Review
Multilocus sequence analysis (MLSA) in prokaryotic taxonomy

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Abstract

To obtain a higher resolution of the phylogenetic relationships of species within a genus or genera within a family, multilocus sequence analysis (MLSA) is currently a widely used method. In MLSA studies, partial sequences of genes coding for proteins with conserved functions (‘housekeeping genes’) are used to generate phylogenetic trees and subsequently deduce phylogenies. However, MLSA is not only suggested as a phylogenetic tool to support and clarify the resolution of bacterial species with a higher resolution, as in 16S rRNA gene-based studies, but has also been discussed as a replacement for DNA–DNA hybridization (DDH) in species delineation. Nevertheless, despite the fact that MLSA has become an accepted and widely used method in prokaryotic taxonomy, no common generally accepted recommendations have been devised to date for either the whole area of microbial taxonomy or for taxa-specific applications of individual MLSA schemes. The different ways MLSA is performed can vary greatly for the selection of genes, their number, and the calculation method used when comparing the sequences obtained. Here, we provide an overview of the historical development of MLSA and critically review its current application in prokaryotic taxonomy by highlighting the advantages and disadvantages of the method's numerous variations. This provides a perspective for its future use in forthcoming genome-based genotypic taxonomic analyses.

Section snippets

Background

Genotypic methods are playing a key role in phylogenetic classification and identification at various taxonomic levels, as well as for diversity analyses of prokaryotic taxa. Tremendous changes have been seen in microbial taxonomy since Sanger et al. [56] described the didesoxy sequencing technology in the 1970s, Woese and Fox [70] and others introduced small subunit ribosomal RNA (rRNA) gene-based phylogeny,1

Origin of MLSA

MLSA is based on multilocus sequence typing (MLST), which was first introduced by Maiden et al. in 1998 as a microbial typing method for epidemiological and population genetic studies of pathogenic bacteria (species) [38]. A high infra-species resolution and the determination of the clonality of strains were key points of these studies [21]. MLST itself is based on the concept of multi-locus enzyme electrophoresis, a molecular typing method applied to populations and epidemiological studies of

Historical overview of the establishment of MLSA in prokaryotic taxonomy

In the statement given by the ad hoc committee for the re-evaluation of the species definition in bacteriology [59], MLST was mentioned as a method mainly applied in epidemiology and population studies, although it was envisaged as a method which had “brought a new dimension into the elucidation of genomic relatedness at the inter- and intra-specific” level. Stackebrandt et al. [59] pointed out that MLST provided microbiologists with a tool to search for phylogenetic markers independently of

Selection of genes

A critical point for MLSA studies is the selection of genes. It is clear that housekeeping genes coding for proteins with important functions should be considered because they are stable with respect to rapid genetic modifications. However, a universal set of genes that allows the hierarchical classification of all prokaryotes must be congruent with the best set of genes in order to distinguish closely related taxa [22]. Therefore, genes are often selected independently for each new taxon

Authors’ recommendations

16S rRNA gene sequence-based phylogenies cannot provide sufficient resolutions down to the species level (and below this taxonomic rank), but should remain the basic approach used in prokaryotic taxonomy because they reflect the overall relationships of prokaryotes. They enable the phylogenetic placement of both cultured and as yet uncultured bacteria, provide an initial genus assignment in most cases, and can reflect overall phylogenetic diversity. Clear recommendations with regard to sequence

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