DNA-DNA hybridizations (DDH) play a key role in microbial species discrimination in cases when 16S rRNA gene sequence similarities are 97 % or higher. Using real-world 16S rRNA gene sequences and DDH data, we here re-investigate whether or not, and in which situations, this threshold value might be too conservative. Statistical estimates of these thresholds are calculated in general as well as more specifically for a number of phyla that are frequently subjected to DDH. Among several methods to infer 16S gene sequence similarities investigated, most of those routinely applied by taxonomists appear well suited for the task. The effects of using distinct DDH methods also seem to be insignificant. Depending on the investigated taxonomic group, a threshold between 98.2 and 99.0 % appears reasonable. In that way, up to half of the currently conducted DDH experiments could safely be omitted without a significant risk for wrongly differentiated species.

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Background Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media.ResultsThe three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon.ConclusionOur findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

The recently isolated strain L21-Fru-ABT represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-ABT represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-ABT and related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.

The whole-genome-sequenced rhizobacterium Bacillus amyloliquefaciens FZB42T (Chen et al., 2007) and other plant-associated strains of the genus Bacillus described as belonging to the species Bacillus amyloliquefaciens or Bacillus subtilis are used commercially to promote the growth and improve the health of crop plants. Previous investigations revealed that a group of strains represented a distinct ecotype related to B. amyloliquefaciens; however, the exact taxonomic position of this group remains elusive (Reva et al., 2004). In the present study, we demonstrated the ability of a group of Bacillus strains closely related to strain FZB42T to colonize Arabidopsis roots. On the basis of their phenotypic traits, the strains were similar to Bacillus amyloliquefaciens DSM 7T but differed considerably from this type strain in the DNA sequences of genes encoding 16S rRNA, gyrase subunit A (gyrA) and histidine kinase (cheA). Phylogenetic analysis performed with partial 16S rRNA, gyrA and cheA gene sequences revealed that the plant-associated strains of the genus Bacillus, including strain FZB42T, formed a lineage, which could be distinguished from the cluster of strains closely related to B. amyloliquefaciens DSM 7T. DNA–DNA hybridizations (DDH) performed with genomic DNA from strains DSM 7T and FZB42T yielded relatedness values of 63.7–71.2 %. Several methods of genomic analysis, such as direct whole-genome comparison, digital DDH and microarray-based comparative genomichybridization (M-CGH) were used as complementary tests. The group of plant-associated strains could be distinguished from strain DSM 7T and the type strain of B. subtilis by differences in the potential to synthesize non-ribosomal lipopeptides and polyketides. Based on the differences found in the marker gene sequences and the whole genomes of these strains, we propose two novel subspecies, designated B. amyloliquefaciens subsp. plantarum subsp. nov., with the type strain FZB42T ( = DSM 23117T = BGSC 10A6T), and B. amyloliquefaciens subsp. amyloliquefaciens subsp. nov., with the type strain DSM 7T( = ATCC 23350T = Fukumoto Strain FT), for plant-associated and non-plant-associated representatives, respecitvely. This is in agreement with results of DDH and M-CGH tests and the MALDI-TOF MS of cellular components, all of which suggested that the ecovars represent two different subspecies.

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The microbiota is generally assumed to have a substantial influence on the biology of multicellular organisms. The exact functional contributions of the microbes are often unclear and cannot be inferred easily from 16S rRNA genotyping, which is commonly used for taxonomic characterization of bacterial associates. In order to bridge this knowledge gap, we here analyzed the metabolic competences of the native microbiota of the model nematode Caenorhabditis elegans. We integrated wholegenome sequences of 77 bacterial microbiota members with metabolic modeling and experimental characterization of bacterial physiology. We found that, as a community, the microbiota can synthesize all essential nutrients for C. elegans. Both metabolic models and experimental analyses revealed that nutrient context can influence how bacteria interact within the microbiota. We identified key bacterial traits that are likely to influence the microbe's ability to colonize C. elegans (i.e., the ability of bacteria for pyruvate fermentation to acetoin) and affect nematode fitness (i.e., bacterial competence for hydroxyproline degradation). Considering that the microbiota is usually neglected in C. elegans research, the resource presented here will help our understanding of this nematode's biology in a more natural context. Our integrative approach moreover provides a novel, general framework to characterize microbiota-mediated functions.