Alternative Phylogenetic Marker Research Articles

Molecular detection of some Gram-negative bacterial species using folp gene sequences

In this study, the folp gene encoding DHPS was tested as a possible alternative phylogenetic marker for more closely related Gram-negative bacterial species. In this new method, 854 bp were implemented for classification instead of 1435 mostly used in 16S gene detection. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including the most important Gram-negative bacterial species. The 16S rRNA-based tree openly portrayed three distinct clusters where cluster 1

Molecular Detection of some Staphylococcal species with the newly developed folp gene sequences

16SrRNA gene sequencing, universally accepted for bacterial identification could not effectively discriminate some closely related Staphylococcal species. For this reason, for bacterial species identification, other DNA sequencing genes are required. In this study, folp gene, encoding DHPS, was tested as a possible and alternative phylogenetic marker for more closely related Staphylococcal species. In this new method, 813 bp were implemented for classification instead of 1447 of 16S rRNA mostly used. Phylogenetic analysis was performed based on DNA sequences obtained from the GenBank including most important Staphylococcal species. As a result, the 16S rRNA-based tree clearly delineated 7 distinct clusters with high bootstrap values (1000) at distance adjusted to 5. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and Staphylococcus saprophyticus were identified in separate clusters. However, 2 mixed clusters were resolved including Staphylococcus epidermidis and Staphylococcus warneri in one cluster, while Staphylococcus pasteuri and Staphylococcus warneri in the other one. Comparatively, the folp gene-based tree yielded 9 clusters with at distance adjusted to 5 and 1000 bootstrap value. Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus ludunensis, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus pasteuri were resolved in separate clusters, however, Staphylococcus warneri and Staphylococcus epidermidis were identified in one mixed cluster. In addition, DNA-DNA relatedness studies indicated high sequence divergence of folp gene exhibiting 61.58-96.48 % interspecies homology compared to 16S rRNA with sequences similarities of 97.09-99.32 %. At the intraspecies level, the nucleotide substitution rates were ranged between 0-25%, mostly at low level for most Staphylococcal species compared to 98.65-99.93 identified in 16S rRNA gene. Moreover, in blind tests, this method was able for correct identification of 13 bacteria isolates of different Staphylococcal species. As a conclusion, folp gene sequences provide better resolution compared to the 16S rRNA gene sequences for both interspecies and interspecies DNA analysis of Staphylococci.

Molecular genetic and chemotaxonomic identification of the bacterium of the genus Ochrobactrum possessing oil-oxidizing and nitrogen-fixing activity

It was shown that analysis of cellular fatty acids using gas chromatography-mass-spectrometry and application of the gene encoding β-subunit of DNA gyrase (gyrB) as an alternative phylogenetic marker supplemented the taxonomic description of the biotechnologically valuable strain Ochrobactrum sp. IB DT-5.3/2 but were ineffective for species identification of the bacterium. However, based on the results of the matrixassisted laser desorption-ionization time of flight mass spectrometry of the protein fractions and our previous studies of the cultural–morphological and biochemical features, as well as sequencing of the 16S rRNA gene, we can conclude that the strain Ochrobactrum sp. IB DT-5.3/2 refers to the species O. intermedium.

The ipdC, hisC1 and hisC2 genes involved in indole-3-acetic production used as alternative phylogenetic markers in Azospirillum brasilense.

Plant growth-promoting bacteria of the genus Azospirillum are present in the rhizosphere and as endophytes of many crops. In this research we studied 40 Azospirillum strains isolated from different plants and geographic regions. They were first characterized by 16S rDNA restriction analysis, and their phylogenetic position was established by sequencing the genes 16S rDNA, ipdC, hisC1, and hisC2. The latter three genes are involved in the indole-3-pyruvic acid (IPyA) biosynthesis pathway of indole-3-acetic acid (IAA). Furthermore, the suitability of the 16S-23S rDNA intergenic spacer sequence (IGS) for the differentiation of closely related Azospirillum taxa and development of PCR protocols allows for specific detection of strains. The IGS-RFLP analysis enabled intraspecies differentiation, particularly of Azospirillum brasilense and Azospirillum lipoferum strains. Results demonstrated that the ipdC, hisC1, and hisC2 genes are highly conserved in all the assessed A. brasilense isolates, suggesting that these genes can be used as an alternative phylogenetic marker. In addition, IAA production determined by HPLC ranged from 0.17 to 98.2μgmg(-1) protein. Southern hybridization with the A. brasilense ipdC gene probe did not show, a hybridization signal with A. lipoferum, Azospirillum amazonense, Azospirillum halopreferans and Azospirillum irakense genomic DNA. This suggests that these species produce IAA by other pathways. Because IAA is mainly synthesized via the IPyA pathway in A. brasilense strains, a species that is used worldwide in agriculture, the identification of ipdC, hisC1, and hisC2 genes by PCR may be suitable for selecting exploitable strains.

Introduction to Establish the Comparative Analysis of 16S rRNA Gene Sequences with amoA and nxrA for Nitrifying Bacteria Isolated from East Kolkata Wetland: an International Ramsar Site

The nitrogen cycle is most complex and very important for the life on earth. Nitrifying bacteria which carried out nitrogen cycling process play a vital role in water quality control, thus creating a genomic fingerprint database for surveillance and monitoring of genetic variability of nitrifying bacteria is very important and also to establish a Biosecurity protocol for the Bheries located in different areas of West Bengal. The currently evolutionary relationships and the natural diversity of Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA) and Nitrite oxidoreductase (NxrA) in the East Kolkata Wetland. This study extended significantly the 16S rRNA and amoA databases for AOB, nxrA databases for NOB. Therefore, either of the functional markers (amoA or nxrA) can be used to trace ammonia oxidizers or structural marker (16S rDNA) can be used to trace specific species in environmental studies. These techniques included the use of 16S rDNA genes to characterize natural AOB and NOB populations and to analyze their taxonomic and phylogenetic features. The current perception of AOB and NOB phylogeny established by comparative 16S rRNA sequence analysis could be confirmed independently by exploiting the gene amoA and nxrA genes and proved as an alternative phylogenetic marker. The aim of our work is to investigate the potential of the ammonia monooxygenase subunit A gene (amoA) and Nitrite oxidoreductase (nxrA) gene as functional markers for AOBs and NOBs along with 16S rDNA sequence analysis in EKW. For ecological surveillance, genes like amoA and nxrA specific for the nitrifying bacteria under present research work could be a more reliable tool.

CloVR-ITS: Automated internal transcribed spacer amplicon sequence analysis pipeline for the characterization of fungal microbiota

BackgroundBesides the development of comprehensive tools for high-throughput 16S ribosomal RNA amplicon sequence analysis, there exists a growing need for protocols emphasizing alternative phylogenetic markers such as those representing eukaryotic organisms.ResultsHere we introduce CloVR-ITS, an automated pipeline for comparative analysis of internal transcribed spacer (ITS) pyrosequences amplified from metagenomic DNA isolates and representing fungal species. This pipeline performs a variety of steps similar to those commonly used for 16S rRNA amplicon sequence analysis, including preprocessing for quality, chimera detection, clustering of sequences into operational taxonomic units (OTUs), taxonomic assignment (at class, order, family, genus, and species levels) and statistical analysis of sample groups of interest based on user-provided information. Using ITS amplicon pyrosequencing data from a previous human gastric fluid study, we demonstrate the utility of CloVR-ITS for fungal microbiota analysis and provide runtime and cost examples, including analysis of extremely large datasets on the cloud. We show that the largest fractions of reads from the stomach fluid samples were assigned to Dothideomycetes, Saccharomycetes, Agaricomycetes and Sordariomycetes but that all samples were dominated by sequences that could not be taxonomically classified. Representatives of the Candida genus were identified in all samples, most notably C. quercitrusa, while sequence reads assigned to the Aspergillus genus were only identified in a subset of samples. CloVR-ITS is made available as a pre-installed, automated, and portable software pipeline for cloud-friendly execution as part of the CloVR virtual machine package (http://clovr.org).ConclusionThe CloVR-ITS pipeline provides fungal microbiota analysis that can be complementary to bacterial 16S rRNA and total metagenome sequence analysis allowing for more comprehensive studies of environmental and host-associated microbial communities.

Analysis of phylogenetic signal in protostomial intron patterns using Mutual Information

Many deep evolutionary divergences still remain unresolved, such as those among major taxa of the Lophotrochozoa. As alternative phylogenetic markers, the intron-exon structure of eukaryotic genomes and the patterns of absence and presence of spliceosomal introns appear to be promising. However, given the potential homoplasy of intron presence, the phylogenetic analysis of this data using standard evolutionary approaches has remained a challenge. Here, we used Mutual Information (MI) to estimate the phylogeny of Protostomia using gene structure data, and we compared these results with those obtained with Dollo Parsimony. Using full genome sequences from nine Metazoa, we identified 447 groups of orthologous sequences with 21,732 introns in 4,870 unique intron positions. We determined the shared absence and presence of introns in the corresponding sequence alignments and have made this data available in "IntronBase", a web-accessible and downloadable SQLite database. Our results obtained using Dollo Parsimony are obviously misled through systematic errors that arise from multiple intron loss events, but extensive filtering of data improved the quality of the estimated phylogenies. Mutual Information, in contrast, performs better with larger datasets, but at the same time it requires a complete data set, which is difficult to obtain for orthologs from a large number of taxa. Nevertheless, Mutual Information-based distances proved to be useful in analyzing this kind of data, also because the estimation of MI-based distances is independent of evolutionary models and therefore no pre-definitions of ancestral and derived character states are necessary.

Reclassification of Xylanibacter oryzae Ueki et al. 2006 as Prevotella oryzae comb. nov., with an emended description of the genus Prevotella

16S rRNA gene sequence information has indicated that Xylanibacter oryzae has a close relationship with the genus Prevotella. To clarify the taxonomic position of X. oryzae, we determined the sequence of hsp60 as it represents an alternative phylogenetic marker for identification and classification of Gram-negative anaerobic rods. On the basis of hsp60 sequences, X. oryzae was located within the genus Prevotella, indicating that the species does not represent a distinct taxon at the genus level. Statistical tests (the Shimodaira-Hasegawa test and the approximately unbiased test) supported the finding that X. oryzae is monophyletic with members of the genus Prevotella and thus belongs to the genus. On the basis of the phylogenetic findings, we propose that X. oryzae should be reclassified as Prevotella oryzae comb. nov.; the type strain is KB3(T) (=JCM 13648(T) =DSM 17970(T)). An emended description of the genus Prevotella is also provided.

Usefulness of the hsp60 gene for the identification and classification of Gram-negative anaerobic rods

The hsp60 gene sequences were determined for 121 strains of Gram-negative anaerobic rods, including the genera Bacteroides, Barnesiella, Butyricimonas, Odoribacter, Parabacteroides, Paraprevotella, Porphyromonas, Prevotella and Tannerella. The mean pairwise hsp60 gene sequence similarity (73.8-97.1 %) between species in each genus, except for the genus Tannerella that comprises one species, was significantly less than that of the 16S rRNA gene sequence (88.3-96.3 %). Only pairwise hsp60 gene sequence similarity (97.1 %) of the genus Paraprevotella was higher than that of the 16S rRNA gene sequence (93.8 %). Each genus formed a distinct clade in the phylogenetic analysis of the hsp60 gene sequence as well as the 16S rRNA gene sequence. The phylogenetic analysis indicated a higher evolutionary rate for the hsp60 gene sequence than the 16S rRNA gene sequence, especially in the genera Porphyromonas and Prevotella. This study suggests that the hsp60 gene is a useful alternative phylogenetic marker for the identification and classification of a broad range of Gram-negative anaerobic rods.

Hsp60 and 16S rRNA gene sequence relationships among species of the genus Bacteroides with the finding that Bacteroides suis and Bacteroides tectus are heterotypic synonyms of Bacteroides pyogenes

hsp60 gene sequences were determined for members of the genus Bacteroides and sequence similarities were compared with those obtained for the 16S rRNA gene. Among the 29 Bacteroides type strains, the mean sequence similarity of the hsp60 gene (84.5 %) was significantly less than that of the 16S rRNA gene (90.7 %), indicating a high discriminatory power of the hsp60 gene. Species of the genus Bacteroides were differentiated well by hsp60 gene sequence analysis, except for Bacteroides pyogenes JCM 6294(T), Bacteroides suis JCM 6292(T) and Bacteroides tectus JCM 10003(T). The hsp60 gene sequence analysis and the levels of DNA-DNA relatedness observed demonstrated that these three type strains are a single species. Consequently, B. suis and B. tectus are heterotypic synonyms of B. pyogenes. This study suggests that the hsp60 gene is an alternative phylogenetic marker for the classification of species of the genus Bacteroides.

Hylodesmus singaporensis gen. et sp. nov., a new autosporic subaerial green alga (Scenedesmaceae, Chlorophyta) from Singapore

The algal flora of subaerial habitats in the tropics remains largely unexplored, despite the fact that it potentially encompasses a wealth of new evolutionary diversity. Here we present a detailed morphological and molecular characterization of an autosporic coccoid green alga isolated from decaying wood in a natural forest in Singapore. Depending on culture conditions, this alga formed globular to irregularly oval solitary cells. Autosporulation was the only mode of reproduction observed. The cell periphery was filled with numerous vacuoles, and a single parietal chloroplast contained a conspicuous pyrenoid surrounded by a bipartite starch envelope. The cell wall was composed of a thick inner layer and a thin trilaminar outer layer, and the cell surface was ornamented with a few delicate ribs. Phylogenetic analyses of 18S rRNA gene sequences placed our strain in the family Scenedesmaceae (Sphaeropleales, Chlorophyceae) as a strongly supported sister branch of the genus Desmodesmus. Analyses of an alternative phylogenetic marker widely used for the Scenedesmaceae, the ITS2 region, confirmed that the strain is distinct from any scenedesmacean alga sequenced to date, but is related to the genus Desmodesmus, despite lacking the defining phenotypic features of Desmodesmus (cell wall with four sporopolleninic layers ornamented with peculiar submicroscopic structures). Collectively, our results establish that we identified a novel, previously undocumented, evolutionary lineage of scenedesmacean algae necessitating its description as a new species in a new genus. We propose it be named Hylodesmus singaporensis gen. et sp. nov. A cryopreserved holotype specimen has been deposited into the Culture Collection of Algae of Charles University in Prague, Czech Republic (CAUP) as CAUP C-H8001.

Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA–DNA hybridization in the Bacillus subtilis group

The Bacillus subtilis group comprises eight closely related species that are indistinguishable from one another by 16S rRNA gene sequence analysis. Therefore, the gyrB gene, which encodes the subunit B protein of DNA gyrase, was selected as an alternative phylogenetic marker. To determine whether gyrB gene sequence analysis could be used for phylogenetic analysis and species identification of members of the B. subtilis group, the congruence of gyrB grouping with both 16S rRNA gene sequencing and DNA-DNA hybridization data was evaluated. Ranges of gyrB nucleotide and translated amino acid sequence similarities among the eight type strains were 75.4-95.0 % and 88.5-99.2 %, respectively, whereas 16S rRNA gene sequence similarities were 98.1-99.8 %. Results showed that gyrB gene sequences provide higher resolution than 16S rRNA gene sequences. The classification achieved by gyrB sequence analysis was in agreement with results obtained with DNA-DNA hybridization. It is concluded that the gyrB gene may be an efficient alternative target for identification and taxonomic analysis of members of the B. subtilis group.

Protein Coding Gene Sequences: Alternative Phylogenetic Markers or Possible Tools to Compare Ecological Diversity in Bacteria?

DNA sequences of protein-coding genes seem to be more effective than 16S rRNA gene sequencing for classifying the ecological diversity of bacteria. This fact, however, may be a consequence of the low evolutionary rate of 16S rRNA genes. While 16S rRNA gene sequence data are useful for placing moderately divergent populations into separate sequence clusters, protein-coding genes provide a more effective tool for distinguishing very closely related, recently evolved, ecological populations. Preliminary investigations using metabolic genes as genetic markers, such as genes coding for malate dehydrogenase or 6-phosphogluconate dehydrogenase, have been performed to study microbial communities. The sequencing of metabolic genes could enable the study of specific biochemical activities in bacteria, the identification of clones within bacterial populations, and the characterisation of specific clones within communities of pathogenic microorganisms. Multilocus sequence typing has been successfully used, e.g. for the identification of the currently circulating hypervirulent meningococcal lineages in epidemiological studies. It has also suggested that sequencing of protein-coding genes would provide a finer phylogenetic resolution when finding geovars possibly belonging to unknown species. It is expected, therefore, that sequencing of protein-coding loci, which usually show a wider sequence variations and are more rapidly evolving than the more conserved 16S rRNA encoding genes, will disclose many previously unknown ecological populations of bacteria in future population surveys. Keywords: protein coding genes, Bioinformatics, Neisseria, meningitidis, campylobacter jejuny, pediococcus acidilactici, lactobacillus delbrueckii, delbrueckii subsp, bulgaricus, lactis, streptococcus

Bacterial phylogeny based on comparative sequence analysis.

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis.

Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.