Species-specific sequences were identified within the V4 variable region of 16S rRNA of two bacterial species capable of aromatic hydrocarbon metabolism, Pseudomonas putida F1 and Burkholderia sp. strain JS150, and a third, Bacillus subtilis ATCC 7003, that can function as a secondary degrader. Fluorescent in situ hybridization (FISH) with species-specific oligonucleotides was used for direct counting of these species throughout a phenol biodegradation experiment in batch culture. Traditional differential plate counting methods could not be used due to the similar metabolism and interactions of the primary degraders and difficulties in selecting secondary degraders in mixed culture. In contrast, the FISH method provided reliable quantitative results without interference from those factors.
Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-μg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.
The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 μM O2. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O2 < 0.1 μM) and low oxygen concentrations (3 μM O2), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.
In Vibrio vulnificus, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.
Why do viruses sometimes not pass through larger pores in track-etch filters? Increasing the salinity (0.8 to 160 mM Na+) decreased φX174 and PRD1 passage through track-etch polycarbonate membranes (sodium dodecyl sulfate coated but not polyvinylpyrrolidone coated) and PRD1 passage through polyester membranes. Undiminished passage when 0.1% Tween 80 was added implied that nonionic virus adsorption occurred and indicated that high levels of salinity decreased virus passage by decreasing electrostatic repulsion that prevented adsorption.
Reduced glutathione (GSH) levels and resistance to chlorine were measured for two isogenic Escherichia coli strains stressed by oxygenation and/or starvation. The E. coli mutant deficient in GSH was not more sensitive to the oxidant than its parent strain when the bacteria were cultured with a low oxygenation rate. Starvation or oxygenation increased the resistance of the parent strain to chlorine, while the resistance of the deficient strain remained unchanged.
The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.
The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using 19F nuclear magnetic resonance (NMR) spectroscopy in combination with 14C radioisotope-detected high-performance liquid chromatography (14C-HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. 14C-HPLC profiles indicated that there were four major biotransformation products, whereas 19F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4′-ol and 4-fluorobiphen-3′-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.
The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.
Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe2+-pyoverdine than for Fe3+-pyoverdine. At pH 7.4, about 90% of Fe3+ was bound by pyoverdine Pa-C after 24 h whereas Fe2+ was bound by the pyoverdine completely in only 5 min. The possibility that Fe2+ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe2+ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe2+ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe3+-specific chelating agent, resulted in the formation of a Fe3+-hydroxyquinoline complex, suggesting that the iron in the Fe2+-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.
Cocultures of Desulfovibrio desulfuricans and Methanococcus maripaludis grew on sulfate-free lactate medium while vigorously methylating Hg2+. Individually, neither bacterium could grow or methylate mercury in this medium. Similar synergistic growth of sulfidogens and methanogens may create favorable conditions for Hg2+ methylation in low-sulfate anoxic freshwater sediments.
The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (clc) from Pseudomonas sp. strain B13 were investigated in activated-sludge microcosms. When the clc genes were transferred into an appropriate recipient bacterium such as Pseudomonas putida F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10−8 per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB+) transconjugants of strain F1 which had acquired the clc genes were detected but only when strain B13 cell densities of more than 105 CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB+ transconjugants reached final cell densities of between 102 and 103 CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm...
The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 μM) and azide (24 μM) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.
Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (Vmax) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (Ks) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, Vmax/Ks, doubled from 4.9 × 10−3 to 9.9 × 10−3 liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the Vmax for TCE was 2.5 nmol/min/mg of protein...
Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.
Fecal droppings of migratory Canada geese, Branta canadensis, collected from nine sites near the Chesapeake Bay (Maryland), were examined for the presence of Cryptosporidium parvum and Giardia spp. Cryptosporidium sp. oocysts were found in feces at seven of nine sites, and Giardia cysts were found at all nine sites. The oocysts from three sites were infectious for mice and molecularly identified as the zoonotic genotype of Cryptosporidium parvum. Waterfowl can disseminate infectious C. parvum oocysts in the environment.
Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the β-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the β-tubulin intron and adjacent exon 2. Isolates carrying different β-tubulin alleles were found. Significantly, one of the β-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different β-tubulin alleles fluctuated during serial passage.
Iodinated X-ray contrast agents are considered to be nondegradable by microorganisms. The decomposition of the ionic X-ray contrast agents Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoic acid) and Iodipamide (3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid) and related triiodinated benzoates (Acetrizoate [3-acetylamino-2,4,6-triiodobenzoic acid] and Aminotrizoate [3-amino-2,4,6-triiodobenzoic acid]) by Trametes versicolor has been investigated. The fungus was able to transform all tested triiodinated benzoates cometabolically. During transformation of these compounds, iodide was released, but deiodination was not complete. T. versicolor liberated traces of 14CO2 from uniformly ring-14C-labeled Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoate). Various extracellular metabolites were detected during transformation of the different substances. In the transformation of Diatrizoate, the three main metabolites were identified as 3,5-di(acetamido)-2,6-diiodobenzoic acid, 3,5-di(acetamido)-2,4-diiodobenzoic acid, and 3,5-di(acetamido)-2-iodobenzoic acid, suggesting reductive deiodinations in steps as initial transformation steps.
The mineralization of phenanthrene in pure cultures of a Pseudomonas fluorescens strain, isolated from soil, was measured in the presence of soil humic fractions and montmorillonite. Humic acid and clay, either separately or in combination, shortened the acclimation phase. A higher mineralization rate was measured in treatments with humic acid at 100 μg/ml. Humic acid at 10 μg/ml stimulated the transformation only in the presence of 10 g of clay per liter. We suggest that sorption of phenanthrene to these soil components may result in a higher concentration of substrate in the vicinity of the bacterial cells and therefore may increase its bioavailability.
The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus...