Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

基于组合探针识别的大规模细菌分类16 S rRNA基因芯片设计

随着16 S rRNA序列资源的不断丰富,以及寡核苷酸微阵列基因芯片技术的不断进步,检测复杂微生物菌落中的微生物种群构成成为可能．现有的序列特异性探针设计算法缺乏足够的覆盖度、灵活性以及效率,不能满足大规模细菌检测基因芯片的设计要求．很多组特异性探针设计算法的思路多局限于针对某个目标序列组设计唯一的组特异性探针．在很多应用场合,设计单个探针检测组内所有目标序列的目标是很难达到的．因此,设计多个探针通过组合方式进行检测是很有必要的．每个探针能特异性地检测组内一部分目标序列,通过组合就能提高覆盖率．然而,在所有可能的探针组合中找到一个优化的探针组合是很耗时的．提出了一个可行的基于相对熵和遗传算法的组合探针设计算法．     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Specific detection of Dehalococcoides species by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.     

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.     

Extensive set of 16S rRNA-based probes for detection of bacteria in human feces

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.     

Identification of Staphylococcus carnosus and Staphylococcus warneri isolated from meat by fluorescent in situ hybridization with 16S rRNA-targeted oligonucleotide probes.

16S rRNA targeted oligonucleotide probes were designed by sequence analysis of an rRNA database to discriminate S. carnosus, S. warneri, and S. saprophyticus species. After establishing hybridization conditions by RNA dot blot hybridization with reference species, our probes were shown to be specific. By in situ hybridization only S-S-S.carno-0440-a-A-23 and S-S-S.war-0180-a-A-23 can specifically detect S. carnosus and S. warneri, respectively. The detection of old cells of S. carnosus 833 was more limited by the permeabilisation than by the low rRNA content. One day old cells could be permeabilized with lysostaphin, whereas young cells were permeabilized with lysozyme.     

Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge.

Laboratory-scale upflow anaerobic sludge-bed reactors were inoculated with industrial granular sludge and fed with either propionate or propionate and sulfate. The population dynamics of the propionate-oxidizing bacteria Desulfobulbus sp. and the syntrophically growing strain SYN7 were studied in reactors by dot blot and in situ hybridization with 16S rRNA-based oligonucleotide probes.     

Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization.

Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S. wolfei LYB was closely related to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, of which the majority was accounted for by the genus Syntrophomonas.     

Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

Detection of different mRnas expressed in the thyro-parathyroid complex of the rat by in situ hybridization using digoxigenin-labelled oligonucleotide probes

The effects have been examined of different methods and regimens for tissue fixation, preservation, permeabilization and immunostaining of different mRNAs detected by in situ hybridization in paraffin-embedded samples. The three main hormone mRNAs expressed in the thyro–parathyroid glands, namely thyroglobulin, calcitonin and parathyroid hormone mRNAs, were chosen as the target nucleic acid sequences to be detected using digoxigenin-labelled probes. Our results suggest that chemical fixation and permeabilization of tissue samples are restrictive steps. Thus, paraformaldehyde fixation provides excellent signal intensities and non-detectable background levels whereas routine formalin and Bouin's solution give unsatisfactory results. A clear linear correlation was also found between signal intensity and proteinase K permeabilization. Moreover, the optimization of immunohistochemical steps, such as anti-digoxigenin antibody concentration and colour development times, enhance the intensity and specificity of hybrid signals. Furthermore, our results show that, in contrast to some data in the literature, paraffin-embedded tissue is suitable for detection of mRNAs by in situ hybridization. It gives equivalent intensities of specific signal and superior histological and cellular resolutions when compared to cryopreserved tissue.     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone     

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.     

Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs ( approximately 20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have described a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here, we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression.     

Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

Detection of FGF-beta mRNA in chondrosarcoma cells by a new in situ hybridization technique with synthetic oligonucleotide probes

Fibroblast growth factor beta (FGF-beta) is a potent mitogenic and angiogenic factor produced by a large number of normal and transformed cells. In this paper we report a new application of the in situ hybridization procedure which has allowed the detection of FGF-beta mRNA in chondrosarcoma cells using 35S-labelled synthetic oligonucleotide probes.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.