Average Sequence Similarity Research Articles

Detection and identification of foot-and-mouth disease O/ME-SA/Ind-2001 virus lineage, Indonesia, 2022

ABSTRACT Foot-and-mouth disease (FMD) is a contagious disease affecting cloven-hoofed animals, which has been well-documented as one of the major animal diseases that causes severe economic loss in livestock sectors. The disease is endemic in many countries, particularly in Asia and Africa. Indonesia has been declared a disease-free country since 1986, and the World Organization of Animal Health (WOAH/OIE) recognized Indonesia as an FMD-free country without vaccination in 1990. However, the FMD virus was detected in many disease outbreaks in cattle and goats in Indonesia in May 2022. This study reports the detection and identification of FMD serotype O viruses in Indonesia. Although these viruses appeared to belong to the ME-SA/Ind-2001e lineage, they formed a unique cluster with 95.3% average nucleotide sequence similarity of the FMD VP1 gene to Ind-2001e viruses from other Asia countries. The illegal trade of live animals from endemic areas in Southeast Asia is one of the possible routes regarding the incursion of FMD in Indonesia, however, it requires further investigation.

Xenorhabdus bovienii subsp. africana subsp. nov., isolated from Steinernema africanum entomopathogenic nematodes.

Four Gram-negative bacterial strains isolated from Steinernema africanum entomopathogenic nematodes were biochemically and molecularly characterized to determine their taxonomic position. Results of 16S rRNA gene sequencing indicated that they belong to the class Gammaproteobacteria, family Morganellaceae, genus Xenorhabdus, and that they are conspecific. The average 16S rRNA gene sequence similarity between the newly isolated strains and the type strain of its more closely related species, Xenorhabdus bovienii T228T, is 99.4 %. We therefore selected only one of them, XENO-1T, for further molecular characterization using whole genome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions show that XENO-1T is closely related to the type strain of X. bovienii, T228T, and to several other strains that are thought to belong to this species. To clarify their taxonomic identities, we calculated average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values. We observed that the ANI and dDDH values between XENO-1T and X. bovienii T228T are 96.3 and 71.2 %, respectively, suggesting that XENO-1T represents a novel subspecies within the X. bovienii species. Noteworthy, the dDDH values between XENO-1T and several other X. bovienii strains are between 68.7 and 70.9 % and ANI values are between 95.8 and 96.4 %, which could be interpreted, in some instances, as that XENO-1T represents a new species. Considering that for taxonomic description the genomic sequences of the type strains are compared, and to avoid future taxonomic conflicts, we therefore propose to assign XENO-1T to a new subspecies within X. bovienii. ANI and dDDH values between XENO-1T and any other of the species with validly published names of the genus are lower than 96 and 70 %, respectively, supporting its novel status. Biochemical tests and in silico genomic comparisons show that XENO-1T exhibit a unique physiological profile that differs from all the Xenorhabdus species with validly published names and from their more closely related taxa. Based on this, we propose that strain XENO-1T represents a new subspecies within the X. bovienii species, for which we propose the name X. bovienii subsp. africana subsp. nov, with XENO-1T (=CCM 9244T=CCOS 2015T) as the type strain.

Genome analysis-based union of the genera Youhaiella and Paradevosia, and proposal that Methyloterrigena soli Kim et al. 2016 is a later heterotypic synonym of Paradevosia shaoguanensis Geng et al. 2015.

Phylogenetic tree analysis of Youhaiella tibetensis F4T, Paradevosia shaoguanensis J5-3T and Methyloterrigena soli M48T based on 16S rRNA gene sequences revealed that the three genera did not form distinct and independent monophyletic clusters. The 16S rRNA gene sequence similarities were all higher than 99 % between each pair of the three type strains. Average nucleotide identity, digital DNA-DNA hybridization, average amino acid identity and 16S rRNA gene sequence similarity between Paradevosia shaoguanensis J5-3T and Methyloterrigena soli M48T showed that they represented the same species. Physiological and biochemical characteristics of these three strains were similar, such as motility by polar flagella, major respiratory quinone, polar lipids, and fatty acids. Polygenetic trees and other characteristic comparative analysis revealed the need to merge the genera Youhaiella and Paradevosia into one genus.

Phylogeny analysis of whole protein-coding genes in metagenomic data detected an environmental gradient for the microbiota.

Environmental factors affect the growth of microorganisms and therefore alter the composition of microbiota. Correlative analysis of the relationship between metagenomic composition and the environmental gradient can help elucidate key environmental factors and establishment principles for microbial communities. However, a reasonable method to quantitatively compare whole metagenomic data and identify the primary environmental factors for the establishment of microbiota has not been reported so far. In this study, we developed a method to compare whole proteomes deduced from metagenomic shotgun sequencing data, and quantitatively display their phylogenetic relationships as metagenomic trees. We called this method Metagenomic Phylogeny by Average Sequence Similarity (MPASS). We also compared one of the metagenomic trees with dendrograms of environmental factors using a comparison tool for phylogenetic trees. The MPASS method correctly constructed metagenomic trees of simulated metagenomes and soil and water samples. The topology of the metagenomic tree of samples from the Kirishima hot springs area in Japan was highly similarity to that of the dendrograms based on previously reported environmental factors for this area. The topology of the metagenomic tree also reflected the dynamics of microbiota at the taxonomic and functional levels. Our results strongly suggest that MPASS can successfully classify metagenomic shotgun sequencing data based on the similarity of whole protein-coding sequences, and will be useful for the identification of principal environmental factors for the establishment of microbial communities. Custom Perl script for the MPASS pipeline is available at https://github.com/s0sat/MPASS.

Nano-RECall provides an integrated pipeline for HIV-1 drug resistance testing from Oxford Nanopore sequence data.

Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing. We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n= 46) and (b) homebrew integrase (IN) (n= 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only. The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings.

Taxonomic Classification of Bacteria Using Common Substrings

For the taxonomic classification of microbes, 16S ribosomal RNA (rRNA) gene sequences are widely used in environmental microbiology as reliable markers. Although the massive sequencing of 16S rRNA gene amplicons encompassing the full length of genes is not easy, because of the limitations of the current sequencing techniques, in databases Greengenes, RDP, and SILVA millions of rRNA gene sequences are uploaded. In this research, first a new similarity measure LCSS, for full length genes is defined. Then it is found that sequences reported for the same bacteria species demonstrate around 53% average sequence similarity in Greengenes and SILVA databases, while average similarity among genes reported for different bacteria species is around 15% only. This is 63%, and 20% respectively at genus level for the three data bases Greengenes, RDP, and SILVA. Hence, species, and genus-specific sequences constitute useful targets for diagnostic assays and other scientific investigations. In the present research, the built in function LongestCommonSubsequence is used repeatedly in computer algebra package MATHEMATICA to create an in silico pipeline for taxonomic classification uploaded new full-length sequences. Conclusions: Our results suggest that LongestCommonSubsequence similarity can be used for taxonomic classification of unknown bacteria through their full 16S ribosomal RNA (rRNA) gene sequences.

Mother\u2212child histocompatibility and risk of rheumatoid arthritis and systemic lupus erythematosus among mothers

The study objective was to test the hypothesis that having histocompatible children increases the risk of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), possibly by contributing to the persistence of fetal cells acquired during pregnancy. We conducted a case control study using data from the UC San Francisco Mother Child Immunogenetic Study and studies at the Inova Translational Medicine Institute. We imputed human leukocyte antigen (HLA) alleles and minor histocompatibility antigens (mHags). We created a variable of exposure to histocompatible children. We estimated an average sequence similarity matching (SSM) score for each mother based on discordant mother−child alleles as a measure of histocompatibility. We used logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals. A total of 138 RA, 117 SLE, and 913 control mothers were analyzed. Increased risk of RA was associated with having any child compatible at HLA-B (OR 1.9; 1.2–3.1), DPB1 (OR 1.8; 1.2–2.6) or DQB1 (OR 1.8; 1.2–2.7). Compatibility at mHag ZAPHIR was associated with reduced risk of SLE among mothers carrying the HLA-restriction allele B*07:02 (n = 262; OR 0.4; 0.2–0.8). Our findings support the hypothesis that mother−child histocompatibility is associated with risk of RA and SLE.

Modeling variation of clinical team processes with multiple sequence alignment.

Our objective was to model process variation of Emergency Medical Service teams responding to simulated pediatric emergencies and determine if sequence alignment distinguishes performance quality. We performed a retrospective process analysis by watching and coding activities in videos from standardized simulations of 42 Emergency Medical Service teams. Teams were classified into high- or low-performing groups based on the Clinical Teamwork Scale™. Activities were coded according to resuscitation tasks, performer, and times. We used ClustalG to align task sequences within and between groups, and measured similarity. Teams within and between performance levels had an average sequence similarity of 52 ± 7% and 50 ± 7%. Teams performed clinically appropriate tasks that varied in prioritization, for example, performing compressions or connecting the EKG monitor early. There was no statistical difference in gross similarity between groups but specific differences in prioritization may have had clinically meaningful implications. Alignment could improve by accounting for task duration and concurrency.

Comparative analyses of whole-genome protein sequences from multiple organisms

Phylogenies based on entire genomes are a powerful tool for reconstructing the Tree of Life. Several methods have been proposed, most of which employ an alignment-free strategy. Average sequence similarity methods are different than most other whole-genome methods, because they are based on local alignments. However, previous average similarity methods fail to reconstruct a correct phylogeny when compared against other whole-genome trees. In this study, we developed a novel average sequence similarity method. Our method correctly reconstructs the phylogenetic tree of in silico evolved E. coli proteomes. We applied the method to reconstruct a whole-proteome phylogeny of 1,087 species from all three domains of life, Bacteria, Archaea, and Eucarya. Our tree was automatically reconstructed without any human decisions, such as the selection of organisms. The tree exhibits a concentric circle-like structure, indicating that all the organisms have similar total branch lengths from their common ancestor. Branching patterns of the members of each phylum of Bacteria and Archaea are largely consistent with previous reports. The topologies are largely consistent with those reconstructed by other methods. These results strongly suggest that this approach has sufficient taxonomic resolution and reliability to infer phylogeny, from phylum to strain, of a wide range of organisms.

Pedobacter solisilvae sp. nov., isolated from forest soil.

The bacteria strain EN12T was isolated from forest soil in the Republic of Korea. The cells were Gram-negative, non-motile and rod-shaped, and the strain was strictly aerobic. Phylogenetic analysis of its 16S rRNA gene sequences showed that strain EN12T belonged to the class Sphingobacteriia of the phylum Bacteroidetes, and its closest relative is Pedobacter namyangjuensis 5G38T, with a sequence similarity of 95.5 %. The average DNA sequence similarity from validly described species within the genus Pedobacter was 92.5±1.3 %. Chemotaxonomic data including major ubiquinones (menaquinone-7), polar lipids (phosphatidylethanolamine and sphingolipid) and fatty acids (iso-C15 : 0, iso-C17 : 0 3-OH, and C16 : 1ω6c/C16 : 1ω7c) also supported an affiliation of strain EN12T with the genus Pedobacter. Genotypic and phenotypic differentiation of strain EN12T from six published Pedobacter species was revealed through DNA-DNA relatedness and physiological/biochemical tests. Results of these phenotypic, phylogenetic and chemotaxonomic analyses indicated that strain EN12T is a novel species in the genus Pedobacter, for which we propose the name Pedobacter solisilvae sp. nov. (=KCTC 42612T=LMG 28820T).

Phylogenomics and comparative genomic studies delineate six main clades within the family Enterobacteriaceae and support the reclassification of several polyphyletic members of the family

The family Enterobacteriaceae harbors many important pathogens, however it has proven difficult to reliably distinguish different members of this family or discern their interrelationships. To understand the interrelationships among the Enterobacteriaceae species, we have constructed two comprehensive phylogenetic trees for 78 genome-sequenced Enterobacteriaceae species based on 2487 core genome proteins, and another set of 118 conserved proteins. The genome sequences of Enterobacteriaceae species were also analyzed for genetic relatedness based on average amino acid identity and 16S rRNA sequence similarity. In parallel, comparative genomic studies on protein sequences from the Enterobacteriaceae have identified 88 molecular markers in the form of conserved signature indels (CSIs) that are uniquely shared by specific members of the family. All of these multiple lines of investigations provide consistent evidence that most of the species/genera within the family can be assigned to 6 different subfamily level clades which are designated as the “Escherichia clade”, “Klebsiella clade”, “Enterobacter clade”, “Kosakonia clade”, “Cronobacter clade” and “Cedecea clade”. The members of the six described clades, in addition to their distinct branching in phylogenetic trees, can now be reliably demarcated in molecular terms on the basis of multiple identified CSIs that are exclusively shared by the group members. Several additional CSIs identified in this work that are either specific for individual genera (viz. Kosakonia, Kluyvera and Escherichia-Shigella), or are present at various taxonomic depths, offer information regarding the interrelationships among the different clades. The described molecular markers provide novel means for diagnostic as well as genetic and biochemical studies on the Enterobacteriaceae species and for resolving the polyphyly of its several genera viz. Escherichia, Enterobacter and Kluyvera. On the bases of our results, we are proposing the reclassification of Escherichia vulneris and Enterobacter massiliensis into two novel genera viz. Pseudescherichia gen. nov. and Metakosakonia gen. nov., respectively. Additionally, our results also support the transfer of “Enterobacter lignolyticus” and “Kluyvera intestini” to the genera Pluralibacter and Metakosakonia, respectively.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

English

To clearly delimit the members of the Bacillus subtilis group (BSG), is difficult using common phenotypic and genotypic methods. This study described the use of pheS and tuf gene as targets for interspecies discrimination within the BSG, and also to develop specific PCR and SNP primers for species and subspecies identification and differentiation. The average sequence similarity values of the pheS and tuf gene among type strains were 85.1 and 94.7%, respectively, and all members of the BSG could be clearly distinguished based on phylogenetic analyses of pheS gene sequence. In addition, the specific primers were designed according to pheS and tuf gene sequence. The primers were shown to specifically identify B. subtilis subsp. subtilis, B. amyloliquefaciens and Bacillus licheniformis, and clearly differentiate the subspecies of B. amyloliquefaciens using specific-PCR, combined with two-plex minisequencing method. In conclusion, we have successfully established a comparative sequence analysis and rapid molecular diagnosis techniques for determination of interspecies within the BSG. Key words: Bacillus subtilis group (BSG), species and subspecies discrimination, comparative sequence analysis, specific Pcr, two-plex minisequencing.

Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5Mb) from 239 clones were obtained, spanning ~4370bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS>LSU>SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR.

A Novel Data Association Algorithm for Unequal Length Fluctuant Sequence

There are quantities of such sensors as radar, ESM, navigator in aerospace areas and the sequence data is the most ordinary data in sensor domain. How to mine the information of these data has attracted a great interest in data mining. But sequence data is easily interfered and produces some fluctuant points. When dealing with these sequences, traditional sequence similarity measurement such as Euclidean distance arises large error, especially for unequal length fluctuant sequence. A novel average weight 1-norm unequal length fluctuant sequence similarity measurement algorithm based on dynamic time warping (DTW) is proposed to solve this problem. It constructs an absolute distance matrix based on DTW firstly, then weight average weight 1-norm and modify it with modifying factor to measure the distance of unequal length fluctuant sequence. It solves the fluctuation sensitivity of maximum distance measurement algorithm. Finally transform distance to similarity as the index of the association, associate the sequence data according to the maximum similarity association rule. Simulation results show the effectiveness of the proposed algorithm when associating unequal length fluctuant sequence, association rate is above 70% and simulate the effect of variation of the sequence length, fluctuant rate and processing time to the proposed algorithm.

Use of highly variable gene (yycH) as DNA marker to resolve interspecific relationships within the Lactobacillus casei group and a target for developing novel species-specific PCR primers

Identifying Lactobacillus species group using only phenotypic and genotypic (16S rDNA sequence homology analysis) techniques yields inaccurate results. In this study, the partial yycH gene sequence was used for species discrimination by direct DNA sequencing and as target in a species-specific PCR assay. All examined Lactobacillus strains were clearly distinguished from the closely related species by comparative sequence analysis of the yycH gene. The average sequence similarity for the yycH gene (78.5 %) among type strains was significantly lower than that of the 16S rRNA sequence (99.0 %). Therefore, the yycH gene can be proposed as an additional molecular marker for Lactobacillus casei group that provides higher resolution than 16S rRNA. In addition, the species-specific primers were also developed based on the yycH gene sequences, which were then employed for PCR using the template DNA of Lactobacter strains. The PCR primer pairs were shown to be specific for Lactobacillus paracasei and Lactobacillus rhamnosus. Our data indicate that the phylogenetic relationships in the L. casei group are easily resolved by direct sequencing of the yycH gene and combined with species-specific PCR assays.

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

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

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.

Subgrouping Automata: Automatic sequence subgrouping using phylogenetic tree-based optimum subgrouping algorithm

Sequence subgrouping for a given sequence set can enable various informative tasks such as the functional discrimination of sequence subsets and the functional inference of unknown sequences. Because an identity threshold for sequence subgrouping may vary according to the given sequence set, it is highly desirable to construct a robust subgrouping algorithm which automatically identifies an optimal identity threshold and generates subgroups for a given sequence set. To meet this end, an automatic sequence subgrouping method, named ‘Subgrouping Automata’ was constructed. Firstly, tree analysis module analyzes the structure of tree and calculates the all possible subgroups in each node. Sequence similarity analysis module calculates average sequence similarity for all subgroups in each node. Representative sequence generation module finds a representative sequence using profile analysis and self-scoring for each subgroup. For all nodes, average sequence similarities are calculated and ‘Subgrouping Automata’ searches a node showing statistically maximum sequence similarity increase using Student's t-value. A node showing the maximum t-value, which gives the most significant differences in average sequence similarity between two adjacent nodes, is determined as an optimum subgrouping node in the phylogenetic tree. Further analysis showed that the optimum subgrouping node from SA prevents under-subgrouping and over-subgrouping.

Utilization of elongation factor Tu gene (tuf) sequencing and species-specific PCR (SS-PCR) for the molecular identification of Acetobacter species complex

The aim of this study was to use tuf gene as a molecular target for species discrimination in the Acetobacter genus, as well as to develop species-specific PCR method for direct species identification of Acetobacter aceti. The results showed that most Acetobacter species could be clearly distinguished, and the average sequence similarity for the tuf gene (89.5%) among type strains was significantly lower than that of the 16S rRNA gene sequence (98.0%). A pair of species-specific primers were designed and used to specifically identify A. aceti, but none of the other Acetobacter strains. Our data indicate that the phylogenetic relationships of most strains in the Acetobacter genus can be resolved using tuf gene sequencing, and the novel species-specific primer pair could be used to rapidly and accurately identify the species of A. aceti by the PCR based assay.