Heavy-Metal Resistance of a France Vineyard Soil Bacterium,Pseudomonas mendocina Strain S5.2, Revealed by Whole-Genome Sequencing

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.     

Plasmid-associated biodegradation of an organophosphorus pesticide, Monocrotophos, by Pseudomonas mendocina

Pseudomonas mendocina isolated from soil degraded an insecticide, Monocrotophos (MCP), by 67% and harbored a 7.4 kb plasmid, designated as pMCP424. On the basis of curing and transformation experiments, MCP degradation by Pseudomonas mendocinawas plasmid-borne and transferable to other bacteria.     

Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.     

Comparison of medium-chain-length polyhydroxyalkanoates synthases from Pseudomonas mendocina NK-01 with the same substrate specificity

The medium-chain-length polyhydroxyalkanoate (PHA_MCL) 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 PHA_MCL-negative strain P. mendocina C7 whose PHA_MCL 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 PHA_MCL production rate but its PHA_MCL had lower molecular weight than that of P. mendocina C7C2. Gas chromatograph/mass spectrometry (GC/MS) analysis revealed that the two PHA_MCL 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 PHA_MCL had the same physical properties. P. mendocina NK-01was the first reported strain whose PHA_MCL synthases PhaC1 and PhaC2 had the same substrate specificity.     

Biotransformation of N-Nitrosodimethylamine by Pseudomonas mendocina KR1

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 H₂¹⁸O 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 α-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.     

Survival in Soil of Different Toluene-Degrading Pseudomonas Strains after Solvent Shock

We assayed the tolerance to solvents of three toluene-degrading Pseudomonas putida strains and Pseudomonas mendocina KR1 in liquid and soil systems. P. putida DOT-T1 tolerated concentrations of heptane, propylbenzene, octanol, and toluene of at least 10% (vol/vol), while P. putida F1 and EEZ15 grew well in the presence of 1% (vol/vol) propylbenzene or 10% (vol/vol) heptane, but not in the presence of similar concentrations of octanol or toluene. P. mendocina KR1 grew only in the presence of heptane. All three P. putida strains were able to become established in a fluvisol soil from the Granada, Spain, area, whereas P. mendocina KR1 did not survive in this soil. The tolerance to organic solvents of all three P. putida strains was therefore assayed in soil. The addition to soil of 10% (vol/wt) heptane or 10% (vol/wt) propylbenzene did not affect the survival of the three P. putida strains. However, the addition of 10% (vol/wt) toluene led to an immediate decrease of several log units in the number of CFU per gram of soil for all of the strains, although P. putida F1 and DOT-T1 subsequently recovered. This recovery was influenced by the humidity of the soil and the incubation temperature. P. putida DOT-T1 recovered from the shock faster than P. putida F1; this allowed the former strain to become established at higher densities in polluted sites into which both strains had been introduced.     

Induction of antioxidant enzymes is involved in the greater effectiveness of a PGPR versus AM fungi with respect to increasing the tolerance of lettuce to severe salt stress

This study investigated the influence of inoculation with a plant growth-promoting rhizobacterium, Pseudomonas mendocina Palleroni, alone or in combination with an arbuscular mycorrhizal (AM) fungus, Glomus intraradices (Schenk & Smith) or Glomus mosseae (Nicol & Gerd.) Gerd. & Trappe, on antioxidant enzyme activities (catalase and total peroxidase), phosphatase activity, solute accumulation, growth and mineral nutrient uptake in leaves of Lactuca sativa L. cv. Tafalla affected by three different levels of salt stress. Salinity decreased lettuce growth, regardless of the biological treatment and of the salt stress level. The plants inoculated with P. mendocina had significantly greater shoot biomass than the control plants at both salinity levels, whereas the mycorrhizal inoculation treatments only were effective in increasing shoot biomass at the medium salinity level. At the highest salinity level, the water content was greater in leaves of plants treated with P. mendocina or G. mosseae. At the medium salinity level, G. intraradices- or G. mosseae-colonised plants showed the highest concentrations of foliar P. The P. mendocina- and G. mosseae-colonised plants presented higher concentrations of foliar K and lower concentrations of foliar Na under high salt conditions. Salt stress decreased sugar accumulation and increased foliar proline concentration, particularly in plants inoculated with the PGPR. Increasing salinity stress raised significantly the antioxidant enzyme activities, including those of total peroxidase and catalase, of lettuce leaves compared to their respective non-stressed controls. The PGPR strain induced a higher increase in these antioxidant enzymes in response to severe salinity. Inoculation with selected PGPR could serve as a useful tool for alleviating salinity stress in salt-sensitive plants.     

Biochemical, molecular characterization and growth promoting effects of phosphate solubilizing Pseudomonas sp. isolated from weeds grown in salt range of Pakistan

Three species of phosphate solubilizing bacteria viz, Pseudomonas mendocina Khsr2, Pseudomonas stutzeri Khsr3 and Pseudomonas putida Khsr4 were isolated from roots of weeds Lactuca dissecta D. Don, Solanum surattense Burm. f and Sonchus arvensis L. respectively growing in Khewra salt range (EC: 2.3 dS m^?1; pH 8.6). Preliminary identification of bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16S-rRNA gene sequencing. The genetic diversity among the isolates was evaluated by Randomly Amplified Polymorphic DNA finger printing and similarity matrix was measured. All the Pseudomonas sp. were capable of solubilizing phosphate, produced phytohormones: Indole-3-acetic acid, Gibberellic acid, Trans-zeatin riboside and Abscisic acid in culture media and were found to be efficient in stimulating root/shoot length and dry weight and proline contents of Zea mays L (advance germplasm line: Islamabad Gold) seedlings grown under normal and NaCl (20 dS m^?1) stress. The strain Pseudomonas stutzeri Khsr3 appears to be a potential candidate as bio-inoculant for saline fields.     

Toluene Monooxygenase-Catalyzed Epoxidation of Alkenes

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.     

Acquisition of Fe from Various Natural Organic Matter Isolates by Aerobic Pseudomonad Bacteria

Iron (Fe) is an essential nutrient to most microorganisms. Aerobic microorganisms exhibit various strategies for acquiring Fe at near-neutral pH conditions, where Fe oxyhydroxides are insoluble. Although much research has focused on microbial acquisition of Fe from minerals, little is known about Fe acquisition from natural organic matter (NOM). Yet, in surface waters, soils and shallow sediments, Fe is often associated with natural organic matter (NOM), and this NOM-associated Fe could represent an important pool of Fe for microorganisms. Here, we investigated the growth of aerobic Pseudomonas mendocina on soil and surface water NOM samples containing Fe, under Fe-limited conditions. In the presence of NOM, bacteria grew to population sizes greater than in no-Fe-added controls, indicating that the bacteria were able to access Fe associated with NOM. Maximum population size correlated with the NOM-associated Fe concentration. In an additional experiment, Pseudomonas putida was able to acquire Fe from an NOM sample, demonstrating that this ability is not limited to P. mendocina. When Fe was added as 30 μ M FeEDTA plus NOM, together in the same reaction flasks, P. mendocina and P. putida growth was less than in the presence of 30 μM FeEDTA alone. The fact that Fe sources are not simply additive and that the presence of NOM inhibits growth in FeEDTA suggests that further study on the responses of bacteria to a combination of Fe sources is needed to understand the complexities of bacterial Fe acquisition in the subsurface.     

Construction of a Genetic Map of Bacteria Pseudomonas mendocina

Based on the results of matings with interrupted conjugation and analysis of marker joint inheritance frequencies, distances between 26 genetic determinants were estimated and a genetic map of Pseudomonas mendocina bacteria was constructed.     

Conjugational Mapping of Bacteria Pseudomonas mendocina

Donor strains of the Hfr type were isolated using plasmid pRK2013 with transposons Tn10 and B21 as a chromosome-mobilizing factor. The isolated strains were shown to promote transfer of donor chromosome from different origins in different directions during isogenic matings of Pseudomonas mendocina bacteria. The created collection of donors and polyauxotrophic recipient bacteria permitted mapping 26 genetic determinants on the bacterial chromosome and identifying the genome of these microorganisms as a circular DNA molecule.     

Efficient production of polyhydroxyalkanoates (PHAs) from Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) as the sole carbon source

Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.     

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

Iron Acquisition from Natural Organic Matter by an Aerobic Pseudomonas mendocina Bacterium: Siderophores and Cellular Iron Status

This research investigated the potential role of siderophores in aerobic microbial Fe acquisition from natural organic matter (NOM; XAD-8 isolate and reverse osmosis concentrate pre- and post-Chelex® treatment) through the use of a siderophore-producing Pseudomonas mendocina wild type (WT) bacterium and an engineered mutant (Mt) that was incapable of siderophore production. NOM had complex effects on microbial growth under Fe-limited conditions as measured by optical density, most likely because of the presence of other toxic (trace) metals such as Al, NOM binding interference with additional trace metal nutrients, and/or biofilm development. However, a bioassay for cellular Fe status showed that both WT and Mt readily acquired Fe naturally associated with NOM. Thus, while siderophores may be useful for Fe acquisition from NOM by P. mendocina, they do not appear to be essential for this process.     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

Effects of an Algicidal Bacterium <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> on the Growth and Antioxidant System of <Emphasis Type="Italic">Aphanizomenon flos-aquae</Emphasis>

Evident effect of an algicidal bacterium Pseudomonas mendocina on the growth and antioxidant system of Aphanizomenon flos-aquae was detected in this experiment. Seven parameters including the chlorophyll a contents, Fv/Fm values, reactive oxygen species (ROS), malonaldehyde (MDA), catalase (CAT), peroxide dismutase (POD), and superoxide dismutase (SOD) were tested in the cyanobacterium A. flos-aquae cells after inoculation with the algicidal bacterium Pseudomonas mendocina DC10. It was shown from the experiment that the growth of the treated cyanobacterium A. flos-aquae was significantly restrained, which was expressed as great reductions in the chlorophyll a contents and Fv/Fm values. At the same time, the treated cyanobacterial cells exhibited an obvious increase in the production of ROS and MDA compared with the control. CAT and POD activities in the treated group kept at high level, however, they both reduced significantly on day 6. SOD activities in the treated A. flos-aquae showed obvious declines after inoculation, and great augmentations on day 3 and 4, thereafter, they kept in a declining tendency. The results showed the oxidative stresses induced by the bacterium could be a killing agent of the cyanobacterium A. flos-aquae cells.     

Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 ± 2 °C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO₂ through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation. Received: 27 November 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999     

Identification of Putative Biomarkers for Toluene-Degrading Burkholderia and Pseudomonads 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.