Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization

Two electrophoretically different forms of superoxide dismutase, one of them containing manganese-protein and the other iron-protein, were detected in eleven different strains of the genus Flavobacterium. The activities of the different strains were similar to those described for other bacteria. The two molecular forms of the enzyme differed clearly with regard to activity, electrophoretic behaviour, sensitivity to cyanide and peroxide, and NaCl requirement. Both molecular forms were isolated from Flavobacterium halmephilum. Molecular mass absorption spectra, metal content, optimum pH, heat-sensitivity and stability were described.     

Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium

Nitrite reductase [nitric-oxide : (acceptor) oxidoreductase,EC 1.7.2.1 [EC] ] from a denitrifying phototrophic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans, was purified. The molecularweight of the enzyme, estimated by gel-filtration, was 80,000.Sodium dodecyl sulfate polyacrylamide gel electrophoresis ofthe purified enzyme showed a single 39,000 molecular weightband, indicating that the enzyme was composed of two subunitsof identical molecular weight. The oxidized form of the enzymeexhibited maximum absorption at 280 nm, 450 nm and 590 nm, andthe reduced form only at 280 nm. The ESR spectrum of a frozensolution of the oxidized enzyme showed a typical spectrum patternof a copper protein, suggesting that two types of Cu²⁺ existedwithin the enzyme. Estimates with an atomic absorption spectrophotometer,revealed two copper atoms per molecule. The optimum pH of theenzyme was 7.0. Km for nitrite was estimated to be 51 µM,and the optimum temperature, 30?C. The enzyme was inhibitedby CO, potassium cyanide and diethyldithiocarbamate and activatedby monoiodoacetate. Phenazine methosulfate, 2,6-dichlorophenolindophenol,horse heart cytochrome c, and cytochrome c₂ from this bacteriumwere suitable electron donors. The enzyme also showed cytochromec oxidase activity. (Received May 4, 1978; )     

Identification and characterization of a pigment-producing denitrifying bacterium

Herein, a denitrifying bacterium that produced greenish fluorescent pigment under aerobic conditions was accidentally isolated from municipal sewage sludge. Using 16S-rDNA sequence analysis, we identified the isolate as Pseudomonas aeruginosa R12, with 100% similarity. We achieved the highest pigment production rate (1.36 mg/L/h) in a 1-L bioreactor under aerobic conditions, using the optimal culture parameters determined in this study: 37°C, pH 8.0, 200 rpm, 5 wm aeration, and medium containing succinate and (NH₄)₂SO₄. The pigment was not a secondary metabolite and had no antibacterial activity on its co-isolates. Under anaerobic conditions, the isolate produced mainly N₂ and behaved as a strong denitrifier, displaying synergistic denitrification with co-isolated denitrifiers. To our knowledge, herein we have described the first instance in which P. aeruginosa R12 produces a fluorescent pigment under aerobic conditions. This newly-isolated strain therefore shows potential as a commercial resource for natural pigment.     

The anaerobic degradation of aromatic compounds by a denitrifying bacterium

Summary Six strains of Rhizobium, present as bacteroids, in Lotus nodules were studied by electron microscopy. Three inclusion bodies frequently detected are described and their distribution among the strains is given. Cytochemical techniques indicated that they have, as principal components, polyphosphate, lipid and neutral polysaccharide, probably glycogen, respectively.     

Metabolism of monofluoro- and monochlorobenzoates by a denitrifying bacterium

Monofluoro- and monochlorobenzoates did not support the growth of Pseudomonas PN-1, either aerobically or anaerobically (nitrate respiration), when supplied as sole sources of carbon and energy. Anaerobic growth yields on nonfluorinated substrates were increased by p-fluorobenzoate (pFBz) with a utilization of pFBz and release of F^-. Cell suspensions grown on p-hydroxybenzoate (pOHBz), either aerobically or anaerobically, only degraded o-fluorobenzoate (oFBz) and pFBz of the monohalogenated benzoates tested. Both compounds were catabolized anaerobically, but not aerobically, with a release of F^-. oFBz was immediately attacked, by cells grown anaerobically on pOHBz, whereas pFBz was only degraded after a lag phase; chloramphenicol inhibited the breakdown of pFBz, but not oFBz, thereby indicating the need for additional enzyme(s) to attack pFBz. o-Chlorobenzoate (oClBz) inhibited the anaerobic, but not aerobic, oxidation of pOHBz and stopped anaerobic growth on pOHBz. A mutant was isolated which metabolized pOHBz in the presence of oClBz but it was defective in its anaerobic metabolism of benzoate (Bz). Comparative studies, of the mutant and Pseudomonas PN-1, indicated that the mutation involved a metabolic site common to Bz, oClBz and the monofluorobenzoates. The dependence of the oxidation rate of Bz and oFBz on their concentrations at a millimolar level, in the mutant but not Pseudomonas PN-1, suggested a defect at the permease level: the uptake of ¹⁴C-labelled Bz by the mutant was also concentration-dependent. The response of the organism to the inhibitory effect of oClBz on pOHBz catabolism is discussed with respect to its significance in the perturbation of natural degradative processes by unnatural chemicals in the environment.Non-common abbreviations Bz benzoate - pOHBz p-hydroxybenzoate - oFBz o-fluorobenzoate - mFBz m-fluorobenzoate - pFBz p-fluorobenzoate - oClBz o-chlorobenzoate     

Anaerobic oxidation of p-cresol by a denitrifying bacterium

Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07. The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions. The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation. Nitrate is required as the external electron acceptor and is reduced to molecular N2. Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor. In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates. The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation. The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments.     

Anaerobic degradation of toluene by a denitrifying bacterium

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

The isolation and characterization of a rumen chitinolytic bacterium

J.S. DICKSON, T.R. MANKE, i.v. WESLEY AND A.L. BAETZ. 1996. Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm² tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log₁₀ cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus , which was non-culturable in broth culture, attained population densities of 10⁹ cells ml^-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm² resulted in increased cell densities.     

Anaerobic oxidation of p-cresol by a denitrifying bacterium.

Metabolism of p-cresol (pCr) under nitrate-reducing conditions is mediated by the denitrifying bacterial isolate PC-07. The methyl substituent of the substrate is oxidized anaerobically by whole-cell suspensions of PC-07 through a series of dehydrogenation and hydration reactions to yield p-hydroxybenzoate (pOHB) in stoichiometric proportions. The partially oxidized intermediates in the pathway p-hydroxybenzyl alcohol and p-hydroxybenzaldehyde can also serve as substrates for pOHB formation. Nitrate is required as the external electron acceptor and is reduced to molecular N2. Reduction of the nitrate is stoichiometric, with pCr serving as the electron donor. In addition, the molar relationship between the electron acceptor (NO3-) reduced to the electron donor oxidized decreased to approximately 2:3 and then to 1:3 when p-hydroxybenzyl alcohol or p-hydroxybenzaldehyde, respectively, served as substrates. The decreased ratios were to be expected when the partially oxidized intermediates served as substrates, because they provided correspondingly less reducing power for pOHB formation. The anaerobic oxidation of pCr by PC-07 demonstrates a mechanism whereby aromatic compounds can be transformed in anoxic environments.     

Anaerobic degradation of 3-halobenzoates by a denitrifying bacterium

A denitrifying bacterium was isolated from a river sediment after enrichment on 3-chlorobenzoate under anoxic, denitrifying conditions. The bacterium, designated strain 3CB-1, degraded 3-chlorobenzoate, 3-bromobenzoate, and 3-iodobenzoate with stoichiometric release of halide under conditions supporting anaerobic growth by denitrification. The 3-halobenzoates and 3-hydroxybenzoate were used as growth substrates with nitrate as the terminal electron acceptor. The doubling time when growing on 3-halobenzoates ranged from 18 to 25 h. On agar plates with 1 mM 3-chlorobenzoate as the sole carbon source and 30 mM nitrate as the electron acceptor, strain 3CB-1 formed small colonies (1–2 mm in diameter) in 2 to 3 weeks. Anaerobic degradation of both 3-chlorobenzoate and 3-hydroxybenzoate was dependent on nitrate as an electron acceptor and resulted in nitrate reduction corresponding to the stoichiometric values for complete oxidation of the substrate to CO₂. 3-Chlorobenzoate was not degraded in the presence of oxygen. 3-Bromobenzoate and 3-iodobenzoate were also degraded under denitrifying conditions with stoichiometric release of halide, but 3-fluorobenzoate was not utilized by the bacterium. Utilization of 3-chlorobenzoate was inducible, while synthesis of enzymes for 3-hydroxybenzoate degradation was constitutively low, but inducible. Degradation was specific to the position of the halogen substituent, and strain 3CB-1 did not utilize 2- or 4-chlorobenzoate. Received: 6 November 1998 / Accepted: 19 January 1999     

Anaerobic degradation of toluene by a denitrifying bacterium.

A denitrifying bacterium, designated strain T1, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain T1. Greater than 50% of the toluene carbon was oxidized to CO2, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain T1 was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 mumol min-1 liter-1 (56 nmol min-1 mg of protein-1) in a cell suspension. Strain T1 was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain T1. This transformation was dependent on the degradation of toluene.     

Growth yield of a denitrifying bacterium, Pseudomonas denitrificans, under aerobic and denitrifying conditions.

The effciency of denitrification, or anaerobic respiration, in Pseudomonas denitrificans was investigated, using growth yield as an index. Glutamate was mainly used as the sole source of energy and carbon. In batch culture, the growth yield per mole of electrons transported through the respiratory system under denitrifying conditions was about half that under aerobic conditions. Similar figures were also obtained in chemostat cultures under glutamate-limited conditions. The decrease in growth yield under denitrifying conditions could be due to the restriction of phosphorylation associated with nitrate reduction to nitrogen gas.     

Population structure of the fish-pathogenic bacterium Flavobacterium psychrophilum

Flavobacterium psychrophilum is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide, and its control mainly relies on antibiotic treatments. To better understand the population structure of this bacterium and its mode of evolution, we have examined the nucleotide polymorphisms at 11 protein-coding loci of the core genome in a set of 50 isolates. These isolates were selected to represent the broadest possible diversity, originating from 10 different host fish species and four continents. The nucleotide diversity between pairs of sequences amounted to fewer than four differences per kilobase on average, corresponding to a particularly low level of diversity, possibly indicative of a small effective-population size. The recombination rate, however, seemed remarkably high, and as a consequence, most of the isolates harbored unique combinations of alleles (33 distinct sequence types were resolved). The analysis also showed the existence of several clonal complexes with worldwide geographic distribution but marked association with particular fish species. Such an association could reflect preferential routes of transmission and/or adaptive niche specialization. The analysis provided no clues that the initial range of the bacterium was originally limited to North America. Instead, the historical record of the expansion of the pathogen may reflect the spread of a few clonal complexes. As a resource for future epidemiological surveys, a multilocus sequence typing website based on seven highly informative loci is available.     

Microbial metabolism of haloaromatics: isolation and properties of a chlorobenzene-degrading bacterium.

A chlorobenzene-degrading bacterium was isolated by continuous enrichment from a mixture of soil and sewage samples. This organism, strain WR1306, was grown in a chemostat on a mineral medium with chlorobenzene being supplied through the vapor phase with a critical Dc value at a dilution rate of 0.55 h-1. Maximum growth rates in batch culture were accomplished at substrate concentrations of less than or equal to 0.5 mM in the culture medium. During growth on chlorobenzene, stoichiometric amounts of chloride were released. Respiration data and enzyme activities in cell extracts as well as the isolation of 3-chlorocatechol from the culture fluid are consistent with the degradation of chlorobenzene via 3-chloro-cis-1,2-dihydroxycyclohexa-3,5-diene, 3-chlorocatechol, 2-chloro-cis,cis-muconate, trans-4-carboxymethylenebut-2-en-4-olide, maleylacetate, and 3-oxoadipate.     

The isolation and preliminary characterization of a halophilic photosynthetic bacterium

Summary The isolation of a photosynthetic bacterium which is apparently an obligate halophile is reported. The organism (strain SL-1) grows in a medium with either thiosulfate of sulfide and bicarbonate containing 22% NaCl but not in one containing 4% NaCl. The morphology is abnormal in media with 8% NaCl. Strain SL-1 is an obligate anaerobe. The major carotenoid pigment is spirilloxanthin. The spectrum of the chlorophyllous pigment is very similar to that of bacteriochlorophyll; however, its identity with bacteriochlorophyll has not been demonstrated.Strain SL-1 is a spirillum. Cells possess a single, polar, sheathed flagellum. The ultrastructure of the cells is depicted.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday.Supported in part by a grant from the National Science Foundation.     

Anaerobic oxidation of 1-n-heptadecene by a marine denitrifying bacterium

Summary A denitrifying bacterium showing typical characteristics of Pseudomonas sp. (Al1) capable of growth on 1-heptadecene as the sole source of carbon and energy has been isolated from a hydrocarbon-polluted marine sediment by using classical enrichment techniques. The generation time for anaerobic growth on 1-heptadecene was 24 h, and the percentage of hydrocarbon degradation under anaerobic conditions ranged from 19 to 23%. The emulsifying capacity was observed and suggested that Al1 cultivated anaerobically on heptadecene produced surface-active agents. Offprint requests to: M. Gilewicz     

Light-activated,-inhibited and-independent denitrification by a denitrifying phototrophic bacterium

Effects of illumination on denitrification by a freshly isolated denitrifying phototrophic bacterium were investigated. Denitrification activity was induced when cells were grown in either light or darkness in the presence of nitrate without oxygen. Denitrification of nitrate with malate as the electron donor by cells at a phase of exponential growth occurred independently of illumination while that by cells in a stationary phase was activated. Effects of illumination on denitrification varied with electron donors. Using malate or succinate, denitrification by cells in a stationary phase was accelerated by illumination, inhibited when glucose or lactate was used, and independent of illumination when pyruvate was used. Denitrification by cells in an exponential phase was independent of illumination when succinate, malate or pyruvate was used and inhibited by it when glucose or lactate was used. Effects of illumination on the denitrification of nitrite were similar to those involving nitrate. Effects of various inhibitors on denitrification were examined in light-succinate and dark-lactate systems. Differences between the two systems are discussed.Abbreviations KCN potassium cyanide - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TTFA 2-thenoyltrifluoroacetone - CCCP carbonyl cyanide-m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996