Multilocus Sequence Analysis, a Rapid and Accurate Tool for Taxonomic Classification, Evolutionary Relationship Determination, and Population Biology Studies of the Genus Shewanella

Abstract

The genus Shewanella comprises a group of marine-dwelling species with worldwide distribution. Several species are regarded as causative agents of food spoilage and opportunistic pathogens of human diseases. In this study, a standard multilocus sequence analysis (MLSA) based on six protein-coding genes (gyrA, gyrB, infB, recN, rpoA, and topA) was established as a rapid and accurate identification tool in 59 Shewanella type strains. This method yielded sufficient resolving power in regard to enough informative sites, adequate sequence divergences, and distinct interspecies branches. The stability of phylogenetic topology was supported by high bootstrap values and concordance with different methods. The reliability of the MLSA scheme was further validated by identical phylogenies and high correlations of genomes. The MLSA approach provided a robust system to exhibit evolutionary relationships in the Shewanella genus. The split network tree proposed twelve distinct monophyletic clades with identical G+C contents and high genetic similarities. A total of 86 tested strains were investigated to explore the population biology of the Shewanella genus in China. The most prevalent Shewanella species was Shewanella algae, followed by Shewanella xiamenensis, Shewanella chilikensis, Shewanella indica, Shewanella seohaensis, and Shewanella carassii The strains frequently isolated from clinical and food samples highlighted the importance of increasing the surveillance of Shewanella species. Based on the combined genetic, genomic, and phenotypic analyses, Shewanella upenei should be considered a synonym of S. algae, and Shewanella pacifica should be reclassified as a synonym of Shewanella japonicaIMPORTANCE The MLSA scheme based on six housekeeping genes (HKGs) (gyrA, gyrB, infB, recN, rpoA, and topA) is well established as a reliable tool for taxonomic, evolutionary, and population diversity analyses of the genus Shewanella in this study. The standard MLSA method allows researchers to make rapid, economical, and precise identification of Shewanella strains. The robust phylogenetic network of MLSA provides profound insight into the evolutionary structure of the genus Shewanella The population genetics of Shewanella species determined by the MLSA approach plays a pivotal role in clinical diagnosis and routine monitoring. Further studies on remaining species and genomic analysis will enhance a more comprehensive understanding of the microbial systematics, phylogenetic relationships, and ecological status of the genus Shewanella.

Keywords: Shewanella; evolutionary relationship; identification; multilocus sequence analysis; population biology; taxonomic classification.

FIG 1

Phylogenetic tree reconstructed by the neighbor-joining method based on six concatenated gene sequences (gyrA, gyrB, infB, recN, rpoA, and topA [4,191 bp]) of 59 Shewanella type strains. The robustness of tree topologies was evaluated with 1,000 bootstrap replications, and values of >70% are shown at the nodes of the branches. The scale bar indicates substitutions per site. The strains Aeromonas hydrophila ATCC 7966^T, Escherichia coli JCM 1649^T, and Vibrio cholerae ATCC 14035^T served as outgroups.

FIG 2

Correlation analysis between similarities of isDDH and MLSA for the genus Shewanella. The vertical line indicates a 70% isDDH threshold, and the horizontal line indicates the corresponding 97.3% MLSA similarity. The four points greater than the species boundary are marked in red.

FIG 3

Concatenated split network tree based on six gene loci. The gyrA, gyrB, infB, recN, rpoA, and topA gene sequences from 56 validated Shewanella species were concatenated and reconstructed using the SplitsTree 4 program. Twelve distinct clades were identified and are indicated in the figure by red outlines.

FIG 4

Phylogenetic tree reconstructed by the neighbor-joining method based on six concatenated gene sequences (gyrA, gyrB, infB, recN, rpoA, and topA, 4191 bp) of 86 Shewanella tested strains and 26 related type strains. The number of tested strains for each compact cluster (black triangle) is shown in parentheses (each of these clusters also contained one type strain). The robustness of tree topologies was evaluated with 1,000 bootstrap replications, and values of >70% are shown at the nodes of the branches. The scale bar indicates the substitutions per site.

FIG 5

Intraspecies and interspecies similarities of 16S rRNA, six HKGs, and MLSA for 56 validated Shewanella species. The ranges of similarity are displayed in black (intraspecies) and gray (interspecies).