Estimation of bacterial species phylogeny through oligonucleotide frequency distances

Classification of bacteria is mainly based on sequence comparisons of certain homologous genes such as 16S rRNA. Recently there are challenges to classify bacteria using oligonucleotide frequency pattern of nonhomologous sequences. However, the evolutionary significance of oligonucleotides longer than tetra-nucleotide is not studied well. We performed phylogenetic analysis by using the Euclidean distances calculated from the di to deca-nucleotide frequencies in bacterial genomes, and compared these oligonucleotide frequency-based tree topologies with those for 16S rRNA gene and concatenated seven genes. When oligonucleotide frequency-based trees were constructed for bacterial species with similar GC content, their topologies at genus and family level were congruent with those based on homologous genes. Our results suggest that oligonucleotide frequency is useful not only for classification of bacteria, but also for estimation of their phylogenetic relationships for closely related species.     

Genome comparison of Mycobacterium tuberculosis and other bacteria

The availability of the complete genome sequence of Mycobacterium tuberculosis allows its phylogenetic analysis based on the whole genome rather than single genes. As a genome-based tree is more representative of whole organisms and less inconsistent than single-gene trees, it could provide a better index for interpretation and inference about the origin and nature of species. The standard bacterial phylogeny based on 16S ribosomal RNA sequence comparison shows that M. tuberculosis is more related to Gram-positive than to Gram-negative bacteria. Our results based on genome comparison in terms of shared orthologous genes challenge this implication. We demonstrate that M. tuberculosis is more related to Gram-negative than to Gram-positive bacteria by a quantitative analysis on the genome tree. The numerical distance data derived from genome comparison and those from 16S rRNA comparison show high significant correlation, implying that conserved gene content carries a strong phylogenetic signature in evolution.     

Discordant phylogenies within the rrn loci of Rhizobia

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of alpha-Proteobacteria by sequencing analysis of three loci within their rrn operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera Bradyrhizobium, Mesorhizobium and Sinorhizobium have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.     

Cellular identification of a novel uncultured marine stramenopile (MAST-12 Clade) small-subunit rRNA gene sequence from a norwegian estuary by use of fluorescence in situ hybridization-scanning electron microscopy

Revealing the cellular identity of organisms behind environmental eukaryote rRNA gene sequences is a major objective in microbial diversity research. We sampled an estuarine oxygen-depleted microbial mat in southwestern Norway and retrieved an 18S rRNA gene signature that branches in the MAST-12 clade, an environmental marine stramenopile clade. Detailed phylogenetic analyses revealed that MAST-12 branches among the heterotrophic stramenopiles as a sister of the free-living Bicosoecida and the parasitic genus Blastocystis. Specific sequence signatures confirmed a relationship to these two groups while excluding direct assignment. We designed a specific oligonucleotide probe for the target sequence and detected the corresponding organism in incubation samples using fluorescence in situ hybridization (FISH). Using the combined FISH-scanning electron microscopy approach (T. Stoeck, W. H. Fowle, and S. S. Epstein, Appl. Environ. Microbiol. 69:6856-6863, 2003), we determined the morphotype of the target organism among the very diverse possible morphologies of the heterotrophic stramenopiles. The unpigmented cell is spherical and about 5 mum in diameter and possesses a short flagellum and a long flagellum, both emanating anteriorly. The long flagellum bears mastigonemes in a characteristic arrangement, and its length (30 mum) distinguishes the target organism from other recognized heterotrophic stramenopiles. The short flagellum is naked and often directed posteriorly. The organism possesses neither a lorica nor a stalk. The morphological characteristics that we discovered should help isolate a representative of a novel stramenopile group, possibly at a high taxonomic level, in order to study its ultrastructure, physiological capabilities, and ecological role in the environment.     

Beta-subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria

The genes encoding the beta-subunits of ATP-synthases (ATPases) from Bacteroides fragilis DSM 2151, Cytophaga lytica DSM 2039 and 'Taxeobacter ocellatus' were cloned. The nucleotide sequences were determined completely for the genes of the first two organisms and to a major part for that of 'T. ocellatus'. The predicted amino acid sequences were compared with previously published amino acid sequences of beta-subunits. Two characteristic insertions were found in genes from organisms belonging to the so-called bacteroides-cytophaga-flavo-bacterium group. The remaining structure shows a high degree of sequence similarity within this group. These data support the conclusions drawn from comparative 16S rRNA sequence analyses that organisms in this phenotypically heterogeneous group are phylogenetically related. A phylogenetic tree was constructed based on a distance matrix of optimally aligned amino acid sequences of beta-subunits of ATPases of various eubacteria, chloroplasts and mitochondria. It is in good agreement with a tree derived from 16S rRNA sequence analyses.     

用序列比对设计的茎环结构探针检测金黄色葡萄球菌

基于金黄色葡萄球菌16S rRNA基因序列,采用序列比对设计了一种茎环结构的寡聚核苷酸探针。探针的环序列即为金黄色葡萄球菌16S rRNA基因序列的其中一个片段,同其他菌种的16S rRNA基因序列误配2个以上的核苷酸,因此能高度专一、灵敏的检测金黄色葡萄球菌16S rRNA。根据分子信标技术和酶联免疫分析的原理,评估一个实验方法,即利用能构象转换的、固定化的茎环结构探针酶联检测靶核酸。由于探针的特异性加强,这个检测系统能有效的排除假阳性即不会出现误配一个核苷酸的情况。采用微量浓度测定分析,最低下限可检测出大约4ng的金葡球菌16SrRNA。这种方法的灵敏度比其他常规检测方法高出了至少一个数量级。     

Cellular Identification of a Novel Uncultured Marine Stramenopile (MAST-12 Clade) Small-Subunit rRNA Gene Sequence from a Norwegian Estuary by Use of Fluorescence In Situ Hybridization-Scanning Electron Microscopy

Revealing the cellular identity of organisms behind environmental eukaryote rRNA gene sequences is a major objective in microbial diversity research. We sampled an estuarine oxygen-depleted microbial mat in southwestern Norway and retrieved an 18S rRNA gene signature that branches in the MAST-12 clade, an environmental marine stramenopile clade. Detailed phylogenetic analyses revealed that MAST-12 branches among the heterotrophic stramenopiles as a sister of the free-living Bicosoecida and the parasitic genus Blastocystis. Specific sequence signatures confirmed a relationship to these two groups while excluding direct assignment. We designed a specific oligonucleotide probe for the target sequence and detected the corresponding organism in incubation samples using fluorescence in situ hybridization (FISH). Using the combined FISH-scanning electron microscopy approach (T. Stoeck, W. H. Fowle, and S. S. Epstein, Appl. Environ. Microbiol. 69:6856-6863, 2003), we determined the morphotype of the target organism among the very diverse possible morphologies of the heterotrophic stramenopiles. The unpigmented cell is spherical and about 5 μm in diameter and possesses a short flagellum and a long flagellum, both emanating anteriorly. The long flagellum bears mastigonemes in a characteristic arrangement, and its length (30 μm) distinguishes the target organism from other recognized heterotrophic stramenopiles. The short flagellum is naked and often directed posteriorly. The organism possesses neither a lorica nor a stalk. The morphological characteristics that we discovered should help isolate a representative of a novel stramenopile group, possibly at a high taxonomic level, in order to study its ultrastructure, physiological capabilities, and ecological role in the environment.     

16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas

The plant-pathogenic mycoplasmalike organisms (MLOs) are so named because they lack cell walls. Many features that are essential to a definitive classification remain uncharacterized, because these organisms have resisted attempts at in vitro culturing. To establish the taxonomic position of the MLOs, the DNA region containing the 16S rRNA gene from a representative of the MLOs has been cloned and sequenced. Sequence comparisons indicate that the MLOs are related to Mycoplasma capricolum and that these two bacteria share their phylogenetic origin with Bacillus subtilis. The low G + C content of this gene and features of its deduced secondary structure further support this grouping. However, the presence of a single tRNAIle gene in the spacer between the 16S rRNA and 23S rRNA genes of the MLOs differentiates the MLOs from other representatives of the mycoplasmas, which indicates an early divergence in the evolution of the members of the class Mollicutes. The presence of certain characteristic oligonucleotides in the 16S rRNA sequence indicates that MLOs may be closely related to acholeplasmas.     

Comparative sequence analysis and oligonucleotide probe design based on 23S rRNA genes of Alphaproteobacteria from North Sea bacterioplankton

Almost complete 23S rRNA gene sequences were obtained from 11 Alphaproteobacteria isolated from marine surface water of the German Bight. Five of the strains belong to the "marine alpha" group, a phylogenetic cluster which encompasses members of the genus Roseobacter and closely related bacteria. Phylogenetic sequence analysis based on 52 published as well as unpublished complete 23S rDNA sequences from Alphaproteobacteria including the newly obtained was in general consistent with the 16S rRNA gene sequence-derived phylogeny. 16S and 23S rRNA based phylogenies both showed a distinct cluster for strains associated with the "marine alpha" group. The suitability of both markers for the design of oligonucleotide probes targeting selected groups of Alphaproteobacteria was systematically evaluated and compared in silico. Six clusters of sequences covering different phylogenetic levels as well as two strains were selected in a case study. To compensate for the quantitative difference in the two data sets, the 16S rRNA dataset was truncated to sequences with an equivalent in the 23S rRNA data set. Our results show, that the overall number of phylogenetically redundant probes available could be more than doubled by extending probe design to the 23S rRNA. For small clusters of high sequence similarity and single strains, up to 8 times more discriminating binding sites were provided by the 23S rRNA.     

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.     

Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea

In this study, we report on first 16S rRNA gene sequences from highly saline brine sediments taken at a depth of 1,515 m in the Kebrit Deep, northern Red Sea. Microbial DNA extracted directly from the sediments was subjected to PCR amplification with primers specific for bacterial and archaeal 16S rRNA gene sequences. The PCR products were cloned, and a total of 11 (6 bacterial and 5 archaeal) clone types were determined by restriction endonuclease digestion. Phylogenetic analysis revealed that most of the cloned sequences were unique, showing no close association with sequences of cultivated organisms or sequences derived from environmental samples. The bacterial clone sequences form a novel phylogenetic lineage (KB1 group) that branches between the Aquificales and the Thermotogales. The archaeal clone sequences group within the Euryarchaeota. Some of the sequences cluster with the group II and group III uncultivated archaea sequence clones, while two clone groups form separate branches. Our results suggest that hitherto unknown archaea and bacteria may thrive in highly saline brines of the Red Sea under extreme environmental conditions. Received: 5 February 1999 / Accepted: 14 July 1999     

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such “heterogeneity hot spots” occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.     

Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat.

Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture. Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism. Individual cells of S. jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples. Therefore the radiolabelled probe was used to monitor the population of S. jonesii introduced into a chemostat which simulated the rumen ecosystem. The S. jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism. After inoculation, S. jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks. This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem.     

The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid.

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.     

Visualization of ribosomal RNA operon copy number distribution

Background  

Results of microbial ecology studies using 16S rRNA sequence information can be deceiving due to differences in rRNA operon copy number and genome size of the detected organisms. It therefore will be useful for investigators to have a better understanding of how these two parameters differ in various organism types. In this study, the number of ribosomal operons and genome size were separately mapped onto a Bacterial phylogenetic tree.