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PubMed2004

Microbial population dynamics during fed-batch operation of commercially available garbage composters.

Microbial populations in terms of quantity, quality, and activity were monitored during 2 months of start-up operation of commercially available composters for fed-batch treatment of household biowaste. All the reactors, operated at a waste-loading rate of 0.7 kg day(-1) (wet wt), showed a mass reduction efficiency of 88-93%. The core temperature in the reactors fluctuated between 31 degrees C and 58 degrees C due to self-heating. The pH declined during the early stage of operation and steadied at pH 7.4-9.3 during the fully acclimated stage. The moisture content was 48-63% early in the process and 30-40% at the steady state. Both direct total counts and plate counts of bacteria increased via two phases (designated phases I, II) and reached an order of magnitude of 10(11) cells g(-1) (dry wt) at the steady state. Microbial community changes during the start-up period were studied by culture-independent quinone profiling and denatured gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA. In all the reactors, ubiquinones predominated during phase I, whereas partially saturated menaquinones became predominant during phase II. This suggested that there was a drastic population shift from ubiquinone-containing Proteobacteria to Actinobacteria during the start-up period. The DGGE analysis of the bacterial community in one of the reactors also demonstrated a drastic population shift during phase I and the predominance of members of the phyla Proteobacteria and Bacteroidetes during the overall period. But this molecular analysis failed to detect actinobacterial clones from the reactor at any stage.

Actinobacteria↗
PubMed2004

Similarity of bacterial communities in sawdust- and straw-amended cow manure composts.

We analyzed bacterial communities in two cow manure composts derived from the same feed manure and composted in the same location, but composted with different carbon amendments, and in peat-based potting mixes amended with these composts. Bacterial communities were characterized by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of extracted DNAs, and population fingerprints generated for each sample were compared. Sequence analyses of dominant DGGE bands revealed that members of the phylum Bacteroidetes were the most dominant bacteria detected in this study (19 of 31 clones). These analyses demonstrate that bacterial community profiles of individual composts were highly similar, as were profiles of compost-amended potting mixes. However, potting mix profiles differed substantially from the original compost profiles and from that of the peat base. These data indicate that highly similar bacterial populations were active in the two composts, and suggest that the effects of the initial carbon amendment on the mature compost bacterial communities were minor, while factors such as the feed manure and composting location may have been more influential.

Animals↗
PubMed2005

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad.

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.

Ascomycota↗
PubMed2006

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments.

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.

Algorithms↗
PubMed2003

Indigenous and contaminant microbes in ultradeep mines.

Rock, air and service water samples were collected for microbial analyses from 3.2 kilometres depth in a working Au mine in the Witwatersrand basin, South Africa. The approximately metre-wide mined zone was comprised of a carbonaceous, quartz, sulphide, uraninite and Au bearing layer, called the Carbon Leader, sandwiched by quartzite and conglomerate. The microbial community in the service water was dominated by mesophilic aerobic and anaerobic, alpha-, beta- and gamma-Proteobacteria with a total biomass concentration approximately 10(4) cells ml(-1), whereas, that of the mine air was dominated by members of the Chlorobi and Bacteroidetes groups and a fungal component. The microorganisms in the Carbon Leader were predominantly mesophilic, aerobic heterotrophic, nitrate reducing and methylotrophic, beta- and gamma-Proteobacteria that were more closely related to service water microorganisms than to air microbes. Rhodamine WT dye and fluorescent microspheres employed as contaminant tracers, however, indicated that service water contamination of most of the rock samples was < 0.01% during acquisition. The microbial contaminants most likely originated from the service water, infiltrated the low permeability rock through and accumulated within mining-induced fractures where they survived for several days before being mined. Combined PLFA and terminal restriction fragment length profile (T-RFLP) analyses suggest that the maximum concentration of indigenous microorganisms in the Carbon Leader was < 10(2) cells g(-1). PLFA, 35S autoradiography and enrichments suggest that the adjacent quartzite was less contaminated and contained approximately 10(3) cells gram(-1) of thermophilic, sulphate reducing bacteria, SRB, some of which are delta-Proteobacteria. Pore water and rock geochemical analyses suggest that these SRB's may have been sustained by sulphate diffusing from the adjacent U-rich, Carbon Leader where it was formed by radiolysis of sulphide.

Air Microbiology↗
PubMed2006

Probing the importance of selected phylum-specific amino acids in sigma A of Bacteroides fragilis, a primary sigma factor naturally devoid of an N-terminal acidic region 1.1.

The sigmaA factor of Bacteroides fragilis is the prototype of a novel subgroup of primary sigma factors that are essential for growth and ensure the initiation of transcription of the housekeeping genes. This subgroup is confined to the phyla Bacteroidetes and Chlorobi. Its members carry a specific amino acid signature and are notably characterized by a short, basic N-terminal segment instead of the typical acidic region 1.1. Using in vitro mutagenesis, we investigated the importance of this basic segment and of several residues of the signature for the function of sigmaA. We have shown that the conserved residues Phe-61 and Lys-265, located in the core binding and DNA binding subregions 2.1 and 4.2, respectively, are critical for full function of the B. fragilis holoenzyme. With respect to the unusual subregion composition of sigmaA, we have shown that truncation of the basic N-terminal segment, or reversion of its charge, strongly affects the overall transcriptional activity of B. fragilis RNA polymerase in vitro. Our results indicate that the presence of the intact basic segment is required for the formation of RNA polymerase (RNAP)-promoter open complexes, the correct architecture of the transcription bubble, and efficient promoter clearance.

Amino Acid Sequence↗
PubMed2005

Petrimonas sulfuriphila gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir.

A mesophilic, anaerobic, fermentative bacterium, strain BN3(T), was isolated from a producing well of a biodegraded oil reservoir in Canada. Cells were Gram-negative, non-motile rods that did not form spores. The temperature range for growth was 15-40 degrees C, with optimum growth at 37-40 degrees C. The strain grew with up 4 % NaCl, with optimum growth in the absence of NaCl. Tryptone was required for growth. Yeast extract and elemental sulfur stimulated growth. Growth was also enhanced during fermentation of glucose, arabinose, galactose, maltose, mannose, rhamnose, lactose, ribose, fructose, sucrose, cellobiose, lactate, mannitol and glycerol. Acetate, hydrogen and CO(2) were produced during glucose fermentation. Elemental sulfur and nitrate were used as electron acceptors and were reduced to sulfide and ammonium, respectively. The G + C content of the genomic DNA was 40.8 mol%. Phylogenetic analyses of the 16S rRNA gene sequence indicated that the strain was a member of the phylum 'Bacteroidetes', distantly related to the genera Bacteroides and Tannerella (similarity values of less than 90 %). The chemotaxonomic data (fatty acids, polar lipids and quinones composition) also indicated that strain BN3(T) could be clearly distinguished from its closest cultivated relatives. This novel organism possesses phenotypic, chemotaxonomic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Petrimonas gen. nov., of which Petrimonas sulfuriphila sp. nov. is the type species. The type strain is BN3(T) (= DSM 16547(T) = JCM 12565(T)).

Acetic Acid↗
PubMed2006

Pedobacter aquatilis sp. nov., isolated from drinking water, and emended description of the genus Pedobacter.

A Gram-negative, rod-shaped, non-spore-forming bacterium (strain AR107(T)) was isolated from the drinking water distribution system of Seville (Spain). A polyphasic taxonomic study of the isolate resulted in its identification as a member of the genus Pedobacter. On the basis of 16S rRNA gene sequence comparisons, strain AR107(T) was shown to belong to the phylum Bacteroidetes, being related to members of the genus Pedobacter. It showed 95.2 % sequence similarity with respect to the type strains of Pedobacter heparinus and Pedobacter piscium, and 94.1 % similarity with respect to the type strain of Pedobacter himalayensis. The predominant fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c), which supports the affiliation of strain AR107(T) to the genus Pedobacter. The DNA G+C content of this strain was 38 mol%. On the basis of the phenotypic, phylogenetic and genotypic results, strain AR107(T) represents a novel species, for which the name Pedobacter aquatilis sp. nov. is proposed. The type strain is AR107(T) (=CCM 7347(T)=CECT 7114(T)=JCM 13454(T)).

Bacteroidetes↗
PubMed2006

Isolation of aerobic, gliding, xylanolytic and laminarinolytic bacteria from acidic Sphagnum peatlands and emended description of Chitinophaga arvensicola Kampfer et al. 2006.

Four aerobic, heterotrophic, yellow-pigmented and flexirubin-producing bacterial strains with gliding motility were isolated from acidic Sphagnum-dominated wetlands of Northern Russia. These bacteria are capable of degrading xylan, laminarin and some other polysaccharides, but not cellulose, pectin or chitin. The four strains possess almost identical 16S rRNA gene sequences and are most closely related (98.9-99.5 % sequence similarity) to the recently reclassified species of the phylum Bacteroidetes, Chitinophaga arvensicola Kämpfer et al. 2006, formerly known as [Cytophaga] arvensicola Oyaizu et al. 1983. However, the novel isolates from Sphagnum peat differed from C. arvensicola DSM 3695(T) in their ability to degrade xylan and starch, by greater tolerance of acidic pH and by their inability to reduce nitrate. An emended description of this species is proposed.

Bacteria, Aerobic↗
PubMed2006

Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand.

Three strains of gliding bacteria, 24(T), 62 and 71, isolated from a marine sponge and algae from the southern coastline of Thailand, were studied using a polyphasic approach to clarify their taxonomic positions. A phylogenetic analysis based on 16S rRNA gene sequences showed that the three isolates formed a distinct lineage within the family 'Saprospiraceae' of the phylum Bacteroidetes and were related to members of the genus Saprospira. The G+C contents of the isolates were in the range 38-39 mol%. The major respiratory quinone was MK-7. The predominant cellular fatty acids were 20 : 4omega6c (arachidonic acid), 16 : 0 and iso-17 : 0. On the basis of morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA hybridization data and 16S rRNA gene sequences, the isolates represent a novel species of a novel genus, for which the name Aureispira marina gen. nov., sp. nov. is proposed. The type strain of Aureispira marina is 24(T) (=IAM 15389(T)=TISTR 1719(T)).

Animals↗
PubMed2005

Marine diatom species harbour distinct bacterial communities.

We examined bacterial dynamics in batch cultures of two axenic marine diatoms (Thalassiosira rotula and Skeletonema costatum). The axenic diatoms were inoculated with natural bacterial assemblages and monitored by 4,6-diamidino-2-phenolindole (DAPI) counts, denaturing gradient gel electrophoresis (DGGE) with subsequent analysis of excised, sequenced 16S rRNA gene fragments, and fluorescence in situ hybridization (FISH) with group-specific 16S rRNA oligonucleotide probes. Our results show that algal growth exhibited pronounced differences in axenic treatments and when bacteria were present. Bacterial abundance and community structure greatly depended on species, growth and physiological status of even closely related algae. Free-living and phytoplankton-associated bacteria were very different from each other and were dominated by distinct phylogenetic groups. The diatom-associated bacteria mainly belonged to the Flavobacteria-Sphingobacteria group of the Bacteroidetes phylum whereas free-living bacteria, which were rather similar in both cultures, comprised mainly of members of the Roseobacter group of alpha-Proteobacteria. Presence and disappearance of specific bacteria during algal growth indicated pronounced differences in environmental conditions over time and selection of bacteria highly adapted to the changing conditions. Tight interactions between marine bacteria and diatoms appear to be important for the decomposition of organic matter and nutrient cycling in the sea.

Alphaproteobacteria↗
PubMed2006

Characterization of acid-tolerant H/CO-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State.

Two methanogenic cultures were enriched from acidic peat soil using a growth medium buffered to c. pH 5. One culture, 6A, was obtained from peat after incubation with H(2)/CO(2), whereas culture NTA was derived from a 10(-4) dilution of untreated peat into a modified medium. 16S rRNA gene clone libraries from each culture contained one methanogen and two bacterial sequences. The methanogen 16S rRNA gene sequences were 99% identical with each other and belonged to the novel "R-10/Fen cluster" family of the Methanomicrobiales, whereas their mcrA sequences were 96% identical. One bacterial 16S rRNA gene sequence from culture 6A belonged to the Bacteroidetes and showed 99% identity with sequences from methanogenic enrichments from German and Russian bogs. The other sequence belonged to the Firmicutes and was identical to a thick rod-shaped citrate-utilizing organism isolated from culture 6A, the numbers of which decreased when the Ti (III) chelator was switched from citrate to nitrilotriacetate. Bacterial clones from the NTA culture clustered in the Delta- and Betaproteobacteria. Both cultures contained thin rods, presumably the methanogens, as the predominant morphotype, and represent a significant advance in characterization of the novel acidiphilic R-10 family methanogens.

Acids↗
PubMed2006

Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions.

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 x 10(9) marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10(5) marker equivalents per liter).

Animals↗
PubMed2005

Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients.

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

Actinobacteria↗
PubMed2006

Effect of nutrient periodicity on microbial community dynamics.

When microbes are subjected to temporal changes in nutrient availability, growth rate and substrate affinity can contribute to competitive fitness and thereby affect microbial community structure. This hypothesis was tested using planktonic bacterial communities exposed to nutrient additions at 1-, 3-, 7-, or 14-day intervals. Growth rates after nutrient addition were inversely proportional to the pulse interval and declined from 0.5 h(-1) to 0.15 h(-1) as the pulse interval increased from 1 to 14 days. The dynamics of community structure were monitored by 16S rRNA gene PCR-denaturing gradient gel electrophoresis. At pulse intervals of more than 1 day, the community composition continued to change over 130 days. Although replicate systems exposed to the same pulse interval were physiologically similar, their community compositions could exhibit as much dissimilarity (Dice similarity coefficients of <0.5) as did systems operated at different intervals. Bacteria were cultivated from the systems to determine if the physiological characteristics of individual members were consistent with the measured performance of the systems. The isolates fell into three bacterial divisions, Bacteroidetes, Proteobacteria, and Actinobacteria. In agreement with community results, bacteria isolated from systems pulsed every day with nutrients had higher growth rates and ectoaminopeptidase specific activities than isolates from systems pulsed every 14 days. However, the latter isolates did not survive starvation longer than those provided with nutrients every day. The present study demonstrates the dynamic nature of microbial communities exposed to even simple and regular environmental discontinuities when a substantial pool of species that can catabolize the limiting substrate is present.

Actinobacteria↗
PubMed2006

Acetylornithine transcarbamylase: a novel enzyme in arginine biosynthesis.

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.

Arginine↗
PubMed2004

Bacterial alpha2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

BACKGROUND: Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor alpha2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. RESULTS: Database searches with metazoan alpha2-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial alpha2-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial alpha2-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial alpha2-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial alpha2-macroglobulins, indicating that bacterial alpha2-macroglobulin is a colonization rather than a virulence factor. CONCLUSIONS: Metazoan alpha2-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. Alpha2-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial alpha2-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial alpha2-macroglobulins might provide useful targets for enhancing vaccine efficacy in combating infections.

Alphaproteobacteria↗