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Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium)

Laboratorium voor Microbiologie (WE10), Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium

Correspondence
Anne Willems
Anne.Willems{at}UGent.be

There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA–DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (atpD, dnaK, gap, glnA, gltA, gyrB, pnp, recA, rpoB and thrC) was performed on 34 representatives of the genus Ensifer. Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA–DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA–DNA hybridization for the assessment of genetic relatedness between Ensifer species. Our MLSA data, confirmed by DNA–DNA hybridizations, support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.

The GenBank/EMBL/DDBJ accession numbers of newly reported sequences are provided in Table 1.

Details of primers and PCR cycling conditions, scatter plots of genetic similarity, various parameters for some of the sequences analysed and results of ILD tests are available as supplementary material with the online version of this paper.