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The family Bifidobacteriaceae constitutes an important phylogenetic group that particularly includes bifidobacterial taxa demonstrating proven or debated positive effects on host health. The increasingly widespread application of probiotic cultures in the twenty-first century requires detailed classification to the level of particular strains. This study aimed to apply the glutamine synthetase class I (glnAI) gene region (717 bp representing approximately 50% of the entire gene sequence) using specific PCR primers for the classification, typing, and phylogenetic analysis of bifidobacteria and closely related scardovial genera. In the family Bifidobacteriaceae, this is the first report on the use of this gene for such purposes. To achieve high-value results, almost all valid Bifidobacteriaceae type strains (75) and 15 strains isolated from various environments were evaluated. The threshold value of the glnAI gene identity among Bifidobacterium species (86.9%) was comparable to that of other phylogenetic/identification markers proposed for bifidobacteria and was much lower compared to the 16S rRNA gene. Further statistical and phylogenetic analyses suggest that the glnAI gene can be applied as a novel genetic marker in the classification, genotyping, and phylogenetic analysis of isolates belonging to the family Bifidobacteriaceae.

The order Lactobacillales represents a morphologically, metabolically, and physiologically diverse group of bacteria. Lactic acid bacteria represent the core of this phylogenetic group. They are a part of epiphytic microflora, fermented dairy, meat, fruit and vegetable products, and the digestive tract of humans and animals. Despite the fact that these bacteria form a phenotypically and genotypically heterogeneous group, their phylogenetic relationship enables to propose a common genetic marker usable in classification, typing, and phylogeny. By creation of consensus sequence based on available genomic sequences of some representatives of order Lactobacillales, a specific primer-pair binding variable region of aspS gene (length of 615 nts) encoding the aspartyl-tRNA synthetase was designed. This gene has not yet been used in classification and phylogeny of the order Lactobacillales, although it meets the requirements of molecular markers (distribution and single copy in bacterial genomes, functional constancy and genetic stability, sequence variability among taxonomic units, irreplaceable role in proteosynthesis). Primers were applied on 54 type and wild Lactobacillales strains. Obtained sequences allowed to provide alignments for purpose of phylogenetic tree reconstructions that uncovered particular phylogenetic clusters of vagococci/enterococci, obligately homofermentative and heterofermentative lactobacilli. Although a relatively short fragment of the aspS gene (approximately 33% of the complete gene sequence) was evaluated, much higher sequence variability (61.8% of pairwise identity) among strains examined compared with 16S rRNA gene (90.7%, length of 1318 nt) provides a relatively simple and effective tool for classification and typing of selected representatives of the order Lactobacillales.

Detailed differentiation, classification, and phylogenetic analysis of the order Lactobacillales are performed using molecular techniques that involve the comparison of whole genomes, multilocus sequence analysis, DNA-DNA hybridisation, and 16S rRNA sequencing. Despite the wide application of the latter two techniques, issues associated with them are extensively discussed. Although complete genomic analyses are the most appropriate for phylogenetic studies, they are time-consuming and require high levels of expertise. Many phylogenetic/identification markers have been proposed for enterococci, lactobacilli, streptococci, and lactobacilli. However, none have been established for vagococci and some genera within the order Lactobacillales. The objective of the study was to find novel alternative housekeeping genes for classification, typing, and phylogenetic analysis of selected genera within the order Lactobacillales. We designed primers flanking variable regions of the infB (504 nt) and rpsB (333 nt) genes and amplified and sequenced them in 56 strains of different genera within the order Lactobacillales. Statistical analysis and characteristics of the gene regions suggested that they could be used for taxonomic purposes. Phylogenetic analyses, including assessment of (in)congruence between individual phylogenetic trees indicated the possibility of using the concatenation of the two genes as an alternative tool for the evaluation of phylogeny compared with the 16S rRNA gene representing the standard phylogenetic marker of prokaryotes. Moreover, infB, rpsB regions and their concatenate were phylogenetically consistent with two widely applied alternative genetic markers in taxonomy of particular Lactobacillales genera encoding the 60 kDa chaperonin protein (GroEL-hsp60) and phenylalanyl-tRNA synthetase, alpha subunit (pheS).

No common, unique genetic markers applicable to classification and phylogenetics for significant genera within the Propionibacteriaceae family have been suggested yet. Therefore, the aim of the study was to propose those genes in the genera Acidipropionibacterium, Cutibacterium, Propionibacterium and Pseudopropionibacterium. These genera were recently elicited from the genus Propionibacterium through whole genomic analyses. Three housekeeping genes, glyS, infB and rplB, were selected from many others according to the requirements for appropriate classification/phylogenetic markers. Concrete fragments of the genes were amplified using specific primers in most of the type (14) and 11 wild strains (originating from dairy products, human skin and the crop of a laying hen) recently classified into the genus Propionibacterium. Sequences obtained from amplicons were used to perform gene statistics and phylogenetic analyses with respect to applicability in classification, typing and phylogeny. The 16S rRNA gene sequences, still considered relevant in spite of its proven shortcomings as a basic tool for evaluation of bacterial phylogeny, were used as a baseline for comparative analyses. The statistics of the gene sequences revealed that the variable regions of all three genes have higher resolution capabilities among strains examined compared to the 16S rRNA gene analysis. Phylogenetic analyses based on individual gene sequences and their concatenate enabled to distinguish clusters of species belonging to the genera Acidipropionibacterium, Cutibacterium and Propionibacterium, which corresponds with a recently reported genomic study. Thus, the crucial importance of this study is the economically advantageous classification and typing of propionibacterial isolates and strains through the three gene regions in contrast to the requirement for whole genomic assays.

An alternative molecular marker with respect to the 16S rRNA gene demonstrating better identification and phylogenetic parameters has not been designed for the whole Bifidobacteriaceae family, which includes the genus Bifidobacterium and scardovial genera. Therefore, the aim of the study was to find such a gene in available genomic sequences, suggest appropriate means and conditions for asmplification and sequencing of the desired region of the selected gene in various strains of the bacterial family and verify the importance in classification and phylogeny. Specific primers flanking the variable region (~800 pb) within the pyrG gene encoding the CTP synthetase were designed by means of gene sequences retrieved from the genomes of strains belonging to the family Bifidobacteriaceae. The functionality and specificity of the primers were subsequently tested on the wild (7) and type strains of bifidobacteria (36) and scardovia (7). Comparative and phylogenetic studies based on obtained sequences revealed actual significance in classification and phylogeny of the Bifidobacteriaceae family. Gene statistics (percentages of mean sequence similarities and identical sites, mean number of nucleotide differences, P- and K-distances) and phylogenetic analyses (congruence between tree topologies, percentages of bootstrap values >50 and 70%) indicate that the pyrG gene represents an alternative identification and phylogenetic marker exhibiting higher discriminatory power among strains, (sub)species, and genera than the 16S rRNA gene. Sequences of the particular gene fragment, simply achieved through specific primers, enable more precisely to classify and evaluate phylogeny of the family Bifidobacteriaceae including, with some exceptions, health-promoting probiotic bacteria., (© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The concept of genome signature allows sequence comparisons without alignment. It relies on the premise that oligonucleotide compositions of DNA segments from the same or closely related genomes tend to be more similar than those from distantly related genomes. This concept has been used in detection of lateral gene transfer, phylogenetic classification of metagenome sequences (binning), and in studies of evolution of viruses and plasmids. The goal of this work is to explore limitations of genome signature in phylogenetic classification of DNA sequences and to identify formal representations of genome signature that expose best the phylogenetic relationships among prokaryotes. We found that genome signatures that best represent phylogenetic relationships are those normalized to factor out differences in G + C content and utilizing the standard A-C-G-T alphabet or the degenerate R-Y (purine-pyrimidine) alphabet. The main limitation of all genome signature representations tested is lack of divergence among some distantly related species. "Crowding" of the genome signature space and absence of molecular clock likely contribute to this phenomenon. We introduce "periodicity signatures"--formal representations of periodic sequence patterns related to DNA curvature--which can discriminate between bacterial and archaeal DNA sequences. Interestingly, archaea of the order Halobacteriaceae have periodic signatures similar to bacteria, possibly due to their early divergence from other archaea, extensive lateral gene transfer, or due to their adaptation to high salt environments. Our results have practical implications for development and application of genome signature-based methods for analysis and classification of DNA sequences.

As we enter the post-genomic era, with the accelerating availability of complete genome sequences, new theoretical approaches and new experimental techniques, our ability to dissect cellular processes at the molecular level continues to expand. Recent advances include the application of RNA interference methods to characterize loss-of-function phenotype genes in higher eukaryotes, comparative analysis of the human and mouse genome sequences, and methods for reconciling contradictory phylogenetic reconstructions. New developments feed into the increasingly rich content of databases such as the COG database. The next phase of research will be increasingly dominated by efforts to integrate the deluge of data into our understanding of biological systems.

Background: Identifying orthologs continues to be an early and imperative step in genome analysis but remains a challenging problem. While synteny (conservation of gene order) has previously been used independently and in combination with other methods to identify orthologs, applying synteny in ortholog identification has yet to be automated in a user-friendly manner. This desire for automation and ease-of-use led us to develop OrthoRefine, a standalone program that uses synteny to refine ortholog identification. Results: We developed OrthoRefine to improve the detection of orthologous genes by implementing a look-around window approach to detect synteny. We tested OrthoRefine in tandem with OrthoFinder, one of the most used software for identification of orthologs in recent years. We evaluated improvements provided by OrthoRefine in several bacterial and a eukaryotic dataset. OrthoRefine efficiently eliminates paralogs from orthologous groups detected by OrthoFinder. Using synteny increased specificity and functional ortholog identification; additionally, analysis of BLAST e-value, phylogenetics, and operon occurrence further supported using synteny for ortholog identification. A comparison of several window sizes suggested that smaller window sizes (eight genes) were generally the most suitable for identifying orthologs via synteny. However, larger windows (30 genes) performed better in datasets containing less closely related genomes. A typical run of OrthoRefine with ~ 10 bacterial genomes can be completed in a few minutes on a regular desktop PC. Conclusion: OrthoRefine is a simple-to-use, standalone tool that automates the application of synteny to improve ortholog detection. OrthoRefine is particularly efficient in eliminating paralogs from orthologous groups delineated by standard methods. [ABSTRACT FROM AUTHOR]