MLST protocol

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
Diversity Analysis of Dairy and Nondairy Lactococcus lactis Isolates, Using a Novel Multilocus Sequence Analysis Scheme and (GTG)5-PCR Fingerprinting.
Applied and Environmental Microbiology, November 2007, p. 7128-7137, Vol. 73, No. 22

Nucleotide sequences

The sequences used in this study were retrieved from GenBank accession numbers:

• rpoA partial gene sequences: EU111131 to EU111178 (codon start 1)
• pheS partial gene sequences: EU111220 to EU111267 (codon start 3)
• atpA partial gene sequences: EU111568 to EU111615 (codon start 3)
• pepX partial gene sequences: EU111309 to EU111354 (codon start 2)
• pepN partial gene sequences: EU111396 to EU111442 (codon start 1)
• bcaT partial gene sequences: EU111483 to EU111529 (codon start 1)

When applied, we removed the first two firsts nucleotides to obtained coding sequences in frame 1.

MLST scheme

The MLST scheme is based on partial DNA sequences of atpA, rpoA, pheS, bcaT, pepN, and pepXP genes obtained from 89 L. lactis subsp. lactis and L. lactis subsp. cremoris isolates.

Primers and temperatures are listed in Table 2.

Excluded positions

Sequences with nucleotide ambiguities and/or nucleotide deletions were obtained for bcaT, pepN, and pepXP. The positions containing ambiguities and or deletions were excluded from the MLST scheme.

Excluded strains

No sequences were obtained of pepN in strains KF7 (NIZO2219) and KF 178 (NIZO2234), of pepXP in strains P7266 and P7304, and of bcaT in strains P7266, KF 178, KW2 (NIZO2247), and AM3 (NIZO2251). Sequences of these isolates were excluded from the MLST scheme.

Excluded gene

One of the six genes, pheS showed different strain groupings than the ones observed with the five other genes and the one based on SSU rRNA gene. The gene was excluded from the MLST scheme.

Final MLST scheme

The MLST scheme is based on five loci (atpA, rpoA, bcaT, pepN, and pepXP) and 83 isolates.