Metagenomics reveals the high polycyclic aromatic hydrocarbon-degradation potential of abundant uncultured bacteria from chronically polluted subantarctic and temperate coastal marine environments.

AIMS: To investigate the potential to degrade polycyclic aromatic hydrocarbons (PAHs) of yet-to-be-cultured bacterial populations from chronically polluted intertidal sediments. METHODS AND RESULTS: A gene variant encoding the alpha subunit of the catalytic component of an aromatic-ring-hydroxylating oxygenase (RHO) was abundant in intertidal sediments from chronically polluted subantarctic and temperate coastal environments, and its abundance increased after PAH amendment. Conversely, this marker gene was not detected in sediments from a nonimpacted site, even after a short-term PAH exposure. A metagenomic fragment carrying this gene variant was identified in a fosmid library of subantarctic sediments. This fragment contained five pairs of alpha and beta subunit genes and a lone alpha subunit gene of oxygenases, classified as belonging to three different RHO functional classes. In silico structural analysis suggested that two of these oxygenases contain large substrate-binding pockets, capable of accepting high molecular weight PAHs. CONCLUSIONS: The identified uncultured micro-organism presents the potential to degrade aromatic hydrocarbons with various chemical structures, and could represent an important member of the PAH-degrading community in these polluted coastal environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides valuable information for the design of environmental molecular diagnostic tools and for the biotechnological application of RHO enzymes.

Aromatic hydrocarbon degradation genes from chronically polluted Subantarctic marine sediments.

AIM: The goal of this study was to identify functional targets to detect polycyclic aromatic hydrocarbon (PAH)-degrading bacterial populations in cold marine ecosystems. METHODS AND RESULTS: We designed a degenerate primer set targeting genes encoding the alpha subunit of PAH-dioxygenases from Gram-positive bacteria. This primer set was used to amplify gene fragments from metagenomic DNA isolated from Subantarctic marine sediments (Ushuaia Bay, Argentina). These gene fragments were cloned and sequenced. We identified 14 distinct groups of genes, most of them showing significant relatedness with dioxygenases from Gram-positive bacteria of the genera Rhodococcus, Mycobacterium, Nocardioides, Terrabacter and Bacillus. The level of identity with these genes, however, was low to moderate (33-62% at the amino acid level). CONCLUSION: These results indicate the presence of a high diversity of hitherto unidentified dioxygenase genes in this cold polluted environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Subantarctic marine ecosystems are particularly vulnerable to hydrocarbon pollution, and the development of environmental restoration strategies for these environments is pressing. The information obtained in this work will be the starting point for the design of quantitative molecular tools to analyse the abundance and dynamics of these aromatic hydrocarbon-degrading bacterial populations in the marine environment.

Effect of microbial species richness on community stability and community function in a model plant-based wastewater processing system.

Microorganisms will be an integral part of biologically based waste processing systems used for water purification or nutrient recycling on long-term space missions planned by the National Aeronautics and Space Administration. In this study, the function and stability of microbial inocula of different diversities were evaluated after inoculation into plant-based waste processing systems. The microbial inocula were from a constructed community of plant rhizosphere-associated bacteria and a complexity gradient of communities derived from industrial wastewater treatment plant-activated sludge. Community stability and community function were defined as the ability of the community to resist invasion by a competitor (Pseudomonas fluorescens 5RL) and the ability to degrade surfactant, respectively. Carbon source utilization was evaluated by measuring surfactant degradation and through Biolog and BD oxygen biosensor community level physiological profiling. Community profiles were obtained from a 16S-23S rDNA intergenic spacer region array. A wastewater treatment plant-derived community with the greatest species richness was the least susceptible to invasion and was able to degrade surfactant to a greater extent than the other complexity gradient communities. All communities resisted invasion by a competitor to a greater extent than the plant rhizosphere isolate constructed community. However, the constructed community degraded surfactant to a greater extent than any of the other communities and utilized the same number of carbon sources as many of the other communities. These results demonstrate that community function (carbon source utilization) and community stability (resistance to invasion) are a function of the structural composition of the community irrespective of species richness or functional richness.

Molecular assessment of ammonia- and nitrite-oxidizing bacteria in full-scale activated sludge wastewater treatment plants.

Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 x 10(13) copies/L and Nitrospira 16S rDNA values were 2-4 x 10(10) copies/L. amoA gene concentrations averaged 1.73 x 10(9) copies/L (municipal) and 1.06 x 10(10) copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.

Quantification of Nitrosomonas oligotropha and Nitrospira spp. using competitive polymerase chain reaction in bench-scale wastewater treatment reactors operating at different solids retention times.

The effect of solids retention time (SRT) on ammonia-and nitrite-oxidizing bacteria was measured by Nitrosomonas oligotropha-like ammonia monooxygenase A and Nitrospira 16S rDNA competitive polymerase chain reaction assays in a complete-mix, bench-scale, activated-sludge system. During steady-state operation, nitrification was complete in the 20- and 10-day SRT reactors, nearly complete in the 5-day SRT reactor, and incomplete in the 2-day SRT reactor (76% ammonia oxidation and 85% nitrite oxidation). Total microbes, measured by dot-blot hybridizations, ranged from 3 x 10(11) to 3 x 10(12) cells/L, and increased with increasing SRTs. The concentration of the ammonia-oxidizer N. oligotropha dropped 100-fold from the 20-day SRT (5 x 10(9) cells/L) to the 2-day SRT (< or = 4 x 10(7) cells/L). Thus, N. oligotropha became a much smaller fraction of the total biomass in the poorly performing 2-day SRT reactor. The concentration of Nitrospira cells also decreased (10-fold) as the SRT was reduced from 20 days to 2 days. However, the number of Nitrospira cells was always greater than the number of N. oligotropha cells measured in each reactor (10- to 60-fold). While Nitrospira comprised 1 to 2% of the biomass, N. oligotropha represented only 0.04 to 0.27% of the total population. This low percentage suggests that N. oligotropha was not a dominant ammonia oxidizer in the bench-scale systems.

Alkali metal ions protect mitochondrial rhodanese against thermal inactivation.

Incubation of bovine liver mitochondrial rhodanese in dilute, reducing solutions at temperatures ranging between 30 and 45 degreesC conduced to a rapid loss of enzymatic activity. This inactivation was substantially reduced in the presence of millimolar concentrations of alkali metal ions, divalent cations (including Mg2+, Ca2+, and Ba2+) were ineffective. The extent of protection afforded by monovalent cations was highly dependent on their ionic radii, with K+ and Na+ ions being the most effective protective agents. The protection afforded by a number of anions, including thiosulfate, could be totally ascribed to the presence of the accompanying monovalent cation. The overall results indicate that K+ and Na+, at concentrations and temperatures within the physiological range, substantially contribute to the stabilization of the functional structure of rhodanese.

Purification and characterization of Chromatium vinosum GroEL and GroES proteins overexpressed in Escherichia coli cells lacking the endogenous groESL operon.

Using an Escherichia coli strain (RF101) in which the endogenous chromosomal groESL operon was removed, we overexpressed the GroEL and GroES chaperonins cloned from the photosynthetic bacterium Chromatium vinosum. The identities of these proteins were confirmed by immunological and N-terminal sequence analyses. The native molecular masses of GroEL and GroES, as determined by size-exclusion chromatography, were 830 and 74 kDa, respectively. This suggests a tetradecameric structure for GroEL and a heptameric structure for GroES. C. vinosum GroEL catalyzed a K+-stimulated ATP hydrolysis with a specific activity at 25 degreesC of 50.2 +/- 3.8 nmol Pi released min-1 mg protein-1. GroEL ATPase was inhibited by GroES, reaching about 50% inhibition at a ratio GroES-7mer/GroEL-14mer of 1 in the presence of 10 mM KCl. The ATPase Vmax increased almost fivefold in the 25 to 65 degreesC temperature range; higher temperatures led to a rapid inactivation of this activity. The chaperone activity of the C. vinosum GroEL/GroES system was characterized by its effect on the refolding of guanidinium chloride-unfolded rhodanese. In the presence of ATP and GroES, C. vinosum GroEL assisted rhodanese refolding. The heterologous combination C. vinosum GroEL/E. coli GroES or E. coli GroEL/C. vinosum GroES was as effective as the homologous complexes. In summary, this strategy allowed the purification at high yields of fully functional, homogenous C. vinosum GroEL and GroES chaperonins from E. coli.

Cooperation of the DnaK and GroE chaperone systems in the folding pathway of plant ferredoxin-NADP+ reductase expressed in Escherichia coli.

The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme. Expression of preFNR in OmpT-protease-deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15537-15541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.

Hemofilosis genital. / Genital haemophilosis

En 1954 tuvo lugar un importante acontecimiento en el campo de la patologia ginecologica: Gardner y Dukes aislaron al Haemophilus vaginales como el microorganismo responsable de la afeccion conocida como "vaginitia inespecifica". Aunque han transcurrido desde entonces muchos anos, no se ha logrado todavia un total acuerdo a proposito de su taxonomia, patogenia y tratamiento. En este trabajo los autores analizan la amplia literatura existente a proposito de este tema, exponiendo la controversia existente en el presente respecto de este germen

Hemofilosis genital. / Genital haemophilosis

En 1954 tuvo lugar un importante acontecimiento en el campo de la patologia ginecologica: Gardner y Dukes aislaron al Haemophilus vaginales como el microorganismo responsable de la afeccion conocida como "vaginitia inespecifica". Aunque han transcurrido desde entonces muchos anos, no se ha logrado todavia un total acuerdo a proposito de su taxonomia, patogenia y tratamiento. En este trabajo los autores analizan la amplia literatura existente a proposito de este tema, exponiendo la controversia existente en el presente respecto de este germen