What can genome-scale metabolic network reconstructions do for prokaryotic systematics?

It has recently been proposed that in addition to Nomenclature, Classification and Identification, Comprehending Microbial Diversity may be considered as the fourth tenet of microbial systematics [Staley JT (2010) The Bulletin of BISMiS, 1(1): 1–5]. As this fourth goal implies a fundamental understanding of microbial speciation, this perspective article argues that translation of bacterial genome sequences into metabolic features may contribute to the development of modern polyphasic taxonomic approaches. Genome-scale metabolic network reconstructions (GSMRs), which are the result of computationally predicted and experimentally confirmed stoichiometric matrices incorporating all enzyme and metabolite components encoded by a genome sequence, provide a platform that can illustrate bacterial speciation. As the topology and the composition of GSMRs are expected to be the result of adaptive evolution, the features of these networks may provide the prokaryotic taxonomist with novel tools for reaching the fourth tenet of microbial systematics. Through selected examples from the Actinobacteria, which have been inferred from GSMRs and experimentally confirmed after phenotypic characterisation, it will be shown that this level of information can be incorporated into modern polyphasic taxonomic approaches. In conclusion, three specific examples are illustrated to show how GSMRs will revolutionize prokaryotic systematics, as has previously occurred in many other fields of microbiology.     

Classification and identification of the Burkholderia cepacia complex: Past, present and future

The Burkholderia cepacia complex is a group of closely related species with conflicting biological properties. Triggered by the devastating effect of pulmonary infections in cystic fibrosis patients, the scientific community generated an unusually large amount of taxonomic data for these bacteria during the past 15 years. This review presents the polyphasic, multilocus and genomic methodology used for the classification and identification of these bacteria. The current state-of-the-art demonstrates that present day taxonomists can replace traditional DNA-DNA hybridizations for species level demarcation and 16S rRNA sequence analysis for studying phylogeny by superior whole genome sequence-based parameters within the framework of polyphasic taxonomic studies.     

Polyphasic approach of bacterial classification — An overview of recent advances

Classification of microorganisms on the basis of traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the ‘polyphasic approach’ for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classified on the basis of the polyphasic approach. Also previously classified organisms, as and when required, can be reclassified on this ground in order to obtain information about their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.     

Phylogenetic Characterization of Two Novel Commensal Bacteria Involved with Innate Immune Homeostasis in Drosophila melanogaster

During a previous study on the molecular interaction between commensal bacteria and host gut immunity, two novel bacterial strains, A911^T and G707^T, were isolated from the gut of Drosophila melanogaster. In this study, these strains were characterized in a polyphasic taxonomic study using phenotypic, genetic, and chemotaxonomic analyses. We show that the strains represent novel species in the family Acetobacteraceae. Strain G707^T, a highly pathogenic organism, represents a new species in the genus Gluconobacter, “Gluconobacter morbifer” sp. nov. (type strain G707 = KCTC 22116^T = JCM 15512^T). Strain A911^T, dominantly present in the normal Drosphila gut community, represents a novel genus and species, designated “Commensalibacter intestini” gen. nov., sp. nov. (type strain A911 = KCTC 22117^T = JCM 15511^T).     

Genome-based taxonomic classification of the closest-to-Comamonadaceae group supports a new family Sphaerotilaceae fam. nov. and taxonomic revisions

Many genera closest to the family Comamonadaceae have not been classified into any family; moreover, some of them are not monophyletic groups beyond the genus level. To resolve the taxonomic uncertainty of the closest-to-Comamonadaceae (CTC) group, we performed 16S rRNA gene- and genome-based phylogenetic analyses combined with genome relatedness indices and phenotypic traits comparison. Phylogenies based on the 16S rRNA gene and genome sequences demonstrated that the CTC group formed a coherent and robust monophyletic lineage and was sister to the family Comamonadaceae, thereby proposing the CTC group as a novel family, Sphaerotilaceae fam. nov. The resolved genus- and species-level taxonomic relationships of this new family were then validated by the phylogenomic reconstruction and comparisons of genome relatedness indices including digital DNA-DNA hybridization and average nucleotide identity (ANI) as well as comprehensive phenotypic analysis for type strains. Finally, we reclassified all misidentified genera and species, resulting in 19 new combinations, and proposed Sphaerotilaceae-specific thresholds of ANI and average amino acid identity for genus delineation. Collectively, this study has established a sound taxonomic framework of the novel family Sphaerotilaceae and will help guide future taxonomic efforts and prevent the propagation of taxonomic errors.     

Alexandriicola marinus gen. nov., sp. nov., a new member of the family Rhodobacteraceae isolated from marine phycosphere

Two yellow-pigmented bacterial strains, LZ-14 ^T and ABI-LZ29, were isolated from the cultivable phycosphere microbiota of the highly toxic marine dinoflagellate Alexandrium catenella LZT09 and demonstrated obvious microalgae growth-promoting potentials toward the algal host. To elucidate the taxonomic status of the two bioactive bacterial strains, they were subjected to a polyphasic taxonomic characterization. Both strains were found to be Gram-negative, aerobic, rod-shaped and motile; to contain Q-10 as the predominant ubiquinone; summed feature 8, C_16:0, C_18:1 ω7c 11-methyl and summed feature 3 as the major fatty acids; and diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and two unidentified phospholipids as the predominant polar lipids. Based on the phylogenetic analysis, phylogenomic inferences and phenotypic characteristics, the strains could be clearly distinguished from phylogenetically closely related species and formed a distinct monophyletic lineage in the family Rhodobacteraceae. The size of the draft genome of strain LZ-14 ^T is 4.615 Mb, with a DNA G?+?C content of 63.3 mol%. It contains ten predicted secondary metabolite biosynthetic gene clusters and core genes for bacterial exopolysaccharide biosynthesis. Therefore, strain LZ-14 ^T (= CCTCC AB 2017230 ^T?=?KCTC 62342 ^T) represents a novel species of a new genus, for which the name Alexandriicola marinus gen. nov., sp. nov., is proposed.

     

Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments of approximately 1 kb from each strain were spotted on microarrays. Genomes from 12 well-characterized fluorescent Pseudomonas strains were labeled with Cy dyes and hybridized to the arrays. Cluster analysis of the hybridization profiles revealed taxonomic relationships between bacterial strains tested at species to strain level resolution, suggesting that this approach is useful for the identification of bacteria as well as determining the genetic distance among bacteria. Since arrays can contain thousands of DNA spots, a single array has the potential for broad identification capacity. In addition, the method does not require laborious cross-hybridizations and can provide an open database of hybridization profiles, avoiding the limitations of traditional DNA-DNA hybridization.     

Genomic taxonomy of vibrios

Background  

Vibrio taxonomy has been based on a polyphasic approach. In this study, we retrieve useful taxonomic information (i. e. data that can be used to distinguish different taxonomic levels, such as species and genera) from 32 genome sequences of different vibrio species. We use a variety of tools to explore the taxonomic relationship between the sequenced genomes, including Multilocus Sequence Analysis (MLSA), supertrees, Average Amino Acid Identity (AAI), genomic signatures, and Genome BLAST atlases. Our aim is to analyse the usefulness of these tools for species identification in vibrios.     

Chromosomal-level assembly of the Leptodermis oblonga (Rubiaceae) genome and its phylogenetic implications

Rubiaceae is the fourth largest and a taxonomically complex family of angiosperms. Many species in this family harbor low reproductive isolation and frequently exhibit inconsistent phenotypic characteristics. Therefore, taxonomic classification and their phylogenetic relationships in the Rubiaceae family is challenging, especially in the genus Leptodermis. Considering the low taxonomic confusion and wide distribution, Leptodermis oblonga is selected as a representative Leptodermis for genome sequencing. The assemblies resulted in 497 Mbp nuclear and 155,100 bp chloroplast genomes, respectively. Using the nuclear genome as a reference, SNPs were called from 37 Leptodermis species or varieties. The phylogenetic tree based on SNPs exhibited high resolution for species delimitation of the complex and well-resolved phylogenetic relationships in the genus. Moreover, 28,987 genes were predicted in the nuclear genome and used for comparative genomics study. As the first chromosomal-level genome of the subfamily Rubioideae in Rubiaceae, it will provide fruitfully evolutionary understanding in the family.     

Prokaryotic systematics in the genomics era

As an essential and basic biological discipline, prokaryotic systematics is entering the era of genomics. This paradigmatic shift is significant not only for understanding molecular phylogeny at the whole genome level but also in revealing the genetic or epigenetic basis that accounts for the phenotypic criteria used to classify and identify species. These developments provide an opportunity and a challenge for systematists to reanalyze the molecular mechanisms underlying the taxonomic characteristics of prokaryotes by drawing the knowledge from studies of genomics and/or functional genomics employing platform technologies and related bioinformatics tools. It is expected that taxonomic books, such as Bergey’s Manual of Systematic Bacteriology may evolve into a systematics library indexed by phylogenomic information with an comprehensive understanding of prokaryotic speciation and associated increasing knowledge of biological phenomena.     

Molecular and evolutionary relationships among enteric bacteria

Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.     

Sphingomonas pokkalii sp. nov., a novel plant associated rhizobacterium isolated from a saline tolerant pokkali rice and its draft genome analysis

Three strains L3B27^T, 3CNBAF, L1A4 isolated from a brackish cultivated pokkali rice rhizosphere were characterised using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA and recA gene sequences revealed that these strains were highly similar among each other and formed a separate monophyletic cluster within the genus Sphingomonas with Sphingomonas pituitosa DSM 13101^T, Sphingomonas azotifigens DSM 18530^T and Sphingomonas trueperi DSM 7225^T as their closest relatives sharing 97.9–98.3% 16S rRNA similarity and 91.3–94.0% recA similarity values, respectively. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA–DNA hybridisation (dDDH) values between L3B27^T (representative of the novel strains) and its phylogenetically closest Sphingomonas species were well below the established cut-off <94% (ANI/AAI) and <70% (dDDH) for species delineation. Further, the novel strains can be distinguished from its closest relatives based on several phenotypic traits. Thus, based on the polyphasic approach, we describe a novel Sphingomonas species for which the name Sphingomonas pokkalii sp. nov (type strain L3B27^T = KCTC 42098^T = MCC 3001^T) is proposed. In addition, the novel strains were characterised for their plant associated properties and found to possess several phenotypic traits which probably explain its plant associated lifestyle. This was further confirmed by the presence of several plant associated gene features in the genome of L3B27^T. Also, we could identify gene features which may likely involve in brackish water adaptation. Thus, this study provides first insights into the plant associated lifestyle, genome and taxonomy of a novel brackish adapted plant associated Sphingomonas.