Yong Joon Cho

INTRODUCING EZTAXON-E: A PROKARYOTIC 16S RRNA GENE SEQUENCE DATABASE WITH PHYLOTYPES THAT REPRESENT UNCULTURED SPECIES

Despite recent advances in commercially optimized identification systems, bacterial identification remains a challenging task in many routine microbiological laboratories, especially in situations where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively for this task when coupled with a well-curated database, such as EzTaxon, containing sequences of type strains of prokaryotic species with validly published names. Although the EzTaxon database has been widely used for routine identification of prokaryotic isolates, sequences from uncultured prokaryotes have not been considered. Here, the next generation database, named EzTaxon-e, is formally introduced. This new database covers not only species within the formal nomenclatural system but also phylotypes that may represent species in nature. In addition to an identification function based on Basic Local Alignment Search Tool (blast) searches and pairwise global sequence alignments, a new objective method of assessing the degree of completeness in sequencing is proposed. All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the identification of cultured and uncultured prokaryotes and offers a valuable means of communication among microbiologists who routinely encounter taxonomically novel isolates. The database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.

Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq

Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia coli grown under four growth conditions. The results clearly showed that our DSN treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods.

The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level

Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.

Profiling bacterial community in upper respiratory tracts

BackgroundInfection by pathogenic viruses results in rapid epithelial damage and significantly impacts on the condition of the upper respiratory tract, thus the effects of viral infection may induce changes in microbiota. Thus, we aimed to define the healthy microbiota and the viral pathogen-affected microbiota in the upper respiratory tract. In addition, any association between the type of viral agent and the resultant microbiota profile was assessed.MethodsWe analyzed the upper respiratory tract bacterial content of 57 healthy asymptomatic people (17 health-care workers and 40 community people) and 59 patients acutely infected with influenza, parainfluenza, rhino, respiratory syncytial, corona, adeno, or metapneumo viruses using culture-independent pyrosequencing.ResultsThe healthy subjects harbored primarily Streptococcus, whereas the patients showed an enrichment of Haemophilus or Moraxella. Quantifying the similarities between bacterial populations by using Fast UniFrac analysis indicated that bacterial profiles were apparently divisible into 6 oropharyngeal types in the tested subjects. The oropharyngeal types were not associated with the type of viruses, but were rather linked to the age of the subjects. Moraxella nonliquefaciens exhibited unprecedentedly high abundance in young subjects aged <6 years. The genome of M. nonliquefaciens was found to encode various proteins that may play roles in pathogenesis.ConclusionsThis study identified 6 oropharyngeal microbiome types. No virus-specific bacterial profile was discovered, but comparative analysis of healthy adults and patients identified a bacterium specific to young patients, M. nonliquefaciens.

Draft Genome Sequence of Mycobacterium abscessus subsp. bolletii BDT

Mycobacterium abscessus subsp. bolletii is an increasing cause of human pulmonary disease and infections of the skin and soft tissues. Consistent reports of human infections indicate that M. bolletii is a highly pathogenic, emerging species of rapidly growing mycobacteria (RGM). Here we report the first whole-genome sequence of M. abscessus subsp. bolletii BD(T).

Genome Sequence of Escherichia coli J53, a Reference Strain for Genetic Studies

Escherichia coli J53 (F(-) met pro Azi(r)) is a derivative of E. coli K-12 which is resistant to sodium azide. This strain has been widely used as a general recipient strain for various conjugation experiments. Here, we report the genome sequence of E. coli J53 (=KACC 16628).

Genome Sequence of Escherichia coli AA86, Isolated from Cow Feces

Escherichia coli AA86 (=KACC 15541) is an enteric bacterium that was isolated from a sample of healthy cow feces. Its genome sequence revealed that it is most closely related to the human fecal strain E. coli SE15 and could be classified under E. coli phylogenetic group B2. Here, we report the genome sequence of E. coli AA86, consisting of 3 contigs and 2 plasmids.

Reclassification of Serpens flexibilis Hespell 1977 as Pseudomonas flexibilis comb. nov., with Pseudomonas tuomuerensis Xin et al. 2009 as a later heterotypic synonym

Serpens flexibilis was proposed in 1977 and approved in 1980 without the 16S rRNA gene sequence information. The sequence of S. flexibilis became available in 2010, after the publication of Pseudomonas tuomuerensis in 2009. Our preliminary phylogenetic analyses indicated that these two strains share high sequence similarity and therefore showed strong potential to be united into a single species. To clarify the taxonomic status of the two species, a polyphasic taxonomy study was conducted including whole genome sequencing. The value of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between the genome sequences of S. flexibilis ATCC 29606(T) and P. tuomuerensis JCM 14085(T) were 98.1% and 89.0%, respectively. The phenotypic and chemotaxonomic properties including enzymatic activities, substrate utilization profiles, and fatty acids, supported that the two taxa have no pronounced difference and should thus constitute a single species. Therefore, we propose to transfer Serpens flexibilis Hespell 1977 to the genus Pseudomonas as Pseudomonas flexibilis comb. nov. (type strain=ATCC 29606(T)), with Pseudomonas tuomuerensis Xin et al. 2009 as a later heterotypic synonym of Pseudomonas flexibilis.

Draft Genome Sequence of Escherichia coli W26, an Enteric Strain Isolated from Cow Feces

An enteric bacterium, Escherichia coli W26 (KACC 16630), was isolated from feces from a healthy cow in South Korea. Here, we report the draft genome sequence of the isolate, which is closely affiliated with commensal strains belonging to E. coli phylogroup B1.

Flavobacterium gilvum sp. nov., isolated from stream water

A cream-colored, non-gliding, aerobic Flavobacterium strain, designated EM1308T, was isolated from stream water. Gene sequence analysis of the 16S rRNA indicated that this isolate is closely related to Flavobacterium glycines NBRC 105008T (97.3 %) and Flavobacterium piscis CCUG 60099T (97.2 %). To evaluate the genomic relatedness of the isolate with its neighbors, the whole genome sequences of strain EM1308T and type strains of F. glycines and F. piscis were determined. The average nucleotide identities revealed that strain EM1308T is independent from other Flavobacterium species. The properties of major cellular fatty acids, polar lipids, menaquinone, and DNA G+C content of the isolate were within the general range for the genus Flavobacterium, but many biochemical and physiological characteristics distinguished the isolate from previously known species. Thus, strain EM1308T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium gilvum sp. nov. is proposed. The type strain is EM1308T (= KACC 18113T = JCM 30144T).