Beta Subclass Of Proteobacteria Research Articles

Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores.

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

In situ probing reveals Aquabacterium commune as a widespread and highly abundant bacterial species in drinking water biofilms

Drinking water biofilm communities originated from distribution systems in Hamburg, Berlin, Mainz and Stockholm were subjected to a top-to-bottom in situ analysis with rRNA-targeted, fluorescently labeled oligonucleotide probes including beta1–8, specific for drinking water bacteria within the beta-subclass of Proteobacteria. Independent from the raw water source used, between 19 and 77% of the total attached bacterial cell counts on polyethylen slides could be hybridized with probes beta1–8 and 19–53% of the bacteria were affiliated to the autochthonous species Aquabacterium commune. To investigate seasonal fluctuations of the bacterial population composition, polyethylen slides exposed during different times of the year were analyzed. Although changes in the species composition of the biofilms could be observed, A. commune was a dominant community member in all of the investigated biofilms. In situ probing with two oligonucleotide probes specific for A. commune revealed that during distinct sampling times an additional yet unknown species was present in the biofilm. Material induced population shifts were studied using glass, polyethylene low and high density and soft-PVC as biofilm substrata. The community composition on soft-PVC differed significantly from the other materials, the dominant species A. commune was replaced by other beta- Proteobacteria hybridizing to an amount of 66% with the Aquabacterium citratiphilum specific probe beta4.

Changes in the community structure of ammonia‐oxidizing bacteria during secondary succession of calcareous grasslands

The community structure of beta-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S rRNA and the AmoA subunit of ammonia monooxygenase were used. Similar shifts were detected in the composition of the ammonia oxidizer communities by both culture-dependent and independent approaches. A predominance of Nitrosospira sequence cluster 3 in early successional fields was replaced by Nitrosospira sequence cluster 4 in late successional fields. The rate of this shift differed between the two areas examined. This shift occurred in a background of relative stability in the dominant bacterial populations in the soil, as determined by domain-level polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Molecular analysis of enrichment cultures obtained using different ammonia concentrations revealed biases towards Nitrosospira sequence cluster 3 or Nitrosospira sequence cluster 4 under high- or low-ammonia conditions respectively. High-ammonia MPNs suggested a decease in ammonia oxidizer numbers with succession, but low-ammonia MPNs and competitive PCR targeting amoA failed to support such a trend. Ammonia turnover rate, not specific changes in plant diversity and species composition, is implicated as the major determinant of ammonia oxidizer community structure in successional chalk grassland soils.

In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 4′,6-diamidino-2-phenylindol (DAPI) at a polyphosphate-probing concentration

Polyphosphate-accumulating bacteria in activated sludge samples from an anaerobic/aerobic batch-type reactor for enhanced biological phosphate removal (EBPR) were identified by dual staining with ribosomal RNA-targeted oligonucleotide probes and 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI) at a polyphosphate-probing concentration. The staining with the oligonucleotide probes showed that there were four major bacterial groups in the sludge: the beta subclass of Proteobacteria, Gram-positive bacteria with a high G+C content, the alpha subclass of Proteobacteria, and bacteria belonging to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum. By staining with DAPI, many of the Gram-positive bacteria with a high G+C content and the alpha subclass of Proteobacteria fluoresced bright yellow. These bacteria were considered to accumulate a large amount of polyphosphate.

Phylogenetic Identification of Two Major Nitrogen-Fixing Bacteria Associated with Sugarcane

Acetobacter diazotrophicus and Herbaspirillum seropedicae were identified by genetic methods based on 16S rRNA sequences. A specific PCR method in combination with probing was developed for A. diazotrophicus. The PCR system includes four primers, of which the primers named AC (CTGTTTCCCGCAAGGGAC) and DI (GCGCCCCATTGCTGGGTT) generated an 445 bp amplicon in all of the 11 A. diazotrophicus strains tested. The phylogenetic position of H. seropedicae was determined. H. seropedicae forms with Oxalobacter formigenes a separate lineage in the beta-subclass of Proteobacteria.

Morphology, In-Situ characterization with rRNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge

The microbial communities in activated sludge acclimated with either acetate or glucose as the major carbon source under phosphorus limited or rich conditions were investigated morphologically, phylogenetically and chemotaxonomically. The sludge with a minimized polyphosphate content was dominated by tetrad shaped bacteria, which were suspected to be ‘glycogen accumulating bacteria (GAOs) or G bacteria’ The sludge containing high polyphosphate was dominated by cluster forming coccus bacteria. Quinone analyses suggested that all the sludge tested contained various ubiquinones and menaquinones, of which the ubiquinones Q-8 and Q-10 were dominant. Analyses with rRNA targeted probes showed that beta sub class of Proteobacteria was most predominant in all sludges tested. Morphological, phylogenetic and chemotaxonomic investigation all indicated that both high and low P sludges are microbiologically diverse.

Thauera linaloolentis sp. nov. and Thauera terpenica sp. nov., Isolated on Oxygen-containing Monoterpenes (Linalool, Menthol, and Eucalyptol and Nitrate

The monoterpenes menthol, linalool, and eucalyptol were recently used as sole electron donor and carbon source for the isolation of three denitrifying bacterial strains 21Mol, 47Lol, and 58Eu. The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism. Monoterpenes were completely mineralised to carbon dioxide, nitrate was reduced to dinitrogen. Strain 47Lol utilised aliphatic monoterpenes, strain 21Mol oxygenated monocyclic monoterpenes, and strain 58Eu the bicyclic eucalyptol and monocyclic monoterpene alkenes. The fatty acid composition of the strains indicated an allocation to the rRNA group III of pseudomonads. Comparative 16S rRNA gene sequence analyses revealed that the new isolates can be assigned as members of the genus Thauera within the beta subclass of Proteobacteria. DNA-DNA hybridisation studies indicated a relateness of 68.5% between strains 21Mol and 58Eu which shared 36.0% and 40.6% DNA similarity with strain 47Lol. The strains are described as new species belonging to the genus Thauera, strain 47Lol (DSM 12138T) as T. linaloolentis sp. nov. and strains 21Mol and 58Eu as T. terpenica sp. nov. with strain 58Eu (DSM 12139T) as type strain.

Alcaligenes defragrans sp. nov., Description of Four Strains Isolated on Alkenoic Monoterpenes ((+)-menthene, α-pinene, 2-carene, and α-phellandrene) and Nitrate

Four pseudomonad strains 51Men, 54Pin, 62Car and 65Phen were recently isolated on the monoterpenes (+)-menthene, alpha-pinene, 2-carene and alpha-phellandrene as sole carbon source and nitrate as electron acceptor. These bacteria were characterised. The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism. Monoterpenes as carbon sources were completely mineralised to carbon dioxide. The physiology of all strains was very similar, but displayed an individual utilisation preference for the isolation substrate. The fatty acid composition of whole cells showed a high degree of similarity to that of Alcaligenes faecalis. Comparative 16S rDNA data analysis placed the isolates into the beta-subclass of Proteobacteria in a common offshoot together with Alcaligenes and Bordetella species. On the basis of these characteristics, the strains are described as a new species belonging to the genus Alcaligenes, A. defragrans sp. nov., with strain 54Pin (DSM 12141T) as type strain.

In situ structure/function studies in wastewater treatment systems

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O2, NH4+, NO2−, and NO3− and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift from an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 μm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Proteobacteria and a thus far unknown nitrite oxidising population.

Morphology, in-situ characterization with rRNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge

The microbial communities in activated sludge acclimated with either acetate or glucose as the major carbon source under phosphorus limited or rich conditions were investigated morphologically, phylogenetically and chemotaxonomically. The sludge with a minimized polyphosphate content was dominated by tetrad shaped bacteria, which were suspected to be ‘glycogen accumulating bacteria (GAOs) or G bacteria’ The sludge containing high polyphosphate was dominated by cluster forming coccus bacteria. Quinone analyses suggested that all the sludge tested contained various ubiquinones and menaquinones, of which the ubiquinones Q-8 and Q-10 were dominant. Analyses with rRNA targeted probes showed that beta sub class of Proteobacteria was most predominant in all sludges tested. Morphological, phylogenetic and chemotaxonomic investigation all indicated that both high and low P sludges are microbiologically diverse.

In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.

Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow).

Microbial assemblages on large organic aggregates (lake snow) of Lake Constance, Germany, were analyzed with rRNA-directed fluorescent oligonucleotide probes specific for the domain Bacteria and the alpha-, beta-, and gamma-subclasses of the class Proteobacteria. Lake snow aggregates were either collected in situ by SCUBA diving or in a sediment trap at 50 m or formed of natural lake water incubated in rolling cylinders under simulated in situ conditions. For the latter aggregates, the time course of the microbial colonization was also examined. The natural aggregates and those made in rolling cylinders were composed of the particulate organic material present in the lake and thus reflected the composition of the ambient plankton community. All types of lake snow aggregates examined were heavily colonized by microbial cells and harbored between 0.5 x 10(6) and > 2 x 10(6) cells aggregate -1. Between 55 and 100% of the microbial cells stained with 4', 6-diamidino-2-phenylindole (DAPI) could be visualized with the domain Bacteria-specific probe. In most samples, beta-subclass proteobacteria dominated the microbial community, constituting 27 to 42% of total cells as counted by DAPI staining, irrespective of the composition of the aggregates. During the time course experiments with the laboratory-made aggregates, the fraction of beta-subclass proteobacteria usually increased over time. Except for a few samples, alpha- and gamma-subclass proteobacteria were far less abundant than beta-subclass proteobacteria, constituting 11 to 25 and 9 to 33% of total cells, respectively. Therefore, we assume that a specific aggregate-adapted microbial community was established on the aggregates. Because the compositions of the microbial assemblages on natural and laboratory-made aggregates were similar, we conclude that aggregates made in rolling cylinders are good model system with which to examine the formation and microbial colonization of macroscopic organic aggregates.

Flow cytometric analysis of activated sludge with rRNA-targeted probes.

Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.

In situ characterization of the microbial consortia active in two wastewater treatment plants

Activated sludge samples from a one-state-system of a municipal sewage plant and a sequencing batch reactor (SBR) treating dairy-sewage were characterized using 16S and 23S ribosomal RNA (rRNA) targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Cell counts were determined by a combination of in situ hybridization with fluorescently-labelled oligonucleotide probes and epifluorescence microscopy. Around 80% of total cells, as determined with the DNA specific fluorochrome DAPI, hybridized with a Bacteria specific probe. This indicated that the majority of microscopically visualized cells were indeed physiologically active. The samples of the municipal sewage plant were dominated by proteobacteria belonging to the beta-subclass, whereas small cells hybridizing with a cytophaga-flavobacterium-specific probe were most abundant in the batch reactor. Comparison of in situ microbial community structures and the heterotrophic saprophyte flora (analysed with standard cultivation techniques) resulted in major discrepancies. The nutrient rich medium favoured growth of gamma-subclass proteobacteria (e.g. Acinetobacter sp.) which were only a minor constituents (< 10%) of both sludge consortia. Dominating groups like the beta-subclass proteobacteria, the cytophagaflavobacteria cluster and the Gram-positive bacteria with a high G + C content of DNA were suppressed. These results suggest that the application of in situ hybridization techniques can yield a more complete understanding of the microbial populations involved in the purification of sewage.

Molecular Systematics of the Genus Zoogloea and Emendation of the Genus

Phylogenetic relationships among strains of Zoogloea and related taxa were determined by 16S rDNA sequencing and genomic DNA hybridization techniques. The 16S rRNA gene was amplified by the polymerase chain reaction with a pair of eubacterial consensus primers and sequenced directly by using an automated fluorescent DNA sequencer. Sequence comparisons and distance matrix tree analysis revealed that Zoogloea ramigera IAM 12136 (= N. C. Dondero 106, type strain) and Zoogloea sp. ATCC 19324 formed a lineage with Rhodocyclus purpureus in the beta subclass of Proteobacteria. Z. ramigera IAM 12670 (= P. R. Dugan 115) was shown to belong to another cluster with Alcaligenes eutrophus and Pseudomonas cepacia in the beta subclass. In contrast, Z. ramigera IAM 12669 (= K. Crabtree I-16-M) proved to be a member of the alpha subclass of the Proteobacteria, closely related to Agrobacterium tumefaciens. Genomic DNA hybridization studies also showed that there is genetic diversity among the strains currently designated Z. ramigera, but typical Zoogloea strains, characterized by their production of rhodoquinones, are highly related to each other and can be regarded as a single species. On the basis of the molecular data, together with the early phenotypic and chemotaxonomic information, we have emended the generic description of Zoogloea.

Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure.

Bacterial community structures in activated sludge samples from aeration tanks of a two-stage system with a high-load first stage and a low-load second stage were analyzed with oligonucleotide probes. The probes were complementary to conserved regions of the rRNA of the alpha, beta, and gamma subclasses of proteobacteria and of all bacteria. Group-specific cell counts were determined by in situ hybridization with fluorescent probe derivatives. Contributions of the proteobacterial subclasses to total bacterial rRNA were quantified by dot blot hybridization with digoxigenin-labeled oligonucleotides. The activated sludge samples were dominated by proteobacteria from the alpha, beta, or gamma subclass. These proteobacteria account for about 80% of all active bacteria found in the activated sludge. For both samples the community structures determined with molecular techniques were compared with the composition of the heterotrophic saprophyte flora isolated on nutrient-rich medium. Probes were used to rapidly classify the isolates and to directly monitor population shifts in nutrient-amended, activated sludge samples. The rich medium favored growth of gamma-subclass proteobacteria (e.g., enterobacteria) and selected against beta-subclass proteobacteria. The culture-dependent community structure analysis of activated sludge produced partial and heavily biased results. A more realistic view will be obtained by using in situ techniques.

Distribution of hydroxypolyamines, amino-propylcadaverine, and spermine in thermophilic, hydrogen-oxidizing pseudomonads.

A slightly thermophilic hydrogen bacterium, Pseudomonas hydrogenothermophila, was found to contain 2-hydroxyputrescine, 2-hydroxyspermidine, diaminopropane, putrescine, cadaverine, spermidine, aminopropylcadaverine and spermine. A mesophilic hydrogen-oxidizing pseudomonad, Pseudomonas hydrogenovora contained putrescine, 2-hydroxyputrescine, and spermidine. The polyamine distribution pattern of the latter is rather common in the beta subclass of Proteobacteria. The occurrence of 2-hydroxyspermidine, aminopropylcadaverine and spermine is unique in the thermophile.

Polyamine Pattern as a Chemotaxonomic Marker within the Proteobacteria

The polyamine pool of 50 Gram-negative, aerobic species belonging to the class Proteobacteria (Stackebrandt et al., 1988), formerly “purple bacteria and relatives” (Woese et al., 1985b; Woese, 1987) was analyzed by high performance liquid chromatography (HPLC) in order to evaluate polyamines as a chemotaxonomic marker within this class. The unusual polyamine 2-hydroxyputrescine emerged as the ubiquitous component of absolute specificity within the beta subclass of Proteobacteria. The species of the alpha subclass had a triamine as the characteristic component, e. g., spermidine in the alpha-3 subclass and sym-homospermidine in the alpha-2 subclass. In fluorescent pseudomonads belonging to the gamma subclass, where genus-specificity of polyamine patterns is emerging, putrescine and spermidine were found as the main components.