Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract.

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1-nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.     

Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1-nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.     

Identification, Isolation and characterization of a novel azoreductase from Clostridium perfringens

Azo dyes are used widely in the textile, pharmaceutical, cosmetic and food industries as colorants and are often sources of environmental pollution. There are many microorganisms that are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced thereby a concern for human health. The field of research on azoreductases is growing, but there is very little information available on azoreductases from strict anaerobic bacteria. In this study, the azoreductase gene was identified in Clostridium perfringens, a pathogen that is commonly found in the human intestinal tract. C. perfringens shows high azoreductase activity, especially in the presence of the common dye Direct Blue 15. A gene that encodes for a flavoprotein was isolated and expressed in Escherichia coli, and further purified and tested for azoreductase activity. The azoreductase (known as AzoC) was characterized by enzymatic reaction assays using different dyes. AzoC activity was highest in the presence of two cofactors, NADH and FAD. A strong cofactor effect was shown with some dyes, as dye reduction occurred without the presence of the AzoC (cofactors alone). AzoC was shown to perform best at a pH of 9, at room temperature, and in an anaerobic environment. Enzyme kinetics studies suggested that the association between enzyme and substrate is strong. Our results show that AzoC from C. perfringens has azoreductase activity.     

Antagonism among the normal anaerobic bacteria of the mouse gastrointestinal tract determined by immunofluorescence.

Strictly anaerobic Bacteroides sp., Eubacterium sp., and Fusobacterium sp. were isolated from the cecum of a conventional mouse. An immunofluorescent method utilizing rabbit antisera specific for each of these three strains was developed to determine their population levels in the gastrointestinal tracts of gnotobiotic mice. Population levels of these anaerobes in groups of gnotobiotic mice colonized with either Bacteroides, Eubacterium, or Fusobacterium were compared with those of gnotobiotes colonized with all three strains. Bacteroides population levels in gnotobiotes colonized with all three strains were 100-fold less than the Bacteroides population level in gnotobiotes colonized with only the Bacteroides strain. Eubacterium or Fusobacterium population levels were not reduced by the presence of the other anaerobic strains. Thus, strictly anaerobic Eubacterium sp. and Fusobacterium sp. that colonized gnotobiotic mice caused a reduction in the in vivo population levels of a strictly anaerobic Bacteroides sp.     

Antagonism among the normal anaerobic bacteria of the mouse gastrointestinal tract determined by immunofluorescence.

Strictly anaerobic Bacteroides sp., Eubacterium sp., and Fusobacterium sp. were isolated from the cecum of a conventional mouse. An immunofluorescent method utilizing rabbit antisera specific for each of these three strains was developed to determine their population levels in the gastrointestinal tracts of gnotobiotic mice. Population levels of these anaerobes in groups of gnotobiotic mice colonized with either Bacteroides, Eubacterium, or Fusobacterium were compared with those of gnotobiotes colonized with all three strains. Bacteroides population levels in gnotobiotes colonized with all three strains were 100-fold less than the Bacteroides population level in gnotobiotes colonized with only the Bacteroides strain. Eubacterium or Fusobacterium population levels were not reduced by the presence of the other anaerobic strains. Thus, strictly anaerobic Eubacterium sp. and Fusobacterium sp. that colonized gnotobiotic mice caused a reduction in the in vivo population levels of a strictly anaerobic Bacteroides sp.     

Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria.

Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spectra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme of E. acidaminophilum, which exhibited by itself a dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346-1354, 1989), the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring flavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the dihydrolipoamide dehydrogenases purified from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to dihydrolipoamide dehydrogenases known from other organisms.     

Coprobacillus catenaformis gen. nov., sp. nov., a new genus and species isolated from human feces

Three strains of Eubacterium-like isolates from human feces were characterized by biochemical tests and 16S rDNA analysis. The phenotypic characteristics of the three strains resembled those of the genus Collinsella transferred from the genus Eubacterium recently. However, Eubacterium-like strains were phylogenetically members of the Clostridium subphylum of gram-positive bacteria, and these showed a specific phylogenetic association with Clostridium ramosum and C. spiroforme. C. ramosum and C. spiroforme are gram-positive, anaerobic, spore-forming bacteria that belong to the genus Clostridium, and the G + C contents are 26.0 and 27.4 mol%, respectively. However, the three Eubacterium-like strains had G + C contents of 32.1 to 33.1 mol% and were non-spore-forming rods. Based on phenotypic characteristics, we can differentiate these species, and furthermore, a 16S rDNA sequence divergence of greater than 9% with a new related genus, Coprobacillus, is proposed for the three strains, with one species, Coprobacillus catenaformis. The type strain of C. catenaformis is JCM 10604T.     

Diversity of cellulolytic bacteria in landfill

Nine of 37 cellulolytic bacterial isolates obtained from landfill waste could be easily differentiated on the basis of gross morphological characteristics. Four isolates were selected for further characterization and on the basis of initial results appear to be previously unidentified cellulolytic species of bacteria. An aerotolerant anaerobic, cellulolytic Clostridium and three obligately anaerobic cellulolytic Eubacterium isolates are described. The Clostridium has an unusually high pH optimum for growth of 7.7. The optimum temperature for growth is 50°C. The pH growth optimum of each of the Eubacterium isolates is around pH 7.0 while temperature optima are 37° 45° and 50°C for LFI, LF4 and LF5 respectively. Most isolates had growth optima in the thermophilic range. The ease with which apparently previously unidentified species could be isolated is a reflection of the unique and highly variable, heterogeneous environment within landfill waste.     

Anaerobic ureolytic bacteria from caecal content and soft faeces of rabbit

Forty strains of ureolytic bacteria were isolated from the caecal content and soft faeces of seven rabbits by the anaerobic roll tube method and were characterized. The isolates were identified with Clostridium coccoides, Cl. innocuum, Peptostreptococcus productus, P. micros, Peptococcus magnus, Fusobacterium russii and Fusobacterium sp. Urease activity of representative strains of the various species was also determined. The study indicated that strongly-ureolytic anaerobic bacteria are present in the caecum of the rabbit.     

Chitinolytic bacteria in the minke whale forestomach

Minke whales consume large amounts of pelagic crustaceans. Digestion of the prey is initiated by indigenous bacteria in a rumen-like forestomach system. A major structural component of the crustacean exoskeleton is chitin, the beta-1,4-linked polymer of N-acetyl-D-glucosamine. The exoskeletons appear to dissolve completely in the non-glandular forestomach. Bacteria in the forestomach fluid of six krill-eating minke whales were enumerated and isolated using an anaerobic habitat-simulating culture medium. Median viable population densities ranged between 6.0 x 10(6) and 9.9 x 10(9) bacterial cells per mL forestomach fluid. Bacterial isolates (n = 44) cultured from the forestomach fluid of one minke whale mainly resembled strains of Eubacterium (25%), Streptococcus (18%), Clostridium (14%), and Bacteroides (11%). As much as 12% of the bacterial isolates were chitinolytic, while beta-N-acetylglucosaminidase activity was demonstrated in 54% of the isolates, and utilisation of N-acetyl-D-glucosamine was observed in 73%. The chitinolytic isolates resembled strains of Bacteroides, Bacteroidaceae, Clostridium, and Streptococcus. Scanning and transmission electron microscopy of partly digested krill from the minke whale forestomach revealed bacteria close to and inside the chitinous exoskeleton. The bacterial chitinase may act on the chitinous crustacean exoskeletons, thereby allowing other bacteria access to the nutritious soft inner tissues of the prey, and thus initiating its degradation and fermentation.     

Anaerobic ureolytic bacteria from caecal content and soft faeces of rabbit

Forty strains of ureolytic bacteria were isolated from the caecal content and soft faeces of seven rabbits by the anaerobic roll tube method and were characterized. The isolates were identified with Clostridium coccoides, Cl. innocuum, Peptostreptococcus productus, P. micros, Peptococcus magnus, Fusobacterium russii and Fusobacterium sp. Urease activity of representative strains of the various species was also determined. The study indicated that strongly-ureolytic anaerobic bacteria are present in the caecum of the rabbit.     

Characterisation of the flavin-free oxygen-tolerant azoreductase from Xenophilus azovorans KF46F in comparison to flavin-containing azoreductases

The flavin-free azoreductase from Xenophilus azovorans KF46F (AzoB), which has been the very first characterized oxygen-tolerant azoreductase, was analyzed in comparison to various recently described flavin-containing azoreductases from different bacterial sources. Sequence comparisons demonstrated that the azoreductase from X. azovorans KF46F is a member of the NmrA family of proteins and demonstrates 30% sequence identity with a NADPH-dependent quinone oxidoreductase from Escherichia coli (encoded by ytfG). In contrast, it was found that the flavin-containing azoreductases from E. coli OY1-2 (AZR), Bacillus sp. OY1-2 (AZR) and related azoreductases all belong to the FMN_red superfamily of enzymes. The substrate specificity of AzoB was reanalyzed in respect to the recently characterized flavin-containing azoreductases, and it was found that purified AzoB converted in addition to different ortho-hydroxy azo compounds [such as Orange II = 1-(4′-sulfophenylazo)-2-naphthol] also the simple non-hydroxylated non-sulfonated azo dye Methyl Red (4′-dimethylaminoazobenzene-2-carboxylic acid), but no indications for the conversion of quinones were obtained. Significant differences were observed in the substrate specificities between AzoB and the flavin-containing azoreductases. The kinetic analysis of the turn-over of Orange II by AzoB suggested an ordered bireactant reaction mechanism which was different from the ping-pong mechanism suggested for the flavin-containing azoreductases.     

Comparison of the azoreductase and nitroreductase from Clostridium perfringens.

The purified azoreductase and nitroreductase of Clostridium perfringens, which have similar electrophoretic properties, both reacted in a Western blot (immunoblot) with a polyclonal antibody raised against the azoreductase. The activity of both enzymes was enhanced by flavin adenine dinucleotide and was inhibited by menadione, o-iodosobenzoic acid, and the antibody against azoreductase. Reduction of the azo dye Direct Blue 15 by the azoreductase was inhibited by nitroaromatic compounds. The apparent Km of the enzyme for reduction of Direct Blue 15 in the presence of 1-nitropyrene was higher than the Km with the azo dye alone, demonstrating competitive inhibition. The data show that the same protein is involved in the reduction of both azo dyes and nitroaromatic compounds.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

The Occurence and Properties of Uric Acid Decomposing Anaerobic Bacteria in the Avian Caecum

S ummary . Anaerobic bacteria which decompose uric acid have been found in the caeca of chickens, turkeys, ducks, pheasants and guinea-fowl at levels between 5.4 × 10⁸ and 1.8 × 10¹⁰/g of caecal material (wet weight). A study has been made of the properties and identity of 35 strains which were present in high numbers in the chicken caecum. The isolates included strains of Bacteroides clostridiiformis var. clostridiiformis, Fusobacterium plauti, Clostridium malenominatum, Peptostreptococcus productus and a number of types which could not be identified with known species. Of these 3 were Bacteroides spp., 3 Eubacterium spp., 2 Peptostreptococcus spp. and 1 was an anaerobic Streptococcus . None of the strains had an absolute requirement for uric acid.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.

A growth medium and test were developed for rapid detection of urease in fermentative anaerobic bacteria. Using nonselective rumen fluid roll-tube agar medium and the new test, it was confirmed that Peptostreptococcus productus is often the most numerous urease-forming species in human feces. Also, some fecal strains of Ruminococcus albus, Clostridium innocuum, and Clostridium beijerinckii produced urease. Single strains of Fusobacterium prausnitzii, Coprococcus catus, and Streptococcus mitis that were strongly ureolytic on isolation later lost this ability. Urease activity was also detected in many strains of nonselectively isolated rumen species. They include Succinivibrio dextrinosolvens, Treponema sp., Ruminococcus bromii (not previously known to be present in the rumen), Butyrivibrio sp., Bifidobacterium sp., Bacteroides ruminicola, and P. productus. Most P. productus strains contain urease; however, the uniformity of this feature in the other species noted above is not known. The urease in many of these species was not detected if the growth medium contained 0.2% or more (each) yeast extract and Trypticase.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

Properties of a cellulolytic Sporolactobacillus and some non-sporing cellulolytic rods, presumptive clostridia, from an anaerobic digester

Anaerobic digesters contain a wide variety of anaerobic cellulolytic bacteria. Many of these are sporing rods. Some isolates from a mesophilic cattle waste anaerobic digester were classified as Sporolactobacillus spp. A further group of bacteria could not be induced to sporulate. In some respects they resembled Eubacterium , but considering all the properties it is suggested that they should be classified as a species of Clostridium.