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Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in Escherichia coli O157:H7

Lisle, John T.; Broadaway, Susan C.; Prescott, Annette M.; Pyle, Barry H.; Fricker, Colin; McFeters, Gordon A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1998 EN
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Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

Occurrence of Male-Specific Bacteriophage in Feral and Domestic Animal Wastes, Human Feces, and Human-Associated Wastewaters

Calci, Kevin R.; Burkhardt, William; Watkins, William D.; Rippey, Scott R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1998 EN
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Male-specific bacteriophage (MSB) densities were determined in animal and human fecal wastes to assess their potential impact on aquatic environments. Fecal samples (1,031) from cattle, chickens, dairy cows, dogs, ducks, geese, goats, hogs, horses, seagulls, sheep, and humans as well as 64 sewerage samples were examined for MSB. All animal species were found to harbor MSB, although the great majority excreted these viruses at very low levels. The results from this study demonstrate that in areas affected by both human and animal wastes, wastewater treatment plants are the principal contributors of MSB to fresh, estuarine, and marine waters.

Molecular Epidemiology of Campylobacter jejuni in Broiler Flocks Using Randomly Amplified Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in Its Transmission

Payne, Randy E.; Lee, Margie D.; Dreesen, David W.; Barnhart, Harold M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/1999 EN
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Poultry has long been cited as a reservoir for Campylobacter spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for Campylobacter jejuni. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded C. jejuni in their cecal droppings through 7 weeks. Two successive flocks from the same Campylobacter-positive broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of C. jejuni in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of C. jejuni. These molecular typing methods demonstrate their usefulness...

Effects of Surfactant Mixtures, Including Corexit 9527, on Bacterial Oxidation of Acetate and Alkanes in Crude Oil

Bruheim, Per; Bredholt, Harald; Eimhjellen, Kjell
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/1999 EN
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Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil by Acinetobacter calcoaceticus ATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation by A. calcoaceticus ATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed for A. calcoaceticus ATCC 31012.

Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134

Schenzle, Andreas; Lenke, Hiltrud; Spain, Jim C.; Knackmuss, Hans-Joachim
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/1999 EN
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Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation

Shigematsu, Toru; Hanada, Satoshi; Eguchi, Masahiro; Kamagata, Yoichi; Kanagawa, Takahiro; Kurane, Ryuichiro
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1999 EN
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The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.

Occurrence of Shewanella algae in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival

Gram, Lone; Bundvad, Anemone; Melchiorsen, Jette; Johansen, Charlotte; Fonnesbech Vogel, Birte
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 EN
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The marine bacterium Shewanella algae, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of S. algae decreased rapidly from 107 CFU/ml to 101 CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 105 to 107 CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that S. algae would survive cold exposure better if in the biofilm state; however...

Identification of Some of the Major Groups of Bacteria in Efficient and Nonefficient Biological Phosphorus Removal Activated Sludge Systems

Bond, Philip L.; Erhart, Robert; Wagner, Michael; Keller, Jürg; Blackall, Linda L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 EN
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To investigate the bacteria that are important to phosphorus (P) removal in activated sludge, microbial populations were analyzed during the operation of a laboratory-scale reactor with various P removal performances. The bacterial population structure, analyzed by fluorescence in situ hybridization (FISH) with oligonucleotides probes complementary to regions of the 16S and 23S rRNAs, was associated with the P removal performance of the reactor. At one stage of the reactor operation, chemical characterization revealed that extremely poor P removal was occurring. However, like in typical P-removing sludges, complete anaerobic uptake of the carbon substrate occurred. Bacteria inhibiting P removal overwhelmed the reactor, and according to FISH, bacteria of the β subclass of the class Proteobacteria other than β-1 or β-2 were dominant in the sludge (58% of the population). Changes made to the operation of the reactor led to the development of a biomass population with an extremely good P removal capacity. The biochemical transformations observed in this sludge were characteristic of typical P-removing activated sludge. The microbial population analysis of the P-removing sludge indicated that bacteria of the β-2 subclass of the class Proteobacteria and actinobacteria were dominant (55 and 35%...

Bacterial Spores Survive Treatment with Commercial Sterilants and Disinfectants

Sagripanti, Jose-Luis; Bonifacino, Aylin
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 EN
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This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.

Enzymatic Combustion of Aromatic and Aliphatic Compounds by Manganese Peroxidase from Nematoloma frowardii

Hofrichter, Martin; Scheibner, Katrin; Schneegaß, Ivonne; Fritsche, Wolfgang
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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The direct involvement of manganese peroxidase (MnP) in the mineralization of natural and xenobiotic compounds was evaluated. A broad spectrum of aromatic substances were partially mineralized by the MnP system of the white rot fungus Nematoloma frowardii. The cell-free MnP system partially converted several aromatic compounds, including [U-14C]pentachlorophenol ([U-14C]PCP), [U-14C]catechol, [U-14C]tyrosine, [U-14C]tryptophan, [4,5,9,10-14C]pyrene, and [ring U-14C]2-amino-4,6-dinitrotoluene ([14C]2-AmDNT), to 14CO2. Mineralization was dependent on the ratio of MnP activity to concentration of reduced glutathione (thiol-mediated oxidation), a finding which was demonstrated by using [14C]2-AmDNT as an example. At [14C]2-AmDNT concentrations ranging from 2 to 120 μM, the amount of released 14CO2 was directly proportional to the concentration of [14C]2-AmDNT. The formation of highly polar products was also observed with [14C]2-AmDNT and [U-14C]PCP; these products were probably low-molecular-weight carboxylic acids. Among the aliphatic compounds tested, glyoxalate was mineralized to the greatest extent. Eighty-six percent of the 14COOH-glyoxalate and 9% of the 14CHO-glyoxalate were converted to 14CO2, indicating that decarboxylation reactions may be the final step in MnP-catalyzed mineralization. The extracellular enzymatic combustion catalyzed by MnP could represent an important pathway for the formation of carbon dioxide from recalcitrant xenobiotic compounds and may also have general significance in the overall biodegradation of resistant natural macromolecules...

Delineating the Specific Influence of Virus Isoelectric Point and Size on Virus Adsorption and Transport through Sandy Soils

Dowd, Scot E.; Pillai, Suresh D.; Wang, Sookyun; Corapcioglu, M. Yavuz
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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Many of the factors controlling viral transport and survival within the subsurface are still poorly understood. In order to identify the precise influence of viral isoelectric point on viral adsorption onto aquifer sediment material, we employed five different spherical bacteriophages (MS2, PRD1, Qβ, φX174, and PM2) having differing isoelectric points (pI 3.9, 4.2, 5.3, 6.6, and 7.3 respectively) in laboratory viral transport studies. We employed conventional batch flowthrough columns, as well as a novel continuously recirculating column, in these studies. In a 0.78-m batch flowthrough column, the smaller phages (MS2, φX174, and Qβ), which had similar diameters, exhibited maximum effluent concentration/initial concentration values that correlated exactly with their isoelectric points. In the continuously recirculating column, viral adsorption was negatively correlated with the isoelectric points of the viruses. A model of virus migration in the soil columns was created by using a one-dimensional transport model in which kinetic sorption was used. The data suggest that the isoelectric point of a virus is the predetermining factor controlling viral adsorption within aquifers. The data also suggest that when virus particles are more than 60 nm in diameter...

Physiological Characterization of a Bacterial Consortium Reductively Dechlorinating 1,2,3- and 1,2,4-Trichlorobenzene

Adrian, Lorenz; Manz, Werner; Szewzyk, Ulrich; Görisch, Helmut
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate...

Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1998 EN
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As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts.

Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

Huang, Ching-Tsan; Xu, Karen D.; McFeters, Gordon A.; Stewart, Philip S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/1998 EN
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The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin...

Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance

Rohr, Ute; Weber, Susanne; Michel, Rolf; Selenka, Fidelis; Wilhelm, Michael
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 EN
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Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53°C.

Isolation and Characterization of Phenol-Degrading Denitrifying Bacteria

van Schie, Paula M.; Young, L. Y.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1998 EN
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Phenol is a man-made as well as a naturally occurring aromatic compound and an important intermediate in the biodegradation of natural and industrial aromatic compounds. Whereas many microorganisms that are capable of aerobic phenol degradation have been isolated, only a few phenol-degrading anaerobic organisms have been described to date. In this study, three novel nitrate-reducing microorganisms that are capable of using phenol as a sole source of carbon were isolated and characterized. Phenol-degrading denitrifying pure cultures were obtained by enrichment culture from anaerobic sediments obtained from three different geographic locations, the East River in New York, N.Y., a Florida orange grove, and a rain forest in Costa Rica. The three strains were shown to be different from each other based on physiologic and metabolic properties. Even though analysis of membrane fatty acids did not result in identification of the organisms, the fatty acid profiles were found to be similar to those of Azoarcus species. Sequence analysis of 16S ribosomal DNA also indicated that the phenol-degrading isolates were closely related to members of the genus Azoarcus. The results of this study add three new members to the genus Azoarcus, which previously comprised only nitrogen-fixing species associated with plant roots and denitrifying toluene degraders.

A Two-Component Monooxygenase Catalyzes Both the Hydroxylation of p-Nitrophenol and the Oxidative Release of Nitrite from 4-Nitrocatechol in Bacillus sphaericus JS905

Kadiyala, Venkateswarlu; Spain, Jim C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1998 EN
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Bacteria that metabolize p-nitrophenol (PNP) oxidize the substrate to 3-ketoadipic acid via either hydroquinone or 1,2,4-trihydroxybenzene (THB); however, initial steps in the pathway for PNP biodegradation via THB are unclear. The product of initial hydroxylation of PNP could be either 4-nitrocatechol or 4-nitroresorcinol. Here we describe the complete pathway for aerobic PNP degradation by Bacillus sphaericus JS905 that was isolated by selective enrichment from an agricultural soil in India. Washed cells of PNP-grown JS905 released nitrite in stoichiometric amounts from PNP and 4-nitrocatechol. Experiments with extracts obtained from PNP-grown cells revealed that the initial reaction is a hydroxylation of PNP to yield 4-nitrocatechol. 4-Nitrocatechol is subsequently oxidized to THB with the concomitant removal of the nitro group as nitrite. The enzyme that catalyzed the two sequential monooxygenations of PNP was partially purified and separated into two components by anion-exchange chromatography and size exclusion chromatography. Both components were required for NADH-dependent oxidative release of nitrite from PNP or 4-nitrocatechol. One of the components was identified as a reductase based on its ability to catalyze the NAD(P)H-dependent reduction of 2...

Vibrio cholerae O1 Strain TSI-4 Produces the Exopolysaccharide Materials That Determine Colony Morphology, Stress Resistance, and Biofilm Formation

Wai, Sun Nyunt; Mizunoe, Yoshimitsu; Takade, Akemi; Kawabata, Shun-Ichiro; Yoshida, Shin-Ichi
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/1998 EN
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Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-d-glucosamine, d-mannose, 6-deoxy-d-galactose, and d-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.

Influence of Pretreatment and Experimental Conditions on Electrophoretic Mobility and Hydrophobicity of Cryptosporidium parvum Oocysts

Brush, Charles F.; Walter, Michael F.; Anguish, Lynne J.; Ghiorse, William C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/1998 EN
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Surface properties of Cryptosporidium parvum oocysts were investigated by using electrophoretic mobility and hydrophobicity measurements. Oocysts purified from calf feces by several sucrose flotation steps and deionized water (DI) washes (DIS method) had an electrophoretic mobility (neutral surface charge) near 0.0 m2 V−1 s−1 over a pH range of 2 to 10. The mean electrophoretic mobility of oocysts stored in DI containing a mixture of antibiotics had a lower standard deviation (ς = 0.36) than that of oocysts stored in DI without antibiotics (ς = 0.53); their electrophoretic mobility remained unchanged up to 121 days after collection. The electrophoretic mobility of oocysts purified on a cold Percoll-sucrose gradient after the feces was defatted with ethyl acetate (EAPS method) varied linearly with pH from 0.0 m2 V−1 s−1 at pH 2.4 to −3.2 × 10−8 m2 V−1 s−1 at pH 10 (ς = 0.52), thus displaying the negative surface charge at neutral pH observed by other researchers. The hydrophobicity of oocysts and two types of polystyrene beads was measured as a function of ionic strength by adhesion to polystyrene. Oocysts were purified by the DIS method. The ionic strength of the suspending solution was varied from 0 to 95 mmol liter−1. Two-week-old oocysts exhibited strong adhesion (∼85%) at ionic strengths of 0 to 10 mmol liter−1 and moderate adhesion (∼20%) at ionic strengths of 20 to 95 mmol liter−1. Two-month-old oocysts exhibited high adhesion (∼60 to 80%) at all ionic strengths. These results show that adhesion properties governed by the electrophoretic mobility of purified C. parvum oocysts can be altered by the method of purification and that hydrophobicity can change as oocysts age.

Phages Infecting Vibrio vulnificus Are Abundant and Diverse in Oysters (Crassostrea virginica) Collected from the Gulf of Mexico

DePaola, Angelo; Motes, Miles L.; Chan, Amy M.; Suttle, Curtis A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/1998 EN
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Phages infecting Vibrio vulnificus were abundant (>104 phages g of oyster tissue−1) throughout the year in oysters (Crassostrea virginica) collected from estuaries adjacent to the Gulf of Mexico (Apalachicola Bay, Fla.; Mobile Bay, Ala.; and Black Bay, La.). Estimates of abundance ranged from 101 to 105 phages g of oyster tissue−1 and were dependent on the bacterial strain used to assay the sample. V. vulnificus was near or below detection limits (<0.3 cell g−1) from January through March and was most abundant (103 to 104 cells g−1) during the summer and fall, when phage abundances also tended to be greatest. The phages isolated were specific to strains of V. vulnificus, except for one isolate that caused lysis in a few strains of V. parahaemolyticus. Based on morphological evidence obtained by transmission electron microscopy, the isolates belonged to the Podoviridae, Styloviridae, and Myoviridae, three families of double-stranded DNA phages. One newly described morphotype belonging to the Podoviridae appears to be ubiquitous in Gulf Coast oysters. Isolates of this morphotype have an elongated capsid (mean, 258 nm; standard deviation, 4 nm; n = 35), with some isolates having a relatively broad host range among strains of V. vulnificus. Results from this study indicate that a morphologically diverse group of phages which infect V. vulnificus is abundant and widely distributed in oysters from estuaries bordering the northeastern Gulf of Mexico.