Gamma Subclass Of Proteobacteria Research Articles

The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge.

The bacteria involved in organic pollutant degradation in pelagic deep-sea environments are largely unknown. In this report, the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was analyzed in deep-sea water on the Southwest Indian Ridge (SWIR). After enrichment with a PAH mixture (phenanthrene, anthracene, fluoranthene, and pyrene), nine bacterial consortia were obtained from depths of 3946–4746 m. While the consortia degraded all four PAHs when supplied in a mixture, when PAHs were tested individually, only phenanthrene supported growth. Thus, degradation of the PAH mixture reflected a cometabolism of anthracene, fluoranthene, and pyrene with phenanthrene. Further, both culture-dependent and independent methods revealed many new bacteria involved in PAH degradation. Specifically, the alpha and gamma subclasses of Proteobacteria were confirmed as the major groups within the communities. Additionally, Actinobacteria, the CFB group and Firmicutes were detected. Denaturing Gradient Gel Electrophoresis (DGGE) analysis showed that bacteria closely affiliated with Alcanivorax, Novosphingobium, and Rhodovulum occurred most frequently in different PAH-degrading consortia. By using general heterotrophic media, 51 bacteria were isolated from the consortia and of these 34 grew with the PAH mixture as a sole carbon source. Of these, isolates most closely related to Alterierythrobacter, Citricella, Erythrobacter, Idiomarina, Lutibacterium, Maricaulis, Marinobacter, Martelella, Pseudidiomarina, Rhodobacter, Roseovarius, Salipiger, Sphingopyxis, and Stappia were found to be PAH degraders. To the best of our knowledge, this is the first time these bacteria have been identified in this context. In summary, this report revealed significant diversity among the PAH-degrading bacteria in the deep-sea water column. These bacteria may play a role in PAH removal in deep-sea environments.

Towards implementation of a benthic microbial fuel cell in lake Furnas (Azores): Phylogenetic affiliation and electrochemical activity of sediment bacteria

This work was conducted to examine the composition and electrochemical activity of the bacterial community inhabiting lake Furnas sediments (Azores). Fingerprinting analysis of the bacterial 16S rRNA gene fragment was done by denaturing gradient gel electrophoresis. The sequences retrieved from lake Furnas sediments were affiliated to Bacteroidetes/Chlorobi group, Chloroflexi, Alfa-, Delta-, and Gamma-subclasses of Proteobacteria, Cyanobacteria, and Gemmatimonadetes. A cyclic voltammetric study was carried out with an enriched sediment bacterial suspension in a standard two chamber electrochemical cell using a carbon paper anode. Cyclic voltammograms (scan rate of 50 mV/s) showed the occurrence of oxidation–reduction reactions at the carbon anode surface. The benthic microbial fuel cell operated with lake Furnas sediments presented a low power density (1 mW/m 2) indicating that further work is required to optimise its power generation. These results suggested that sediment bacteria, probably from the Delta- and Gamma-subclasses of Proteobacteria, were electroactive under tested conditions.

Microbial community analysis of a full-scale membrane bioreactor treating industrial wastewater

A Kubotatrade mark submerged membrane bio-reactor was applied to treat wastewater from a sugar manufacturing industry. To achieve optimal results, fundamental and extended understanding of the microbiology is important. Fluorescence in situ hybridization was used to evaluate the microbial community present. The majority of cells visualized in the sludge flocs by staining with the DNA fluorochrome DAPI, hybridized strongly with a bacterial probe. Probes specific for the alpha-, beta-, and gamma-subclasses of proteobacteria and high G + C Gram positive bacteria were used to characterize the community structures by in situ hybridization. Sampling was carried out over 12 weeks and samples were fixed with 4% paraformaldehyde for gram positive organisms and ice cold ethanol for gram negative organisms. The activated sludge population usually constitutes about 80 to 90% of proteobacteria. However, in this study it was found that a relatively small amount of proteobacteria was present within the system. No positive hybridization signal was observed with any of the applied eubacterial family- level probes.

Chemolithoautotrophic oxidation of thiosulfate and phylogenetic distribution of sulfur oxidation gene (soxB) in rhizobacteria isolated from crop plants

Twenty-one thiosulfate-oxidizing bacteria were isolated from rhizosphere soils and 16S rRNA analysis revealed that the isolates were affiliated with seven different phylogenetic groups within the Beta and Gamma subclasses of Proteobacteria and Actinobacteria. Among these, five genera, including Dyella, Burkholderia, Alcaligenes, Microbacterium and Leifsonia sp., represented new sulfur oxidizers in rhizosphere soils. The thiosulfate-oxidizing Dyella, Burkholderia, Alcaligenes, Microbacterium, Leifsonia and Pandoraea were able to grow chemolithotrophically with a medium containing thiosulfate and exhibited growth coupled with thiosulfate oxidation. They accumulated intermediate products such as sulfur, sulfite and trithionate in the spent medium during the time course of thiosulfate oxidation, and these products were finally oxidized into sulfate. Furthermore, they possessed thiosulfate-metabolizing enzymes such as rhodanese, thiosulfate oxidase, sulfite oxidase and trithionate hydrolase, suggesting that these bacteria use the ‘S4 intermediate’ (S4I) pathway for thiosulfate oxidation. Phylogenetic analysis of the soxB gene revealed that Pandoraea sp. and Pandoraea pnomenusa strains formed a separate lineage within Betaproteobacteria.

Bacterium–bacterium inhibitory interactions among psychrotrophic bacteria isolated from Antarctic seawater (Terra Nova Bay, Ross Sea)

One hundred and forty bacteria isolated from Antarctic seawater samples were examined for their ability to inhibit the growth of indigenous isolates and their sensitivity to antibacterial activity expressed by one another. On the basis of 16S rRNA gene sequencing and analysis, bacterial isolates were assigned to five phylogenetically different taxa, Actinobacteria, alpha and gamma subclasses of Proteobacteria, Bacillaceae, and Bacteroidetes. Twenty-one isolates (15%), predominantly Actinobacteria, exhibited antagonistic properties against marine bacteria of Antarctic origin. Members of Bacteroidetes and Firmicutes did not show any inhibitory activity. Differences were observed among inhibition patterns of single isolates, suggesting that their activity was more likely strain-specific rather than dependent on phylogenetic affiliation. A novel analysis based on network theory confirmed these results, showing that the structure of this population is probably robust to perturbations, but also that it depends strongly on the most active strains. The determination of plasmid incidence in the bacterial strains investigated revealed that there was no correlation between their presence and the antagonistic activity. The data presented here provide evidence for the antagonistic interactions within bacterial strains inhabiting Antarctic seawater and suggest the potential exploitation of Antarctic bacteria as a novel source of antibiotics.

Molecular Characterization of an Oil-Degrading Cyanobacterial Consortium

Recent studies have shown that the cyanobacterium Microcoleus chthonoplastes forms a consortium with heterotrophic bacteria present within the cyanobacterial sheath. These studies also show that this consortium is able to grow in the presence of crude oil, degrading aliphatic heterocyclic organo-sulfur compounds as well as alkylated monocyclic and polycyclic aromatic hydrocarbons. In this work, we characterize this oil-degrading consortium through the analysis of the 16S rRNA gene sequences. We performed the study in cultures of Microcoleus grown in mineral medium and in cultures of the cyanobacterium grown in mineral medium supplemented with crude oil. The results indicate that most of the clones found in the polluted culture correspond to well-known oil-degrading and nitrogen-fixing microorganisms, and belong to different phylogenetic groups, such as the Alpha, Beta, and Gamma subclasses of Proteobacteria, and the Cytophaga/Flavobacteria/Bacteroides group. The control is dominated by one predominant organism (88% of the clones) closely affiliated to Pseudoxanthomonas mexicana (similarity of 99.8%). The presence of organisms closely related to well-known nitrogen fixers such as Rhizobium and Agrobacterium suggests that at least some of the cyanobacteria-associated heterotrophic bacteria are responsible for nitrogen fixation and degradation of hydrocarbon compounds inside the polysaccharidic sheath, whereas Microcoleus provides a habitat and a source of oxygen and organic matter.

Monitoring microbial community in a subsurface soil contaminated with hydrocarbons by quinone profile

A natural attenuation experiment was carried out using a lysimeter for 308 days after contaminating the subsoil with hydrocarbons (HCs) and the changes in the structures of microbial community in the hydrocarbon (HC) contaminated subsoil were monitored by quinone profile analysis. The residues of HCs remained for 217 days in the subsoil after the contamination. The amount of total quinones (TQ), an indicator of microbial biomass, significantly increased in the HC contaminated subsoil for 217 days, comparing with that of the background subsoil or the subsoil before the addition of HCs. The major quinone species and the quinone composition, indicators of community structure, were significantly different between the HC contaminated soil and the background soil for 217 days. The major increased quinine species in the HC contaminated soil were menaquinone-8(H4), menaquinone-9(H2) and ubiquinone-9, indicating the propagation of Gram-positive bacteria with high guanine and cytosine content and gamma-subclass of Proteobacteria and fungi. There was no significant difference in the diversity of the quinone species (DQ), an indicator of taxonomic diversity of microbial community, except for the decrease in DQ in the shallow subsoil after 35 days when a high concentration of HCs was detected. After 308 days when the HCs in the subsoil disappeared, TQ returned to the level of the background soil, and no significant difference in quinone composition were observed between the HC contaminated soil and the background soil. The results suggested that respiratory quinones are effective biomarkers for characterizing the temporal changes of microbial community in the HC contaminated subsoil.

Isolation and characterization of bacteria capable of degrading phenol and reducing nitrate under low-oxygen conditions.

Nitrate-reducing bacteria capable of degrading phenol were isolated from natural and contaminated environments under low-oxygen conditions with nitrate-containing media, using phenol as a sole carbon and energy source. A total of 27 bacteria able to degrade phenol and reduce nitrate under low-oxygen conditions were isolated from all of the inoculum samples, regardless of previous phenol contamination. For all of these bacteria, oxygen was an essential requirement for phenol degradation. Nitrate reduction by 19 of the strains was insensitive to 10 mM sodium azide, and these strains were placed into the alpha- and beta-subclasses of Proteobacteria and two were Gram-positive bacteria. To date, the order of Rhizobiales has hardly been reported to have an ability to degrade aromatic compounds. Interestingly, our study showed that all isolates that were placed into the alpha-subclass of Proteobacteria are in the order of Rhizobiales. Furthermore, the genus Agrobacterium was isolated from most inoculum samples and one genus of Gram-positive bacteria, Staphylococcus, was also isolated. In the case of the remaining eight strains, nitrate reduction was inhibited by 10 mM sodium azide. Of these strains, seven were placed into the gamma-subclass of Proteobacteria.

A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures.

Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.

Microbial Diversity in an in situ Reactor System Treating onochlorobenzene 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.

Formation and bacterial composition of young, natural biofilms obtained from public bank-filtered drinking water systems

In Germany, bank-filtered raw water and ground water is mainly used for drinking water conditioning. Microorganisms, which are neither retarded by the subsoil passage of the bank-filtration, nor by the different filtration and disinfection steps at the water works, cause the growth of biofilms on different materials originally used in drinking water distribution systems. The development, the phylogenetical diversity and the bacterial metabolic activities of biofilms on polyethylen (PE-HD), polyvinylchlorid (PVC), steel and copper were analyzed at different sampling points. The incubation experiments were performed under natural conditions using a flow device technique. The devices were installed after the activated carbon filters and disinfection step at the water works and at two different house branch connections within the distribution system of the conditioned drinking water of the water works. The synthetic materials were colonized very rapidly within a few days in significant higher densities than steel and copper. The total bacterial cell counts of the biofilms were measured by DAPI (4′,6-diamidino-2-phenylindole) staining. The metabolic activities of the bacteria were quantified by the use of the redox dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride), which is frequently described as an indicator for respiration. The highest respiratory activities were observed in biofilms from synthetic materials grown after the activated carbon filters at the water works. A significant reduction of the total bacterial cell counts and respiratory activities of about 80% was measured due to the disinfection at the water works. A time dependent growth of bacteria in biofilms was observed at the two sampling points within the distribution system, whereas the percentage of CTC-reducing cells stabilized at 35%. The in situ-hybridizations with fluorescence labelled, group-specific rRNA targeted oligonucleotide probes revealed the following: (i) bacteria of the Beta- and Gamma-subclass of Proteobacteria were found most frequently within the biofilm population, (ii) the percentage of the different subclasses depended on the used material, (iii) there were no significant changes in bacterial subclass composition of the biofilms taken from the water works and house branch connections. In addition, polymerase chain reaction (PCR), southern blot hybridization and in situ hybridization were used to detect facultative pathogenic bacteria in biofilms. Non-pneumophila Legionella were found in a relative high percentage up to about 7% in many biofilms, whereas fecal streptococci were detected only in few biofilms of the drinking water distribution systems

Physiological characterization of a bacterial consortium reductively dechlorinating 1,2,3- and 1,2,4-trichlorobenzene.

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO2, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.

Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture.

Culturable bacteria that were numerically important members of a marine enrichment community were identified and characterized phylogenetically. Selective and nonselective isolation methods were used to obtain 133 culturable bacterial isolates from model marine communities enriched with the high-molecular-weight (lignin-rich) fraction of pulp mill effluent. The culture collection was screened against community DNA from the lignin enrichments by whole-genome hybridization methods, and three marine bacterial isolates were identified as being numerically important in the communities. One isolate was in the alpha-subclass of Proteobacteria, and the other two were in the gamma-subclass of Proteobacteria. Isolate-specific 16S rRNA oligonucleotide probes designed to precisely quantify the isolates in the lignin enrichment communities indicated contributions ranging from 2 to 32% of enrichment DNA, values nearly identical to those originally obtained by the simpler whole-genome hybridization method. Two 16S rRNA sequences closely related to that of one of the isolates, although not identical, were amplified via PCR from the seawater sample originally used to inoculate the enrichment medium. Partial sequences of 14 other isolates revealed significant phylogenetic diversity and unusual sequences among the culturable lignin enrichment bacteria, with the Proteobacteria, Cytophaga-Flavobacterium, and gram-positive groups represented.

The genus Methylophaga, a new line of descent within phylogenetic branch γ of proteobacteria

The genus Methylophaga with two species, M. marina and M. thalassica, comprises halophilic methylotrophic bacteria. These organisms utilise C1 compounds through the ribulose monophosphate pathway and are unable to grow on methane. Nearly complete 16S rRNA sequences were obtained for both Methylophaga species by directly sequencing the amplified 16S rRNA gene. These sequences were compared with published 16S rRNA sequences of methylotrophic strains and a large number of marine bacterial strains including several members of the alpha, beta and gamma subclasses of Proteobacteria. Phylogenetic trees were inferred using both parsimony and distance matrix methods. Each topology was analysed by bootstrap. The genus Methylophaga was found to be clearly separated from other methylotrophic bacteria and formed a distinct branch within the gamma subclass of Proteobacteria.

In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy

Bacteria of the family Legionellaceae form a monophyletic group within the gamma-subclass of Proteobacteria. Based on comparative sequence analysis we constructed two oligonucleotide probes complementary to regions of 16S rRNA characteristic for Legionellaceae. Probe specificities were tested by whole-cell or dot-blot hybridization against 14 serogroups of Legionella pneumophila, 22 different Legionella spp. and 72 non-legionellae reference strains. Using optimized conditions both probes hybridized to all tested strains of L. pneumophila. Probes LEG226 and LEG705 hybridized to 71% and 90% of the Legionella species tested, respectively. With the exception of Methylomonas alba none of the non-target strains showed complete sequence homology within the target molecule. In a preliminary evaluation the results of classical techniques employing selective media, immunofluorescence and the probe assay were in good accordance for routine environmental and clinical isolates. L. pneumophila suspended in drinking water at approximately 10(3)-10(4) c.f.u. ml-1 could be rapidly detected by a combination of membrane filtration on polycarbonate filters and whole-cell hybridization. Even after incubation for 1 year a proportion of the released cells was still detectable. In situ hybridization also facilitated visualization of Legionella spp, cells in model biofilms. A combination of in situ hybridization and confocal laser scanning microscopy (CLSM) was used to analyse the three-dimensional arrangement of L. pneumophila within cells of the ciliated protozoan Tetrahymena pyriformis. Whole-cell probing with 16S rRNA-targeted oligonucleotides could, in the future, complement established techniques like immunofluorescence and PCR in ecological and epidemiological studies of Legionellaceae.

The phylogenetic structure of the genus Acinetobacter

16S rDNA sequence analysis was performed on the type strains of all validly described Acinetobacter species and five unnamed Acinetobacter strains. The phylogenetic analyses confirm that Acinetobacter is a coherent genus within the gamma subclass of Proteobacteria and that the species are phylogenetically well defined. A. calcoaceticus, A. lwoffii, A. johnsonii and A. haemolyticus form one cluster of closely related species, the pair A. junii and A. baumannii forms a second cluster. A. radioresistens stands phylogenetically isolated. The study reveals that three undescribed strains can be assigned to individually described species, while strains DSM 30009 and DSM 590 may represent two novel Acinetobacter species.

Anaerobic oxidation of ferrous iron by purple bacteria, a new type of phototrophic metabolism.

Anoxic iron-rich sediment samples that had been stored in the light showed development of brown, rusty patches. Subcultures in defined mineral media with ferrous iron (10 mmol/liter, mostly precipitated as FeCO3) yielded enrichments of anoxygenic phototrophic bacteria which used ferrous iron as the sole electron donor for photosynthesis. Two different types of purple bacteria, represented by strains L7 and SW2, were isolated which oxidized colorless ferrous iron under anoxic conditions in the light to brown ferric iron. Strain L7 had rod-shaped, nonmotile cells (1.3 by 2 to 3 microns) which frequently formed gas vesicles. In addition to ferrous iron, strain L7 used H2 + CO2, acetate, pyruvate, and glucose as substrate for phototrophic growth. Strain SW2 had small rod-shaped, nonmotile cells (0.5 by 1 to 1.5 microns). Besides ferrous iron, strain SW2 utilized H2 + CO2, monocarboxylic acids, glucose, and fructose. Neither strain utilized free sulfide; however, both strains grew on black ferrous sulfide (FeS) which was converted to ferric iron and sulfate. Strains L7 and SW2 grown photoheterotrophically without ferrous iron were purple to brownish red and yellowish brown, respectively; absorption spectra revealed peaks characteristic of bacteriochlorophyll a. The closest phototrophic relatives of strains L7 and SW2 so far examined on the basis of 16S rRNA sequences were species of the genera Chromatium (gamma subclass of proteobacteria) and Rhodobacter (alpha subclass), respectively. In mineral medium, the new isolates formed 7.6 g of cell dry mass per mol of Fe(II) oxidized, which is in good agreement with a photoautotrophic utilization of ferrous iron as electron donor for CO2 fixation. Dependence of ferrous iron oxidation on light and CO2 was also demonstrated in dense cell suspensions. In media containing both ferrous iron and an organic substrate (e.g., acetate, glucose), strain L7 utilized ferrous iron and the organic compound simultaneously; in contrast, strain SW2 started to oxidize ferrous iron only after consumption of the organic electron donor. Ferrous iron oxidation by anoxygenic phototrophs is understandable in terms of energetics. In contrast to the Fe3+/Fe2+ pair (E0 = +0.77 V) existing in acidic solutions, the relevant redox pair at pH 7 in bicarbonate-containing environments, Fe(OH)3 + HCO3-/FeCO3, has an E0' of +0.2 V. Ferrous iron at pH 7 can therefore donate electrons to the photosystem of anoxygenic phototrophs, which in purple bacteria has a midpoint potential around +0.45 V. The existence of ferrous iron-oxidizing anoxygenic phototrophs may offer an explanation for the deposition of early banded-iron formations in an assumed anoxic biosphere in Archean times.

The phylogenetic structure of the genus Acinetobacter.

16S rDNA sequence analysis was performed on the type strains of all validly described Acinetobacter species and five unnamed Acinetobacter strains. The phylogenetic analyses confirm that Acinetobacter is a coherent genus within the gamma subclass of Proteobacteria and that the species are phylogenetically well defined. A. calcoaceticus, A. lwoffii, A. johnsonii and A. haemolyticus form one cluster of closely related species, the pair A. junii and A. baumannii forms a second cluster. A. radioresistens stands phylogenetically isolated. The study reveals that three undescribed strains can be assigned to individually described species, while strains DSM 30009 and DSM 590 may represent two novel Acinetobacter species.

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.

Phylogenetic Oligodeoxynucleotide Probes for the Major Subclasses of Proteobacteria: Problems and Solutions

Summary Based on comparative analyses of 16S and 23S ribosomal RNA sequences we have located sites specific for the alpha-, beta-, and gamma-subclasses of Proteobacteria. Short oligodeoxynucleotides complementary to these signature regions were evaluated as potential nucleic acid probes for the differentiation of the major subclasses of Proteobacteria. Hybridization conditions were optimized by the addition of form-amide to the hybridization buffer and high stringency post-hybridization washing. Single-mismatch discrimination of probes was further improved by blocking nontarget probe binding sites with competitor oligonucleotides. Nonisotopic dot-blot hybridization to reference strains demonstrated the expected probe specificities, whole cell hybridization with fluorescent probe derivatives allowed the classification of individual microbial cells. The probes will be useful for determinative studies and for the in situ monitoring of population distribution and dynamics in microbial communities.