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Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium.

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.

Base Composition↗

Trichloroethylene biodegradation by a methane-oxidizing bacterium.

Trichloroethylene (TCE), a common groundwater contaminant, is a suspected carcinogen that is highly resistant to aerobic biodegradation. An aerobic, methane-oxidizing bacterium was isolated that degrades TCE in pure culture at concentrations commonly observed in contaminated groundwater. Strain 46-1, a type I methanotrophic bacterium, degraded TCE if grown on methane or methanol, producing CO(2) and water-soluble products. Gas chromatography and C radiotracer techniques were used to determine the rate, methane dependence, and mechanism of TCE biodegradation. TCE biodegradation by strain 46-1 appears to be a cometabolic process that occurs when the organism is actively metabolizing a suitable growth substrate such as methane or methanol. It is proposed that TCE biodegradation by methanotrophs occurs by formation of TCE epoxide, which breaks down spontaneously in water to form dichloroacetic and glyoxylic acids and one-carbon products.

Journal Article↗

Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium.

A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid. This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions. As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry. The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation. The presence of 0.1% yeast extract increased both cell growth and DDV degradation. The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized. Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species. The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme. The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively.

Animals↗

Corky root of lettuce caused by strains of a gram-negative bacterium from muck soils of Florida, new york, and wisconsin.

Slow-growing bacteria similar to the bacterium causing lettuce corky root (CR) in California (strain CA1) were isolated from muck soils of Florida, New York, and Wisconsin, using lettuce seedlings as bait. All strains were tested for reaction with polyclonal antibodies produced against strain CA1 and for pathogenicity on CR-susceptible (Salinas) and CR-resistant (Green Lake) lettuce cultivars in a greenhouse. Five strains from Florida, three from New York, and three from Wisconsin induced severe CR symptoms on Salinas and mild symptoms on Green Lake. All strains were gram-negative, aerobic, oxidase positive, and catalase positive and reduced nitrate to ammonia. Whole-cell fatty acid compositions were similar for all strains and resembled that of Pseudomonas paucimobilis. Since this fatty acid pattern is unique, it is suggested that CR of lettuce is caused by strains of the same bacterium in Florida, New York, Wisconsin, and California.

Journal Article↗

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin.

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.

Journal Article↗

Isolation and characterization of a mo -reducing bacterium.

A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.

Journal Article↗

Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures.

Base Sequence↗

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense.

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot) strain and a motility-deficient (Mot) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.

Journal Article↗

Anaerobic degradation of propionate by a mesophilic acetogenic bacterium in coculture and triculture with different methanogens.

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO(2) in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H(2) plus CO(2) (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.

Journal Article↗

Description of a new polymer-secreting bacterium from a deep-sea hydrothermal vent, Alteromonas macleodii subsp. fijiensis, and preliminary characterization of the polymer.

A deep-sea, aerobic, mesophilic, heterotrophic bacterium was isolated from fluid collected near an active hydrothermal vent. On the basis of phenotypic and phylogenetic analyses and DNA-DNA relatedness, strain ST716 could be assigned to the species Alteromonas macleodii as a new subspecies. This bacterium secreted an unusual high-molecular-weight polysaccharide in the presence of glucose in batch cultures. The viscosity of this exopolysaccharide is of the same order of magnitude as that of xanthan, another bacterial polysaccharide of industrial interest. This polysaccharide, produced during the stationary phase, contained glucose, mannose, pyruvated mannose, and galactose along with galacturonic acid and glucuronic acid.

Base Composition↗

Isolation and characterization of a novel toluene-degrading, sulfate-reducing bacterium.

A novel sulfate-reducing bacterium isolated from fuel-contaminated subsurface soil, strain PRTOL1, mineralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. The mineralization of 80% of toluene carbon to CO2 was demonstrated in experiments with [ring-U-14C]toluene; 15% of toluene carbon was converted to biomass and nonvolatile metabolic by-products, primarily the former. The observed stoichiometric ratio of moles of sulfate consumed per mole of toluene consumed was consistent with the theoretical ratio for mineralization of toluene coupled with the reduction of sulfate to hydrogen sulfide. Strain PRTOL1 also transforms o- and p-xylene to metabolic products when grown with toluene. However, xylene transformation by PRTOL1 is slow relative to toluene degradation and cannot be sustained over time. Stable isotope-labeled substrates were used in conjunction with gas chromatography-mass spectrometry to investigate the by-products of toluene and xylene metabolism. The predominant by-products from toluene, o-xylene, and p-xylene were benzylsuccinic acid, (2-methylbenzyl)succinic acid, and 4-methylbenzoic acid (or p-toluic acid), respectively. Metabolic by-products accounted for nearly all of the o-xylene consumed. Enzyme assays indicated that acetyl coenzyme A oxidation proceeded via the carbon monoxide dehydrogenase pathway. Compared with the only other reported toluene-degrading, sulfate-reducing bacterium, strain PRTOL1 is distinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marine environment.

Acetyl Coenzyme A↗

Purification and characterization of a novel antibacterial protein from the marine bacterium D2.

A biofilm-forming marine bacterium, D2, isolated from the surface of the tunicate Ciona intestinalis, was found to produce a novel, 190-kDa protein with antibacterial activity. The protein contained at least two subunits of 60 and 80 kDa, joined together by noncovalent bonds, and was shown to be released by D2 cells into the surrounding medium during stationary phase. N-terminal sequence analysis revealed no close similarity of this protein to any other proteins within the Swiss Prot database. Bacteriocidal activity against a wide variety of marine and medical bacterial isolates was observed, 77% of the strains tested being sensitive to the protein. Bacterial strains varied in their resistance to the D2 protein, with D2 itself being among the most sensitive with an MBC in liquid suspension of 4 micrograms/ml. An apparent increased resistance of D2 to the protein as the cells progressed further into stationary phase was observed and seen as a possible explanation for its survival despite the production of an autoinhibitory factor. The ability of the D2 bacterium to produce an antibacterial factor in addition to its inhibitory effects on marine invertebrates and algae (S. Egan et al., unpublished data) indicates that D2 has the potential to greatly affect the survival of a range of colonizers of the marine surface environment.

Amino Acid Sequence↗

Novel detoxification of the trichothecene mycotoxin deoxynivalenol by a soil bacterium isolated by enrichment culture.

A mixed microbial culture capable of metabolizing deoxynivalenol was obtained from soil samples by an enrichment culture procedure. A bacterium (strain E3-39) isolated from the enrichment culture completely removed exogenously supplied deoxynivalenol from culture medium after incubation for 1 day. On the basis of morphological, physiological, and phylogenetic studies, strain E3-39 was classified as a bacterium belonging to the Agrobacterium-Rhizobium group. Thin-layer chromatographic analysis indicated the presence of one major and two minor metabolites of deoxynivalenol in ethyl acetate extracts of the E3-39 culture filtrates. The main metabolite was identified as 3-keto-4-deoxynivalenol by mass spectroscopy and 1H and 13C nuclear magnetic resonance analysis. The immunosuppressive toxicity of 3-keto-4-deoxynivalenol was evaluated by means of a bioassay based on the mitogen-induced and mitogen-free proliferations of mouse spleen lymphocytes. This compound exhibited a remarkably decreased (to less than one tenth) immunosuppressive toxicity relative to deoxynivalenol, indicating that the 3-OH group in deoxynivalenol is likely to be involved in exerting its immunosuppressive toxicity. Strain E3-39 was also capable of transforming 3-acetyldeoxynivalenol but not nivalenol and fusarenon-X.

Animals↗

Glycogen biosynthesis via UDP-glucose in the ruminal bacterium Prevotella bryantii B1(4).

Prevotella bryantii is an important amylolytic bacterium in the rumen that produces considerable amounts of glycogen when it is grown on maltose. Radiolabel studies indicated that glucose-1-phosphate was converted to UDP-glucose, and this latter intermediate served as the immediate precursor for glycogen synthesis. High levels of UDP-glucose pyrophosphorylase activities (> 1,492 nmol/min/mg of protein) were detected in cells grown on maltose, cellobiose, glucose, or sucrose, and activity was greatly stimulated (by approximately 60-fold) by the addition of fructose-1,6-bis phosphate (half-maximal activation concentration was 240 microM). However, ADP-glucose pyrophosphorylase activity was not detected in any of the cultures. Glycogen synthase activity in maltose-grown cultures (48 nmol/min/mg of protein) was higher than that in cellobiose-, sucrose-, and glucose-grown cultures (< 26 nmol/min/mg of protein). This is the first report of a bacterium that exclusively uses UDP-glucose to synthesize glycogen. The elucidation of this unique glycogen biosynthesis pathway provides information necessary to further investigate the role of bacterial glycogen accumulation in rumen metabolism.

Allosteric Regulation↗

Elemental sulfur and thiosulfate disproportionation by Desulfocapsa sulfoexigens sp. nov., a new anaerobic bacterium isolated from marine surface sediment.

A mesophilic, anaerobic, gram-negative bacterium, strain SB164P1, was enriched and isolated from oxidized marine surface sediment with elemental sulfur as the sole energy substrate in the presence of ferrihydrite. Elemental sulfur was disproportionated to hydrogen sulfide and sulfate. Growth was observed exclusively in the presence of a hydrogen sulfide scavenger, e.g., ferrihydrite. In the absence of a scavenger, sulfide and sulfate production were observed but no growth occurred. Strain SB164P1 grew also by disproportionation of thiosulfate and sulfite. With thiosulfate, the growth efficiency was higher in ferrihydrite-supplemented media than in media without ferrihydrite. Growth coupled to sulfate reduction was not observed. However, a slight sulfide production occurred in cultures incubated with formate and sulfate. Strain SB164P1 is the first bacterium described that grows chemolithoautotrophically exclusively by the disproportionation of inorganic sulfur compounds. Comparative 16S rDNA sequencing analysis placed strain SB164P1 into the delta subclass of the class Proteobacteria. Its closest relative is Desulfocapsa thiozymogenes, and slightly more distantly related are Desulfofustis glycolicus and Desulforhopalus vacuolatus. This phylogenetic cluster of organisms, together with members of the genus Desulfobulbus, forms one of the main lines of descent within the delta subclass of the Proteobacteria. Due to the common phenotypic characteristics and the phylogenetic relatedness to Desulfocapsa thiozymogenes, we propose that strain SB164P1 be designated the type strain of Desulfocapsa sulfoexigens sp. nov.

Base Composition↗

Sequencing and characterization of the xyl operon of a gram-positive bacterium, Tetragenococcus halophila.

The xyl operon of a gram-positive bacterium, Tetragenococcus halophila (previously called Pediococcus halophilus), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, and xylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally in Escherichia coli. XylE transported D-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, and gnt operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon in T. halophila.

Amino Acid Sequence↗

Pseudomonas sp. strain 273, an aerobic alpha, omega-dichloroalkaneDegrading bacterium.

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genus Pseudomonas. After induction with 1,10-DCD, Pseudomonas sp. strain 273 released stoichiometric amounts of chloride from C5 to C12 alpha, omega-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C9 to C12 chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degrading Pseudomonas species did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts of Pseudomonas sp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.

Aerobiosis↗

Isolation from the Sorghum bicolor mycorrhizosphere of a bacterium compatible with arbuscular mycorrhiza development and antagonistic towards soilborne fungal pathogens.

A gram-positive bacterium with antagonistic activity towards soilborne fungal pathogens has been isolated from the mycorrhizosphere of Sorghum bicolor inoculated with Glomus mosseae. It has been identified as Paenibacillus sp. strain B2 based on its analytical profile index and on 16S ribosomal DNA analysis. Besides having antagonistic activity, this bacterium stimulates mycorrhization.

Base Sequence↗