16S rRNA Gene-based Phylogeny Research Articles

Roseinatronobacter alkalisoli sp. nov., an alkaliphilic bacterium isolated from soda soil, and genome-based reclassification of the genera Rhodobaca and Roseinatronobacter.

The genera Rhodobaca and Roseinatronobacter are phylogenetically related genera within the family Paracoccaceae. Species of these genera were described using 16S rRNA gene-based phylogeny and phenotypic characteristics. However, the 16S rRNA gene identity and phylogeny reveal the controversy of the taxonomic status of these two genera. In this work, we examined the taxonomic positions of members of both genera using 16S rRNA gene phylogeny, phylogenomic analysis and further validated using overall genome-related indexes, including digital DNA-DNA hybridization, average nucleotide identity, average amino acid identity and percentage of conserved proteins. Based on phylogenetic and phylogenomic results, the current four species of the two genera clustered tightly into one clade with high bootstrap values, suggesting that the genus Rhodobaca should be merged with Roseinatronobacter. In addition, a novel species isolated from a soda soil sample collected from Anda City, PR China, and designated as HJB301T was also described. Phenotypic, chemotaxonomic, genomic and phylogenetic properties suggested that strain HJB301T (=CCTCC AB 2021113T=KCTC 82977T) represents a novel species of the genus Roseinatronobacter, for which the name Roseinatronobacter alkalisoli sp. nov. is proposed.

Identification and phylogenetic analysis of various termite species distributed across southern Haryana, India

Termites are highly abundant and vital insects that directly and indirectly influence local soils. The present study investigated the morphological and molecular phylogenetics of termite species collected from study fields in southern Haryana, India, from 2020 to 2021. Samples were subjected to integrated systematic analyses, taking into account the mandible features of soldiers for morphological systematics and 16S rRNA gene-based phylogeny for molecular systematics. Based on the external phenotype and relations to reference sequences in NCBI GenBank, 21 species were identified; these included five species each from Odontotermes and Microcerotermes, four species from Coptotermes, two species each from Microtermes and Eremotermes, and one species each from Amitermes, Angulitermes, and Neotermes. 16S rRNA gene sequences were utilized to construct phylogenetic trees to explore the relatedness among identified species. The results of the different molecular phylogenetic approaches including maximum parsimony, maximum likelihood, and neighbor-joining revealed nearly identical topological relations between the species and grouping of individuals in relation to their genera. The maximum parsimony tree based on mandible features has been found to be effective in clustering multiple species of a given genus in a clade in at least three termite genera.

Closed genomes uncover a saltwater species of Candidatus Electronema and shed new light on the boundary between marine and freshwater cable bacteria

Cable bacteria of the Desulfobulbaceae family are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera: Candidatus Electronema, typically found in freshwater environments, and Candidatus Electrothrix, typically found in saltwater environments. This taxonomic framework is based on both 16S rRNA gene sequences and metagenome-assembled genome (MAG) phylogenies. However, most of the currently available MAGs are highly fragmented, incomplete, and thus likely miss key genes essential for deciphering the physiology of cable bacteria. Also, a closed, circular genome of cable bacteria has not been published yet. To address this, we performed Nanopore long-read and Illumina short-read shotgun sequencing of selected environmental samples and a single-strain enrichment of Ca. Electronema aureum. We recovered multiple cable bacteria MAGs, including two circular and one single-contig. Phylogenomic analysis, also confirmed by 16S rRNA gene-based phylogeny, classified one circular MAG and the single-contig MAG as novel species of cable bacteria, which we propose to name Ca. Electronema halotolerans and Ca. Electrothrix laxa, respectively. The Ca. Electronema halotolerans, despite belonging to the previously recognized freshwater genus of cable bacteria, was retrieved from brackish-water sediment. Metabolic predictions showed several adaptations to a high salinity environment, similar to the “saltwater” Ca. Electrothrix species, indicating how Ca. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.

Genes of NAD<sup>+</sup>-Dependent Formate Dehydrogenases in Taxonomy of Aerobic Methylotrophic Bacteria of the Genus <i>Ancylobacter</i>

Abstract—Comparative phylogenetic analysis of NAD+-dependent formate dehydrogenases (NAD+–FDH) genes, which have been detected in all available genomes of methylotrophs of the genera Ancylobacter, Starkeya and Angulomicrobium, as well as in other members of the family Xanthobacteraceae (Xanthobacter, Aquabacter, Azorhizobium), was carried out. The position of Xanthobacteraceae on the tree constructed based on comparison of NAD+–FDH amino acid sequences was found to correlate with the 16S rRNA gene-based phylogeny. The sequences of the NAD+–FDH proteins of the genera Ancylobacter, Starkeya, and Angulomicrobium exhibited 87.8–98.3% identity, indicating that this protein is very conservative within this group of methylotrophs. For the first time, analysis of the NAD+–FDH functional genes is recommended as a supplementary criterion for interspecies differentiation between methylotrophic bacteria of the genus Ancylobacter.

Diversity of Cultivable Bacteria in A Saline Desert of Little Rann of Kutch, India: A Phylogenetic Perspective

Deserts in general, and Indian deserts in particular, are less attended for microbial diversity. The Little Rann of Kutch (LRK), a coastal saline desert, is characterized by a unique combination of both dry and wet features. This study represents the first report on the extensive isolation, spatial distribution, 16S rRNA gene-based phylogeny, and identification of novel taxa. A total of 87 isolates were obtained from three different study sites in LRK. Based on the full 16S rRNA gene sequences, the isolates were grouped into 44 different phylotypes of four phyla: Firmicutes, Proteobacteria, Actinobacteria, and Euryarchaeota. These in turn were represented by 19 different genera. Halomonas, Gracilibacillus, Thalassobacillus, Piscibacillus, Salimicrobium, Alkalibacillus, Bhargavaea, Proteus, Marinobacter, Pseudomonas, Kocuria, Corynebacterium, Planococcus, Micrococcus and Natronococcus identified in this study had never before been reported from this habitat. A majority of the isolates displayed broad salt and pH tolerance. The bacterial diversity of Venasar and Jogad closely resembled with each other. While Bacillus, Virgibacillus, Gracillibacillus, and Bhargavaea were common genera in all sites, six putative novel taxa of different phylogenetic groups were identified. Available nitrogen, pH, Organic carbon, TDS, and EC were the main environmental variables affecting the microbial diversity. Analysis of the geographical distribution revealed that a majority of the phylotypes had cosmopolitan distribution, followed by the saline and marine distribution, while ∼13% were affiliated with only LRK. The phylotypes associated with marine distribution decreased with increasing distance from the Gulf of Kutch, suggesting their endemism to marine environments. The study established the taxonomic novelty and prospects for the discovery of unique products and metabolites.

New human-associated species of the family Atopobiaceae and proposal to reclassify members of the genus Olsenella.

Five novel bacterial strains, Marseille-P1476T (=CSURP1476T=DSM 100642T), Marseille-P3256T (=CSURP3256T=CECT 9977T), Marseille-P2936T (=CSURP2936T=DSM 103159T), Marseille-P2912T (=CSURP2912T=DSM 103345T) and Marseille-P3197T (=CSURP3197T=CCUG 71847T), were isolated from various human specimens. These five strains were not identified at the species level by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Following 16S rRNA gene sequence comparisons with the GenBank database, the highest nucleotide sequence similarities of all studied strains were obtained to members of the paraphyletic genus Olsenella. A polyphasic taxono-genomic strategy (16S rRNA gene-based and core genome-based phylogeny, genomic comparison, phenotypic and biochemical characteristics) enabled us to better classify these strains and reclassify Olsenella species. Among the studied strains, Marseille-P1476T, Marseille-P2936T and Marseille-P3197T belonged to new species of the genus Olsenella for which we propose the names Olsenella massiliensis sp. nov., Olsenella phocaeensis sp. nov. and Olsenella urininfantis sp. nov., respectively. Strains Marseille-P2912T and Marseille-P3256T belonged to a new genus for which the names Thermophilibacter provencensis gen. nov., sp. nov. and Thermophilibacter mediterraneus gen. nov., sp. nov. are proposed, respectively. We also propose the creation of the genera Parafannyhessea gen. nov., Tractidigestivibacter gen. nov. and Paratractidigestivibacter gen. nov. and the reclassification of Olsenella umbonata as Parafannyhessea umbonata comb. nov., Olsenella scatoligenes as Tractidigestivibacter scatoligenes comb. nov., and Olsenella faecalis as Paratractidigestivibacter faecalis comb. nov.

Assessment of phylo-functional coherence along the bacterial phylogeny and taxonomy

In this report we use available curated phylogenies, taxonomy, and genome annotations to assess the phylogenetic and gene content similarity associated with each different taxon and taxonomic rank. Subsequently, we employ the same data to assess the frontiers of functional coherence along the bacterial phylogeny. Our results show that within-group phylogenetic and gene content similarity of taxa in the same rank are not homogenous, and that these values show extensive overlap between ranks. Functional coherence along the 16S rRNA gene-based phylogeny was limited to 44 particular nodes presenting large variations in phylogenetic depth. For instance, the deep subtree affiliated to class Actinobacteria presented functional coherence, while the shallower family Enterobacteriaceae-affiliated subtree did not. On the other hand, functional coherence along the genome-based phylogeny delimited deep subtrees affiliated to phyla Actinobacteriota, Deinococcota, Chloroflexota, Firmicutes, and a subtree containing the rest of the bacterial phyla. The results presented here can be used to guide the exploration of results in many microbial ecology and evolution research scenarios. Moreover, we provide dedicated scripts and files that can be used to continue the exploration of functional coherence along the bacterial phylogeny employing different parameters or input data (https://git.io/Jec5U).

Rouxiella aceris sp. nov., isolated from tree sap and the emended description of the genus Rouxiella.

Polyphasic taxonomic studies were performed for the seven strains, which were isolated from sap extracted from Acer pictum in Mt. Halla in Jeju, Republic of Korea. Cells of all the isolates were Gram-reaction-negative, facultatively anaerobic, short rods and contained the major isoprenoid quinone of Q-8, the predominant fatty acids of C16:0 and C17:0 cyclo and the major polar lipids including phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. The G + C contents of the genomic DNAs were 50.6-51.3%.The 16S rRNA gene-based phylogeny exhibited that the seven isolates formed two distinct sublines within the family Yersiniaceae. In the 92 core gene analysis, strain SAP-1T formed a subline at the base of radiation of the genus Rouxiella and its assignment to the genus Rouxiella was supported by high amino acid identity values (82.0-83.4%), albeit with sharing low 16S rRNA gene identities (96.0-96.9%). The average nucleotide identity and digital DNA-DNA hybridisation values together with phenotypic differences showed that strains SAP-1T, SAP-7, SAP-8 and SAP-13 belonged to a new species of the genus Rouxiella, while strains SAP-2, SAP-3 and SAP-27 were strains of Rouxiella silvae. On the basis of data obtained here, Rouxiella aceris sp. nov. (type strain, SAP-1T = KCTC 72599T = CCM 9078T) is proposed, with the emended description of the genus Rouxiella.

Phylogenomic analysis and characterization of carbon monoxide utilization genes in the family Phyllobacteriaceae with reclassification of Aminobacter carboxidus (Meyer et al. 1993, Hördt et al. 2020) as Aminobacter lissarensis comb. nov. (McDonald et al. 2005)

The monotypic carboxydophilic genus Carbophilus has recently been transferred to the genus Aminobacter within the family Phyllobacteriaceae, and Carbophilus carboxidus was renamed Aminobacter carboxidus (comb. nov.) [Hördt et al. 2020]. Due to the poor resolution of the 16S rRNA gene-based phylogeny, an extensive phylogenomic analysis of the family Phyllobacteriaceae was conducted, with particular focus on the genus Aminobacter. Whole genome-based analyses of Phyllobacteriaceae type strains provided evidenced that the genus Aminobacter forms a monophyletic cluster, clearly demarcated from all other members of the family. Close relatedness between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T was inferred from core proteome phylogeny, shared gene content, and multilocus sequence analyses. ANI and GGDC provided genetic similarity values above the species demarcating threshold for these two type strains. Metabolic profiling and cell morphology analysis corroborated the phenotypic identity between A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Search for the presence of carbon monoxide dehydrogenase (CODH) genes in Phyllobacteriaceae genomes revealed that the form II CODH is widespread in the family, whereas form I CODH was detected in few Mesorhizobium type strains, and in both A. carboxidus DSM 1086T and A. lissarensis DSM 17454T. Results of phylogenomic, chemotaxonomic, and morphological investigations, combined with the presence of similarly arranged CODH genes, indicate that A. carboxidus DSM 1086T and A. lissarensis DSM 17454T are distinct strains of the same species. Hence A. carboxidus is a later subjective heterotypic synonym of A. lissarensis.

Casting Light on the Adaptation Mechanisms and Evolutionary History of the Widespread Sumerlaeota.

Sumerlaeota is a mysterious, putative phylum-level lineage distributed globally but rarely reported. As such, their physiology, ecology, and evolutionary history remain unknown. The 16S rRNA gene survey reveals that Sumerlaeota is frequently detected in diverse environments globally, especially cold arid desert soils and deep-sea basin surface sediments, where it is one dominant microbial group. Here, we retrieved four Sumerlaeota metagenome-assembled genomes (MAGs) from two hot springs and one saline lake. Including another 12 publicly available MAGs, they represent six of the nine putative Sumerlaeota subgroups/orders, as indicated by 16S rRNA gene-based phylogeny. These elusive organisms likely obtain carbon mainly through utilization of refractory organics (e.g., chitin and cellulose) and proteinaceous compounds, suggesting that Sumerlaeota act as scavengers in nature. The presence of key bidirectional enzymes involved in acetate and hydrogen metabolisms in these MAGs suggests that they are acetogenic bacteria capable of both the production and consumption of hydrogen. The capabilities of dissimilatory nitrate and sulfate reduction, nitrogen fixation, phosphate solubilization, and organic phosphorus mineralization may confer these heterotrophs great advantages to thrive under diverse harsh conditions. Ancestral state reconstruction indicated that Sumerlaeota originated from chemotrophic and facultatively anaerobic ancestors, and their smaller and variably sized genomes evolved along dynamic pathways from a sizeable common ancestor (2,342 genes), leading to their physiological divergence. Notably, large gene gain and larger loss events occurred at the branch to the last common ancestor of the order subgroup 1, likely due to niche expansion and population size effects.IMPORTANCE In recent years, the tree of life has expanded substantially. Despite this, many abundant yet uncultivated microbial groups remain to be explored. The candidate phylum Sumerlaeota is widely distributed in various harsh environments. However, their physiology, adaptation mechanisms, and evolution remain elusive due to a lack of pure cultures and limited available genomes. Here, we used genomes from uncultivated members of Sumerlaeota to disclose why these taxa can thrive under diverse harsh conditions and how they evolved from a chemotrophic and facultatively anaerobic common ancestor. This study deeply explored the biology of Sumerlaeota and provided novel insights into their possible roles in global biogeochemical cycles, adaptation mechanisms, ecological significance, and evolutionary history.

Taxogenomic and Comparative Genomic Analysis of the Genus Saccharomonospora Focused on the Identification of Biosynthetic Clusters PKS and NRPS.

Actinobacteria are prokaryotes with a large biotechnological interest due to their ability to produce secondary metabolites, produced by two main biosynthetic gene clusters (BGCs): polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). Most studies on bioactive products have been carried out on actinobacteria isolated from soil, freshwater or marine habitats, while very few have been focused on halophilic actinobacteria isolated from extreme environments. In this study we have carried out a comparative genomic analysis of the actinobacterial genus Saccharomonospora, which includes species isolated from soils, lake sediments, marine or hypersaline habitats. A total of 19 genome sequences of members of Saccharomonospora were retrieved and analyzed. We compared the 16S rRNA gene-based phylogeny of this genus with evolutionary relationships inferred using a phylogenomic approach obtaining almost identical topologies between both strategies. This method allowed us to unequivocally assign strains into species and to identify some taxonomic relationships that need to be revised. Our study supports a recent speciation event occurring between Saccharomonospora halophila and Saccharomonospora iraqiensis. Concerning the identification of BGCs, a total of 18 different types of BGCs were detected in the analyzed genomes of Saccharomonospora, including PKS, NRPS and hybrid clusters which might be able to synthetize 40 different putative products. In comparison to other genera of the Actinobacteria, members of the genus Saccharomonospora showed a high degree of novelty and diversity of BGCs.

Taxogenomics Resolves Conflict in the Genus Rhodobacter: A Two and Half Decades Pending Thought to Reclassify the Genus Rhodobacter.

The genus Rhodobacter is taxonomically well studied, and some members are model organisms. However, this genus is comprised of a heterogeneous group of members. 16S rRNA gene-based phylogeny of the genus Rhodobacter indicates a motley assemblage of anoxygenic phototrophic bacteria (genus Rhodobacter) with interspersing members of other genera (chemotrophs) making the genus polyphyletic. Taxogenomics was performed to resolve the taxonomic conflicts of the genus Rhodobacter using twelve type strains. The phylogenomic analysis showed that Rhodobacter spp. can be grouped into four monophyletic clusters with interspersing chemotrophs. Genomic indices (ANI and dDDH) confirmed that all the current species are well defined, except Rhodobacter megalophilus. The average amino acid identity values between the monophyletic clusters of Rhodobacter members, as well as with the chemotrophic genera, are less than 80% whereas the percentage of conserved proteins values were below 70%, which has been observed among several genera related to Rhodobacter. The pan-genome analysis has shown that there are only 1239 core genes shared between the 12 species of the genus Rhodobacter. The polyphasic taxonomic analysis supports the phylogenomic and genomic studies in distinguishing the four Rhodobacter clusters. Each cluster is comprised of one to seven species according to the current Rhodobacter taxonomy. Therefore, to address this taxonomic discrepancy we propose to reclassify the members of the genus Rhodobacter into three new genera, Luteovulum gen. nov., Phaeovulum gen. nov. and Fuscovulum gen. nov., and provide an emended description of the genus Rhodobacter sensu stricto. Also, we propose reclassification of Rhodobacter megalophilus as a sub-species of Rhodobacter sphaeroides.

Whole-Genome Sequences of a Cluster of 14 Unidentified Related Veillonella sp. Strains from Human Clinical Samples and Type Strains of 3 Veillonella Validated Species.

We report 17 draft genomes for 14 unidentified Veillonella sp. strains closely related in 16S rRNA gene-based phylogeny and type strains of 3 Veillonella species with the aims of deciphering relationships between related species, evaluating the accuracy of current thresholds for species delineation, and robustly describing new species in the genus.

Phylogeny and genomics of SAUL, an enigmatic bacterial lineage frequently associated with marine sponges.

Many marine sponges contain dense and diverse communities of associated microorganisms. Members of the 'sponge-associated unclassified lineage' (SAUL) are frequently recorded from sponges, yet little is known about these bacteria. Here we investigated the distribution and phylogenetic status of SAUL. A meta-analysis of the available literature revealed the widespread distribution of this clade and its association with taxonomically varied sponge hosts. Phylogenetic analyses, conducted using both 16S rRNA gene-based phylogeny and concatenated marker protein sequences, revealed that SAUL is a sister clade of the candidate phylum 'Latescibacteria'. Furthermore, we conducted a comprehensive analysis of two draft genomes assembled from sponge metagenomes, revealing novel insights intothe physiology of this symbiont. Metabolic reconstruction suggested that SAUL members are aerobic bacteria with facultative anaerobic metabolism, with the capacity to degrade multiple sponge- and algae-derived carbohydrates. We described for the first time in a sponge symbiont the putative genomic capacity to transport phosphate into the cell and to produce and store polyphosphate granules, presumably constituting a phosphate reservoir for the sponge host in deprivation periods. Our findings suggest that the lifestyle of SAUL is symbiotic with the host sponge, and identify symbiont factors which may facilitate the establishment and maintenance of this relationship.

Microbial lipids reveal carbon assimilation patterns on hydrothermal sulfide chimneys.

Sulfide 'chimneys' characteristic of seafloor hydrothermal venting are diverse microbial habitats. ¹³C/¹²C ratios of microbial lipids have rarely been used to assess carbon assimilation pathways on these structures, despite complementing gene- and culture-based approaches. Here, we integrate analyses of the diversity of intact polar lipids (IPL) and their side-chain δ¹³C values (δ¹³ C(lipid)) with 16S rRNA gene-based phylogeny to examine microbial carbon flow on active and inactive sulfide structures from the Manus Basin. Surficial crusts of active structures, dominated by Epsilonproteobacteria, yield bacterial δ¹³C(lipid) values higher than biomass δ¹³C (total organic carbon), implicating autotrophy via the reverse tricarboxylic acid cycle. Our data also suggest δ¹³C(lipid) values vary on individual active structures without accompanying microbial diversity changes. Temperature and/or dissolved substrate effects - likely relating to variable advective-diffusive fluxes to chimney exteriors - may be responsible for differing ¹³C fractionation during assimilation. In an inactive structure, δ¹³C(lipid) values lower than biomass δ¹³C and a distinctive IPL and 16S rRNA gene diversity suggest a shift to a more diverse community and an alternate carbon assimilation pathway after venting ceases. We discuss here the potential of IPL and δ¹³C(lipid) analyses to elucidate carbon flow in hydrothermal structures when combined with other molecular tools.

<i>Candidatus</i> Methanogranum caenicola: a Novel Methanogen from the Anaerobic Digested Sludge, and Proposal of <i>Methanomassiliicoccaceae</i> fam. nov. and <i>Methanomassiliicoccales</i> ord. nov., for a Methanogenic Lineage of the Class <i>Thermoplasmata</i>

The class Thermoplasmata harbors huge uncultured archaeal lineages at the order level, so-called Groups E2 and E3. A novel archaeon Kjm51a affiliated with Group E2 was enriched from anaerobic sludge in the present study. Clone library analysis of the archaeal 16S rRNA and mcrA genes confirmed a unique archaeal population in the enrichment culture. The 16S rRNA gene-based phylogeny revealed that the enriched archaeon Kjm51a formed a distinct cluster within Group E2 in the class Thermoplasmata together with Methanomassiliicoccus luminyensis B10T and environmental clone sequences derived from anaerobic digesters, bovine rumen, and landfill leachate. Archaeon Kjm51a showed 87.7% 16S rRNA gene sequence identity to the closest cultured species, M. luminyensis B10T, indicating that archaeon Kjm51a might be phylogenetically novel at least at the genus level. In fluorescence in situ hybridization analysis, archaeon Kjm51a was observed as coccoid cells completely corresponding to the archaeal cells detected, although bacterial rod cells still coexisted. The growth of archaeon Kjm51a was dependent on the presence of methanol and yeast extract, and hydrogen and methane were produced in the enrichment culture. The addition of 2-bromo ethanesulfonate to the enrichment culture completely inhibited methane production and increased hydrogen concentration, which suggested that archaeon Kjm51a is a methanol-reducing hydrogenotrophic methanogen. Taken together, we propose the provisional taxonomic assignment, named Candidatus Methanogranum caenicola, for the enriched archaeon Kjm51a belonging to Group E2. We also propose to place the methanogenic lineage of the class Thermoplasmata in a novel order, Methanomassiliicoccales ord. nov.

RpoA is a useful gene for identification and classification of Streptococcus pneumoniae from the closely related viridans group streptococci

Streptococcus pneumoniae, the leading etiological agent of pneumonia, shares a high degree of DNA sequence homology with the viridans group of streptococci. The clinical and pathological manifestations may present with different features, and discrimination between S. pneumoniae and its close viridans cocci relatives, such as Streptococcus mitis and Streptococcus oralis, is still quite difficult. The 445-bp sequences of the N-terminal region of rpoA from nine S. pneumoniae, seven S. mitis, ten S. oralis, and two related strains were determined and compared with their respective 16S rRNA gene sequences to establish their usefulness in phylogenetic analysis. Pairwise comparisons of rpoA sequences among the species showed higher rates of evolution with lower similarities (92.3-100%) than those of 16S rRNA genes (96.8-100%). The rpoA-based phylogeny generated deeper branches and presented improved discriminatory resolution than the 16S rRNA gene-based phylogeny. These results show that rpoA sequences represent a consistent, but more discriminating, genomic marker than 16S rRNA gene sequences for investigating the evolutionary relationships of Streptococcus at the species level. rpoA could be a useful marker for identifying and classifying S. pneumoniae, S. mitis, and S. oralis from closely related taxa.

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Phylogeny, diversity and host specialization in the phylum Synergistetes with emphasis on strains and clones of human origin

Members of the phylum Synergistetes have been demonstrated in several environmental ecosystems and mammalian microflorae by culture-independent methods. In the past few years, the clinical relevance of some uncultivated phylotypes has been demonstrated in endodontic infections, and uncultured Synergistetes have been demonstrated in human mouth, gut and skin microbiota. However, Synergistetes are rarely cultured from human samples, and only 17 isolates are currently reported. Twelve members of Synergistetes isolated in the course of various infectious processes, including 3 Jonquetella anthropi, 2 Cloacibacillus evryensis, 2 Pyramidobacter piscolens and 5 unidentified strains, as well as 56 clones obtained by specific PCR from the normal vaginal microflora, were studied. 16S rRNA gene-based phylogeny showed that the clones were grouped into 3 clusters, corresponding to the genus Jonquetella, P. piscolens and one novel Synergistetes taxon. The presence and diversity of Synergistetes were reported for the first time in the vaginal microflora. Synergistetes were found in healthy patients, suggesting that they could play a functional role in human microflorae, but may also act as opportunistic pathogens. Studying the phylogenetic relationships between environmental and mammalian strains and clones revealed clearly delineated independent lineages according to the origin of the sequences.

DnaJ is a useful phylogenetic marker for alphaproteobacteria

In the past, bacterial phylogeny relied almost exclusively on 16S rRNA gene sequence analysis. More recently, multilocus sequence analysis has been used to infer organismal phylogenies. In this study, the dnaJ chaperone gene was investigated as a marker for phylogeny studies in alphaproteobacteria. Preliminary analysis of G+C contents and G+C3s contents (the G+C content of the synonymous third codon position) showed no clear evidence of horizontal transfer of this gene in proteobacteria. dnaJ-based phylogenies were then analysed at three taxonomic levels: the Proteobacteria, the Alphaproteobacteria and the genus Mesorhizobium. Dendrograms based on DnaJ and 16S rRNA gene sequences revealed the same topology described previously for the Proteobacteria. These results indicate that the DnaJ phylogenetic signal is able to reproduce the accepted relationships among the five classes of the Proteobacteria. At a lower taxonomic level, using 20 alphaproteobacteria, the 16S rRNA gene-based phylogeny is distinct from the one based on DnaJ sequence analysis. Although the same clusters are generated, only the topology of the DnaJ tree is consistent with broader phylogenies from recent studies based on concatenated alignments of multiple core genes. For example, the DnaJ tree shows the two clusters within the Rhizobiales as closely related, as expected, while the 16S rRNA gene-based phylogeny shows them as distantly related. In order to evaluate the phylogenetic performance of dnaJ at the genus level, a multilocus analysis based on five housekeeping genes (atpD, gapA, gyrB, recA and rplB) was performed for ten Mesorhizobium species. In contrast to the 16S rRNA gene, the DnaJ sequence analysis generated a tree similar to the multilocus dendrogram. For identification of chickpea mesorhizobium isolates, a dnaJ nucleotide sequence-based tree was used. Despite different topologies, 16S rRNA gene- and dnaJ-based trees led to the same species identification. This study suggests that the dnaJ gene is a good phylogenetic marker, particularly for the class Alphaproteobacteria, since its phylogeny is consistent with phylogenies based on multilocus approaches.