Downloaded from www.microbiologyresearch.org by IP: 54.161.102.96 On: Fri, 09 Sep 2016 08:47:29 Genome diversity in the genera Fructobacillus, Leuconostoc and Weissella determined by physical and genetic mapping Ivo M. Chelo,3 Lı ´bia Ze ´ -Ze ´ 4 and Roge ´ rio Tenreiro Correspondence Ivo M. Chelo imchelo@igc.gulbenkian.pt Centro de Biodiversidade, Geno ´ mica Integrativa e Funcional (BioFIG), Faculdade de Cie ˆ ncias da Universidade de Lisboa, Edificio ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal Received 13 February 2009 Revised 19 October 2009 Accepted 2 November 2009 Pulsed-field gel electrophoresis analysis of chromosomal single and double restriction profiles of 17 strains belonging to three genera of ‘Leuconostocaceae’ was done, resulting in physical and genetic maps for three Fructobacillus, six Leuconostoc and four Weissella strains. AscI, I-CeuI, NotI and SfiI restriction enzymes were used together with Southern hybridization of selected probes to provide an assessment of genomic organization in different species. Estimated genome sizes varied from 1408 kb to 1547 kb in Fructobacillus, from 1644 kb to 2133 kb in Leuconostoc and from 1371 kb to 2197 kb in Weissella. Other genomic characteristics of interest were analysed, such as oriC and terC localization and rrn operon organization. The latter seems markedly different in Weissella, in both number and disposition in the chromosome. Comparisons of intra- and intergeneric features are discussed in the light of chromosome rearrangements and genomic evolution. INTRODUCTION The genera Fructobacillus, Leuconostoc and Weissella are composed of obligate heterofermentative bacterial species that, with species from the genus Oenococcus, constitute a single line of descent, the Leuconostoc group (Yang & Woese, 1989; Martinez-Murcia & Collins, 1990). Like other lactic acid bacteria (LAB) they are Gram-positive, non- spore-forming, inhabit nutrient-rich environments such as milk, meat, vegetable products and fermented drinks (Kandler & Weiss, 1986) and have lactic acid as their main end product. The phylogenetic structure of these genera has been defined based on the analysis of 16S rRNA gene sequences (Martinez-Murcia & Collins, 1990; Collins et al., 1993; Endo & Okada, 2008) and in studies involving different molecular markers (Chelo et al., 2007; De Bruyne et al., 2007; Endo & Okada, 2008). However, some taxonomic issues remain to be resolved, including the ‘temporary’ polyphyly of Leuconostoc as a result of the newly formed genus Fructobacillus (Endo & Okada, 2008). As a supra-generic group, the Leuconostocs are phylogen- etically related to Lactobacillus and Pediococcus (Vandamme et al., 1996; Makarova & Koonin, 2007). Although there is currently high interest in LAB genomics (Klaenhammer et al., 2002, 2005; Makarova et al., 2006; Makarova & Koonin, 2007) only three Leuconostoc genome sequences have been published so far, those of Oenococcus oeni PSU-1, Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T and Leuconostoc citreum KM20 (Makarova et al., 2006; Kim et al., 2008); four others (Leuconostoc mesenteroides, Leuconostoc gasicomitatum, Oenococcus oeni and Weissella paramesenteroides strains) are being sequenced (data from the ENTREZ Genome Project database at NCBI). The L. mesenteroides and W. paramesenteroides strains are relatively new additions to the ongoing sequencing projects and in the case of Weissella the only available information regarding genomic features such as chromosome size or number of rrn operons comes from the physical and genetic map of W. paramesenteroides DSM 20288 T (Chelo et al., 2004). Perhaps due to this scarcity of information, comparative genomic analyses in the Leuconostoc group have been restricted to compar- isons with species from other genera (Makarova et al., 2006; Makarova & Koonin, 2007; Marcobal et al., 2007) or are limited to a single species (Ze ´-Ze ´ et al., 2000, 2008). In this study we analysed the chromosomes of 17 strains of the genera Fructobacillus, Leuconostoc and Weissella by restriction with AscI, I-CeuI, NotI and SfiI and electro- phoretic separation by PFGE. Together with Southern Abbreviation: LAB, lactic acid bacteria. 3Present address: Instituto Gulbenkian de Cie ˆ ncia, Apartado 14, P- 2781-901 Oeiras, Portugal. 4Present address: Centro de Estudos de Vectores e Doenc ¸as Infecciosas Dr Francisco Cambournac, Instituto Nacional de Sau ´de Dr Ricardo Jorge I.P., Av. Liberdade no. 5, 2965-575 A ´ guas de Moura, Portugal. Supplementary material is available with the online version of this paper. Microbiology (2010), 156, 420–430 DOI 10.1099/mic.0.028308-0 420 028308 G 2010 SGM Printed in Great Britain