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PubMed2006

Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

Bacteria↗
PubMed2005

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil.

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.

Bacteria↗
PubMed2006

Bacterial diversity in organically-enriched fish farm sediments.

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.

Animal Husbandry↗
PubMed2006

Microbial diversity of cold-seep sediments in Sagami Bay, Japan, as determined by 16S rRNA gene and lipid analyses.

Microbial communities in Calyptogena sediment and microbial mats of Sagami Bay, Japan, were characterized using 16S rRNA gene sequencing and lipid biomarker analysis. Characterization of 16S rRNA gene isolated from these samples suggested a predominance of bacterial phylotypes related to Gammaproteobacteria (57-64%) and Deltaproteobacteria (27-29%). The Epsilonproteobacteria commonly found in cold seeps and hydrothermal vents were only detected in the microbial mat sample. Significantly different archaeal phylotypes were found in Calyptogena sediment and microbial mats; the former contained only Crenarchaeota clones (100% of the total archaeal clones) and the latter exclusively Euryarchaeota clones, including the anaerobic oxidation of methane archaeal groups ANME-2a and ANME-2c. Many of these lineages are as yet uncultured and undescribed groups of bacteria and archaea. Phospholipid fatty acid analysis suggested the presence of sulphate-reducing and sulphur-oxidizing bacteria. Results of intact glyceryl dialkyl glyceryl tetraether lipid analysis indicated the presence of nonthermophilic marine planktonic archaea. These results suggest that the microbial community in the Sagami Bay seep site is distinct from previously characterized cold-seep environments.

Bacteroides↗
PubMed2006

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir.

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.

Bacteria↗
PubMed2004

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea.

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

Bacteria↗
PubMed2006

Cultivation of denitrifying bacteria: optimization of isolation conditions and diversity study.

An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.

Culture Media↗
PubMed2004

Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways.

BACKGROUND: Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. Recently, it has been proposed that autoinducer-2 (AI-2), a furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine (SAH) to homocysteine, serves as a universal signal for interspecies communication. RESULTS: In this study, 138 completed genomes were examined for the genes involved in the synthesis and detection of AI-2. Except for some symbionts and parasites, all organisms have a pathway to recycle SAH, either using a two-step enzymatic conversion by the Pfs and LuxS enzymes or a one-step conversion using SAH-hydrolase (SahH). 51 organisms including most Gamma-, Beta-, and Epsilonproteobacteria, and Firmicutes possess the Pfs-LuxS pathway, while Archaea, Eukarya, Alphaproteobacteria, Actinobacteria and Cyanobacteria prefer the SahH pathway. In all 138 organisms, only the three Vibrio strains had strong, bidirectional matches to the periplasmic AI-2 binding protein LuxP and the central signal relay protein LuxU. The initial two-component sensor kinase protein LuxQ, and the terminal response regulator luxO are found in most Proteobacteria, as well as in some Firmicutes, often in several copies. CONCLUSIONS: The genomic analysis indicates that the LuxS enzyme required for AI-2 synthesis is widespread in bacteria, while the periplasmic binding protein LuxP is only present in Vibrio strains. Thus, other organisms may either use components different from the AI-2 signal transduction system of Vibrio strains to sense the signal of AI-2, or they do not have such a quorum sensing system at all.

Adenosylhomocysteinase↗
PubMedDate not supplied

[Highest level of division in classification of organisms. 3. Monodermata and Didermata].

The deepening our knowledge and embrassing the larger array of the investigated organisms leads to replacement of typological classifications with phylogenetic ones. This process seems to be the main stream of modern systematics. But typological classifications have not lost the value, remaining the important tool of the description of phylogeny. It is especially obvious today when molecular reconstructions are using so widely. However resulted phylogenetic classifications are difficult for understandable interpretation. Therefore phylogeneticist is interested in elaboration of typological classifications that can help to explain the results. As an example the phylogenetic classifications of organisms proposed recently by Cavalier-Smith (1998, 2002) and Gupta (1998, 2000) are considered. The modified system of Gupta is the most adequate description of organism phylogeny. Basal clostridia and togobacteria have to the greatest degree kept features of a common ancestor of organisms. From this common ancestor evolution spread by two phyletic lines. One of them included Gram-negative bacteria. The main groups of them have branched of from a common ancestor in the following order: (Deinococci, Chloroflexi) --> (Cyanobacteria) --> (Chlamydia, CFB, Fibrobacteria, Chlorobia) --> (Aquificae) --> --> (Epsilonproteobacteria, Deltaproteobacteria) --> (Alfaproteobacteria) --> (Betaproteobacteria) --> --> (Gammaproteobacteria). In other phyletic line the main groups were separated in the following order: (Thermotogae) --> (Clostridia, Fusobacteria) --> (Bacillae) --> (Actinobacteria). Exact position of archaebacteria and eukaryotes related to this line remains unclear. Typological division of organisms into Didermata and Monodermata (Gupta, 1998) corresponds to these two branches of a cladogram. The cell of the diderm organisms is covered with two membranes, plasmatic and outer. The cell of the monoderm organisms has only one plasmatic membrane. Development of the cellular organization at the earliest stages of evolution of a life went through use of non-lamellar (non-bilayer) lipids which could give a cell with one membrane (not two membranes as in the scenario of Cavalier-Smith (2001)). Membranes appeared at the earliest stages of the evolution of life. Therefore their distinction is quite logical to take as a principle the first typological division of organisms. At the same time the typological classifications considered beyond the framework of phylogenetics, have no independent value. Typological classifications do not give monothetic division into groups. Always there are exceptions. So, among Monodermata there are Gram-negative forms (Acidaminococcaceae, Syntrophomonadaceae, some Thermoanaerobacteriaceae), which are didermic.

Bacteria↗
PubMed2002

A non-dechlorinating strain of Dehalospirillum multivorans: evidence for a key role of the corrinoid cofactor in the synthesis of an active tetrachloroethene dehalogenase.

A strain of Dehalosprillum multivorans, designated strain N, was isolated from the same source as the formerly described tetrachloroethene (PCE)-dechlorinating D. multivorans, herein after referred to as strain K. Neither growing cells nor cell extracts of strain N were able to dechlorinate PCE. The pceA and pceB genes encoding for the PCE-reductive dehalogenase were detected in cells of strain N; and they were 100% homologous to the corresponding genes of strain K. Since the PCE dehalogenase of D. multivorans strain K contains a corrinoid cofactor, the corrinoids of strain N cells were extracted. Analysis of the corrinoids revealed the absence of the specific corrinoid, which is the cofactor of the PCE dehalogenase of strain K cells. RT-PCR of mRNA indicated that the pceA gene was transcribed in strain N cells to a far lower extent than the pceA gene of strain K under the same experimental conditions. Western blot analysis of crude extracts of strain N showed that, if at all, an insignificant amount of the apoprotein of the PCE dehalogenase was present. The results indicate that the inability of strain N to dechlorinate is due to the absence of the corrinoid cofactor of the enzyme mediating PCE dechlorination.

Chromatography, High Pressure Liquid↗
PubMed2003

Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans.

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene ( fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe(4)/S(4) cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Amino Acid Sequence↗
PubMed2006

Growth substrate dependent localization of tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans.

Sulfurospirillum multivorans is a dehalorespiring organism, which is able to utilize tetrachloroethene as terminal electron acceptor in an anaerobic respiratory chain. The localization of the tetrachloroethene reductive dehalogenase in dependence on different growth substrates was studied using the freeze-fracture replica immunogold labeling technique. When the cells were grown with pyruvate plus fumarate, a major part of the enzyme was either localized in the cytoplasm or membrane associated facing the cytoplasm. In cells grown on pyruvate or formate as electron donors and tetrachloroethene as electron acceptor, most of the enzyme was detected at the periplasmic side of the cytoplasmic membrane. These results were confirmed by immunoblots of the enzyme with and without the twin arginine leader peptide. Trichloroethene exhibited the same effect on the enzyme localization as tetrachloroethene. The data indicated that the localization of the enzyme was dependent on the electron acceptor utilized.

Bacterial Proteins↗
PubMed2005

Continuous enrichment culture and molecular monitoring to investigate the microbial diversity of thermophiles inhabiting deep-sea hydrothermal ecosystems.

The microflora developing during a continuous enrichment culture from a hydrothermal chimney sample was investigated by molecular methods. The culture was performed in a gas-lift bioreactor under anaerobic conditions, at 90 degrees C and pH 6.5, on a complex medium containing sulfur as the terminal electron acceptor. Archaeal and bacterial diversity was studied. Microorganisms affiliated with the genera Pyrococcus, Marinitoga, and Bacillus were detected through DGGE analysis of 16S rDNA. Additional sequences phylogenetically related to Thermococcus and epsilon-Proteobacteria were detected by cloning and sequencing of 16S rDNA from two samples of the enrichment culture. In comparison, the sequences retrieved from cloning analysis from an enrichment culture performed in a flask (batch condition) using the same culture medium showed that only members of the genus Thermococcus were cultivated. Therefore, continuous enrichment culture using the gas-lift bioreactor can be considered as an efficient and improved method for investigating microbial communities originating from deep-sea hydrothermal vents.

Atlantic Ocean↗
PubMed2007

Continuous enrichment culturing of thermophiles under sulfate and nitrate-reducing conditions and at deep-sea hydrostatic pressures.

A continuous culture bioreactor was developed to enrich for nitrate and sulfate reducing thermophiles under in situ deep-sea pressures. The ultimate objective of this experimental design was to be able to study microbial activities at chemical and physical conditions relevant to seafloor hydrothermal vents. Sulfide, sulfate and oxide minerals from sampled seafloor vent-chimney structures [East Pacific Rise (9 degrees 46'N)] served as source mineral and microbial inoculum for enrichment culturing using nitrate and sulfate-enriched media at 70 and 90 degrees C and 250 bars. Changes in microbial diversity during the continuous reaction flow were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rRNA gene fragments. Time series changes in fluid chemistry were also monitored throughout the experiment to assess the feedback between mineral-fluid reaction and metabolic processes. Data indicate a shift from the dominance of epsilon Proteobacteria in the initial inoculum to the several Aquificales-like phylotypes in nitrate-reducing enrichment media and Thermodesulfobacteriales in the sulfate-reducing enrichment media. Methanogens were detected in the original sulfide sample and grew in selected sulfate-enriched experiments. Microbial interactions with anhydrite and pyrrhotite in the chimney material resulted in measurable changes in fluid chemistry despite a fluid residence time only 75 min in the reactor. Changes in temperature rather than source material resulted in greater differences in microbial enrichments and mediated geochemical reactions.

Biodiversity↗
PubMed2004

The fdh operon of Sulfurospirillum multivorans.

The complete single copy fdh operon (approximately 5.7 kb) encoding the formate dehydrogenase subunits of the gram negative, reductively dehalogenating anaerobe Sulfurospirillum multivorans was sequenced and analyzed. The gene fdhA encoding the catalytically active periplasmic subunit is part of an operon (fdhEABCD) containing additional structural genes. The genes fdhEABCD were cotranscribed as indicated by RT-PCR and primer extension experiments. Two mRNAs for fdhEABCD and fdhABCD were either transcribed independently from two transcription start sites upstream of fdhE and fdhA or might result from posttranscriptional processing of the full-length fdhEABCD mRNA. The operon shows a high degree of similarity to the fdh operons of Campylobacter jejuni and Wolinella succinogenes in terms of architecture and putative cofactor binding motifs of the gene products.

Amino Acid Sequence↗
PubMed2005

Complex pattern formation of marine gradient bacteria explained by a simple computer model.

We report on the formation of conspicuous patterns by the sulfide-oxidizing bacterium Thiovulum majus and a recently described vibrioid bacterium. These microaerophilic bacteria form mucus veils on top of sulfidic marine sediment exhibiting regular spaced bacterial patterns (honeycombs, interwoven bands, or inverse honeycombs). A simple qualitative computer model, based on chemotaxis towards oxygen and the ability of the bacteria to induce water advection when attached, can explain the formation of the observed patterns. Our study shows that complex bacterial patterns in nature can be explained in terms of chemotaxis and resource optimisation without involvement of cell-cell signalling or social behavior amongst bacteria.

Bacterial Physiological Phenomena↗
PubMed2005

Displacement of epsilon-proteobacterial core genes by horizontally transferred homologous genes.

The introduction of novel genes by horizontal gene transfer (HGT) is considered an alternative mechanism for genetic adaptation, leading to diversification and speciation. The goal of this study was to determine which genes that are present in all sequenced epsilon-proteobacterial genomes were acquired by HGT. In our approach we used BLAST analysis to reduce the number of genes that subsequently needed to be analysed using more in-depth phylogenetic methods, including neighbour-joining and maximum likelihood. Among the 991 core genes found in all five completed epsilon-proteobacterial genome sequences, we identified 30 genes that were probably acquired by HGT. It is proposed that these genes displaced an ancestral core gene with a similar function. Although it was not possible to identify putative donor taxa for all acquired genes, it was clear that genes were acquired from a wide range of Bacteria, including Spirochaetes, Firmicutes, Actinobacteria, mycoplasmas and several subdivisions of the Proteobacteria. We did not observe HGT from Archaea to the epsilon-Proteobacteria. The majority of acquired genes were operational genes involved in transport, metabolism, signal transduction and energy production and conversion.

Computational Biology↗
PubMed2000

Bacterial cytochrome c nitrite reductase: new structural and functional aspects.

Cytochrome c nitrite reductase catalyzes the six-electron reduction of nitrite to ammonia as a key step within the biological nitrogen cycle. Most recently, the crystal structure of the soluble enzyme from Sulfurospirillum deleyianum could be solved to 1.9 A resolution. This set the basis for new experiments on structural and functional aspects of the pentaheme protein which carries a Ca(2+) ion close to the active site heme. In the crystal, the protein was a homodimer with ten hemes in very close packing. The strong interaction between the nitrite reductase monomers also occurred in solution according to the dependence of the activity on the protein concentration. Addition of Ca(2+) to the enzyme as isolated had a stimulating effect on the activity. Ca(2+) could be removed from the enzyme by treatment with chelating agents such as EGTA or EDTA which led to a decrease in activity. In addition to nitrite, the enzyme converted NO, hydroxylamine and O-methyl hydroxylamine to ammonia at considerable rates. With N2O the activity was much lower; most likely dinitrogen was the product in this case. Cytochrome c nitrite reductase exhibited a remarkably high sulfite reductase activity, with hydrogen sulfide as the product. A paramagnetic Fe(II)-NO, S = 1/2 adduct was identified by rapid freeze EPR spectroscopy under turnover conditions with nitrite. This potential reaction intermediate of the reduction of nitrite to ammonia was also observed with PAPA NONOate and Spermine NONOate.

Binding Sites↗