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Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Bacterial Proteins↗

Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant.

'Sphingomonas (formerly Pseudomonas) elodea' produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from 'S. elodea' and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.

Amino Acid Sequence↗

Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26.

Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18-20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 8.9. The crystals diffract to at least 1.60 A using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 A. The asymmetric unit contains one molecule of the enzyme.

Crystallization↗

Use of monoclonal antibodies against dibenzo-p-dioxin degrading Sphingomonas sp. strain RW1.

A monoclonal antibody prepared against surface antigen of Sphingomonas sp. strain RW1 was used for the direct detection of RW1-like organisms in environmental samples by epifluorescence microscopy and subsequent confirmation by Western blot. Of the 76 samples collected from various sources and probed using epifluorescence, only one sample, effluent from paper and pulp processing, gave a positive result. The effluent was cultured and yielded an organism which, by Western blot analysis, was shown to contain the 28 kDa protein recognized by the monoclonal antibody.

Animals↗

Evidence for the role of 2-hydroxychromene-2-carboxylate isomerase in the degradation of anthracene by Sphingomonas yanoikuyae B1.

Sphingomonas yanoikuyae B1 is extremely versatile in its catabolic ability. An insertional mutant strain, S. yamoikuyae EK504, which is unable to grow on naphthalene due to the loss of 2-hydroxychromene-2-carboxylate isomerase activity, was utilized to investigate the role of this enzyme in the degradation of anthracene by S. yanoikuyae B1. Although EK504 is unable to grow on anthracene, this strain could transform anthracene to some extent. A metabolite in the degradation of anthracene by EK504 was isolated by high-pressure liquid chromatography (HPLC) and was identified as 6,7-benzocoumarin by UV-visible, gas-chromatographic, HPLC/mass-spectrometric, and 1H nuclear magnetic resonance spectral techniques. The identification of 6,7-benzocoumarin provides direct chemical and genetic evidence for the involvement of nahD in the degradation of anthracene by S. yanoikuyae B1.

Anthracenes↗

The role of the Sphingomonas species UG30 pentachlorophenol-4-monooxygenase in p-nitrophenol degradation.

Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.

Biodegradation, Environmental↗

Biodegradation of diphenyl ether and its monohalogenated derivatives by Sphingomonas sp. strain SS3.

The bacterium Sphingomonas sp. strain SS3, which utilizes diphenyl ether and its 4-fluoro, 4-chloro, and (to a considerably lesser extent) 4-bromo derivatives as sole sources of carbon and energy, was enriched from soil samples of an industrial waste deposit. The bacterium showed cometabolic activities toward all other isomeric monohalogenated diphenyl ethers. During diphenyl ether degradation in batch culture experiments, phenol and catechol were produced as intermediates which were then channeled into the 3-oxoadipate pathway. The initial step in the degradation follows the recently discovered mechanism of 1,2-dioxygenation, which yields unstable phenolic hemiacetals from diphenyl ether structures. Oxidation of the structure-related dibenzo-p-dioxin yielded 2-(2-hydroxyphenoxy)-muconate upon ortho cleavage of the intermediate 2,2',3-trihydroxydiphenyl ether. Formation of phenol, catechol, halophenol, and halocatechol from the conversion of monohalogenated diphenyl ethers gives evidence for a nonspecific attack of the dioxygenating enzyme system.

Adipates↗

Biodegradation and transformation of 4,4'- and 2,4-dihalodiphenyl ethers by Sphingomonas sp. strain SS33.

The bacterium Sphingomonas sp. strain SS33, obtained from parent diphenyl ether-mineralizing strain SS3 (S. Schmidt, R.-M. Wittich, D. Erdmann, H. Wilkes, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:2744-2750, 1992) after several weeks of adaptation on 4,4'-difluorodiphenyl ether as the new target compound, also utilized 4,4'-dichlorodiphenyl ether for growth. Intermediary halocatechols were also mineralized via the ortho pathway by type I enzymes. 4,4'-Dibromodiphenyl ether was not used as a carbon source although transformation by resting cells yielded mononuclear haloaromatic compounds, such as 4-bromophenol and 4-bromocatechol. The same was true for the conversion of 2,4-dichlorodiphenyl ether, which yielded the respective (halo-) phenols and (halo-) catechols.

Bacteria↗

Studies on a Viscous, Gel-Forming Exopolysaccharide from Sphingomonas paucimobilis GS1.

A new strain, Sphingomonas paucimobilis GS1, accumulated 6.5 g of a highly viscous exopolysaccharide per liter, using sucrose as a substrate. The anionic heteropolysaccharide contained the following, in grams per gram: glucose, 0.7; galacturonic acid, 0.11; glucuronic acid, 0.07; and acetate, 0.12. The viscosity of the exopolysaccharide (4.0 g/liter; 4,200 cP) was 5.5 times that of xanthan gum and was stable over a wide pH and temperature range as well as in the presence of NaCl. Deacetylated polymer produced a clear, agarlike, thermoreversible gel in the presence of cations. The gel strength of the modified polymer was four times that of agar and could withstand autoclaving.

Journal Article↗

Metabolism of Hydroxydibenzofurans, Methoxydibenzofurans, Acetoxydibenzofurans, and Nitrodibenzofurans by Sphingomonas sp. Strain HH69.

The metabolism of 11 substituted dibenzofurans by the dibenzofuran-degrading Sphingomonas sp. strain HH69 was investigated. Strain HH69 utilizes 2-, 3-, and 4-acetoxydibenzofuran as well as 2-, 3-, and 4-hydroxydibenzofuran as sole sources of carbon and energy. The degradation of acetoxydibenzofurans is initiated by hydrolysis of the ester bonds, yielding the corresponding hydroxydibenzofurans and acetate. Strain HH69 grew on 2-methoxydibenzofuran only after it was adapted to the utilization of 5-methoxysalicylic acid, whereas 3- and 4-methoxydibenzofuran as well as 2- and 3-nitrodibenzofuran were only cooxidized. During the breakdown of all eight hydroxy-, methoxy-, and nitrodibenzofurans studied here, the corresponding substituted salicylic acids accumulated in the culture broth. In the cases of 2- and 3-hydroxydibenzofuran as well as 2- and 3-nitrodibenzofuran, salicylic acid was also formed. Those four dibenzofurans which did not serve as carbon sources for strain HH69 were converted to a nonutilizable salicylic acid derivative. From turnover experiments with the mutant HH69/II, which is deficient in meta-cleavage, 2,2(prm1),3,4(prm1)-tetrahydroxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-methoxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-nitrobiphenyl, and 2,2(prm1),3-trihydroxy-4(prm1)-nitrobiphenyl were isolated as the main products formed from 3-hydroxydibenzofuran, 2-methoxydibenzofuran, and 2- and 3-nitrodibenzofuran, respectively. These results indicate significant regioselectivity for the dioxygenolytic cleavage of the ether bond of these monosubstituted dibenzofurans, with a preference for the nonsubstituted aromatic nucleus. Substituted trihydroxybiphenyls are converted further by meta-cleavage followed by the removal of the side chain of the resulting product. A stepwise degradation of this side chain was found to be involved in the metabolism of 2-hydroxydibenzofuran.

Journal Article↗

Complete microbial degradation of both enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in an enantioselective manner by Sphingomonas herbicidovorans sp. nov.

Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.

2-Methyl-4-chlorophenoxyacetic Acid↗

Accelerated Mineralization of Pentachlorophenol in Soil upon Inoculation with Mycobacterium chlorophenolicum PCP1 and Sphingomonas chlorophenolica RA2.

Mineralization of pentachlorophenol (PCP) was studied in nonsterile soil from a PCP-contaminated site upon inoculation with two PCP-degrading bacterial strains. At spiked [(sup14)C]PCP concentrations of 30 and 100 mg/kg, the effects of organism type, different inoculation techniques, including structural amendment with sawdust and cell attachment to polyurethane (PU), as well as the effect of different inoculum sizes of 10(sup4) to 10(sup8) cells per g (dry weight) of soil were compared with PCP mineralization by indigenous bacteria. Gas chromatographic analysis was used to monitor PCP disappearance and to check mass balances. The survival and activity of the released bacteria were examined by immunofluorescence microscopy and respiking experiments. Noninoculated soil completely mineralized 30 mg of PCP per kg within 7 months but showed no or only low degradation activity at 100 mg/kg in the same period. Structural amendment with PU or sawdust initiated slow mineralization after half a year. Soil inoculation with Sphingomonas chlorophenolica RA2 shortened the mineralization time drastically to 1 month at 30 mg of PCP per kg using 10(sup8) cells per g, with approximately 80% of the added radioactivity being converted to CO(inf2). The inoculated cells disappeared rapidly, with a count of 2 x 10(sup6) cells per g after 2.3 months and nondetectability after 7 months. At 100 mg/kg, mineralization was slower because of PCP toxicity but approached completion within 7.5 months. The inhibition could be overcome by addition of sawdust (1 g/kg of soil), resulting in a mineralization rate of 3 to 4 mg/kg(middot)d. PU had the opposite effect. Lower inoculum densities resulted in prolonged lag phases and lower rates, although mineralization was still enhanced over the background level. At 30 mg of PCP per kg, inoculation with Mycobacterium chlorophenolicum PCP1 increased mineralization slightly over the indigenous bacterial activity, regardless of inoculum size, but only when the organisms were attached to PU. At 100 mg of PCP per kg, only 27% were mineralized within 7.5 months. After 7 months, the original strain PCP1 inoculum of 10(sup8) cells per g was recovered at 5 x 10(sup6) to 3 x 10(sup7) cells per g, depending on the PCP concentration, but independent of PU amendment. Amendment with sawdust had no effect on the performance of this organism. Possible reasons for the poor performance of this strain include its sensitivity to PCP and its preference for slightly acidic soil conditions.

Journal Article↗

Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains.

The substrate oxidation profiles of Sphingomonas yanoikuyae B1 biphenyl-2,3-dioxygenase and cis-biphenyl dihydrodiol dehydrogenase activities were examined with 1,2-dihydronaphthalene and various cis-diols as substrates. m-Xylene-induced cells of strain B1 oxidized 1,2-dihydronaphthalene to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2-3,4-tetrahydronaphthalene as the major product (73% relative yield). Small amounts of (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (15%), naphthalene (6%), and alpha-tetralone (6%) were also formed. Strain B8/36, which lacks an active cis-biphenyl dihydrodiol dehydrogenase, formed (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene (51%), in addition to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (44%) and (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (5%). The cis-biphenyl dihydrodiol dehydrogenase of strain B1 oxidized both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene, but only the (+)-(1S,2R)-enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene. The results show that biphenyl dioxygenase expressed by S. yanoikuyae catalyzes dioxygenation, monooxygenation, and desaturation reactions with 1,2-dihydronaphthalene as the substrate, and cis-biphenyl dihydrodiol dehydrogenase catalyzes the enantioselective dehydrogenation of (+)-(1S,2R)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (+)-(1S,2R)-cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene.

Naphthalenes↗

Degradation of Chlorinated Dibenzofurans and Dibenzo-p-Dioxins by Sphingomonas sp. Strain RW1.

The ability of the dibenzofuran- and dibenzo-p-dioxin-mineralizing bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) to oxidize chlorinated derivatives of dibenzofuran and dibenzo-p-dioxin was analyzed. Strain RW1 degraded several mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins, but it did not degrade more highly chlorinated congeners. Most mono- and dichlorinated dibenzofurans and dibenzo-p-dioxins investigated in this study were degraded to the corresponding mono- and dichlorinated salicylates and catechols, respectively, together with salicylate and catechol. This indicates an initial dioxygenolytic attack on the substituted as well as on the nonsubstituted aromatic nucleus of most of the target compounds. Strain RW1 could not grow at the expense of monochlorinated dibenzo-p-dioxins and dibenzofurans as carbon sources, with the exception of 4-chlorodibenzofuran, which was stoichiometrically converted to 3-chlorosalicylate.

Journal Article↗

Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas sp. Strain RB2256.

Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay in Alaska and possibly represents the dominant bacterial species in some oligotrophic marine environments. Strain RB2256 has a high-affinity nutrient uptake system when growing under nutrient-limiting conditions, and growing cells are very small (<0.08 (mu)m(sup3)). These characteristics indicate that RB2256 is highly evolved for withstanding nutrient limitations and grazing pressure by heterotrophic nanoflagellates. In this study, strain RB2256 was subjected to nutrient starvation and other stresses (high temperature, ethanol, and hydrogen peroxide). It was found that growing cells were remarkably resistant, being able to survive at a temperature of 56(deg)C, in 25 mM hydrogen peroxide, or in 20% ethanol. In addition, growing cells were generally as resistant as starved cells. The fact that vegetative cells of this strain are inherently resistant to such high levels of stress-inducing agents indicates that they possess stress resistance mechanisms which are different from those of other nondifferentiating bacteria. Only minor changes in cell volume (0.03 to 0.07 (mu)m(sup3)) and maximum specific growth rate (0.13 to 0.16 h(sup-1)) were obtained for cells growing in media with different organic carbon concentrations (0.8 to 800 mg of C per liter). Furthermore, when glucose-limited, chemostat-grown cultures or multiple-nutrient-starved batch cultures were suddenly subjected to excess glucose, maximum growth rates were reached immediately. This immediate response to nutrient upshift suggests that the protein-synthesizing machinery is constitutively regulated. In total, these results are strong evidence that strain RB2256 possesses novel physiological and molecular strategies that allow it to predominant in natural seawater.

Journal Article↗

Mutational analysis of pcpA and its role in pentachlorophenol degradation by Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723.

Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723 degrades pentachlorophenol (PCP) through a catabolic pathway encoded by multiple genes. One gene required for PCP degradation is pcpA, which encodes information for a 30-kDa polypeptide, PcpA, found in the periplasm of the bacterium. The biological role of PcpA has remained unknown. We disrupted pcpA by replacing it with a defective copy through homologous recombination. The pcpA recombinant, mutant strains accumulated 2,6-dichlorohydroquinone (2,6-DiCH) as a metabolite of PCP. This work confirms that pcpA is essential for degradation of PCP by S. chlorophenolica ATCC 39723 and suggests that it encodes a protein involved in hydrolytic dehalogenation of 2,6-DiCH, an already established primary metabolite of the PCP catabolic pathway.

Bacterial Proteins↗

Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. strain CF06.

A bacterial strain (CF06) that mineralized both the carbonyl group and the aromatic ring of the insecticide carbofuran and that is capable of using carbofuran as a sole source of carbon and nitrogen was isolated from a soil in Washington state. Phospholipid fatty acid and 16S rRNA sequencing analysis indicate that CF06 is a Sphingomonas sp. CF06 contains five plasmids, at least some of which are required for metabolism of carbofuran. Loss of the plasmids induced by growth at 42 degrees C resulted in the inability of the cured strain to grow on carbofuran as a sole source of carbon. Introduction of the plasmids confers on Pseudomonas fluorescens M480R the ability to use carbofuran as a sole source of carbon for growth and energy. Of the five plasmids, four are rich in insertion sequence elements and contain large regions of overlap. Rearrangements, deletions, and loss of individual plasmids that resulted in the loss of the carbofuran-degrading phenotype were observed following introduction of Tn5.

Carbofuran↗

Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS.

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.

Journal Article↗