Species demarcation in Bacteria and Archaea is mainly based on overall genome relatedness, which serves a framework for modern microbiology. Current practice for obtaining these measures between two strains is shifting from experimentally determined similarity obtained by DNA-DNA hybridization (DDH) to genome-sequence-based similarity. Average nucleotide identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values between two genome sequences may be different from each other when reciprocal calculations are compared. We compared 63 690 pairs of genome sequences and found that the differences in reciprocal ANI values are significantly high, exceeding 1 % in some cases. To resolve this problem of not being symmetrical, a new algorithm, named OrthoANI, was developed to accommodate the concept of orthology for which both genome sequences were fragmented and only orthologous fragment pairs taken into consideration for calculating nucleotide identities. OrthoANI is highly correlated with ANI (using BLASTn) and the former showed approximately 0.1 % higher values than the latter. In conclusion, OrthoANI provides a more robust and faster means of calculating average nucleotide identity for taxonomic purposes. The standalone software tools are freely available at http://www.ezbiocloud.net/sw/oat.

OrthoANI: An improved algorithm and software for calculating average nucleotide identity.

This research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 311975. This publication reflects the views only of the author, and the European Union cannot be held responsible for any use which may be made of the information contained therein.

JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison.

Trabajo presentado en la 113th General Meeting of the American Society for Microbiology, celebrada en Denver, Estados Unidos, del 18 al 21 de mayo de 2013

Species concept for prokaryotes

The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies.

/pdf/phylogenomics-and-systematics-in-pseudomonas-4z06titkab.pdf

Phylogenomics and systematics in Pseudomonas

Acute hepatopancreatic necrosis disease (AHPND) has extended rapidly, causing alarming shrimp mortalities. Initially, the only known causative agent was Vibrio parahaemolyticus carrying a plasmid coding for the mortal toxins PirVP. Recently, it has been found that the plasmid and hence the disease, could be transferred among members of the Harveyi clade. The current study performs a genomic characterization of an isolate capable of developing AHPND in shrimp. Mortality studies and molecular and histopathological analyses showed the infection capacity of the strain. Multilocus sequence analysis placed the bacteria as a member of the Orientalis clade, well known for containing commensal and even probiotic bacteria used in the shrimp industry. Further whole genome comparative analyses, including Vibrio species from the Orientalis clade, and phylogenomic metrics (TETRA, ANI and DDH) showed that the isolate belongs to a previously unidentified species, now named Vibrio punensis sp. nov. strain BA55. Our findings show that the gene transfer capacity of Vibrio species goes beyond the clade classification, demonstrating a new pathogenic capacity to a previously known commensal clade. The presence of these genes in a different Vibrio clade may contribute to the knowledge of the Vibrio pathogenesis and has major implications for the spread of emerging diseases.

/pdf/pirvp-genes-causing-ahpnd-identified-in-a-new-vibrio-species-4jm9m933hs.pdf

PirVP genes causing AHPND identified in a new Vibrio species ( Vibrio punensis ) within the commensal Orientalis clade

In the original proposal of Wayne et al. (Int J Syst Bacteriol 37:463–464, 1987), two measures of genetic relatedness were proposed to set the boundary for prokaryotic species. The first was the change in the melting temperature (ΔTm) of heteroduplex DNA and the second was the extent of DNA–DNA hybridization (DDH). While this approach was justified given the experimental error inherent in these methods, genomic sequencing has the potential to measure both parameters with great precision. The average nucleotide identity (ANIb), a surrogate for the ΔTm, and the calculated DDH (cDDH) were determined from the complete genomes of representatives of 17 genera of prokaryotes. When the ANIb was >75 %, the ratio (100-cDDH)/(100-ANIb) was 3.69 ± 0.93 (± SD) and varied from about 2.35 to 4.59 between genera. The differences among genera was highly significant (p < 0.001) but not correlated with specific phylogenetic or physiological groups. Moreover, the ANIm was a poor measure of ANIb when ANIb was <75 %. Because the ANIb and cDDH provide different measures of relatedness, it is no longer appropiate to consider both when delineating species. For these reasons, measures of relatedness based upon sequence identity should be used for delineating species in the future.

The relationship of the whole genome sequence identity to DNA hybridization varies between genera of prokaryotes.

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The relationship of the whole genome sequence identity to DNA hybridization varies between genera of prokaryotes.