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Research in Microbiology May 2010Pseudomonas mendocina carrying a novel class 1 integron containing an IMP-8 gene was isolated from an inanimate surface in a female ward sanitary facility of the...
Pseudomonas mendocina carrying a novel class 1 integron containing an IMP-8 gene was isolated from an inanimate surface in a female ward sanitary facility of the Hospital Infante D. Pedro, Aveiro, Portugal. Hybridization with the integrase gene (intI1) and 16S rDNA revealed that the integron is chromosomally located. Here we report for the first time the presence of an IMP-8 metallo-beta-lactamase gene in the Pseudomonas genus.
Topics: Bacterial Proteins; Equipment and Supplies, Hospital; Humans; Integrons; Molecular Sequence Data; Portugal; Pseudomonas mendocina; beta-Lactamases
PubMed: 20381610
DOI: 10.1016/j.resmic.2010.03.004 -
Applied and Environmental Microbiology Apr 2010In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of...
In aerobic, circumneutral environments, the essential element Fe occurs primarily in scarcely soluble mineral forms. We examined the independent and combined effects of a siderophore, a reductant (ascorbate), and a low-molecular-weight carboxylic acid (oxalate) on acquisition of Fe from the mineral hematite (alpha-Fe(2)O(3)) by the obligate aerobe Pseudomonas mendocina ymp. A site-directed DeltapmhA mutant that was not capable of producing functional siderophores (i.e., siderophore(-) phenotype) did not grow on hematite as the only Fe source. The concentration of an added exogenous siderophore (1 microM desferrioxamine B [DFO-B]) needed to restore wild-type (WT)-like growth kinetics to the siderophore(-) strain was approximately 50-fold less than the concentration of the siderophore secreted by the WT organism grown under the same conditions. The roles of a reductant (ascorbate) and a simple carboxylic acid (oxalate) in the Fe acquisition process were examined in the presence and absence of the siderophore. Addition of ascorbate (50 microM) alone restored the growth of the siderophore(-) culture to the WT levels. A higher concentration of oxalate (100 microM) had little effect on the growth of a siderophore(-) culture; however, addition of 0.1 muM DFO-B and 100 muM oxalate restored the growth of the mutant to WT levels when the oxalate was prereacted with the hematite, demonstrating that a metabolizing culture benefits from a synergistic effect of DFO-B and oxalate.
Topics: Aerobiosis; Ascorbic Acid; Ferric Compounds; Gene Knockout Techniques; Iron; Mutagenesis, Site-Directed; Oxalates; Pseudomonas mendocina; Siderophores
PubMed: 20118367
DOI: 10.1128/AEM.02349-09 -
Water Research Feb 2009Biofouling and virus penetration are two significant obstacles in water treatment membrane filtration. Biofouling reduces membrane permeability, increases energy costs,...
Biofouling and virus penetration are two significant obstacles in water treatment membrane filtration. Biofouling reduces membrane permeability, increases energy costs, and decreases the lifetime of membranes. In order to effectively remove viruses, nanofiltration or reverse osmosis (both high energy filtration schemes) must be used. Thus, there is an urgent demand for low pressure membranes with anti-biofouling and antiviral properties. The antibacterial properties of silver are well known, and silver nanoparticles (nAg) are now incorporated into a wide variety of consumer products for microbial control. In this study, nAg incorporated into polysulfone ultrafiltration membranes (nAg-PSf) exhibited antimicrobial properties towards a variety of bacteria, including Escherichia coli K12 and Pseudomonas mendocina KR1, and the MS2 bacteriophage. Nanosilver incorporation also increased membrane hydrophilicity, reducing the potential for other types of membrane fouling. XPS analysis indicated a significant loss of silver from the membrane surface after a relatively short filtration period (0.4 L/cm2) even though ICP analysis of digested membrane material showed that 90% of the added silver remained in the membrane. This silver loss resulted in a significant loss of antibacterial and antiviral activity. Thus, successful fabrication of nAg-impregnated membranes needs to allow for the release of sufficient silver ions for microbial control while preventing a rapid depletion of silver.
Topics: Anti-Bacterial Agents; Antiviral Agents; Bacterial Adhesion; Biofilms; Environmental Restoration and Remediation; Escherichia coli; Membranes, Artificial; Metal Nanoparticles; Polymers; Pseudomonas mendocina; Silver; Spectrum Analysis; Sulfones; Suspensions; Ultrafiltration; Viruses
PubMed: 19046755
DOI: 10.1016/j.watres.2008.11.014 -
Environmental Microbiology Dec 2008Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of...
Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C(1)-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems.
Topics: Bacteria; Biodiversity; DNA, Bacterial; DNA, Ribosomal; Dimethylamines; Genes, rRNA; Geologic Sediments; London; Mesylates; Methane; Methanol; Methyl Methanesulfonate; Methylamines; Molecular Sequence Data; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Rivers; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid
PubMed: 18681896
DOI: 10.1111/j.1462-2920.2008.01711.x -
Journal of Applied Microbiology Jul 2008The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms.
AIMS
The aim of the study was to isolate a novel lipolytic enzyme from the activated sludge of uncultured micro-organisms.
METHODS AND RESULTS
The metagenomic DNA was directly extracted from the activated sludge, and a metagenomic library was constructed by using the pUC vector. The library was screened for lipolytic enzyme activity on 1% tributyrin agar plate. A clone among c. 100 000 recombinant libraries showed the lipolytic activity. The putative lipolytic gene encoding lipo1 from the metagenomic library was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The expressed recombinant enzyme was purified by Ni-nitrilotriacetic acid affinity chromatography and characterized using general substrates of lipolytic property. The gene consisted of 972 bp encoding a polypeptide of 324 amino acids with a molecular mass of 35.6 kDa. Typical residues essential for lipolytic activity such as penta-peptide (GXSXG) and catalytic triad sequences (Ser166, Asp221 and His258) were detected. The deduced amino acid sequence of lipo1 showed low identity with amino acid sequences of esterase/lipase (32%, ZP_01528487) from Pseudomonas mendocina ymp and esterase (31%, AAY45707) from uncultured bacterium. This lipolytic enzyme exhibited the highest activity at pH 7.5 and 10 degrees C. At thermal stability analysis, lipo1 was more unstable at 40 degrees C than 10 degrees C.
CONCLUSIONS
An activity based strategy has been an effective method for fishing out a low-temperature adapted lipolytic enzyme from the metagenomic library. This lipo1 enzyme can be considered to belong to the hormone-sensitive lipase family due to the enzyme's oxyanion hole by the sequence HGGG.
SIGNIFICANCE AND IMPACT OF THE STUDY
Lipo1 is a novel psychrophilic esterase obtained directly from the metagenomic library. Owing its support of significant activity at low temperature, this enzyme is expected to be useful for potential application as a biocatalyst in organic chemistry.
Topics: Adaptation, Physiological; Amino Acid Sequence; Base Sequence; Carboxylic Ester Hydrolases; Cloning, Molecular; Cold Temperature; Detergents; Environmental Microbiology; Genome, Bacterial; Genomic Library; Hydrogen-Ion Concentration; Lipolysis; Molecular Sequence Data; Sequence Alignment; Sequence Analysis, DNA; Sewage
PubMed: 18248379
DOI: 10.1111/j.1365-2672.2007.03717.x -
Applied and Environmental Microbiology Mar 2008Enantiopure sulfoxides are valuable asymmetric starting materials and are important chiral auxiliaries in organic synthesis. Toluene monooxygenases (TMOs) have been... (Comparative Study)
Comparative Study
Enantiopure sulfoxides are valuable asymmetric starting materials and are important chiral auxiliaries in organic synthesis. Toluene monooxygenases (TMOs) have been shown previously to catalyze regioselective hydroxylation of substituted benzenes and phenols. Here we show that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides. Mutagenesis of position V106 in the alpha-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and the analogous position I100 in toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 improved both rate and enantioselectivity. Variant TomA3 V106M of TOM oxidized methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increased from 51% for the wild type to 88% for this mutant. Similarly, T4MO variant TmoA I100G increased the wild-type oxidation rate by 1.7-fold, and the enantiomeric excess rose from 86% to 98% (pro-S). Both wild-type enzymes showed lower activity with methyl para-tolyl sulfide as a substrate, but the improvement in the activity and enantioselectivity of the mutants was more dramatic. For example, T4MO variant TmoA I100G oxidized methyl para-tolyl sulfide 11 times faster than the wild type did and changed the selectivity from 41% pro-R to 77% pro-S. A correlation between regioselectivity and enantioselectivity was shown for TMOs studied in this work. Using in silico homology modeling, it is shown that residue I100 in T4MO aids in steering the substrate into the active site at the end of the long entrance channel. It is further hypothesized that the main function of V106 in TOM is the proper positioning or docking of the substrate with respect to the diiron atoms. The results from this work suggest that when the substrate is not aligned correctly in the active site, the oxidation rate is decreased and enantioselectivity is impaired, resulting in products with both chiral configurations.
Topics: Base Sequence; Burkholderia cepacia; DNA Primers; Gene Library; Mixed Function Oxygenases; Models, Molecular; Molecular Sequence Data; Mutagenesis; Oxidation-Reduction; Protein Engineering; Pseudomonas mendocina; Sequence Analysis, DNA; Sulfoxides
PubMed: 18192418
DOI: 10.1128/AEM.01849-07 -
Biometals : An International Journal on... Jun 2008Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization....
Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization. Pseudomonas mendocina ymp was isolated from the Yucca Mountain Site for long-term nuclear waste storage. Its ability to solubilize a variety of Fe-containing minerals under aerobic conditions has been previously investigated but its molecular and genetic potential remained uncharacterized. Here, we have shown that the organism produces a hydroxamate and not a catecholate-based siderophore that is synthesized via non-ribosomal peptide synthetases. Gene clustering patterns observed in other Pseudomonads suggested that hybridizing multiple probes to the same library could allow for the identification of one or more clusters of syntenic siderophore-associated genes. Using this approach, two independent clusters were identified. An unfinished draft genome sequence of P. mendocina ymp indicated that these mapped to two independent contigs. The sequenced clusters were investigated informatically and shown to contain respectively a potentially complete set of genes responsible for siderophore biosynthesis, uptake, and regulation, and an incomplete set of genes with low individual homology to siderophore-associated genes. A mutation in the cluster's pvdA homolog (pmhA) resulted in a siderophore-null phenotype, which could be reversed by complementation. The organism likely produces one siderophore with possibly different isoforms and a peptide backbone structure containing seven residues (predicted sequence: Acyl-Asp-Dab-Ser-fOHOrn-Ser-fOHorn). A similar approach could be applied for discovery of Fe- and siderophore-associated genes in unsequenced or poorly annotated organisms.
Topics: Bacterial Proteins; Base Sequence; Iron; Microbial Viability; Multigene Family; Open Reading Frames; Pseudomonas mendocina; Sequence Analysis
PubMed: 18058194
DOI: 10.1007/s10534-007-9124-5 -
Applied and Environmental Microbiology May 2007Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations....
Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.
Topics: Ferric Compounds; Iron; Pseudomonas mendocina; Reducing Agents; Solubility
PubMed: 17384310
DOI: 10.1128/AEM.02586-06 -
Applied and Environmental Microbiology Oct 2006N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been...
N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of 18O2 and H(2)18O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O2. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH3OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial alpha-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.
Topics: Biotransformation; Dimethylnitrosamine; Nitroso Compounds; Pseudomonas mendocina; Water Pollutants, Chemical
PubMed: 16950909
DOI: 10.1128/AEM.01535-06 -
Antimicrobial Agents and Chemotherapy Nov 2006Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic...
Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Biofilms; Cell Membrane Permeability; Drug Synergism; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Tobramycin
PubMed: 16940063
DOI: 10.1128/AAC.00509-06