Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions

A denitrifying Pseudomonas sp. (strain KC) capable of transforming carbon tetrachloride (CT) was isolated from groundwater aquifer solids. Major products of the transformation of 14C-labeled CT by Pseudomonas strain KC under denitrification conditions were 14CO2 and an unidentified water-soluble fraction. Little or no chloroform was produced. Addition of dissolved trace metals, notably, ferrous iron and cobalt, to the growth medium appeared to enhance growth of Pseudomonas strain KC while inhibiting transformation of CT. It is hypothesized that transformation of CT by this organism is associated with the mechanism of trace-metal scavenging.     

Transformation of carbon tetrachloride by Pseudomonas sp. strain KC under denitrification conditions.

A denitrifying Pseudomonas sp. (strain KC) capable of transforming carbon tetrachloride (CT) was isolated from groundwater aquifer solids. Major products of the transformation of 14C-labeled CT by Pseudomonas strain KC under denitrification conditions were 14CO2 and an unidentified water-soluble fraction. Little or no chloroform was produced. Addition of dissolved trace metals, notably, ferrous iron and cobalt, to the growth medium appeared to enhance growth of Pseudomonas strain KC while inhibiting transformation of CT. It is hypothesized that transformation of CT by this organism is associated with the mechanism of trace-metal scavenging.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Transformation of Dibenzo-p-Dioxin by Pseudomonas sp. Strain HH69

Dibenzo-p-dioxin was oxidatively cleaved by the dibenzofuran-degrading bacterium Pseudomonas sp. strain HH69 to produce minor amounts of 1-hydroxydibenzo-p-dioxin and catechol, while a 2-phenoxy derivative of muconic acid was formed as the major product. Upon acidic methylation, the latter yielded the dimethylester of cis, trans-2-(2-hydroxyphenoxy)-muconic acid.     

Transformation of carbon tetrachloride via sulfur and oxygen substitution by Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC transforms carbon tetrachloride into carbon dioxide and nonvolatile products, without chloroform as an intermediate. To define the pathway for hydrolysis, nonvolatile products were analyzed. Condensation products containing the carbon atom of carbon tetrachloride as carbonyl and thioxo moieties were identified, indicating the intermediacy of phosgene and thiophosgene in the pathway.     

Isolation and Characterization of Pseudomonas sp. Strain ONBA-17 Degrading o-Nitriobenzaldehyde

Aerobic bacteria degrading o-nitrobenzaldehyde (ONBA) were isolated from activated sludges. One of the isolates, ONBA-17, was identified as Pseudomonas sp. The isolate could grow on ONBA as its sole source of carbon and nitrogen. Further studies demonstrated that the strain was a moderately halophilic bacterium and capable of degrading benzoic acid, 2-nitrophenol, 2-aminophenol, 4-hydroxybenzoic acid, and 4-dimetylaminobenzaldehyde. It could completely degrade 100 mg L⁻¹ ONBA at a range of pH 6–8 in 48 h at 30°C, and up to 400 mg L⁻¹ after 288 h. The strain showed potential to be a good candidate for biotreatment of industrial wastewaters containing ONBA due to its salt-tolerance ability, multiresistance to some heavy metals and antibiotics, and the abilities of degradation of aromatic compounds. These findings may help in developing a process for ONBA-containing industrial wastewater treatment.     

Transformation of Substituted Fluorenes and Fluorene Analogs by Pseudomonas sp. Strain F274

Pseudomonas sp. strain F274, previously shown to catabolize fluorene via fluorenone and its angular dioxygenation, 2(prm1),3(prm1)-dihydroxy-2-carboxybiphenyl, phthalate, and protocatechuate, was examined for its ability to transform substituted fluorenes and S- and N-heterocyclic analogs. Halogen- and methyl-substituted fluorenes were metabolized to correspondingly substituted phthalates via attack on the unsubstituted ring. In the case of 1-methylfluorene, initial oxidation of the methyl group to carboxyl prevented all other transformations but 9-monooxygenation. This strain also oxidized the S-heteroatoms and benzylic methylenic groups of fluorene analogs. No angular dioxygenation of S- and N-heterocycles was observed.     

Phenotypic and Phylogenetic Characterization of Burkholderia (Pseudomonas) sp. Strain LB400

The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp. strain LB400 and B. cepacia ATCC 25416^T were compared to determine the degree to which these two strains might be related. Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B. cepacia are plant and human pathogens. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia. The 16S rRNA gene sequence showed that this strain was not as closely related to B. cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B. phenazinium, B. caribensis, B. graminis, and three unnamed Burkholderia spp. not known to be pathogenic. Received: 16 August 2000 / Accepted: 27 September 2000     

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,Pseudomonas sp. Strain DO-1

Five microbial strains that removed hydrogen sulfide (H₂S) or methylmercaptan (CH₃SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it’s immobilized cells removed 1 or 10 ppm of H₂S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H₂ S gas without preliminary feeding or exposure to sulfur com-pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.     

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α₂ structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The K_m values for coniferyl aldehyde and NAD⁺ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH₂-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD⁺-dependent aldehyde dehydrogenases from different sources.     

Chromatographic Characterization and Phytotoxic Activity of Fusarium oxysporum f. sp. albedinis and Saprophytic Strain Toxins

The bayoud disease, vascular fusariosis of date palm tree (Phoenix dactylifera L.), is caused by the pathogenic fungus Fusarium oxysporum f. sp. albedinis. The characteristic symptoms of the bayoud disease were elicited on detached leaves of F. oxysporum f. sp. albedinis‐susceptible cultivars of date palm trees, which were treated either with the F_II (F. oxysporum f. sp. albedinis) fraction purified from the organic extracts of a F. oxysporum f. sp. albedinis liquid culture, or with a solution of fusaric acid. Enniatins, which are secreted by several Fusarium species, were tested at different concentrations and were not capable of inducing symptoms on such detached leaves. The F_II (F. oxysporum f. sp. albedinis) fraction was unable to induce necrosis of potato slices, which indicates that it does not contain significant amounts of enniatins. The high‐performance liquid chromatography (HPLC) profiles of the F_II (F. oxysporum f. sp. albedinis) fraction showed toxic peaks different from fusaric acid. A fraction, named F_II (AZ₄), was obtained from culture filtrates of a saprophytic Fusarium strain maintained in the same cultural conditions as for the F. oxysporum f. sp. albedinis. The HPLC profile of the F_II (AZ₄) fraction did not show the characteristic phytotoxic peaks present in the F_II (F. oxysporum f. sp. albedinis) fraction. This finding well agrees with the fact that the F_II (AZ₄) fraction is not toxic to detached date palm leaves. Moreover, the HPLC profiles of F_II fractions obtained from other special forms of F. oxysporum are different the F_II (F. oxysporum f. sp. albedinis) profile. The phytotoxic compounds purified from the F_II (F. oxysporum f. sp. albedinis) fraction are probably new molecules that may help in understanding the pathogenesis of bayoud disease.     

Catechol 2,3-Dioxygenase from Pseudomonas sp. Strain ND6: Gene Sequence and Enzyme Characterization

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5α using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.     

Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.     

Characterization and implications of the cell surface reactivity of Calothrix sp. strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK(a)] between 5.0 and 6.2) and amine groups (mean pK(a) of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK(a) spectra similarly dominated by carboxyls (pK(a) of 4.6 to 6.1) and amines (pK(a) of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK(a) values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 x 10(-5) and 12.3 x 10(-5) mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Characterization of a Pseudomonas sp. Capable of Aniline Degradation in the Presence of Secondary Carbon Sources

Pseudomonas strain K1 is a gram-negative rod which grows aerobically on minimal media containing aniline with a doubling time of 2 h at 30°C. The half-saturation parameter for aniline metabolism by aniline-grown cells was 3.8 μmol · liter⁻¹. Concentrations of aniline as low as 50 nM were metabolized. Neither substituted anilines nor other aromatic compounds (other than aromatic amino acids) supported growth. Cells grew as fast on aniline as on nonaromatic substrates such as lactate. The aromatic ring was cleaved via the meta pathway. Catechol 2,3-oxygenase activity was induced by aniline, even in cultures containing alternative carbon sources such as lactate. Cultures grown on a mixture of aniline and lactate mineralized aniline in the presence of the second substrate. Lactate-grown cultures lacked catechol oxygenase activity, and resting cells from these cultures did not respire aniline. Resting cells from aniline-grown cultures exhibited high respiratory activity upon the addition of aniline or catechol, some activity with toluidine, and no activity after addition of a wide variety of other aromatic compounds, including dihydroxybenzylamine, chloroanilines, ethylanilines, aminophenols, aminobenzoates, and dihydroxybenzoates. Although substituted anilines were not metabolized, 3-or 4-chloroaniline did induce the enzymes for aniline oxidation.     

Phosphonate Utilization by the Glyphosate-Degrading Pseudomonas sp. Strain PG2982

The glyphosate-degrading Pseudomonas sp. strain PG2982 was found to utilize each of 10 organophosphonate compounds as a sole phosphorus source. Representative compounds tested included alkylphosphonates, 1-amino-substituted alkylphosphonates, amino-terminal phosphonates, and an arylphosphonate. This report demonstrates that PG2982 is capable of utilizing a wider range of structurally different organophosphonate compounds than any organism described to date.     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.