Isolation and biodiversity of hitherto undescribed soil bacteria related to Bacillus niacini.

The hitherto largely not described phylogenetic neighborhood of Bacillus niacini has been explored by a comprehensive cultivation experiment and genomic variety studies. Previous culture-independent studies demonstrated that approximately 15% of all Bacillus 16S rDNA directly extracted from soils worldwide was affiliated to B. niacini. Seven different media were inoculated with soil suspensions in serial dilutions and incubated at different temperatures. Then, bacterial colonies were picked and analyzed by sequencing. A mineral medium with acetate as carbon source yielded a B. niacini rate of >3% of all picked colonies. Other media were less efficient but also successful. Applying this culturing approach, we succeeded in obtaining 64 isolates from different Dutch soils. The isolates turned out to be diverse, although closely related to B. niacini as revealed by 16S rDNA sequencing. Close matches with environmental clones were also found, thus demonstrating much more diversity beyond previously known 16S rDNA sequences. The rep-PCR fingerprinting method revealed a high genomic variety, redundancy could not be observed among our isolates. Hence, the hitherto neglected B. niacini lineage, apparently among the most abundant soil Bacillus, was accessible to our cultivation approach.

Enrichment and immobilization of quinone-respiring bacteria in anaerobic granular sludge.

The capacity of an anaerobic granular sludge for serving as an immobilizing mechanism for quinone-respiring bacteria was evaluated. The inoculum was continuously fed with a basal medium containing the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as a terminal electron acceptor. Complete reduction of AQDS was achieved by the granular sludge for a prolonged period in an anaerobic bioreactor provided with a mixture of volatile fatty acids as a substrate. Phylogenetic analysis revealed the enrichment and immobilization of AQDS-respiring bacteria appearing as dominant organisms in the microbial population of the AQDS-supplemented reactor, compared to a reactor control operated under methanogenic conditions. The consistent quinone-reducing capacity observed in the consortium indicates that it is feasible to apply quinone-reducing microorganisms in continuous bioreactors and this ability can potentially be important in wastewaters rich in humic substances. The quinone reducing activity could also be applied to accelerate the conversion of xenobiotics susceptible to reductive biotransformations such as azo dyes and polychlorinated compounds in continuous bioreactors.

Image analysis, methanogenic activity measurements, and molecular biological techniques to monitor granular sludge from an EGSB reactor fed with oleic acid.

Morphological changes in anaerobic granular sludge fed with increasing loads of oleic acid were quantified by image analysis. The combination of this technique with data on the accumulation of adsorbed long chain fatty acid and with the molecular characterization of microbial community gave insight into the mechanisms of sludge disintegration, flotation and washout. It was found that the bacterial domain was more affected than the archaeal domain during this process. However, no acetoclastic activity and onlya residual hydrogenotrophic activity were detected in the sludge at the end of the operation.

Polymerase chain reaction and denaturing gradient gel electrophoresis monitoring of fecal bifidobacterium populations in a prebiotic and probiotic feeding trial.

A culture-independent approach based on genus-specific PCR and denaturing gradient gel electrophoresis (DGGE) was used to monitor qualitative changes in fecal bifidobacterial communities in a human feeding trial. DNA was extracted directly from feces and bifidobacterial 16S rDNA sequences were amplified using genus-specific PCR. The PCR fragments were subsequently separated in a sequence-specific manner by DGGE in order to obtain a profile of bifidobacterial fragments. The DGGE profiles revealed that in general, administration for two weeks of galactooligosaccharide and/or Bifidobacterium lactis Bb-12 (8 g and 3 x 10(10) cfu per day, respectively) did not affect the qualitative composition of the indigenous Bifidobacterium population, while B. lactis Bb-12 transiently colonised the gut.

Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis.

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density-dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid-log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis-activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S-P. Structural analysis suggested GBAP to be an 11-residue cyclic peptide containing a lactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C-terminal part of a 242-residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB-fsrC encoding FsrB and a putative histidine kinase FsrC and gelE-sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two-component regulatory system.

Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis.

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074(T) exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract.

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.

Spatial distribution of 16S rRNA levels from uncultured acidobacteria in soil.

The activity of uncultured acidobacteria was monitored in Dutch grassland soils by quantifying their ribosomes. These bacteria were detectable by five different 16S rRNA RT-PCR products in temperature gradient gel electrophoresis fingerprints. The ribosomes in surface soil samples were quantified with multiple competitive RT-PCR along a 1.5-km transect through the grassland. In total, the five members of the acidobacteria were estimated to contribute 4 x 1010 to 1 x 1011 ribosomes g soil-1, representing 7-14% of all bacterial ribosomes. These results indicate that ribosomes from acidobacteria are continuously present and abundant in soil and might contribute significantly to microbial activity in soil.

Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes.

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the alpha subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.

Bacterial community changes and enrichment of Burkholderia-like bacteria induced by chlorinated benzoates in a peat-forest soil-microcosm.

Bacterial community shifts in a peat-forest soil spiked with 3-chlorobenzoate (3CBA) or 2,5-dichlorobenzoate (2,5DCB) were monitored by PCR-amplification of the V6 to V8 regions of the 16S rRNA and rDNA, followed by separation of the amplicons by temperature gradient gel electrophoresis. 3CBA disappeared to non-detectable levels after 15 days by a biologically mediated process, while 2,5DCB remained at the initial concentration values. The experiments were conducted under microcosms systems. Addition of the chlorinated benzoates to the soil resulted in a rapid decrease of the microbial diversity, as judged by a time-dependent reduction in the number of amplicons detected by temperature gradient gel electrophoresis. Few amplicons specifically enriched in the spiked soils were cloned and characterised by sequence analysis. The identity of the cloned DNA and the corresponding soil amplicons was confirmed by hybridisation with a radioactively labelled V6-probe. Analysis of the 16S rDNA sequences indicated that Burkholderia-related bacteria dominated the enriched soil populations under 3CBA stress. In addition, enrichment cultures growing on 3CBA as sole C-source were obtained from the respective spiked soil, which were found to contain bacteria with identical 16S rDNA sequences as those induced by 3CBA stress in soil.

A molecular view of the intestinal ecosystem.

This review describes the state of the art as well as the initial results of molecular methodologies used to study the ecology of the complex microflora of the human intestinal tract. The detection and identification of many of these organisms has largely been hampered by the incomplete knowledge of their culture conditions. Many of the molecular methodologies are rooted in the use of ribosomal RNA (rRNA) and its encoding genes to describe the relationship between the bacteria in such communities and their individual identity. This approach permits the elucidation both qualitatively as well as quantitatively of the abundance of bacterial species and how their presence interacts with diet and health. Emphasis is given to the analysis of complex communities rather than detection of individual groups of bacteria. The potential of novel advances in molecular technologies such as DNA arrays for analysis of the intestinal ecosystem are also discussed.

Application of temperature-gradient gel electrophoresis in taxonomy of coryneform bacteria.

Strains belonging to the Gram-positive coryneform soil bacteria were screened genotypically by temperature-gradient gel electrophoresis (TGGE). This method allows the sequence-specific separation of amplified fragments of 16S rRNA genes. A total of 115 reference strains representing the majority of the species of the genera Aeromicrobium, Agromyces, Arthrobacter, Aureobacterium, Cellulomonas, Curtobacterium, Nocardioides and Terrabacter were characterized. Depending on the genus investigated, the resolution limit of the technique appeared to be at the species or genus level or intermediate between the two. Aberrant TGGE profiles of strains within particular taxa revealed genomic heterogeneity and generic misclassification of nine strains studied. Beyond that, indications of 16S rRNA gene heterogeneity were found within the genomes of three Curtobacterium strains. The misclassifications revealed by TGGE were confirmed using whole-cell fatty acid methyl ester analysis and subsequent comparison with a database. TGGE has been demonstrated to be a useful tool in bacterial taxonomy.

Syntrophobacter fumaroxidans sp. nov., a syntrophic propionate-degrading sulfate-reducing bacterium.

A syntrophic propionate-oxidizing bacterium, strain MPOBT, was isolated from a culture enriched from anaerobic granular sludge. It oxidized propionate syntrophically in co-culture with the hydrogen- and formate-utilizing Methanospirillum hungateii, and was able to oxidize propionate and other organic compounds in pure culture with sulfate or fumarate as the electron acceptor. Additionally, it fermented fumarate. 16S rRNA sequence analysis revealed a relationship with Syntrophobacter wolinii and Syntrophobacter pfennigii. The G + C content of its DNA was 60.6 mol%, which is in the same range as that of other Syntrophobacter species. DNA-DNA hybridization studies showed less than 26% hybridization among the different genomes of Syntrophobacter species and strain MPOBT. This justifies the assignment of strain MPOBT to the genus Syntrophobacter as a new species. The name Syntrophobacter fumaroxidans is proposed; strain MPOBT (= DSM 10017T) is the type strain.

Phenotypic, genotypic and pathogenic variation among streptomycetes implicated in common scab disease.

This study examines the diversity of streptomycetes causing potato scab using phenotypic identification, 16S rRNA sequence, and pathogenicity measurements. Pathogenicity did not correlate well with taxonomic identification, nor with phylogenetic position. This result suggests that neither phenotypic nor 16S characteristics are diagnostic for scab-causing strains. Additionally, phenotypic characterizations were not fully consistent with phylogenetic relationships. This incongruency highlights the difficulties of taxonomy within the streptomycetes and affirms the need for better integration of genetic and phenotypic characters in the systematics of this group.

Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.

In situ detection of an uncultured predominant bacillus in Dutch grassland soils.

Uncultured predominant Bacillus ribotype DA001 in Dutch Drentse A grassland soils, as revealed by its 16S rRNA sequence, was detected in soil by fluorescent whole-cell in situ hybridization. A prominent rod-shaped cell type was identified in bacterial suspensions prepared from soil by a multiple 16S rRNA probing approach.

Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription-PCR in temperature gradient gel electrophoresis fingerprints.

A novel approach was developed to quantify rRNA sequences in complex bacterial communities. The main bacterial 16S rRNAs in Drentse A grassland soils (The Netherlands) were amplified by reverse transcription (RT)-PCR with bacterium-specific primers and were separated by temperature gradient gel electrophoresis (TGGE). The primer pair used (primers U968-GC and L1401) was found to amplify with the same efficiency 16S rRNAs from bacterial cultures containing different taxa and cloned 16S ribosomal DNA amplicons from uncultured soil bacteria. The sequence-specific efficiency of amplification was determined by monitoring the amplification kinetics by kinetic PCR. The primer-specific amplification efficiency was assessed by competitive PCR and RT-PCR, and identical input amounts of different 16S rRNAs resulted in identical amplicon yields. The sequence-specific detection system used for competitive amplifications was TGGE, which also has been found to be suitable for simultaneous quantification of more than one sequence. We demonstrate that this approach can be applied to TGGE fingerprints of soil bacteria to estimate the ratios of the bacterial 16S rRNAs.

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria.

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.

Prominent occurrence of ribosomes from an uncultured bacterium of the Verrucomicrobiales cluster in grassland soils.

An uncultured bacterium of the Verrucomicrobiales cluster was identified by its 16S rDNA sequence as a major bacterium in Dutch Drentse A grassland soils. Potential metabolic activity of the according organism was estimated by applying direct ribosome isolation from soil and partial amplification of the 16S rRNA via RT-PCR using bacteria-specific primers. Temperature gradient gel electrophoresis separated the amplicons sequence specifically into reproducible fingerprints. One of the fingerprint bands matched with the signal of clone DA101. Southern blot hybridization with a DA101-specific V6 probe confirmed sequence identity. It is the first time that an organism of the Verrucomicrobiales cluster has been indicated as a potential major metabolizer in environmental microbial communities.

Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands).

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass.