Group-specific Oligonucleotide Probes Research Articles

‘Candidatus Arthromitus’ revised: segmented filamentous bacteria in arthropod guts are members of Lachnospiraceae

The name Arthromitus has been applied collectively to conspicuous filamentous bacteria found in the hindguts of termites and other arthropods. First observed by Joseph Leidy in 1849, the identity of these filaments has remained contentious. While Margulis and colleagues declared them to be a life stage of Bacillus cereus, others have assumed them to belong to the same lineage as the segmented filamentous bacteria (SFB) from vertebrate guts, a group that has garnered much attention due to their unique ability to specifically modulate their host's immune response. Both SFB and Arthromitus filaments from arthropod guts were grouped under provisional name 'Candidatus Arthromitus' by Snel and colleagues as they share a striking similarity in terms of their morphology and close contact to the host gut wall. While SFB form a distinct lineage within the family Clostridiaceae, the identity of the filaments from arthropod guts remains elusive. Using whole-genome amplification of single filaments capillary picked from termite guts and fluorescence in situ hybridization of 16S rRNA with group-specific oligonucleotide probes, we show that they represent a monophyletic lineage within the family Lachnospiraceae distinct from that of SFB. Therefore, 'Candidatus Arthromitus' can no longer be used for both groups. Given the historic precedence, we propose to reserve this name for the filaments that were originally described by Leidy. For the SFB from vertebrate guts, we propose the provisional name 'Candidatus Savagella' in honour of the American gut microbiologist Dwayne C. Savage, who was the first to describe that important bacterial group.

Design and application of group-specific oligonucleotide probes for detecting and monitoring mouse Clostridia

Clostridia dominate the rodent intestinal bacterial community and play an important role in physiological functions of the host. However, their ecology and diversity are still unclear. In our previous report, we showed that phylogenetically novel groups of clostridia inhabit the mouse intestine and contribute to the normalization of germfree mice. In this study, five new oligonucleotide probes were designed and applied to detect these clostridial groups that are essential for the normalization of germfree mice. Faecal microbiota of conventional mouse strains and specific pathogen-free mice from different breeding colonies were analysed by fluorescence in situ hybridization using these five probes. Our results showed that the composition of clostridia differed among mouse strains and also among mouse groups of the same inbred strain from different breeding colonies. These five new probes for mouse clostridia were able to detect the difference in clostridial diversity in each mouse group. In addition to Clostridium, we also analysed Bacteroides and Lactobacillus using previously described probes and the number or the frequency of occurrence of Bacteroides was shown to be different among mouse groups analysed. The oligonucleotide probe set including our newly developed and previously described probes used in this study can be applied to monitoring of significant groups of mouse intestinal microbiota.

Comprehensive and relaxed search for oligonucleotide signatures in hierarchically clustered sequence datasets

PCR, hybridization, DNA sequencing and other important methods in molecular diagnostics rely on both sequence-specific and sequence group-specific oligonucleotide primers and probes. Their design depends on the identification of oligonucleotide signatures in whole genome or marker gene sequences. Although genome and gene databases are generally available and regularly updated, collections of valuable signatures are rare. Even for single requests, the search for signatures becomes computationally expensive when working with large collections of target (and non-target) sequences. Moreover, with growing dataset sizes, the chance of finding exact group-matching signatures decreases, necessitating the application of relaxed search methods. The resultant substantial increase in complexity is exacerbated by the dearth of algorithms able to solve these problems efficiently. We have developed CaSSiS, a fast and scalable method for computing comprehensive collections of sequence- and sequence group-specific oligonucleotide signatures from large sets of hierarchically clustered nucleic acid sequence data. Based on the ARB Positional Tree (PT-)Server and a newly developed BGRT data structure, CaSSiS not only determines sequence-specific signatures and perfect group-covering signatures for every node within the cluster (i.e. target groups), but also signatures with maximal group coverage (sensitivity) within a user-defined range of non-target hits (specificity) for groups lacking a perfect common signature. An upper limit of tolerated mismatches within the target group, as well as the minimum number of mismatches with non-target sequences, can be predefined. Test runs with one of the largest phylogenetic gene sequence datasets available indicate good runtime and memory performance, and in silico spot tests have shown the usefulness of the resulting signature sequences as blueprints for group-specific oligonucleotide probes. Software and Supplementary Material are available at http://cassis.in.tum.de/.

Fixation-free fluorescence in situ hybridization for targeted enrichment of microbial populations

We modified the standard ribosomal RNA-targeted fluorescence in situ hybridization (FISH) protocol by removing the fixation steps to allow recovery of unmodified nucleic acids. Using this method, hybridized cells could be visualized in two bioreactor sludges and termite hindgut samples by epifluorescence microscopy. We then targeted one bacterial and one archaeal population in the sludge samples with group-specific oligonucleotide probes using in-solution fixation-free FISH and sorted hybridized populations using fluorescence-activated cell sorting (FACS). We could show that sorted populations were highly enriched for the target organisms based on 16S rRNA gene sequencing, thus confirming probe specificity using the modified FISH protocol. This approach should facilitate subsequent genomic sequencing and analysis of targeted populations as DNA is not compromised by crosslinking during fixation.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.

Extent of Precipitation and Sorption During Copper Removal from Synthetic Wastewater in the Presence of Sulfate-Reducing Bacteria

A sulfate-reducing bacterial culture was successfully enriched from the seed sludge of anaerobic sludge digester. Sulfate-reducing bacteria (SRB) in the enriched culture (around 42%) were characterized by fluorescence in situ hybridization (FISH) with group and genus specific 16S rRNA-targeted oligonucleotide probes. Desulfosarcina, Desulfococcus, Desulfofaba, and Desulfofrigus spp. were identified as the dominant species of the enriched SRB. Subsequently, batch experiments were conducted at initial copper and sulfate concentrations of 10 and 300 mg/L, respectively, to quantify the ability of the enriched SRB in simultaneous sulfate reduction and copper removal. Sulfate reduction efficiencies of the culture in the absence (biotic system without copper, BS-1) and the presence (biotic system with copper, BS-2) of copper were 96.8 and 98.8%, respectively, after 6 days. In BS-2, 99.2% copper removal was observed after 1 day. However, 67% of copper was removed by chemical precipitation and bioaccumulation. No significant inhibition of bacterial growth was observed at the copper concentration studied, that is, 10 mg/L. Chemical precipitation as hydroxide/carbonate caused a copper removal of 44% in AS-1 (abiotic system without lactate) and 36% in AS-2 (abiotic system with lactate), after 6 days. However, the majority of copper was removed as copper sulfide well before the occurrence of copper hydroxide precipitation. Energy dispersive X-ray spectroscopy (EDS) analysis of the precipitates obtained from biotic and abiotic systems confirmed the origin of copper sulfide as the result of SRB. As a whole, the results of this study could be useful to understand the mechanisms of copper removal, i.e. chemical precipitation, bioprecipitation and biosorption/bioaccumulation during sulfate reduction by SRB.

A MCMC strategy for group-specific 16S rRNA probe design

Revealing biodiversity in microbial communities is essential in metagenomics researches. With thousands of sequenced 16S rRNA gene available, and advancements in oligonucleotide microarray technology, the detection of microor-ganisms in microbial communities consisting of hundreds of species may be possible. Many of the existing strategies developed for oligonucleotide probe design are dependent on the result of global multiple sequences alignment, which is a time-consuming task. We present a novel program named OligoSampling that uses MCMC method to design group-specific oli-gonucleotide probes. The probes generated by OligoSampling are group specific with weak crosshybridization potentials. Furthermore a high coverage of target sequences can be obtained. Our method does not need to globally align target sequences. Locally aligning target sequences iteratively based on a Gibbs sampling strategy has the same effect as globally aligning sequences in the process of seeking group-specific probes. OligoSampling provides more flexibility and speed than other software programs based on global multiple sequences alignment.

Multivariate analysis of microbial communities in the River Elbe (Germany) on different phylogenetic and spatial levels of resolution

The microbial communities of three different habitat types and from two sediment depths in the River Elbe were investigated by fluorescence in situ hybridization at various levels of complexity. Differences in the microbial community composition of free-flowing river water, water within the hyporheic interstitial and sediment-associated bacteria were quantitatively analyzed using domain- and group-specific oligonucleotide probes. Qualitative data on the presence/absence of specific bacterial taxa were gathered using genus- and species-specific probes. The complete data set was statistically processed by univariate statistical approaches, and two-dimensional ordinations of nonmetric multidimensional scaling. The analysis showed: (1) that the resolution of microbial community structures at microenvironments, habitats and locations can be regulated by targeted application of oligonucleotides on phylogenetic levels ranging from domains to species, and (2) that an extensive qualitative presence/absence analysis of multiparallel hybridization assays enables a fine-scale apportionment of spatial differences in microbial community structures that is robust against apparent limitations of fluorescence in situ hybridization such as false positive hybridization signals or inaccessibility of in situ oligonucleotide probes. A general model for the correlation of the phylogenetic depth of focus and the relative spatial resolution of microbial communities by fluorescence in situ hybridization is presented.

Evaluating Children in the Ukraine for Colonization With the Intestinal Bacterium Oxalobacter formigenes, Using a Polymerase Chain Reaction-based Detection System.

Background: Oxalobacter formigenes is a recently discovered anaerobic bacterium residing in the gastrointestinal tracts of most vertebrates, including humans. Evidence suggests that this bacterium plays an important symbiotic relationship with its hosts by regulating oxalic acid homeostasis. Oxalic acid is a ubiquitous toxic by-product of metabolism associated with numerous pathologic conditions, including hyperoxaluia, cardiac myopathy and conductance disorders, kidney stones, and even death. Despite the potential importance of O. formigenes in several major health disorders, the difficulty in culturing, isolating, and identifying this fastidious anaerobe has limited research of its disease associations. Because O. formigenes must use two unique enzymes to catabolize oxalic acid, this bacterium appeared to be a suitable model for DNA-based identification, thereby circumventing the labor-intensive procedures currently used. Methods and Results: In this study, genus- and group-specific oligonucleotide sequences were designed corresponding to homologous regions residing in the oxc gene that enodes for oxalyl-coenzyme A decarboxylase. A polymerase chain reaction (PCR)-based amplification of the 5'end of this gene directly from genomic DNA isolated from various strains of O. formigenes was used to show that the genus- and group-specific oligonucleotide probes could identify and subgroup the bacterium. Field testing of this PCR-based detection system with 100 fecal cultures collected from children aged 0-12 years demonstrated the ease and efficacy with which O. formigenes can now be identified. Furthermore, these latter data provide a profile for the natural colonization of a human population with this intestinal bacterium. Conclusions: Development and use of this PCR-based detection system permit the rapid identification and classification of the gut-associated bacterium O. formigenes, thereby circumventing the need for the more labor-intensive and lengthy method currently used. The first field test of this detection system indicates that humans apparently do not become colonized with O. formigenes until they begin crawling about in the environment. Furthermore, studies investigating the association between several disorders (eg, kidney stones, irritable bowel syndrome, and hyperoxaluria) and the absence of the bacterium from the gut will now prove far easier.

Comparison of Microbial Community Compositions of Two Subglacial Environments Reveals a Possible Role for Microbes in Chemical Weathering Processes

Viable microbes have been detected beneath several geographically distant glaciers underlain by different lithologies, but comparisons of their microbial communities have not previously been made. This study compared the microbial community compositions of samples from two glaciers overlying differing bedrock. Bulk meltwater chemistry indicates that sulfide oxidation and carbonate dissolution account for 90% of the solute flux from Bench Glacier, Alaska, whereas gypsum/anhydrite and carbonate dissolution accounts for the majority of the flux from John Evans Glacier, Ellesmere Island, Nunavut, Canada. The microbial communities were examined using two techniques: clone libraries and dot blot hybridization of 16S rRNA genes. Two hundred twenty-seven clones containing amplified 16S rRNA genes were prepared from subglacial samples, and the gene sequences were analyzed phylogenetically. Although some phylogenetic groups, including the Betaproteobacteria, were abundant in clone libraries from both glaciers, other well-represented groups were found at only one glacier. Group-specific oligonucleotide probes were developed for two phylogenetic clusters that were of particular interest because of their abundance or inferred biochemical capabilities. These probes were used in quantitative dot blot hybridization assays with a range of samples from the two glaciers. In addition to shared phyla at both glaciers, each glacier also harbored a subglacial microbial population that correlated with the observed aqueous geochemistry. These results are consistent with the hypothesis that microbial activity is an important contributor to the solute flux from glaciers.

Structure and Topology of Microbial Communities in the Major Gut Compartments of Melolontha melolontha Larvae (Coleoptera: Scarabaeidae)

Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O2, H2, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and gamma-Proteobacteria were restricted to the lumen, whereas clones related to the beta- and delta-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5'-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.

Empirical Establishment of Oligonucleotide Probe Design Criteria

Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of < or =85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (< or =15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than -30 kcal/mol for 50-mer probes or -40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.

Sulfate Reduction Potential in Sediments in the Norilsk Mining Area, Northern Siberia

The purpose of this study was to characterize the distribution and activity of sulfate-reducing bacteria in tailings and sediments impacted by effluents from mining and smelting operations in the Norilsk area in northern Siberia. The Norilsk mining complex involves three smelter operations, a hydrometallurgical plant, and extensive tailings areas located in the permafrost zone. Sulfate reduction rates measured with a 35SO4 2− tracer technique under various in-situ conditions ranged from 0.05 to 30 nmol S cm−3 day−1. Acetate and glucose addition greatly stimulated sulfate reduction, whereas lactate had less effect. The most pronounced stimulation of sulfate reduction (6.5-fold) was observed with phosphate amendment. Most-probable-number (MPN) counts of sulfate-reducing bacteria in media with glucose, ethanol, lactate, and acetate as electron donors were generally highest at around 107 cells ml−1. The actual MPN counts varied with the sample, electron donor, and incubation conditions (pH 7.2 vs. pH 3.5; 28°C vs. 4°C). Enrichment cultures of sulfate-reducing bacteria were established from a sample that showed the highest rate of sulfate reduction. After multiple serial transfers, the dominant sulfate-reducers were identified by fluorescence in situ hybridization using genus and group-specific 16S rRNA-targeted oligonucleotide probes. Desulfobulbus spp. prevailed in ethanol and lactate enrichments and the Desulfosarcina-Desulfococcus group dominated in acetate and benzoate enrichments. Psychrophilic Desulfotalea-Desulfofustis and moderately psychrophilic Desulforhopalus spp. were identified in enrichments incubated at 4°C, but they were also found in mesophilic enrichments.

Phylogenetic diversity, abundance, and axial distribution of bacteria in the intestinal tract of two soil-feeding termites (Cubitermes spp.).

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (TERMITIDAE: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.

Dynamics of microcystin-degrading bacteria in mucilage of Microcystis.

To reveal the process of degradation of hepatotoxic microcystin produced in Microcystis cells during the Microcystis bloom period, we used fluorescence in situ hybridization (FISH) to analyze the population dynamics of microcystin-degrading bacteria in Microcystis mucilage. We designed and applied an oligonucleotide probe targeted to the 16S rRNA sequence of strain Y2 of a microcystin-degrading bacterium (MCD-bacterium), which was isolated from Lake Suwa, Japan. In both the 1998 and 1999 tests, FISH clearly showed that MCD-bacteria existed in the mucilage and that, when a high concentration of cell-bound microcystin was detected, MCD-bacteria exceeded 10% of the sum of bacteria hybridized with group-specific probes. The concentration of MCD-bacteria was highest in summer 1998, when a toxic species, M. viridis, was dominant. There was a high correlation between the number of MCD-bacteria in the mucilage and the concentration of cell-bound microcystin in the lake. Our results suggest that MCD-bacteria responded to changes in the concentration of microcystin and degraded the microcystin when it was released from Microcystis cells. We also analyzed changes in the bacterial community structure associated with the Microcystis colonies by using domain- and group-specific oligonucleotide probes. Changes in the concentrations of the Cytophaga/Flavobacterium group and delta-Proteobacteria, which can degrade macromolecules derived from Microcystis cells, were synchronized with changes in the concentration of Microcystis. The results not only suggest the significant role of MCD-bacteria in detoxification, but also demonstrate a possible sequence of degradation from Microcystis cells to microcystin maintained in the cell, which is then carried out by bacterial consortia in the mucilage.

Design and evaluation of group-specific oligonucleotide probes for quantitative analysis of intestinal ecosystems: their application to assessment of equine colonic microflora

Nine oligonucleotide probes complementary to conserved regions of small subunit rRNA from phylogenetically defined clusters of intestinal anaerobic bacteria were designed and evaluated for use in quantitative analysis of intestinal microflora. Optimum wash temperatures ( T w) were determined according to the temperature of dissociation ( T d) of each probe and target group specificity was demonstrated by comparing hybridisation to target and non-target rRNA immobilised on nylon membranes. Three probes are targeted to phylogenetic clusters of Clostridiaceae, clusters III, IV and IX, with three probes designed to target previously undefined clusters within the low %G+C Gram-positive phyla. The remaining three probes encompass the Cytophaga-Flexibacter-Bacteroides assemblage, the Bacillus-Lactobacillus-Streptococcus group and the Spirochaetaceae. Application of these probes, alongside available probes targeted to other intestinal bacterial groups, against rRNA extracted from equine colonic samples has provided the first quantitative data on the predominant bacterial populations inhabiting the equine large intestine. Results show the Spirochaetaceae, the Cytophaga-Flexibacter-Bacteroides assemblage, the Eubacterium rectale-Clostridium coccoides group, and ‘Unknown cluster C’ of the Clostridiaceae to be the largest populations in the equine gut, each comprising 10–30% of the total microflora in each horse sampled. Other detected notable populations were the Bacillus-Lactobacillus-Streptococcus group, Fibrobacter and ‘Unknown cluster B’, each comprising 1–10% of the total flora. Average coverage using the suite of probes described here exceeded 75% of the total microbial community.

Characterization of the goldfish fecal microflora by the fluorescent in situ hybridization method

: Bacterial populations in goldfish feces were characterized by the fluorescent in situ hybridization (FISH) method. A total of nine different group-specific rRNA-targeted oligonucleotide probes were used. Approximately half of the microbial cells (57.8 ± 16.7%) were detected with a probe EUB338 and found to be bacteria. The microbial cells in 33–35 of the 35 samples from five specimens strongly hybridized with probes ALF1b, BET42a and GAM42a, suggesting that goldfish intestinal bacteria are mainly composed of α, β and γ-subclasses of Proteobacteria. The fact that a probe AER66 reacted with 25.6 ± 14.2% of the total microbial cells in all 35 samples, demonstrated that genus Aeromonas was the dominant species in the goldfish intestines. Genus Bacteroides including Bacteroides type A detected with a probe BAC303 was observed in 15 of 35 samples while other taxonomic groups determined with HGC69a, CF39a and P72 were detected in 6–11 of 35 samples. These results strongly suggest that Bacteroides shows the greatest daily fluctuation and interindividual variation in the intestines of goldfish. Moreover, the FISH method was proven to be useful for rapid enumeration of taxonomic groups of fish intestinal bacteria.

Seasonal patterns of the bacterioplankton community composition in a large mesotrophic lake

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 31:211-225 (2003) - doi:10.3354/ame031211 Seasonal patterns of the bacterioplankton community composition in a large mesotrophic lake Walter Zwisler, Natascha Selje, Meinhard Simon* Institute for Chemistry and Biology of the Marine Environment, University of Oldenburg, 26111 Oldenburg, Germany *Corresponding author. Email: m.simon@icbm.de ABSTRACT: The composition of the bacterioplankton community in mesotrophic Lake Constance was studied from April 1996 to December 1997 by fluorescence in situ hybridization (FISH) with group specific oligonucleotide probes and denaturing gradient gel electrophoresis (DGGE). Between 10 and 59% of the DAPI-stained cells were detected as Bacteria with means of 38 ± 12, 27 ± 10 and 23 ± 6% at 3, 20, and 50 m depth, respectively. In the euphotic zone at 3 m depth, β-Proteobacteria always constituted highest proportions of the bacterial community detected by FISH and constituted 6 to 23% of the DAPI-stained cells with a mean of 12%. Proportions of bacteria belonging to the Cytophaga/Flavobacteria (CF) cluster and the a-Proteobacteria at this depth were 5.4 ± 2.5 and 4.1 ± 3.5%, respectively. Proportions of b-Proteobacteria decreased at 20 and 50 m, whereas that of a-Proteobacteria remained constant and that of the CF-cluster increased to 7.4%, such that they comprised similar proportions as b-Proteobacteria at 50 m depth. γ Proteobacteria were always of minor importance and constituted only 2.2 ± 1.3% of the DAPI cell counts. Cumulative proportions of all detected bacterial groups constituted 75% of the Bacteria counts at 3 m, 84% at 20 m and 99% at 50 m. The DGGE analysis of the bacterioplankton community showed seasonal and vertical variations of the banding patterns even though several bands occurred throughout the study period and at all depths. A cluster analysis revealed distinct bacterial clusters of the phytoplankton spring bloom and the later period and of epilimnetic and hypolimnetic waters. The sequence analysis of the excised bands indicated that all groups which were detected by FISH were represented in the patterns. Sequence similarities of the retrieved sequences to known isolates were <90 to 96.1%, and to other phylotypes as close as 99%. Phylotypes of b-Proteobacteria and Actinobacteria of the typical banding patterns affiliated closely with recently described clusters of typical freshwater bacteria. In addition, we identified phylotypes of the Holophaga/Acidobacterium and Nitrospira phylum during the stratified period in samples from 50 m and during mixing from all 3 depths. KEY WORDS: Bacteria · DGGE · Fluorescence in situ hybridization · Lake Constance · Actinobacteria · Nitrospira · Holophaga Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 31, No. 3. Online publication date: April 03, 2003 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2003 Inter-Research.

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

Characterization of microbial community in granular sludge treating brewery wastewater

The diversity and distribution of microbes within brewery-degrading anaerobic sludge granules were studied using various molecular techniques. Molecular cloning of small-subunit rRNA gene sequences indicated that all archaeal clones were affiliated with Methanosaeta concillii (>99% sequence similarity), and the bacterial clones were mostly affiliated with a not-yet-cultured Clostridium cluster (48 out of 99 clones) in the low G+C gram-positive group, Xanthomonas spp. in the γ-subclass of Proteobacteria (30 clones), and Desulfovibrio spp. (16 clones) in the δ-subclass of Proteobacteria. Slot-blot hybridization indicated that archaeal cells from the Methanomicrobiales (58.4% of total rRNA), Methanobacterials (3.3%) and Methanococcales (1.0%) accounted for 62.4% of the total community rRNA. The rest of the microbial populations were the clostridial cluster (27.3% of total rRNA) and Desulfovibrio spp. (9.4%). Fluorescence in-situ hybridization with domain and group-specific oligonucleotide probes further revealed a multi-layer granular architecture. On the surface layer, the hydrolytic clostridial species and hydrogenotrophic Methanobacteriales were the predominant. In the middle layer, mostly H 2-producing acetogens from the δ- Proteobacteria (i.e., Desulfovibrio spp.), hydrogenotrophic Methanobacteriales and aceticlastic Methanosaeta were observed to presumably form a syntrophic association. Finally, the center core consisted of microcolonies of Methanosaeta cells.