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AEM Accepts, published online ahead of print on 21 September 2007
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AEM.01017-07v1
73/22/7128    most recent
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Appl. Environ. Microbiol. doi:10.1128/AEM.01017-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diversity analysis of Lactococcus lactis of dairy and non-dairy isolates with a novel multilocus sequence analysis (MLSA) scheme and (GTG)5-PCR fingerprinting

Jan L.W. Rademaker, Hélène Herbet, Marjo J.C. Starrenburg, Sabri M. Naser, Dirk Gevers, William J. Kelly, Jeroen Hugenholtz, Jean Swings, and Johan E.T. van Hylckama Vlieg*

NIZO food research, Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 20, 6710 BA Ede, The Netherlands; Laboratory of Microbiology and BCCMTM/LMG Bacteria Collection, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium; AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand

* To whom correspondence should be addressed. Email: Johan.van.Hylckama.Vlieg{at}nizo.nl.


  Abstract

The diversity of a collection of 102 lactococci including 91 Lactococcus lactis isolates of dairy and non-dairy origin was explored using partial Small SubUnit (SSU) rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG)5-PCR fingerprinting, and a novel Multi-Locus Sequence Analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both subspecies cremoris and lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of non-dairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only.

MLSA of six partial genes (atpA encoding ATP synthase alpha subunit; pheS encoding phenylalanine tRNA synthetase; rpoA encoding RNA polymerase alpha chain; bcaT encoding branched chain amino acid aminotransferase, pepN encoding aminopeptidase N and pepX encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length: 1970 bases) among 89 L. lactis subsp. cremoris and lactis isolates with unique sequence types for most isolates. This allowed high resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis based lineages, indicating a disparate acquisition route for this gene.


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