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Microbiological Research May 2013The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid... (Comparative Study)
Comparative Study
The medium-chain-length polyhydroxyalkanoate (PHAMCL) synthase genes phaC1 and phaC2 of Pseudomonas mendocina NK-01 were cloned and inserted into expression plasmid pBBR1MCS-2 to form pBBR1MCS-C1 and pBBR1MCS-C2 which were expressed respectively in the PHAMCL-negative strain P. mendocina C7 whose PHAMCL synthesis operon was defined knock out. P. mendocina C7 derivatives P. mendocina C7C1 and C7C2 carrying pBBR1MCS-C1 and pBBR1MCS-C2 respectively were constructed. Fermentation and gel permeation chromatography (GPC) revealed that P. mendocina C7C1 had higher PHAMCL production rate but its PHAMCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHAMCL had similarity in monomer composition with 3HD as the favorite monomer i.e. PhaC1 and PhaC2 had the same substrate specificity. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) also revealed that the two PHAMCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHAMCL synthases PhaC1 and PhaC2 had the same substrate specificity.
Topics: Acyltransferases; Amino Acid Sequence; Bacterial Proteins; Molecular Sequence Data; Polyhydroxyalkanoates; Pseudomonas mendocina; Sequence Alignment; Substrate Specificity
PubMed: 23238264
DOI: 10.1016/j.micres.2012.11.003 -
Infection and Immunity Feb 2013The importance of our inner microbial communities for proper immune responses against invading pathogens is now well accepted, but the mechanisms underlying this...
The importance of our inner microbial communities for proper immune responses against invading pathogens is now well accepted, but the mechanisms underlying this protection are largely unknown. In this study, we used Caenorhabditis elegans to investigate such mechanisms. Since very little is known about the microbes interacting with C. elegans in its natural environment, we began by taking the first steps to characterize the C. elegans microbiota. We established a natural-like environment in which initially germfree, wild-type larvae were grown on enriched soil. Bacterial members of the adult C. elegans microbiota were isolated by culture and identified using 16S rRNA gene sequencing. Using pure cultures of bacterial isolates as food, we identified two, Bacillus megaterium and Pseudomonas mendocina, that enhanced resistance to a subsequent infection with the Gram-negative pathogen Pseudomonas aeruginosa. Whereas protection by B. megaterium was linked to impaired egg laying, corresponding to a known trade-off between fecundity and resistance, the mechanism underlying protection conferred by P. mendocina depended on weak induction of immune genes regulated by the p38 MAPK pathway. Disruption of the p38 ortholog, pmk-1, abolished protection. P. mendocina enhanced resistance to P. aeruginosa but not to the Gram-positive pathogen Enterococcus faecalis. Furthermore, protection from P. aeruginosa was similarly induced by a P. aeruginosa gacA mutant with attenuated virulence but not by a different C. elegans-associated Pseudomonas sp. isolate. Our results support a pivotal role for the conserved p38 pathway in microbiota-initiated immune protection and suggest that similarity between microbiota members and pathogens may play a role in such protection.
Topics: Animals; Bacillus megaterium; Bacterial Infections; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Metagenome; Pseudomonas aeruginosa; Pseudomonas mendocina; Soil Microbiology; Virulence; p38 Mitogen-Activated Protein Kinases
PubMed: 23230286
DOI: 10.1128/IAI.00653-12 -
Journal of Bacteriology Nov 2012Here we present the draft genome of Pseudomonas mendocina strain S5.2, possessing tolerance to a high concentration of copper. In addition to being copper resistant, the...
Here we present the draft genome of Pseudomonas mendocina strain S5.2, possessing tolerance to a high concentration of copper. In addition to being copper resistant, the genome of P. mendocina strain S5.2 contains a number of heavy-metal-resistant genes known to confer resistance to multiple heavy-metal ions.
Topics: Agriculture; Animals; France; Genome, Bacterial; Metals, Heavy; Molecular Sequence Data; Pseudomonas mendocina; Soil Microbiology; Vitis
PubMed: 23105092
DOI: 10.1128/JB.01702-12 -
Journal of Bacteriology Nov 2012Pseudomonas mendocina DLHK is an aerobic bacterium isolated from a biotrickling reactor which can remove nitric oxide, a common air pollutant from combustion exhaust...
Pseudomonas mendocina DLHK is an aerobic bacterium isolated from a biotrickling reactor which can remove nitric oxide, a common air pollutant from combustion exhaust gas. Here, we present the draft genome of Pseudomonas mendocina DLHK.
Topics: Bioreactors; Genome, Bacterial; Molecular Sequence Data; Pseudomonas mendocina
PubMed: 23105066
DOI: 10.1128/JB.01618-12 -
Journal of Applied Microbiology Apr 2012Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was...
AIMS
Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next-generation sequencing 454 GS-FLX technology to elucidate the microbial community structure of cattle milk.
METHODS AND RESULTS
Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web-based tool MG-RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt-zinc-cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed.
CONCLUSIONS
The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows.
SIGNIFICANCE AND IMPACT OF THE STUDY
The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Cattle; Crosses, Genetic; Female; High-Throughput Nucleotide Sequencing; Mastitis, Bovine; Milk
PubMed: 22277077
DOI: 10.1111/j.1365-2672.2012.05244.x -
Microbial Biotechnology Jul 2012The two-component system TmoS/TmoT controls the expression of the toluene-4-monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of P(tmoX) activity. The...
The two-component system TmoS/TmoT controls the expression of the toluene-4-monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of P(tmoX) activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (K(D) = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p-chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of P(tmoX) and P(todX) (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family.
Topics: Calorimetry; Gene Expression Regulation, Bacterial; Histidine Kinase; Hydrocarbons, Aromatic; Oxygenases; Phosphorylation; Protein Binding; Protein Kinases; Pseudomonas mendocina; Signal Transduction; Substrate Specificity
PubMed: 22212183
DOI: 10.1111/j.1751-7915.2011.00322.x -
The Israel Medical Association Journal... Jun 2011
Topics: Adult; Anti-Bacterial Agents; Diagnosis, Differential; Hematologic Tests; Humans; Male; Microbial Sensitivity Tests; Pseudomonas Infections; Pseudomonas mendocina; Sepsis
PubMed: 21809738
DOI: No ID Found -
Applied and Environmental Microbiology Jul 2011Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of...
Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.
Topics: Bacteria, Aerobic; Burkholderia; Carbon; Choline; Energy Metabolism; Enzyme Inhibitors; Metabolic Networks and Pathways; Nitrogen; Pseudomonas; Sarcosine; Sinorhizobium meliloti
PubMed: 21602374
DOI: 10.1128/AEM.00504-11 -
Journal of Bacteriology Jul 2011Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and alginate oligosaccharides (AO) simultaneously from glucose under...
Pseudomonas mendocina NK-01 can synthesize medium-chain-length polyhydroxyalkanoate (PHA(MCL)) and alginate oligosaccharides (AO) simultaneously from glucose under conditions of limited nitrogen. Here, we report the complete sequence of the 5.4-Mbp genome of Pseudomonas mendocina NK-01, which was isolated from farmland soil in Tianjin, China.
Topics: Alginates; China; DNA, Bacterial; Genome, Bacterial; Glucose; Glucuronic Acid; Hexuronic Acids; Molecular Sequence Data; Oligosaccharides; Polyhydroxyalkanoates; Pseudomonas mendocina; Sequence Analysis, DNA; Soil Microbiology
PubMed: 21551299
DOI: 10.1128/JB.05068-11 -
Bioscience, Biotechnology, and... 2010The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and...
Identification of putative biomarkers for toluene-degrading Burkholderia and pseudomonas by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting.
The use of peptide mass fingerprinting with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was demonstrated to identify and phenotypically characterize toluene-degrading bacteria via biomarkers of degradation and taxonomical classification. Pseudomonas putida F1, P. mendocina KR1, and Burkholderia sp. JS150 were grown on toluene, extracted, electrophoretically separated, and analyzed by MALDI-TOF MS. Catabolic enzymes were identified and results substantiated using tandem MS.
Topics: Biodegradation, Environmental; Biomarkers; Burkholderia; Electrophoresis, Polyacrylamide Gel; Peptide Mapping; Pseudomonas putida; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Toluene
PubMed: 20622441
DOI: 10.1271/bbb.100064