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Applied and Environmental Microbiology, November 2007, p. 7128-7137, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01017-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting{triangledown}

Jan L. W. Rademaker,1 Hélène Herbet,1 Marjo J. C. Starrenburg,1 Sabri M. Naser,2 Dirk Gevers,2 William J. Kelly,3 Jeroen Hugenholtz,1 Jean Swings,2 and Johan E. T. van Hylckama Vlieg1*

NIZO food research, Kluyver Centre for Genomics of Industrial Fermentation, P.O. Box 20, 6710 BA Ede, The Netherlands,1 Laboratory of Microbiology and BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium,2 AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand3

Received 7 May 2007/ Accepted 10 September 2007

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit 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 multilocus 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 L. lactis subsp. cremoris and L. lactis subsp. 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 nondairy 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, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. 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.

* Corresponding author. Mailing address: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31 318 659 511. Fax: 31 318 650 400. E-mail: Johan.van.Hylckama.Vlieg{at}nizo.nl

{triangledown} Published ahead of print on 21 September 2007.

Applied and Environmental Microbiology, November 2007, p. 7128-7137, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01017-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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