Isolation and Characterization of Plasmid pUOl Mediating Dehalogenation of Haloacetate and Mercury Resistance in Moraxella sp. B

Plasmid pUO1 which specifies haloacetate dehalogenase H-1 and H-2 and mercuric reductase was isolated from a fluoroacetate-assimilating Moraxella strain B. From the spontaneous mutant deficient in H-2 enzyme derived from strain B, plasmid pUO11 specifying H-1 enzyme and mercuric reductase was also isolated. The molecular sizes of pUO1 and pUO11 were estimated to be 43.7 ± 1.6 Mdal and 40.1 ± 1.3 Mdal, respectively, by electron microscopy. These values were in good agreement with those estimated by electrophoretic analyses of the cccDNA and its restriction endonuclease digests. The digestion patterns of both plasmid DNAs were analogous, suggesting that plasmid pUO11 was the deletion mutant derived from pUO1. It could be presumed that the deleted DNA segment had a molecular size of about 3.6 Mdal.     

Genetic Rearrangement of Plasmids: In Vivo Recombination between a Dehalogenation Plasmid and Multiple-Resistance Plasmid RP4 in Pseudomonas sp

When Moraxella plasmid pUO1 encoding haloacetate dehalogenase and mercury resistance coexisted with IncP-1 plasmid RP4 in Pseudomonas sp., genetic exchange between the plasmids often occurred, probably by site-specific recombination. The recombinant plasmids obtained were classified into four groups on the basis of phenotype. Representative plasmids for each group were analyzed for DNA composition and function, and the mechanism for the formation of these plasmids was sought. They were inherited stably in Escherichia coli and a Pseudomonas sp.     

Sequence Analysis of the Plasmid pGY1 Harbored in Salmonella enterica Serovar Paratyphi A

The cryptic plasmid pGY1, which is harbored by a clinical isolate of Salmonella enterica serovar Paratyphi A, was identified in a 9-year-old girl with paratyphoid fever in 2005, and its DNA sequence was determined. It is 3592 bp in length and had a G+C content of 43.3%. Three ORFs were predicted that share low similarity with hypothetical proteins in the GenBank database. pGY1 shared 36.6% sequence homology with the cryptic plasmid pIMVS1 from Salmonella typhimurium. Its unique sequence makes it attractive for further study to obtain insight into the evolutionary relationship of this plasmid with other Enterobacteriaceae plasmids.     

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD⁺, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.     

Metabolism of 2-hydroxyphenylglyoxylate by Moraxella sp. strain VS1

A bacterium, designated as Moraxella sp., was enriched with 2-hydroxyphenylglyoxylate (2HPGA) as sole source of carbon and energy. Identified metabolites and enzyme activities determined with whole cells and extracts indicated that 2HPGA was degraded by an inducible sequence of enzymes via salicylaldehyde, salicylate, and gentisate; only minute amounts of salicylate were converted to catechol. Further evidence was obtained that permeases were necessary for the uptake of most aromatic compounds utilized for growth. For the direct determination of 2HPGA decarboxylase activity, an enzyme assay involving high-performance liquid chromatography for quantitation of the substrate was developped to study the initial step of the degradative pathway.     

Reductive Dehalogenation of Chlorobenzene Congeners in Cell Extracts of Dehalococcoides sp. Strain CBDB1

Enzymatic reductive dehalogenation of tri-, tetra-, penta-, and hexachlorobenzenes was demonstrated in cell extracts with low protein concentration (0.5 to 1 μg of protein/ml) derived from the chlorobenzene-respiring anaerobe Dehalococcoides sp. strain CBDB1. 1,2,3-trichlorobenzene dehalogenase activity was associated with the membrane fraction. Light-reversible inhibition by alkyl iodides indicated the presence of a corrinoid cofactor.     

Reductive Dehalogenation of Trichloroacetic Acid by Trichlorobacter thiogenes gen. nov., sp. nov.

A bacterium able to grow via reductive dechlorination of trichloroacetate was isolated from anaerobic soil enrichments. The isolate, designated strain K1, is a member of the δ proteobacteria and is related to other known sulfur and ferric iron reducers. In anaerobic mineral media supplemented with acetate and trichloroacetate, its doubling time was 6 h. Alternative electron donor and acceptors were acetoin and sulfur or fumarate, respectively. Trichloroacetate dehalogenation activity was constitutively present, and the dechlorination product was dichloroacetate and chloride. Trichloroacetate conversion seemed to be coupled to a novel sulfur-sulfide redox cycle, which shuttled electrons from acetate oxidation to trichloroacetate reduction. In view of its unique physiological characteristics, the name Trichlorobacter thiogenes is suggested for strain K1.     

Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation

We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates.     

Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in Acinetobacter sp. Strain 4-CB1

[This corrects the article on p. 1386 in vol. 58.].     

Enzymic Dehalogenation of 4-Chlorobenzoyl Coenzyme A in Acinetobacter sp. Strain 4-CB1

4-Chlorobenzoate degradation in cell extracts of Acinetobacter sp. strain 4-CB1 occurs by initial synthesis of 4-chlorobenzoyl coenzyme A (4-chlorobenzoyl CoA) from 4-chlorobenzoate, CoA, and ATP. 4-Chlorobenzoyl CoA is dehalogenated to 4-hydroxybenzoyl CoA. Following the dehalogenation reaction, 4-hydroxybenzoyl CoA is hydrolyzed to 4-hydroxybenzoate and CoA. Possible roles for the CoA moiety in the dehalogenation reaction are discussed.     

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Identification of an antifreeze lipoprotein from Moraxella sp. of Antarctic origin

We found six bacteria capable of producing antifreeze protein (AFP) from Ross Island, Antarctica. Among these AFP-producing bacteria, strain No. 82 had the highest antifreeze activity and was identified as Moraxella sp. The optimum temperature and pH for the production of AFP were 5 degrees C and 7.0, respectively. After partially purifying the AFP from the culture supernatant using 60% saturation of ammonium sulfate, only the 52-kDa protein band (100 microg/ml) which eluted from SDS-PAGE indicated antifreeze activity by the formation of hexagonal crystals. Furthermore, we confirmed that this AFP was a lipoprotein by the lipid stain test and treatment with some enzymes and that it had no ice-nucleating activity. Also, the N-terminal amino acid sequence of this AFP had high similarity with that of outer membrane proteins from Moraxella (Branhamella) catarrhalis. This is the first report of AFP-producing bacteria in Antarctica and an antifreeze lipoprotein (AFLP) from Moraxella sp.     

Dehalogenation of a mixture of chloroaromatics by immobilized Pseudomonas sp. US1 ex cells

Summary Pseudomonas sp. US1 ex entrapped in calcium alginate could dehalogenate a mixture of isomeric monochlorobenzoates and 2,4-dichlorophenoxyacetic acid. Rates of dehalogenation by the immobilized cells were found to be comparable to those of free cells. Conditions for optimum dehalogenation of chloroaromatics by immobilized cells and their reusability were investigated. Preliminary attempts were made to set up a continuous system for dehalogenation of chloroaromatics using a fluidized bed column reactor.Offprint requests to: V. Modi     

Novel transformations of i-cholesterol and 6 beta-methoxy-i-cholesterol by Moraxella sp

A soil microorganism was isolated by the enrichment culture technique using cholesterol as the sole source of carbon. The organism has been identified as belonging to the genus Moraxella. With this organism two novel biotransformations of sterols were observed viz. (1) isomerization of 3 alpha,5 alpha-cyclocholestan-6 beta-ol (i-cholesterol) to cholesterol, (2) demethylation of 6 beta-methoxy-3 alpha,5 alpha-cyclocholestane (6 beta-methoxy-i-cholesterol) to i-cholesterol with subsequent isomerization to cholesterol. The enzymes responsible for these transformations were shown to be inducible. The pH optimum of the partially purified i-cholesterol isomerase was found to be 8.4. The apparent Km value for i-cholesterol was 1.43 microM. A plausible mechanism for the i-cholesterol isomerization has been discussed.     

Occurrence of two different forms of protocatechuate 3,4-dioxygenase in a Moraxella sp.

Two alternative forms of protocatechuate 3,4-dioxygenase (PCase) have been purified from Moraxella sp. strain GU2, a bacterium that is able to grow on guaiacol or various other phenolic compounds as the sole source of carbon and energy. One of these forms (PCase-P) was induced by protocatechuate and had an apparent molecular weight of 220,000. The second form (PCase-G) was induced by guaiacol or other phenolic compounds, such as 2-ethoxyphenol or 4-hydroxybenzoate. It appeared to be smaller (Mr 158,000), and its turnover number was about double that of the former enzyme. Both dioxygenases had similar properties and were built from the association of equal amounts of nonidentical subunits, alpha and beta, which were estimated to have molecular weights of 29,500 and 25,500, respectively. The (alpha beta)3 and (alpha beta)4 structures were suggested for PCases G and P, respectively. On the basis of two-dimensional gel electrophoresis, the alpha and beta polypeptides of PCase-G differed from those of PCase-P. Amino acid analysis supported this conclusion. Both PCases, however, had several other properties in common. It is proposed that both isoenzymes were generated from different sets of alpha and beta subunits, and the significance of these data is discussed.     

Purification and properties of cytochrome P-450 from Moraxella sp

A cytochrome P-450 has been purified to homogeneity from a Moraxella species that is able to grow on guaiacol as the sole source of carbon and energy. The pure cytochrome was a monomeric protein of about 52 kDa, with no catalytic activity towards guaiacol. The difference in mM extinction coefficients between 450 and 490 nm in the CO-difference spectrum was 89.5 mM-1.cm-1. The typical shift of the Soret band from 415 to 390 nm that is attributed to the high-spin state of the cytochrome was observed in the presence of guaiacol and other 2-alkoxyphenols with up to 5 carbons in the side chain. It was also obtained with anisole. The maximum difference in mM extinction coefficients between 390 and 420 nm in the P-450 + ligand minus P-450 spectrum was 65 mM-1.cm-1 in all instances. The dissociation constants of the complexes formed between the pure protein and various O-alkoxyphenols were measured, and ranged from 0.1 microM (guaiacol) to 24 microM (2-butoxyphenol). The dissociation constants were 1 microM for anisole, and over 90 microM for phenol. Catechol induced no spectral change in cytochrome P-450 and appeared to be a weak inhibitor of guaiacol binding. The same spectral shift as induced by guaiacol was observed at high P-450 concentration over 1 microM in the absence of any added ligand and disappeared after dilution. The reduction of pure P-450 by dithionite was immediate, but became very slow, and was complete after 10 min or more at 25 degrees C in the presence of guaiacol. This effect was also obtained with the 2 isomers, 3- and 4-methoxyphenols, and with metyrapone, an inhibitor of guaiacol binding that induced the low-spin state. Preliminary experiments using the crude cell lysate or a reconstructed system with purified P-450 and a protein fraction indicated NADH-dependent guaiacol degradation. This was in agreement with the former hypothesis of Moraxella P-450 acting as a monooxygenase in the demethylation of guaiacol. However, cis, cis-muconate rather than catechol was obtained from the substrate, most likely a consequence of the potent catechol 1,2-dioxygenase activity present in the non-purified protein fractions used.     

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate. Received: 22 March 1996 / Accepted: 8 May 1996     

Plasmid localization and organization of melamine degradation genes in Rhodococcus sp. strain Mel

Rhodococcus sp. strain Mel was isolated from soil by enrichment and grew in minimal medium with melamine as the sole N source with a doubling time of 3.5 h. Stoichiometry studies showed that all six nitrogen atoms of melamine were assimilated. The genome was sequenced by Roche 454 pyrosequencing to 13× coverage, and a 22.3-kb DNA region was found to contain a homolog to the melamine deaminase gene trzA. Mutagenesis studies showed that the cyanuric acid hydrolase and biuret hydrolase genes were clustered together on a different 17.9-kb contig. Curing and gene transfer studies indicated that 4 of 6 genes required for the complete degradation of melamine were located on an ～265-kb self-transmissible linear plasmid (pMel2), but this plasmid was not required for ammeline deamination. The Rhodococcus sp. strain Mel melamine metabolic pathway genes were located in at least three noncontiguous regions of the genome, and the plasmid-borne genes encoding enzymes for melamine metabolism were likely recently acquired.