Identification of Actinobacillus actinomycetemcomitans using species-specific 16S rDNA primers

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 16S ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC 33384T. Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.     

Phylogenetic assessment of heterotrophic bacteria from a water distribution system using 16S rDNA sequencing

Determination of a heterotrophic plate count (HPC) for drinking-water samples alone is not enough to assess possible health hazards associated with sudden changes in the bacterial count. Speciation is very crucial to determine whether the population includes pathogens and (or) opportunistic pathogens. Most of the isolates recovered from drinking water samples could not be allocated to a specific phylogenetic branch based on the use of conventional diagnostic methods. The present study had to use phylogenetic analysis, which was simplified by determining and using the first 500-bp sequence of the 16S rDNA, to successfully identify the type and species of bacteria found in the samples. Gram-positive bacteria alpha-, beta-, and gamma-Proteobacteria were found to be the major groups representing the heterotrophic bacteria in drinking water. The study also revealed that the presence of sphingomonads in drinking water supplies may be much more common than has been reported so far and thus further studies are merited. The intermittent mode of supply, mainly characterized by water stagnation and flow interruption associated possibly with biofilm detachment, raised the possibility that the studied bacterial populations in such systems represented organisms coming from 2 different niches, the biofilm and the water column.     

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

The presence of latent bacteria is a serious problem in plant tissue cultures. While endophytes are generally beneficial to plants in situ, they may affect culture growth under the modified conditions in vitro. The present study was undertaken to identify and characterize endophytic bacteria associated with the medicinal plant Echinacea in tissue culture. Based on classical microbiological tests and 16S rRNA analyses, it was found that endophytic bacteria associated with aseptically micropropagated Echinacea plantlets are representatives of several genera, Acinetobacter, Bacillus, Pseudomonas, Wautersia (Ralstonia) and Stenotrophomonas. Based on TLC and HPLC analyses, we found that Pseudomonas stutzeri P3 strain produces plant hormone, auxin (indole-3-acetic acid, IAA). Antibiotic resistance was also assessed as a virulence factor. The majority of endophytic bacteria were resistant to the antibiotic kanamycin, but susceptible to chloramphenicol. Recommendations for propagating Echinacea in vitro cultures involve the addition of chloramphenicol, tetracycline, and ampicillin, antibiotics that cause no side effects on these plant species.     

Direct sequencing of double-stranded polymerase chain reaction-amplified 16S rDNA.

A number of different procedures have been developed for direct sequence analysis of PCR products. These methods rely on the cumbersome isolation of specific PCR products from agarose gels or the production of single-stranded template DNAs. In the approach presented here, we describe primers for the amplification of 16-S rDNA and a simple preparation of PCR product for sequencing.     

Identification and Phylogenetic analysis of thermophilic sulfate-reducing bacteria in oil field samples by 16S rDNA gene cloning and sequencing

Thermophilic sulfate-reducing bacteria (SRB) have been recognized as an important source of hydrogen sulfide (H2S) in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples (sandstone core, drilling mud, and production water) were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low G+C content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low G+C content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.     

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing Was developed by a synthesis method(DP-SBS)for high-throughput DNA sequencing,in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays.In this method,adifferent set of degenerate primers containing a give nnumber(n)of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface.The nucleotides(n 1)on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides.The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately.The main advanmge of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension.From the present study,it is found that the DP-SBS method is reliable,simple,and cost-effective for laboratory-sequencing a large amount of short DNA fragments.     

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Enterococcus sp. was isolated from the midgut of silkworm against the germination ofNosema bombycis spores. Identification was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes ofEnterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose.Enterococcus sp. was clustered together withEnterococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes ofEnterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe ofE. mundtii. So, we can make conclusion the 16S rDNA segment ofEnterococcus sp. can hybridise with species-specific probe ofE. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm.     

Polygenic analysis of ammonia-oxidizing bacteria using 16S rDNA, amoA, and amoB genes.

Finding a unique molecular marker capable of quickly providing rigorous and useful phylogenetic information would facilitate assessing the diversity of ammonia-oxidizing bacteria in environmental samples. Since only one of several available markers can be used at a time in these kinds of studies, the 16S rDNA, amoA and amoB genes were evaluated individually and then compared in order to identify the one that best fits the information provided by the composite dataset. Distance-based neighbor-joining and maximum parsimony trees generated using the sequences of the three mentioned genes were analyzed with respect to the combined polygenic trees. Maximum parsimony trees were found to be more accurate than distance-based ones, and the polygenic topology was shown to best fit the information contained in the sequences. However, the taxonomic and phylogenetic information provided by the three markers separately was also valid. Therefore, either of the functional markers (amoA or amoB) can be used to trace ammonia oxidizers in environmental studies in which only one gene can be targeted.     

Construction of species-specific primers for Pseudomonas andropogonis based on 16S rDNA sequences

Approximately 94% of the total 16S rDNA of Pseudomonas andropogonis strain ACH 01053A was sequenced and compared with that of strain ATCC 23061 obtained from the GenBank database. The two sequences were highly homologous with 1·3% difference. Alignment of sequences with those from closely related bacteria revealed two possible regions for design of a specific primer suitable for detection of Ps. andropogonis . Using primers designed to these variable regions in a PCR test, an amplification product of approximately 410 bp was specifically produced by 40 strains of Ps. andropogonis . No other bacterial species showed an amplification product under optimized PCR conditions. As few as 1000 cells per reaction were detected.     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

Identification of the green alga, Chlorella vulgaris (SDC1) using cyanobacteria derived 16S rDNA primers: targeting the chloroplast

We have tested a set of oligonucleotide primers originally developed for the specific amplification of 16S rRNA gene segments from cyanobacteria, in order to determine their versatility as an identification tool for phototrophic eucaryotes. Using web-based bioinformatics tools we determined that these primers not only targeted cyanobacterium sequences as previously described, but also 87% of sequences derived from phototrophic eucaryotes. In order to qualify our finding, a type culture and environmental strain from the freshwater unicellular, green algae genus Chlorella Beijerinck, were selected for further study. Subsequently, we sequenced a 578-bp fragment of the 16S rRNA gene, which proved to be present within the chloroplast genome, performed sequence analysis and positively identified our solvent-degrading environmental strain (SDC1) as Chlorella vulgaris.     

Phylogenetic affiliations ofRhodoferax fermentans and related species of phototrophic bacteria as determined by automated 16S rDNA sequencing

16S rDNA sequences of strains ofRhodoferax fermentans were analyzed and compared with those of species of the generaRubrivivax andRhodocyclus. Approximately 1.5-kb fragments of 16S rDNA from crude cell lysates were amplified by the polymerase chain reaction (PCR) and sequenced directly by usingTth DNA polymerase with the linear PCR sequencing protocol, followed by on-line detection with an automated laser fluorescent DNA sequencer. Pairwise sequence comparisons and distance matrix tree analysis showed thatRhodoferax fermentans, Rubrivivax gelatinosus, andRhodocyclus species belong to three separate lineages within the beta subclass of theProteobacteria, thereby confirming the phylogenetic validity of the genusRhodoferax, as well as of the generaRubrivivax andRhodocyclus.     

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.     

Effects of tillage managements and maize straw returning on soil microbiome using 16S rDNA sequencing

Agricultural practices could affect bacterial diversity and community structure by altering soil physical and chemical properties.Straw returning and tillage practices are widely used in agriculture,however,the effects of these agricultural practices on microbiomes are still unclear.In the present study,we compared the 18 bacterial communities of soil with different straw retu rning and tillage treatment combinations.The V3-V4 regions of the 16 S ribosomal RNA were amplified and analyzed by high-throughput sequencing technology.The results showed that the bacterial communities were consistently dominated by Acidobacteria,Proteobacteria,Actinobacteria,and Chloroflexi.Short-term straw returning and tillage practices significantly altered the diversity,relative abundance and functions of the soil microbiome.Soil subjected to rotary tillage and straw re turning(RTS)combination possessed the highest bacterial diversity and lowest ratio of G+/G-bacteria,indicating that RTS could be an efficient integrated manageme nt system to improve microbiome in the short term.Double verifications based on relative abundance and network analysis,revealed close relationships of Mycobacterium and Methylibium with RTS,indicating they could serve as biomarkers for RTS.Investigating microbial changes under different agricultural practices will provide valuable foundations for land sustainable utilization and increase crop yields.     

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.