MLST protocol
Affiliation to subspecies lactis or cremoris
Taxonomically, this mesophilic Gram-positive species related to the Streptococcaceae is divided into three subspecies, L. lactis subsp. hordniae, L. lactis subsp. cremoris, and L. lactis subsp. lactis (that includes the biovar diacetylactis).
At the genotypic level, the two subspecies cremoris and lactis have 99.37% 16S rDNA sequence identity (Salama et al., 1991, Appl Environ Microbiol. 57:1313-1318), but display an average of 85% DNA sequence identity between CDSs (Wegmann et al., 2007, J Bacteriol. 189:3256-3270). Therefore, it is necessary to define the subspecies status of any L. lactis isolate before performing the MLST analysis because:
- some Enterococcus, Leuconostoc and Lactobacillus species are sometimes misidentified as Lactococcus lactis,
- strains with subspecies cremoris genotype may have the subspecies lactis phenotype [e.g. MG1363 strain],
- PCR and sequencing primers are different for the two subspecies,
- each subspecies must be analyzed separately when performing population studies.
- E8_F (5'-AGAGTTTGATCCTGGCTCAG-3')
- E807_R (5'-TGGACTACCAGGGTATCTAATC-3')
Sequence alignment of 16S rDNA region V1V4 in clustalW format.
Genes
Our L. lactis subsp. lactis MLST scheme uses fragments of the following six loci:
- Branched-chain-amino-acid aminotransferase(bcaT)
- Serine hydroxymethyltransferase (glyA)
- Pyrimidine-nucleoside phosphorylase (pdp)
- X-prolyl-dipeptidyl aminopeptidase (pepXP)
- Phosphoglycerate kinase (pgk)
- ATPase involved in DNA repair (recN)
PCR amplification
Reaction conditions for all PCR amplifications are: initial denaturation at 94°C for 3 min; 30 cycles at 94°C for 45 s, 55°C for 30 s min, 72°C for 1 min, final elongation step 72°C 5 min in a 25 or 50 μl mixture containing 10 ng of genomic DNA (purified using the "DNeasy Blood & Tissue Kit" (Qiagen), including the "Pretreatment for Gram-Positive Bacteria" step), 200 μM of each dNTP, 0.3 μM of each primer, 1 U Taq polymerase in 1x PCR buffer. Before sequencing, PCR products were purified using the "QIAquick PCR Purification Kit" (Qiagen).
Alternatively, direct colony PCR can be performed using the following protocol (by courtesy of Nathalie Campo): part of a fresh L. lactis colony is picked from (G)M17 plate using a micropipette filter tip and transferred in a PCR tube (or 96 well PCR plate). 25μl of PCR mix (see its composition above) is added and PCR is performed using the following conditions: initial denaturation at 95°C for 10 min; 30 cycles at 95°C for 45 s, 55°C for 1 min, 72°C for 2 min; final elongation step 72°C 7 min.
Primers
Primers specific for subspecies cremoris strains are available upon request
Primers for PCR
| Gene | Name | Positiona | Amplicon size (pb) | Primer sequence |
|---|---|---|---|---|
| bcaT | bcaT_PCR_F | 1,322,807 | 978 | 5'-AATTTAgACTgggAAAATTTAgg-3' |
| bcaT_PCR_R | 1,321,829 | 5'-CAACATCACCAAACTgAATg-3' | ||
| glyA | glyA_PCR_F | 590,943 | 1134 | 5'-ATgATTTTTgATAAAgAAgATTTTgA-3' |
| glyA_PCR_R | 592,077 | 5'-CTTCAACTTCTTTAAATCCTCT-3' | ||
| pdp | pdp_PCR_F | 1,465,707 | 1210 | 5'-ATggTTgATCTCATTCAAAAgAA-3' |
| pdp_PCR_R | 1,464,497 | 5'-TTCAgTAACTAATTCgTCAg-3' | ||
| pepXP | pepXP_PCR_F | 2,135,739 | 1224 | 5'-ggCTTATggTgCTgCTACTACT-3' |
| pdp_PCR_R | 2,136,963 | 5'-CACACTTTCAATAggAATAATgAg-3' | ||
| pgk | pgk_PCR_F | 242,797 | 1191 | 5'-TggCAAAATTgACTgTAAAAgA-3' |
| pgk_PCR_R | 243,988 | 5'-TTTTCAgTCAAAgCTgCAAgTC-3' | ||
| recN | recN_PCR_F | 883,409 | 863 | 5'-TTgTCgAATCAATggTCAAATgg-3' |
| recN_PCR_R | 884,272 | 5'-TCCATATAAAgCTCAgAAAgTTCg-3' |
a, position of the first 5' base of the primer on IL1403 chromosome (GenBanK accession number: NC_002662)
Primers for sequencing
The nucleotide sequences are determined using distinct sequencing primers internal to the amplified fragment. Such primers produce a higher quality of nucleotide sequence data and eliminate the sequencing of nonspecific amplification products.
| Gene | Name | Primer sequence | Sequence lenght (bp) |
|---|---|---|---|
| bcaT | bcaT_SEQ_F | 5'-AGGATTCAGCTATCGGAACTTAC-3' | 516 |
| bcaT_SEQ_R | 5'-TTTAGTATGTGTGCTTGGGTCAA-3' | ||
| glyA | glyA_SEQ_F | 5'-GTTATGGCAGCACAAGGTTC-3' | 453 |
| glyA_SEQ_R | 5'-GTTGTTGTAACGACATCAGCATAAG-3' | ||
| pdp | pdp_SEQ_F | 5'-TCACGATGGCAATGGTTCATTC-3' | 492 |
| pdp_SEQ_R | 5'-TCATTTCATTTCGATTTCCGATAGC-3' | ||
| pepXP | pepXP_SEQ_F | 5'-CTGGAATGTTACACCCGAACA-3' | 504 |
| pepXP_SEQ_R | 5'-AGGAAGTTCAACCAAATCATCTAACA-3' | ||
| pgk | pgk_SEQ_F | 5'-CAAAGCTGGTAAATCACTTGCAC-3' | 480 |
| pgk_SEQ_R | 5'-TGTGGTCAAGTGGCAAAATCAA-3' | ||
| recN | recN_SEQ_F | 5'-CGTCAACAGCTCAATTTACGAC-3' | 480 |
| recN_SEQ_R | 5'-GCAATTTTATGGCGCGCTTC-3' |
Both strands of each PCR-amplified locus is sequenced