Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.     

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.     

Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates

We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization

The bacterial community structure in the winter cover and pelagic zone of a high mountain lake was analyzed by in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes. Cells fixed on membrane filters were hybridized with a probe specific for the domain Bacteria as well as with probes for the alpha, beta, and gamma subclasses of the class Proteobacteria and the Cytophaga-Flavobacterium group. The fraction of bacteria detectable after hybridization with the bacterial probe EUB ranged from 40 to 81% of 4(prm1),6-diamidino-2-phenylindole (DAPI) counts. The bacterial assemblage varied considerably between and within different habitats (snow, slush, and lake water) but was in most cases dominated by members of the beta subclass (6.5 to 116% of bacteria detectable with probe EUB). The sum of bacteria hybridizing with group-specific probes was usually lower than the fraction detectable with probe EUB. Image analysis was used to characterize morphology and the size-specific biomass distribution of bacterial assemblages, which clearly separated the three habitats. Although the measured secondary production parameters and the fraction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride-reducing bacteria varied by more than an order of magnitude in the different slush and pelagic layers, detectability with the fluorescent probe EUB was constantly high. Physiological strategies of bacteria under nutrient limitation and at low temperatures are discussed in the context of the ribosome content of single cells. This study confirms the suitability of fluorescently labeled rRNA-targeted probes for the characterization of bacterial population structures even in oligotrophic habitats.     

Evaluation of group-specific, 16S rRNA-targeted scissor probes for quantitative detection of predominant bacterial populations in dairy cattle rumen

AIMS: To develop a suite of group-specific, rRNA-targeted oligonucleotide scissor probes for the quantitative detection of the predominant bacterial groups within the ruminal microbial community with the rRNA cleavage reaction-mediated microbial quantification method. METHODS AND RESULTS: Oligonucleotides that complement the conserved sites of the 16S rRNA of phylogenetically defined groups of bacteria that significantly contribute to the anaerobic fermentation of carbohydrates in ruminal ecosystems were selected from among published probes or were newly designed. For each probe, target-specific rRNA cleavage was achieved by optimizing the formamide concentration in the reaction mixture. The set of scissor probes was then used to analyse the bacterial community in the rumen fluids of four healthy dairy cows. In the rumen fluid samples, the genera Bacteroides/Prevotella and Fibrobacter and the Clostridium coccoides-Eubacterium rectale group were detected in abundance, accounting for 44-48%, 2.9-10%, and 9.1-10% of the total 16S rRNA, respectively. The coverage with the probe set was 71-78% of the total bacterial 16S rRNA. CONCLUSIONS: The probe set coupled with the sequence-specific small-subunit rRNA cleavage method can be used to analyse the structure of a ruminal bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The probe set developed in this study provides a tool for comprehensive rRNA-based monitoring of the community members that dominate ruminal ecosystems. As the ruminal microbial community can be perturbed, it is important to track its dynamics by analysing microbiological profiles under specific conditions. The method described here will provide a convenient approach for such tracking.     

The use of alkaline phosphatase-labelled oligonucleotide probes as culture confirmation reagents for the identification of commercially important bacteria

A range of rRNA-targeted alkaline phosphatase-labelled oligonucleotide probes was tested for use as culture confirmation reagents for the rapid identification of micro-organisms. The probes were specific to clinically important bacteria ( Helicobacter pylori and Mycobacterium tuberculosis ), fish and shellfish pathogens ( Renibacterium salmoninarum and Vibrio vulnificus ), food spoilage bacteria ( Listeria spp. and L. monocytogenes ), for bacteria of biotechnological importance ( Streptomyces spp.) and for bacteria associated with the oil industry (Sulphate-reducing bacteria, SRB). A universal bacterial probe and a eukaryotic probe were included in the study as positive and negative controls, respectively. A total of 93 bacterial strains was screened. With the exception of a large number of cross-reactions of the SRB probe (specificity value of 29·4%) and a single cross-reaction of the R. salmoninarum probe (specificity value of 97·7%), dot blot analysis indicated that each probe hybridized 100% specifically to the organisms tested. A simple culture confirmation method was then developed using these probes to enable the identification of bacterial colonies using a simple hybridization procedure.     

Hydrothermal vent gastropods from the same family (Provannidae) harbour epsilon- and gamma-proteobacterial endosymbionts

The discovery of new hydrothermal vent systems in the back-arc basins of the Western Pacific revealed chemosynthesis-based faunal communities distinct from those of other vents. These vents are dominated by two related gastropods (Alviniconcha spp. and Ifremeria nautilei) that harbour symbiotic bacteria in their gills. We used comparative 16S ribosomal RNA (rRNA) gene sequencing and in situ hybridization with rRNA-targeted probes to characterize the bacterial symbionts of Alviniconcha sp. and I. nautilei from the Manus Basin in the Western Pacific. The analyses revealed that these two gastropod species, although affiliated with the same family, harbour phylogenetically distant chemosymbionts, suggesting independent origins of these endosymbioses. The I. nautilei endosymbiont clusters with sulfur-oxidizing bacteria within the gamma-Proteobacteria, as is the case for all previously characterized endosymbionts from a wide diversity of host taxa harbouring thioautotrophic prokaryotes. In contrast, the Alviniconcha endosymbiont is affiliated with sulfur-oxidizing bacteria within the epsilon-Proteobacteria. These results show that bacteria from the epsilon-Proteobacteria are also capable of forming endosymbiotic associations with marine invertebrates from chemosynthetic environments. More generally, the endosymbiotic lifestyle is now shown to be distributed throughout all recognized classes of the Proteobacteria.     

New degenerate Cytophaga-Flexibacter-Bacteroides-specific 16S ribosomal DNA-targeted oligonucleotide probes reveal high bacterial diversity in River Taff epilithon.

River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the Cytophaga-Flexibacter-Bacteroides division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria), gamma-Proteobacteria, gram-positive bacteria, Cyanobacteria, beta-Proteobacteria, delta-Proteobacteria, and the Prosthecobacter group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups Cytophaga, Sphingobacterium, Lewinella, and Cytophaga aurantiaca. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.     

Characterization of microbial communities of biofilters by phospholipid fatty acid analysis and rRNA targeted oligonucleotide probes

The microbial community of a biofilter for waste gas treatment of an animal rendering plant was characterized by the analyses of the phospholipid fatty acids (PLFAs) of the filter material. For these analyses five samples of one filter were taken in intervals between one and two months. The main components of the PLFA profiles were straight chain saturated, monounsaturated and cyclopropyl fatty acids. Terminally branched and 10-methyl branched fatty acids were present in minor amounts. The structure and succession of the microbial community was interpreted by the presence and quantitative changes of diagnostic fatty acids. The stability of diagnostic fatty acids in relation to varying incubation parameters was tested for a number of bacterial isolates from biofilters representing different phylogenetic branches. For two samples, the data from the PLFA-analyses were compared with data obtained by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes specific for the alpha-, beta- and gamma-subclass of the Proteobacteria, the Actinobacteria (Firmicutes with high G+C content) and the Firmicutes with low G+C content. These data indicated a dominating number of Proteobacteria (54% and 35% of DAPI-stained cells), in which the gamma-Proteobacteria represented the main fraction. Actinobacteria were detected in minor amounts, the number of Firmicutes with low G+C content was near the detection limit of the method. About half of the cells detected with a probe specific for Bacteria did not hybridize with the probes specific for the alpha-, beta- and gamma subclass of the Proteobacteria and the two subgroups of the Firmicutes. The results of both methods, the fluorescence in situ hybridization (FISH) and the PLFA analyses corresponded well and were best suited to confirm and complement each other.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall. Received: 17 May 1999 / Accepted: 16 September 1999     

Whole-cell versus total RNA extraction for analysis of microbial community structure with 16S rRNA-targeted oligonucleotide probes in salt marsh sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% +/- 3.7% [mean +/- standard deviation]) relative to direct DAPI (4',6'-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r(2) = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization

To investigate the population structure of the predominant phylogenetic groups within the human adult fecal microbiota, a new oligonucleotide probe designated S-G-Clept-1240-a-A-18 was designed, validated, and used with a set of five 16S rRNA-targeted oligonucleotide probes. Application of the six probes to fecal samples from 27 human adults showed additivity of 70% of the total 16S rRNA detected by the bacterial domain probe. The Bacteroides group-specific probe accounted for 37% +/- 16% of the total rRNA, while the enteric group probe accounted for less than 1%. Clostridium leptum subgroup and Clostridium coccoides group-specific probes accounted for 16% +/- 7% and 14% +/- 6%, respectively, while Bifidobacterium and Lactobacillus groups made up less than 2%.     

Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode.

The phylogenetic relationship of chemoautotrophic, sulfur-oxidizing, ectosymbiotic bacteria growing on a marine nematode, a Laxus sp. (formerly a Catanema sp.), to known endosymbionts and free-living bacteria was determined. Comparative 16S rRNA sequencing was used to investigate the unculturable nematode epibionts, and rRNA-targeted oligonucleotide hybridization probes were used to identify the ectosymbionts in situ. Both analyses revealed a remarkably specific and stable symbiosis. Unique hybridization of a specific probe to the ectosymbionts indicated that only one species of bacteria was present and growing on the cuticle of the nematode. Distance and parsimony methods used to infer phylogenetic trees both placed the nematode ectosymbionts at the base of a branch containing chemoautotrophic, sulfur-oxidizing endosymbionts of three bivalve families and of the tube worm Riftia pachyptila. The most closely related free-living bacteria were chemoautotrophic sulfur oxidizers belonging to the genus Thiomicrospira. Furthermore, our results suggested that a second, only distantly related group of thioautotrophic endosymbionts has as its deepest branch surface-colonizing bacteria belonging to the genus Thiothrix, some of which are capable of sulfur-oxidizing chemoautotrophic growth.     

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.