Understanding of the phylogeny and interrelationships of the genera within the order 'Enterobacteriales' has proven difficult using the 16S rRNA gene and other single-gene or limited multi-gene approaches. In this work, we have completed comprehensive comparative genomic analyses of the members of the order 'Enterobacteriales' which includes phylogenetic reconstructions based on 1548 core proteins, 53 ribosomal proteins and four multilocus sequence analysis proteins, as well as examining the overall genome similarity amongst the members of this order. The results of these analyses all support the existence of seven distinct monophyletic groups of genera within the order 'Enterobacteriales'. In parallel, our analyses of protein sequences from the 'Enterobacteriales' genomes have identified numerous molecular characteristics in the forms of conserved signature insertions/deletions, which are specifically shared by the members of the identified clades and independently support their monophyly and distinctness. Many of these groupings, either in part or in whole, have been recognized in previous evolutionary studies, but have not been consistently resolved as monophyletic entities in 16S rRNA gene trees. The work presented here represents the first comprehensive, genome-scale taxonomic analysis of the entirety of the order 'Enterobacteriales'. On the basis of phylogenetic analyses and the numerous identified conserved molecular characteristics, which clearly distinguish members of the order 'Enterobacteriales' and the seven reported clades within this order, a proposal is made here for the order Enterobacterales ord. nov. which consists of seven families: Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov.

The genus Burkholderia contains large number of diverse species which include many clinically important organisms, phytopathogens, as well as environmental species. However, currently, there is a paucity of biochemical or molecular characteristics which can reliably distinguish different groups of Burkholderia species. We report here the results of detailed phylogenetic and comparative genomic analyses of 45 sequenced species of the genus Burkholderia. In phylogenetic trees based upon concatenated sequences for 21 conserved proteins as well as 16S rRNA gene sequence based trees, members of the genus Burkholderia grouped into two major clades. Within these main clades a number of smaller clades including those corresponding to the clinically important Burkholderia cepacia complex (BCC) and the Burkholderia pseudomallei groups were also clearly distinguished. Our comparative analysis of protein sequences from Burkholderia spp. has identified 42 highly specific molecular markers in the form of conserved sequence indels (CSIs) that are uniquely found in a number of well-defined groups of Burkholderia spp. Six of these CSIs are specific for a group of Burkholderia spp. (referred to as Clade I in this work) which contains all clinically relevant members of the genus (viz. the BCC and the B. pseudomallei group) as well as the phytopathogenic Burkholderia spp. The second main clade (Clade II), which is composed of environmental Burkholderia species, is also distinguished by 2 identified CSIs that are specific for this group. Additionally, our work has also identified multiple CSIs that serve to clearly demarcate a number of smaller groups of Burkholderia spp. including 3 CSIs that are specific for the B. cepacia complex, 4 CSIs that are uniquely found in the B. pseudomallei group, 5 CSIs that are specific for the phytopathogenic Burkholderia spp. and 22 other CSI that distinguish two groups within Clade II. The described molecular markers provide highly specific means for the demarcation of different groups of Burkholderia spp. and they also offer novel and useful targets for the development of diagnostic assays for the clinically important members of the BCC or the pseudomallei groups. Based upon the results of phylogenetic analyses, the identified CSIs and the pathogenicity profile of Burkholderia species, we are proposing a division of the genus Burkholderia into two genera. In this new proposal, the emended genus Burkholderia will correspond to the Clade I and it will contain only the clinically relevant and phytopathogenic Burkholderia species. All other Burkholderia spp., which are primarily environmental, will be transferred to a new genus Paraburkholderia gen. nov.

The genus Borrelia contains two groups of organisms: the causative agents of Lyme disease and their relatives and the causative agents of relapsing fever and their relatives. These two groups are morphologically indistinguishable and are difficult to distinguish biochemically. In this work, we have carried out detailed comparative genomic analyses on protein sequences from 38 Borrelia genomes to identify molecular markers in the forms of conserved signature inserts/deletions (CSIs) that are specifically found in the Borrelia homologues, and conserved signature proteins (CSPs) which are uniquely present in Borrelia species. Our analyses have identified 31 CSIs and 82 CSPs that are uniquely shared by all sequenced Borrelia species, providing molecular markers for this group of organisms. In addition, our work has identified 7 CSIs and 21 CSPs which are uniquely found in the Lyme disease Borrelia species and eight CSIs and four CSPs that are specific for members of the relapsing fever Borrelia group. Additionally, 38 other CSIs, in proteins which are uniquely found in Borrelia species, also distinguish these two groups of Borrelia. The identified CSIs and CSPs provide novel and highly specific molecular markers for identification and distinguishing between the Lyme disease Borrelia and the relapsing fever Borrelia species. We also report the results of average nucleotide identity (ANI) analysis on Borrelia genomes and phylogenetic analysis for these species based upon 16S rRNA sequences and concatenated sequences for 25 conserved proteins. These analyses also support the distinctness of the two Borrelia clades. On the basis of the identified molecular markers, the results from ANI and phylogenetic studies, and the distinct pathogenicity profiles and arthropod vectors used by different Borrelia spp. for their transmission, we are proposing a division of the genus Borrelia into two separate genera: an emended genus Borrelia, containing the causative agents of relapsing fever and a novel genus, Borreliella gen. nov., containing the causative agents of Lyme disease.

The genus Mycoplasma, including species earlier classified in the genera Eperythrozoon and Haemobartonella, contains ~ 120 species and constitutes an extensively polyphyletic assemblage of bacteria within the phylum Tenericutes. Due to their small genome sizes and lack of unique characteristics, the relationships among the mycoplasmas/Tenericutes are not reliably discerned. Using genome sequences for 140 Tenericutes, their evolutionary relationships were examined using multiple independent approaches. Phylogenomic trees were constructed for 63 conserved proteins, 45 ribosomal proteins, three main subunits of RNA polymerase and 16S rRNA gene sequences. In all of these trees, Tenericutes species reliably grouped into four main clades designated as the "Acholeplasma", "Spiroplasma", "Pneumoniae" and "Hominis" clusters. These clades are also distinguished based on a similarity matrix constructed based on 16S rRNA gene sequences. Mycoplasma species were dispersed across 3 of these 4 clades highlighting their extensive polyphyly. In parallel, our comparative genomic analyses have identified > 100 conserved signature indels (CSIs) and 14 conserved signature proteins (CSPs), which are uniquely shared by the members of four identified clades, strongly supporting their monophyly and identifying them in molecular terms. Mycoplasma mycoides, the type species of the genus Mycoplasma, and a small number of other Mycoplasma species, formed a strongly supported clade within the "Spiroplasma" cluster. Nine CSIs and 14 CSPs reliably distinguish this clade from all other Mycoplasmatales species. The remainder of the Mycoplasmatales species are part of the "Pneumoniae" and "Hominis" clusters, which group together in phylogenetic trees. Here we are proposing that the order Mycoplasmatales should be emended to encompass only the Mycoplasma species within the "Spiroplasma" cluster and that a new order, Mycoplasmoidales ord. nov., should be created to encompass the other Mycoplasma species. The "Pneumoniae" and the "Hominis" clusters are proposed as two new families, Mycoplasmoidaceae fam. nov., which includes the genera Eperythrozoon, Ureaplasma, and the newly proposed genera Malacoplasma and Mycoplasmoides, and Metamycoplasmataceae fam. nov. to contain the newly proposed genera Metamycoplasma, Mycoplasmopsis, and Mesomycoplasma. The results presented here allow reliable discernment, both in phylogenetic and molecular terms, of the members of the two proposed families as well as different described genera within these families including members of the genus Eperythrozoon, which is comprised of uncultivable organisms. The taxonomic reclassifications proposed here, which more accurately portray the genetic diversity among the Tenericutes/Mycoplasma species, provide a new framework for understanding the biological and clinical aspects of these important microbes.

The species of the class Coriobacteriia are currently distinguished from other bacteria primarily on the basis of their branching in the 16S rRNA gene trees. No reliable molecular marker is known that distinguishes the bacteria of this class from other organisms. We report here the results of detailed phylogenetic and comparative analyses on 22 sequenced genomes from members of the class Coriobacteriia . Detailed comparative analyses on protein sequences from these genomes, reported here, have identified 66 conserved signature inserts or deletions (i.e. indels) (CSIs) in widely distributed proteins that are specific for a number of different clades of the class Coriobacteriia at multiple phylogenetic levels, which are also supported by phylogenetic analyses. A set of 24 CSIs in different proteins are specific for all sequenced members of the class Coriobacteriia , providing novel molecular markers distinguishing and delimiting this class. One additional CSI is uniquely present in all members of the class Coriobacteriia and the phylum Actinobacteria supporting their placement within this bacterial phylum. A set of 16 CSIs in divergent proteins are uniquely found in the genomes of all species for which sequences are available from the glucose-fermenting genera Coriobacterium , Collinsella , Atopobium and Olsenella , but they are not present in any other bacteria. The species from these genera also form a strongly supported clade (Clade I) in the phylogenetic trees based upon concatenated protein sequences and the 16S rRNA. An additional 10 CSIs in different proteins are specifically present in all members of the asaccharolytic genera Eggerthella , Cryptobacterium , Slackia and Gordonibacter for which sequence data is available. A clade consisting of these genera (Clade II) is also supported by our phylogenetic analyses. Within Clade I, two smaller clades, one consisting of the genera Coriobacterium and Collinsella and the other containing the genera Atopobium and Olsenella , are independently supported by multiple CSIs (eight and seven respectively) and our phylogenetic analyses. Based upon the results of phylogenetic studies and the identified molecular markers, which clearly distinguish and demarcate the above indicated clades of the class Coriobacteriia at different phylogenetic depths, we propose division of the class Coriobacteriia into two orders (viz. Coriobacteriales and Eggerthellales ord. nov.) and three families (viz. Coriobacteriaceae , Atopobiaceae fam. nov. and Eggerthellaceae fam. nov.). Additionally, descriptions of the class Coriobacteriia , the order Coriobacteriales and the family Coriobacteriaceea are also emended.