András Táncsics

Investigation of catechol 2,3-dioxygenase and 16S rRNA gene diversity in hypoxic, petroleum hydrocarbon contaminated groundwater.

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of the contaminants under aerobic conditions. In this study, we investigated the diversity and distribution of Comamonadaceae family (Betaproteobacteria) related catechol 2,3-dioxygenase genes, which belong to the I.2.C subfamily of extradiol dioxygenase genes. These catabolic genes encode enzymes supposed to function under hypoxic conditions as well, and may play a notable role in BTEX degradation in oxygen limited environments. Therefore, their diversity was analyzed in oxygen limited, petroleum hydrocarbon contaminated groundwater by terminal restriction fragment length polymorphism and cloning. Subfamily I.2.C related catechol 2,3-dioxygenase genes were detected in every investigated groundwater sample and a dynamic change was observed in the case of the structure of C23O gene possessing bacterial communities. To link the metabolic capability to the microbial structure, 16S rRNA gene-based clone libraries were generated and it was concluded that Betaproteobacteria were abundant in the bacterial communities of the contaminated samples. These results support the opinion that Betaproteobacteria may play a significant role in BTEX degradation under hypoxic conditions.

Applicability of the functional gene catechol 1,2-dioxygenase as a biomarker in the detection of BTEX-degrading Rhodococcus species

Aims:  Catechol 1,2‐dioxygenase is a key enzyme in the degradation of monoaromatic pollutants. The detection of this gene is in focus today but recently designed degenerate primers are not always suitable. Rhodococcus species are important members of the bacterial community involved in the degradation of aromatic contaminants and their specific detection could help assess functions and activities in the contaminated environments. To reach this aim, specific PCR primer sets were designed for the detection of Rhodococcus related catechol 1,2‐dioxygenase genes.

Quantification of Subfamily I.2.C Catechol 2,3-Dioxygenase mRNA Transcripts in Groundwater Samples of an Oxygen-Limited BTEX-Contaminated Site

Low dissolved oxygen concentration of subsurface environments is a limiting factor for microbial aromatic hydrocarbon degradation, and to date, there are only a limited number of available reports on functional genes and microbes that take part in the degradation of aromatic hydrocarbons under hypoxic conditions. Recent discoveries shed light on the prevalence of subfamily I.2.C catechol 2,3-dioxygenases in petroleum hydrocarbon contaminated hypoxic groundwaters, and their considerable environmental importance was suggested. Here, we report on a Hungarian aromatic hydrocarbon (methyl-substituted benzene derivatives, mostly xylenes) contaminated site where we investigated this presumption. Groundwater samples were taken from the center and the edge of the contaminant plume and beyond the plume. mRNA transcripts of subfamily I.2.C catechol 2,3-dioxygenases were detected in considerable amounts in the contaminated samples by qPCR analysis, while activity of subfamily I.2.A, which includes the largest group of extradiol dioxygenases described by culture-dependent studies and thought to be widely distributed in BTEX-contaminated environments, was not observed. Bacterial community structure analyses showed the predominance of genus Rhodoferax related species in the contaminated samples.

The single-nucleotide primer extension (SNuPE) method for the multiplex detection of various DNA sequences: from detection of point mutations to microbial ecology.

Methods based on SNuPE (single-nucleotide primer extension) have become invaluable tools for the rapid and highly specific detection of point mutations and single-nucleotide polymorphisms in the field of human genetics. In the primer extension reaction, a DNA polymerase is used to label a specific primer hybridized to the target sequence by incorporating a single labelled ddNTP (dideoxynucleotide). This labelling provides not only information about the complementary nucleotide of interest in the opposite strand but also a semiquantitative analysis of the sequence targeted by the primer. Since several subdisciplines of microbiology increasingly require cultivation-independent molecular screening tools for elucidating differences between either strains or community structures based on sequence variations of marker genes, SNuPE offers a promising alternative to the existing tool box. The present review describes the method in detail and reports the state-of-the-art applications of this technique both in the field of nucleic acid detections in human genetics and in microbiology.

Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems

ABSTRACT It has recently been suggested that oxygenic dismutation of NO into N2 and O2 may occur in the anaerobic methanotrophic “Candidatus Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to “Ca. Methylomirabilis oxyfera” and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from “Ca. Methylomirabilis oxyfera” and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinol-dependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 × 107 to 5.2 × 1010 copies · g−1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems. IMPORTANCE NO dismutation into N2 and O2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium “Ca. Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future.

Evaluation of single-nucleotide primer extension for detection and typing of phylogenetic markers used for investigation of microbial communities.

ABSTRACT Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3′-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23–25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don’t show homology to each other, but all of them are built up from 3–6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70–75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9–1.7mM of KM and 80–120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

Investigation of mineral water springs of Miercurea Ciuc (Csíkszereda) region (Romania) with cultivation-dependent microbiological methods

Water samples of ten mineral water springs at Miercurea Ciuc (Csíkszereda) region (Romania) were examined during 2005-2006 using cultivation-dependent microbiological methods. The results of standard hygienic bacteriological tests showed that the Hargita Spring had perfect and five other springs had microbiologically acceptable water quality (Zsögöd-, Nagy-borvíz-, Taploca-, Szentegyháza- and Lobogó springs). The water of Borsáros Spring was exceptionable (high germ count, presence of Enterococcus spp.).Both standard bacteriological and molecular microbiological methods indicated that the microbiological water quality of the Szeltersz-, Nádasszék- and Délo springs was not acceptable. Bad water quality resulted from inadequate spring catchment and hygiene (low yield, lack of runoff, negligent usage of the springs, horse manure around the spring).The 16S rRNA gene-based identification of strains isolated on standard meat-peptone medium resulted in the detection of typical aquatic organisms such as Shewanella baltica, Aeromonas spp., Pseudomonas veronii, Psychrobacter sp,. Acinetobacter spp. and allochthonous microbes, like Nocardia, Streptomyces, Bacillus, Microbacterium , and Arthrobacter strains indicating the impact of soil. Other allochthonous microbes, such as Staphylococcus spp., Micrococcus sp., Lactococcus sp., Clostridium butyricum, Yersinia spp., Aerococcus sp., may have originated from animal/human sources.