Vertical Distribution and Seasonal Patterns of Candidatus Nitrotoga in a Sub-Alpine Lake.

The nitrite oxidizing bacterial genus Ca. Nitrotoga was only recently discovered to be widespread in freshwater systems; however, limited information is currently available on the environmental factors and seasonal effects that influence its distribution in lakes. In a one-year study in a dimictic lake, based on monthly sampling along a vertical profile, the droplet digital PCR quantification of Ca. Nitrotoga showed a strong spatio-temporal patchiness. A correlation ana-lysis with environmental parameters revealed that the abundance of Ca. Nitrotoga correlated with dissolved oxygen and ammonium, suggesting that the upper hypolimnion of the lake is the preferred habitat.

Primer evaluation and development of a droplet digital PCR protocol targeting amoA genes for the quantification of Comammox in lakes.

To date, little is known about the ecological significance of Comammox (COMplete AMMonia OXidizers) Nitrospira in the water column of freshwater lakes. Water samples collected along depth profiles were used to investigate the distribution of Comammox in 13 lakes characterized by a wide range of physicochemical properties. Several published primers, which target the α-subunit of the ammonia monooxygenase, generated non-specific PCR products or did not amplify target genes from lake water and other habitats. Therefore, a new primer set has been designed for specific detection of Comammox in lakes. The high specificity of the PCR assay was confirmed by sequencing analysis. Quantification of Comammox amoA genes in lake water samples based on droplet digital PCR (ddPCR) revealed very low abundances (not exceeding 85 amoA copies ml-1), which suggest that Comammox is of minor importance for the nitrification process in the water column of the study sites. Surprisingly, samples taken from the sediment/water-interface along an oxygen gradient in dimictic Piburger See showed Comammox abundances three to four magnitudes higher than in the pelagic realm of the lake, which indicates a preference of Comammox to a particle-attached lifestyle.

Spatiotemporal dynamics of different CO2 fixation strategies used by prokaryotes in a dimictic lake.

The Calvin-Benson-Bassham (CBB) cycle and the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle are two inorganic carbon assimilation pathways widely used by prokaryotic autotrophs in lakes. We investigated the effect of mixing periods and stable water stratification patterns on the trajectories of both CO2 fixation strategies in a dimictic lake (Piburger See), because information on the spatiotemporal dynamics of prokaryotes using these pathways in freshwater ecosystems is far from complete. Based on a quantitative approach (droplet digital PCR) of genes coding for key enzymes in different CO2 assimilation pathways, nine depths covering the entire water column were investigated on a monthly basis for one year. Our data show that the abundance of photoautotrophs and obligate chemolithoautotrophs preferentially using form IA RubisCO was determined by seasonal variations. Highest numbers were observed in summer, while a strong decline of prokrayotes using RubisCO form IA was measured between December and May, the period where the lake was mostly covered by ice. The spatiotemporal distribution patterns of genes coding for RubisCO form IC genes, an enzyme usually used by facultative autotrophs for CO2 assimilation, were less pronounced. Bacteria harboring RubisCO form II were dominating the oxygen limited hypolimnion, while nitrifying Thaumarchaeota using the HP/HB cycle were of minor importance in the lake. Our data reveal that the seasonal heterogeneity, which is determined by the dimictic thermal regime of the lake, results in pronounced spatiotemporal changes of different CO2 assimilation pathways with depth-dependent environmental parameters as key factors for their distribution.

Autotrophic carbon fixation strategies used by nitrifying prokaryotes in freshwater lakes.

Niche specialization of nitrifying prokaryotes is usually studied with tools targeting molecules involved in the oxidation of ammonia and nitrite. The ecological significance of diverse CO2 fixation strategies used by nitrifiers is, however, mostly unexplored. By analyzing autotrophy-related genes in combination with amoA marker genes based on droplet digitial PCR and CARD-FISH counts targeting rRNA, we quantified the distribution of nitrifiers in eight stratified lakes. Ammonia oxidizing (AO) Thaumarchaeota using the 3-hydroxypropionate/4-hydroxybutyrate pathway dominated deep and oligotrophic lakes, whereas Nitrosomonas-related taxa employing the Calvin cycle were important AO bacteria in smaller lakes. The occurrence of nitrite oxidizing Nitrospira, assimilating CO2 with the reductive TCA cycle, was strongly correlated with the distribution of Thaumarchaeota. Recently discovered complete ammonia-oxidizing bacteria (comammox) belonging to Nitrospira accounted only for a very small fraction of ammonia oxidizers (AOs) present at the study sites. Altogether, this study gives a first insight on how physicochemical characteristics in lakes are associated to the distribution of nitrifying prokaryotes with different CO2 fixation strategies. Our investigations also evaluate the suitability of functional genes associated with individual CO2 assimilation pathways to study niche preferences of different guilds of nitrifying microorganisms based on an autotrophic perspective.

Specific detection of form IA RubisCO genes in chemoautotrophic bacteria.

The analysis of RubisCO genes is a highly useful instrument to explore the diversity of chemoautotrophic bacteria using the Calvin-Benson-Bassham cycle for CO2 fixation. However, because of the wide taxonomic distribution of phylogenetically related RubisCO forms, environmental studies targeting chemoautotrophs are hampered in habitats dominated by phototrophs. Here, we report the development of a gene marker that specifically detects form IA RubisCO genes in bacteria, excluding photoautotrophic representatives. The high specificity of the PCR assay was confirmed by sequence analysis of DNA obtained from the photic zone of six lakes, were chemoautotrophs are outnumbered by Cyanobacteria also using form IA RubisCO for CO2 assimilation.

CO2 assimilation strategies in stratified lakes: Diversity and distribution patterns of chemolithoautotrophs.

While mechanisms of different carbon dioxide (CO2 ) assimilation pathways in chemolithoautotrohic prokaryotes are well understood for many isolates under laboratory conditions, the ecological significance of diverse CO2 fixation strategies in the environment is mostly unexplored. Six stratified freshwater lakes were chosen to study the distribution and diversity of the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the recently discovered archaeal 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) pathway. Eleven primer sets were used to amplify and sequence genes coding for selected key enzymes in the three pathways. Whereas the CBB pathway with different forms of RubisCO (IA, IC and II) was ubiquitous and related to diverse bacterial taxa, encompassing a wide range of potential physiologies, the rTCA cycle in Epsilonproteobacteria and Chloribi was exclusively detected in anoxic water layers. Nitrifiying Nitrosospira and Thaumarchaeota, using the rTCA and HP/HB cycle respectively, are important residents in the aphotic and (micro-)oxic zone of deep lakes. Both taxa were of minor importance in surface waters and in smaller lakes characterized by an anoxic hypolimnion. Overall, this study provides a first insight on how different CO2 fixation strategies and chemical gradients in lakes are associated to the distribution of chemoautotrophic prokaryotes with different functional traits.

Prokaryotic community structure in deep bedrock aquifers of the Austrian Central Alps.

The bacterial and archaeal diversity of deep groundwater systems was investigated based on 16S rRNA-SSCP (single strand conformation polymorphism) fingerprints. The study site included five boreholes along the projected Brenner Base Tunnel in the central Alps of Tyrol, Austria. To obtain representative samples, packer-sealed fractures were sampled at specific depths between 105 and 780 m below surface. Sequence analysis of SSCP bands obtained from 13 samples showed that between 29 and 62 % of the phylotypes belonged to a variety of Proteobacteria including representatives of typical freshwater bacteria of the genera Acidovorax, Aquabacterium, and Sphingomonas. Bacteroidetes (especially Flavobacterium), Firmicutes (Acetobacterium), and candidate division OP3-related sequences were observed in the majority of the analysed groundwaters. On average, 14 % of the detected prokaryotic phylotypes were affiliated with Archaea, comprising the phyla Euryarchaeota, Crenarchaeota and Thaumarchaeota. Most of the archaeal sequences showed low similarities to known cultivated species, with exception of two sequences having 98 % similarity to Methanosaeta sp. A considerable number of thaumarchaeal sequences belonged to two groups related to Nitrososphaera and Nitrosopumilus phylotypes. An environmental clustering of the groundwater samples, based on the bacterial and archaeal phylogeny, revealed a clear distribution pattern of the samples (sites and depths) reflecting the hydrochemical characteristics and underlying geologies.

Genetic evidence for bacterial chemolithoautotrophy based on the reductive tricarboxylic acid cycle in groundwater systems.

Geologically and chemically distinct aquifers were screened for the presence of two genes coding for key enzymes of the reverse tricarboxylic acid (rTCA) cycle in autotrophic bacteria, 2-oxoglutarate : ferredoxin oxidoreductase (oorA) and the beta subunit of ATP citrate lyase enzymes (aclB). From 42 samples investigated, aclB genes were detected in two and oorA genes in six samples retrieved from polluted and sulfidic aquifers. aclB genes were represented by a single phylotype of almost identical sequences closely affiliated with chemolithoautotrophic Sulfurimonas species. In contrast, sequences analysis of oorA genes revealed diverse phylotypes mainly related to sequences from cultivation-independent studies.

Diversity and expression of different forms of RubisCO genes in polluted groundwater under different redox conditions.

Groundwater polluted with methyl-tert-butyl ether (MTBE) and ammonium was investigated for chemolithoautotrophic CO(2) fixation capabilities based on detailed analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes. Samples retrieved from a groundwater conditioning unit, characterized by different redox conditions, were examined for the presence of form IA, form IC (cbbL) and form II (cbbM) RubisCO genes and transcripts obtained from DNA- and RNA-extracts. Form IA RubisCO sequences, which revealed a complex and distinct variety in different sampling stations, were expressed in the original groundwater and in samples amended with oxygen, but not in the aquifer groundwater enriched with nitrate. Form IC RubisCO genes were exclusively detected in groundwater supplied with oxygen and sequences were affiliated with cbbL genes in nitrifying bacteria. cbbM genes were not expressed in the oxygen-amended groundwater, probably due to the low CO(2) /O(2) substrate specificity of this enzyme. Most form II RubisCO transcripts were affiliated with RubisCO genes of denitrifiers, which are important residents in the groundwater supplied with nitrate. The distinct distribution pattern and diversity of RubisCO genes and transcripts obtained in this study suggest that the induction of different RubisCO enzymes is highly regulated and closely linked to the actual environmental conditions.

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.

Distribution and diversity of autotrophic bacteria in groundwater systems based on the analysis of RubisCO genotypes.

A molecular approach, based on the detection of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes, was applied to investigate the distribution and diversity of autotrophic bacteria in groundwater systems. DNA extracts from 48 sampling stations, including a variety of pristine and polluted, shallow and deep-subsurface groundwater samples obtained from Germany and Austria, served as a template for the PCR amplification of form I (cbbL) and form II (cbbM) large subunit RubisCO genes. The majority of the samples (>80%) contained two different forms of RubisCO. In 17 samples, all three forms of RubisCO were identified. PCR products from four selected groundwater habitats containing all three forms of RubisCO were used to construct clone libraries. Based on restriction fragment length polymorphism (RFLP) analysis, 109 RubisCO-clone-inserts were subjected to sequencing and phylogenetic analysis. With the exception of a form IA RubisCO sequence cluster obtained from deep subsurface samples, which was identical to the RubisCO genes described for Ralstonia metallidurans CH34, most sequences were distantly related to a variety of RubisCO species in chemolithoautotrophic Proteobacteria. Several sequences occurred in isolated lineages. These findings suggest that autotrophic bacteria with the capability to assimilate CO2 via the Calvin Cycle pathway are widespread inhabitants of groundwater systems.

Helicobacter pylori determination in non-municipal drinking water and epidemiological findings.

BACKGROUND: Studies conducted in Europe as well as in North and South America have tried to link Helicobacter pylori colonization with the drinking water supply, especially since H. pylori is known to survive quite well in water. METHODS: In 2000, a cohort of 1884 grade-two children from two rural counties surrounding the city of Leipzig, Germany (77.4% of the 1991/1992 birth cohort) were tested for H. pylori colonization using the [13C]urea breath test. A parent-completed questionnaire elicited details on living conditions and lifestyle habits including questions on the children's drinking water from sources other than public water supplies, swimming in natural waters, etc. In a second independent study, samples of well water, taken from 157 private wells still used in the two counties, were being tested for the presence of H. pylori, using polymerase chain reaction (PCR) method to determine relevant target DNA fragments of H. pylori. RESULTS: In county I, 5.7% of the children and in county II 6.6% tested H. pylori-positive. Cluster analyses of the questionnaire data in both counties pointed to 'drinking water from other than municipal sources', as the closest H. pylori-associated cluster variable. The cluster estimations were supported by odds ratio (OR) calculations with an OR=16.4 (95% confidence interval (CI) 3.1,...,88.5) for county I and OR=4.0 (95% CI 1.3,...,12.4) for county II. The PCR analyses showed H. pylori DNA fragments in 10.8% of the wells in county I and 9.2% in county II. The detection limit was set at 10 DNA copies corresponding to 125 bacteria/L, the average infestation of these wells was 931 bacteria/L. CONCLUSION: Despite the fact that the microbiological and epidemiological data do not correspond except that both studies were conducted in the same geographical areas, the independent findings of H. pylori in well water in the same general areas where children do seem to drink water other than from the public water supply suggests that water may be an important source of H. pylori infection.

Microbial degradation of chlorobenzene under oxygen-limited conditions leads to accumulation of 3-chlorocatechol.

Five bacterial strains (Acidovorax facilis B517, Cellulomonas turbata B529, Pseudomonas veronii B547, Pseudomonas veronii B549, and Paenibacillus polymyxa B550) isolated on chlorobenzene as the sole source of carbon and energy were screened for the accumulation of the putative metabolic intermediate 3-chlorocatechol during growth on chlorobenzene under oxygen-limited conditions in the presence and absence of nitrate (1 mM). 3-Chlorocatechol accumulated in the growth media of all five strains, but accumulation was significantly less in cultures of A. facilis B517 compared to the other four strains. The presence of nitrate did not influence the biological conversion pattern. However, biologically produced nitrite reacted with 3-chlorocatechol chemically, a reaction that masked the accumulation of 3-chlorocatechol. For P. veronii B549, a clear relationship between the presence of 3-chlorocatechol in the medium and low oxygen concentrations was demonstrated. The assumption is made that accumulation of 3-chlorocatechol is due to the low enzymatic turnover of the 3-chlorocatechol cleaving enzyme, catechol-1,2-dioxygenase, at low oxygen concentrations.

Bioremediation of chlorobenzene-contaminated ground water in an in situ reactor mediated by hydrogen peroxide.

New in situ reactive barrier technologies were tested nearby a local aquifer in Bitterfeld, Saxonia-Anhalt, Germany, which is polluted mainly by chlorobenzene (CB), in concentrations up to 450 microM. A reactor filled with original aquifer sediment was designed for the microbiological remediation of the ground water by indigenous bacterial communities. Two remediation variants were examined: (a) the degradation of CB under anoxic conditions in the presence of nitrate; (b) the degradation of CB under mixed electron acceptor conditions (oxygen+nitrate) using hydrogen peroxide as the oxygen-releasing compound. Under anoxic conditions, no definite degradation of CB was observed. Adding hydrogen peroxide (2.94 mM) and nitrate (2 mM) led to the disappearance of CB (ca. 150 microM) in the lower part of the reactor, accompanied by a strong increase of the number of cultivable aerobic CB degrading bacteria in reactor water and sediment samples, indicating that CB was degraded mainly by productive bacterial metabolism. Several aerobic CB degrading bacteria, mostly belonging to the genera Pseudomonas and Rhodococcus, were isolated from reactor water and sediments. In laboratory experiments with reactor water, oxygen was rapidly released by hydrogen peroxide, whereas biotic-induced decomposition reactions of hydrogen peroxide were almost four times faster than abiotic-induced decomposition reactions. A clear chemical degradation of CB mediated by hydrogen peroxide was not observed. CB was also completely degraded in the reactor after reducing the hydrogen peroxide concentration to 880 microM. The CB degradation completely collapsed after reducing the hydrogen peroxide concentration to 440 microM. In the following, the hydrogen peroxide concentrations were increased again (to 880 microM, 2.94 mM, and 880 microM, respectively), but the oxygen demand for CB degradation was higher than observed before, indicating a shift in the bacterial population. During the whole experiment, nitrate was uniformly reduced during the flow path in the reactor.

Expression of chlorocatechol 1,2-dioxygenase and chlorocatechol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples.

In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H(2)O(2). Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from Pseudomonas chlororaphis RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene cbzE from Pseudomonas putida GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis.

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.