78％,9.89％,2.20％,9.89％,7.69％,and 2.22％ respectively,resistance rates to amikacin and polymyxin were both 0.Conclusion P.putida infection mainly occurs in elderly patients with underlying diseases,mainly respiratory tract infection,resistance rates to most antimicrobial agents were ＜ 10 ％.

Bioconversion of mixed free fatty acids to poly-3-hydroxyalkanoates by Pseudomonas putida BET001 and modeling of its fermentation in shake flasks

Background: The paper reports on the utilization of palm kernel oil (PKO) as a low cost renewable substrate for medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) production by Pseudomonas putida BET001. Investigation on the effects of selected key variables on growth, mixed free fatty acids consumption and mcl-PHA production by the bacterial culture in the shaken flask system were carried out along with its kinetic modeling. Results: The biomass production, fatty acids consumption and mcl-PHA production were found favorable when the strain was cultured in mineral medium at pH 6-7,28°C, aeration surface-to-volume ratio of 0.4 x 10(6) m-1, 250 rpm agitation rate for 48 h. Mcl-PHA production by this strain showed mixed growth and non-growth associated components as described by Luedeking-Piret kinetic model. Conclusion: The findings of this study provided add to the literature on key variables in for achieving good microbial growth and mcl-PHA production in shake flasks culture. In addition, suitable kinetic model to describe cultivation in this system was also presented.

Influences of extracellular polymeric substances (EPS) for autoaggregation of Pseudomonas putida CP1 during growth on mono-chlorophenol

Aims: Pseudomonas putida CP1 exhibits substrate-dependent autoaggregation during the degradation of 100 ppm 2- chlorophenol, 100 ppm 3-chlorophenol and 200 ppm 4-chlorophenol. This study discussed the production of extracellular polymeric substances (EPS) by the organism for the formation of aggregates. Methodology and results: Aggregation was accompanied by the production of extracellular polymeric substances (EPS). The extent of EPS production and the size of the aggregates increased with increasing stress as did the aggregation index and the hydrophobicity of the cells. A biochemical analysis of the EPS showed that the main constituents were carbohydrate (40% w/v) and protein (50% w/v) together with lower levels of DNA (<10% w/v). Conclusion, significance and impact study: Given that the aggregated form of the bacterium has shown potential for use in bioaugmentation, an in-depth understanding of the phenomenon could enhance the use of this organism in biological wastewater treatment systems.

Screening and optimization of low-cost medium for Pseudomonas putida Rs-198 culture using RSM

The plant growth-promoting rhizobacterial strain Pseudomonas putida Rs-198 was isolated from salinized soils from Xinjiang Province. We optimized the composition of the low-cost medium of P. putida Rs-198 based on its bacterial concentration, as well as its phosphate-dissolving and indole acetic acid (IAA)-producing capabilities using the response surface methodology (RSM), and a mathematical model was developed to show the effect of each medium component and its interactions on phosphate dissolution and IAA production. The model predicted a maximum phosphate concentration in medium containing 63.23 mg/L inorganic phosphate with 49.22 g/L corn flour, 14.63 g/L soybean meal, 2.03 g/L K2HPO4, 0.19 g/L MnSO4 and 5.00 g/L NaCl. The maximum IAA concentration (18.73 mg/L) was predicted in medium containing 52.41 g/L corn flour, 15.82 g/L soybean meal, 2.40 g/L K2HPO4, 0.17 g/L MnSO4 and 5.00 g/L NaCl. These predicted values were also verified through experiments, with a cell density of 10(13) cfu/mL, phosphate dissolution of 64.33 mg/L, and IAA concentration of 18.08 mg/L. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The study aims to provide a basis for industrialized fermentation using P. putida Rs-198.

Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp: ONBA-17 and deduction on its metabolic pathway

A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation.

Biodegradation kinetics of o-cresol by Pseudomonas putida DSM 548 (pJP4) and o-cresol removal in a batch-recirculation bioreactor system

The biodegradation kinetics of o-cresol was examined by acclimatized P. putida DSM 548 (pJP4) in batch experiments at varying initial o-cresol concentrations (from 50 to 500 mg/L). The kinetic parameters of o-cresol aerobic biodegradation were estimated by using the Haldane substrate inhibition equation. The biodegradation kinetics of o-cresol was investigated. In batch culture reactors, the Maximum specific growth rate (μmax), Monod constant (Ks) and the inhibition constant (Ki) were established as 0.519 h-1, 223.84 mg/L and 130.883 mg/L, respectively. o-cresol biodegradation in a batch-recirculation bioreactor system by immobilized P. putida was also studied. The recycled packed bed reactor system, which was composed of Ca-alginate beads and pumice on which cells immobilized, has been performed to determine possible stability for further developments.

Effect of culture medium on biocalcification by Pseudomonas Putida, Lysinibacillus Sphaericus and Bacillus Subtilis

The objective of this study is to investigate the efficiency of calcium carbonate bioprecipitation by Lysinibacillus sphaericus, Bacillus subtilis and Pseudomonas putida, obtained from the Coleção de Culturas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS), as a first step in determining their potential to protect building materials against water uptake. Two culture media were studied: modified B4 containing calcium acetate and 295 with calcium chloride. Calcium consumption in the two media after incubation with and without the bacterial inoculum was determined by atomic absorption analysis. Modified B4 gave the best results and in this medium Pseudomonas putida INQCS 113 produced the highest calcium carbonate precipitation, followed by Lysinibacillus sphaericus INQCS 414; the lowest precipitation was produced by Bacillus subtilis INQCS 328. In this culture medium XRD analysis showed that Pseudomonas putida and Bacillus subtilis precipitated calcite and vaterite polymorphs while Lysinibacillus sphaericus produced only vaterite. The shape and size of the crystals were affected by culture medium, bacterial strain and culture conditions, static or shaken. In conclusion, of the three strains Pseudomonas putida INQCS 113 in modified B4 medium gave the best results precipitating 96 percent of the calcium, this strain thus has good potential for use on building materials.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 x 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 x 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.

Detection of a New Type of Aminoglycoside-modifying Enzymes in a Pan-resistant Pseudomonas putida / 中华医院感染学杂志

OBJECTIVE To study resistant mechanisms in a pan-resistant Pseudomonas putida strain.METHODS To detect the susceptibility of antimicrobial agents by MIC,6 aminoglycoside-modifying enzymes(AMEs) genes of 1 strain of P.putida were measured by PCR,and verified by DNA sequencing and similarity searches of DNA sequencing data banks using BLAST.RESULTS In the strain,there were 4 positive kinds of AMEs resistant genes(aac(6′)-Ⅰb,aac(3)-Ⅱ,ant(3″)-Ⅰand ant(2″)-Ⅰ).Sequence analysis revealed that the 4 PCR products were AMEs,the aac(6′)-Ⅰb was a new type(GenBank EU 137667).CONCLUSIONS The mechanisms of resistance to aminoglycosides of the pan-resistant P.putida are mainly related to 4 kinds of AMEs,the aac(6′)-Ⅰb is a new type.

Isolation, Identification and Growth Characteristics of Pseudomonas putida Strain M6 with Malachite Green Decolorization / 微生物学通报

Six bacterial strains with malachite green decolorization ability were isolated from a sediment of aquaculture pond, and strain M6 was selected by further enrichment culture in nutrition broth with malachite green and decolorization rate comparison. The decolorization rate of strain M6 to malachite green was 97.14% in the conditon of 30?C and 150 r/min, and its morphology was observed by gram stain and electronmicroscopy, its physiological and biochemical characteristic was studied by ATB bacteria identification in-strument for identification of bacteria, and its 16S rDNA sequence was determined following PCR amplifi-cation, the sequence was aligned and the phylogenic tree was instructed with those bacterial strains of high identity with strain M6. In addition, its growth characteristics was also studied. The experimental results showed that strain M6 was gram negative and bacilliform with a flagellum at one end. Its size was 0.45 ?m ?0.84 ?m. Its colony produced on common agar plate appeared as round, light blue, dense, hard to choose; 16S rDNA sequence of strain M6 had high identity of 98%～99% with Pseudomonas sp. located in GenBank and strain M6 had the most close relative relation to Pseudomonas putida OW-16 (Locus number: DQ112328.1). Combined the results of the traditional morphological, physiological, biochemical character-istics and 16S rDNA sequence analysis, strain M6 was identified as Pseudomonas putida (Locus number: EU348741.1). Additionally, its growth curve in the condition of 30?C and 150 r/min was as follows: lag phase was 0～4 h, log phase was 4 h～64 h, stationary phase was 64 h～80 h, decline phase was after 80 h. Its best growth conditions were pH 7 and 30?C,and in the rotational speed of 50 r/min to 250 r/min. Its concen-tration increased with the increase in rotational speed.