Resolving phylogenetic history of Enterococcus mundtii using all enterococci core protein sequences

ESTEBAN, L; BLANCATO, VS; QUINTANA, I; MAGNI, C; ESPARIZ, M

Rosario

Congreso; 4to Congreso Argentino de Bioinformática y Biología Computacional; 2013

Asociación Argentina de Bioinformática y Biología Computacional

Background Enterococcus genera include commensal, environmental and human pathogen bacteria. For that reason, accurate identification is indispensable for both environmental and clinical studies. While phylogenetic trees based on comparative analysis of the 16S rDNA sequences is a standard identification and classification procedure, it was previously stated that some environmental strains of enterococci were incorrectly identified to the species level using this method [1]. E. mundtii CRL1656 is a pigmented bacterium that was isolated from stripping milk of an Argentinean cow and its genome was recently sequenced [2]. The aim of this study was to properly infer the phylogenic history of the strain. Materials and methods ANI Plot: ANI values were calculated based on pairwise alignment of the genome stretches using JSpecies software and shared genes using the Compare Metabolic Reconstruction tool from RAST server. Phylogenetic studies: Orthologous proteins were assigned using OrthoMCL tool, concatenated using an ad hoc Perl script, aligned using ClustalX, trimmed using GBlock 0.91b resulting in a dataset with a total of 214,922 positions. Finally, the evolutionary history was inferred by using the Randomized Axelerated Maximum Likelihood (RAxML) algorithm based on DCMUT with empirical base frequencies and GAMMA distribution model. The reliability of the inferred tree was tested by bootstrapping with 1000 replicates. Results We observed that E. mundtii is more related to E. faecium species (Figure 1A). Noteworthy, other pigmented species, such as E. casseliflavus are phylogenetically more distant. To quantify the relationships among these enterococcal species, we generated average nucleotide identity (ANI) plots (Figure 1B), which have been used to query and refine prokaryotic species definitions. As expected, ANI values were lower than the 94% accepted as a threshold for species designation. E. faecium strains showed the highest ANI values (around 76 %), with a shared gene content between 87 and 89%. With respect to other enterococcal species, ANI varies within a narrow range (approximately 72%) but shared gene content is more disperse (75 to 82%). Figure 1: (A) Phylogenetic analysis of E. mundtii CRL1556. The tree with the highest log likelihood (-1983927.3) is shown. Lactococcus lactis SK11 was included as outgroup species. (B) ANI plot. Each point represents a comparison between E. mundtii and the following bacterial genomes: L. lactis SK11 (1), L. lactis IL1403 (2), S. uberis 01405 (3), E. italicus DSM15952 (4), E. casseliflavus ATCC12755 (5), E. faecalis 62 (6), E. faecalis V583 (7), E. saccharolyticus 30_1 (8), E. gallinarum EG2 (9), E. faecium Com15 (10), and E. faecium DO (11). Conclusions Our study reveals that E. mundtii shares a higher number of gene functions with E. faecium. With respect to other enterococcal species analyzed, all of them share a similar ANI with E. mundtii, however E. faecalis strains showed the lower percentage of shared genes, which might indicate that the niche occupied by E. mundtii might be closer to that were environmental strains are present. Reference [1] Nayak, B.S., Badgley, B, Harwood, V.J. Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing. Appl Environ Microbiol 2011, 77(14):5050-5055 [2] Magni, C. et al: Draft genome sequence of Enterococcus mundtii CRL1656. J Bacteriol 2012, 194:550.