The recent advent of DNA sequencing technologies facilitates the use of genome sequencing data that provide means for more informative and precise classification and identification of members of the Bacteria and Archaea. Because the current species definition is based on the comparison of genome sequences between type and other strains in a given species, building a genome database with correct taxonomic information is of paramount need to enhance our efforts in exploring prokaryotic diversity and discovering novel species as well as for routine identifications. Here we introduce an integrated database, called EzBioCloud, that holds the taxonomic hierarchy of the Bacteria and Archaea, which is represented by quality-controlled 16S rRNA gene and genome sequences. Whole-genome assemblies in the NCBI Assembly Database were screened for low quality and subjected to a composite identification bioinformatics pipeline that employs gene-based searches followed by the calculation of average nucleotide identity. As a result, the database is made of 61 700 species/phylotypes, including 13 132 with validly published names, and 62 362 whole-genome assemblies that were identified taxonomically at the genus, species and subspecies levels. Genomic properties, such as genome size and DNA G+C content, and the occurrence in human microbiome data were calculated for each genus or higher taxa. This united database of taxonomy, 16S rRNA gene and genome sequences, with accompanying bioinformatics tools, should accelerate genome-based classification and identification of members of the Bacteria and Archaea. The database and related search tools are available at www.ezbiocloud.net/.

Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.

Evolution of Protein Molecules

Among available genome relatedness indices, average nucleotide identity (ANI) is one of the most robust measurements of genomic relatedness between strains, and has great potential in the taxonomy of bacteria and archaea as a substitute for the labour-intensive DNA–DNA hybridization (DDH) technique. An ANI threshold range (95–96 %) for species demarcation had previously been suggested based on comparative investigation between DDH and ANI values, albeit with rather limited datasets. Furthermore, its generality was not tested on all lineages of prokaryotes. Here, we investigated the overall distribution of ANI values generated by pairwise comparison of 6787 genomes of prokaryotes belonging to 22 phyla to see whether the suggested range can be applied to all species. There was an apparent distinction in the overall ANI distribution between intra- and interspecies relationships at around 95–96 % ANI. We went on to determine which level of 16S rRNA gene sequence similarity corresponds to the currently accepted ANI threshold for species demarcation using over one million comparisons. A twofold cross-validation statistical test revealed that 98.65 % 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species, which is consistent with previous suggestions (98.2–99.0 %) derived from comparative studies between DDH and 16S rRNA gene sequence similarity. Our findings should be useful in accelerating the use of genomic sequence data in the taxonomy of bacteria and archaea.

Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes

Genomic information has already been applied to prokaryotic species definition and classification. However, the contribution of the genome sequence to prokaryotic genus delimitation has been less studied. To gain insights into genus definition for the prokaryotes, we attempted to reveal the genus-level genomic differences in the current prokaryotic classification system and to delineate the boundary of a genus on the basis of genomic information. The average nucleotide sequence identity between two genomes can be used for prokaryotic species delineation, but it is not suitable for genus demarcation. We used the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance. A comprehensive genomic survey indicated that the POCP can serve as a robust genomic index for establishing the genus boundary for prokaryotic groups. Basically, two species belonging to the same genus would share at least half of their proteins. In a specific lineage, the genus and family/order ranks showed slight or no overlap in terms of POCP values. A prokaryotic genus can be defined as a group of species with all pairwise POCP values higher than 50%. Integration of whole-genome data into the current taxonomy system can provide comprehensive information for prokaryotic genus definition and delimitation.

A Proposed Genus Boundary for the Prokaryotes Based on Genomic Insights

The polyphasic approach used today in the taxonomy and systematics of the Bacteria and Archaea includes the use of phenotypic, chemotaxonomic and genotypic data. The use of 16S rRNA gene sequence data has revolutionized our understanding of the microbial world and led to a rapid increase in the number of descriptions of novel taxa, especially at the species level. It has allowed in many cases for the demarcation of taxa into distinct species, but its limitations in a number of groups have resulted in the continued use of DNA-DNA hybridization. As technology has improved, next-generation sequencing (NGS) has provided a rapid and cost-effective approach to obtaining whole-genome sequences of microbial strains. Although some 12,000 bacterial or archaeal genome sequences are available for comparison, only 1725 of these are of actual type strains, limiting the use of genomic data in comparative taxonomic studies when there are nearly 11,000 type strains. Efforts to obtain complete genome sequences of all type strains are critical to the future of microbial systematics. The incorporation of genomics into the taxonomy and systematics of the Bacteria and Archaea coupled with computational advances will boost the credibility of taxonomy in the genomic era. This special issue of International Journal of Systematic and Evolutionary Microbiology contains both original research and review articles covering the use of genomic sequence data in microbial taxonomy and systematics. It includes contributions on specific taxa as well as outlines of approaches for incorporating genomics into new strain isolation to new taxon description workflows.

Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea.

A Gram-reaction-negative, aerobic, yellowish-orange-pigmented bacterial strain, designated JC2436(T), was isolated from tidal-flat sediment of Oi Island in Korea. Comparative 16S rRNA gene sequence analysis indicated its close affiliation to Vitellibacter vladivostokensis, with 96 % sequence similarity to the type strain. Cells grew with 2-6 % NaCl and at 10-41 degrees C. Orange flexirubin pigments were present. The major isoprenoid quinone was MK-6, the DNA G+C content was 48.7 mol% and the predominant fatty acids (>10 %) were iso-C(15 : 0) and iso-C(17 : 0) 3-OH. The data obtained from this polyphasic study support the classification of this isolate within a novel species in the genus Vitellibacter, for which the name Vitellibacter aestuarii sp. nov. is proposed. The type strain is JC2436(T) (=IMSNU 14137(T) =KACC 13727(T) =KCTC 22361(T) =JCM 15496(T)).

Vitellibacter aestuarii sp. nov., isolated from tidal-flat sediment, and an emended description of the genus Vitellibacter.

Global warming has accelerated glacial retreat in the high Arctic. The exposed glacier foreland is an ideal place to study chronosequential changes in ecosystems. Although vegetation succession in the glacier forelands has been studied intensively, little is known about the microbial community structure in these environments. Therefore, this study focused on how glacial retreat influences the bacterial community structure and its relationship with soil properties. This study was conducted in the foreland of the Midtre Lovenbreen glacier in Svalbard (78.9°N). Seven soil samples of different ages were collected and analyzed for moisture content, pH, soil organic carbon and total nitrogen contents, and soil organic matter fractionation. In addition, the structure of the bacterial community was determined via pyrosequencing analysis of 16S rRNA genes. The physical and chemical properties of soil varied significantly along the distance from the glacier; with increasing distance, more amounts of clay and soil organic carbon contents were observed. In addition, Cyanobacteria, Firmicutes, and Actinobacteria were dominant in soil samples taken close to the glacier, whereas Acidobacteria were abundant further away from the glacier. Diversity indices indicated that the bacterial community changed from homogeneous to heterogeneous structure along the glacier chronosequence/distance from the glacier. Although the bacterial community structure differed on basis of the presence or absence of plants, the soil properties varied depending on soil age. These findings suggest that bacterial succession occurs over time in glacier forelands but on a timescale that is dif ferent from that of soil development.

Soil development and bacterial community shifts along the chronosequence of the Midtre Lovenbreen glacier foreland in Svalbard

The genes and intergenic regions of the amoCAB operon were analyzed to establish their potential as molecular markers for analyzing ammonia-oxidizing betaproteobacterial (beta-AOB) communities. Initially, sequence similarity for related taxa, evolutionary rates from linear regressions, and the presence of conserved and variable regions were analyzed for all available sequences of the complete amoCAB operon. The gene amoB showed the highest sequence variability of the three amo genes, suggesting that it might be a better molecular marker than the most frequently used amoA to resolve closely related AOB species. To test the suitability of using the amoCAB genes for community studies, a strategy involving nested PCR was employed. Primers to amplify the whole amoCAB operon and each individual gene were tested. The specificity of the products generated was analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The fragments obtained showed different grades of sequence identity to amoCAB sequences in the GenBank database. The nested PCR approach provides a possibility to increase the sensitivity of detection of amo genes in samples with low abundance of AOB. It also allows the amplification of the almost complete amoA gene, with about 300 bp more sequence information than the previous approaches. The coupled study of all three amo genes and the intergenic spacer regions that are under different selection pressure might allow a more detailed analysis of the evolutionary processes, which are responsible for the differentiation of AOB communities in different habitats.

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Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the amoCAB operon

One important pathway of the nitrogen cycle in aquatic environments is the oxidation of ammonia to nitrite by ammonia-oxidizing bacteria (AOB). In this study the composition of AOB communities was compared between fresh (lakes Plusssee and Schohsee) and brackish (Baltic Sea) water at two different levels: i) between environments and ii) within different depths in each environment. Changes in the community structure were studied by denaturing gradient gel electrophoresis (DGGE) and clone libraries of PCR products of 16S rRNA genes (rDNA) from AOB of the beta subclass of proteobacteria. Each environment displayed a particular DGGE band pattern. In Plusssee and the Baltic Sea, the differentiation of communities in epi- and metalimnion from those in hypolimnion coincided with a distinct stratification of the water column. In Schohsee with an aerobic hypolimnion, the communities at all depths were similar. AOB communities in sediments were different from those in the water column. The composition of clone libraries showed the presence of specific Nitrosomonas and Nitrosospira-like sequences in each environment and habitat.

Comparative analysis of ammonia-oxidizing bacterial communities in two lakes in North Germany and the Baltic Sea

Although the beneficial effects of endophytic bacteria on their host are significant, the investigation of the microbial diversity in any Antarctic moss has been neglected. In this study, we investigate the endophytic bacterial diversity of the upper green part and the lower brown part of Sanionia uncinata through 16S rRNA genes using pyrosequencing. Proteobacteria was the most dominant phylum with 65.6%, followed by Bacteroidetes (29.1%) and Actinobacteria (11.7%). The different distribution of Alphaproteobacteria between the upper green (2%) and lower brown (22.2%) parts of the moss was significant. Furthermore, dominant and diverse species were detected and closely related to the environmental sequences. These findings suggest that there are likely to be specific relationships between endophytes and host Antarctic moss species.

Ok-Sun Kim

Papers

Vitellibacter aestuarii sp. nov., isolated from tidal-flat sediment, and an emended description of the genus Vitellibacter.

Soil development and bacterial community shifts along the chronosequence of the Midtre Lovenbreen glacier foreland in Svalbard

Community analysis of betaproteobacterial ammonia-oxidizing bacteria using the amoCAB operon

Comparative analysis of ammonia-oxidizing bacterial communities in two lakes in North Germany and the Baltic Sea

Endophytic bacterial diversity of an Antarctic moss, Sanionia uncinata