Chromosomal diversity in Lactococcus lactis and the origin of dairy starter cultures

Abstract

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment.

FIG. 1.—

PFGE patterns of SmaI-digested genomic DNA from Lactococcus lactis subsp. cremoris SK11 and related strains. The mixed strain starter isolates (MSS1–4) were isolated from various mixed strain dairy starter cultures.

FIG. 2.—

(A) PFGE patterns of SmaI digests of genomic DNA from Cit⁺ Lactococcus lactis strains. (B) Southern blot of (A) hybridized with a PCR-amplified product of the left hand junction between the IL1403 chromosome and the prophage bIL309.

FIG. 3.—

(A) Locations of I-CeuI recognition sites on the Lactococcus lactis IL1403 chromosome. I-CeuI cleaves at sites within the six 23S rRNA genes whose map positions are indicated. The resulting restriction fragments are designated Ce1 through Ce6. Their order in IL1403 and the majority of other strains is Ce2-Ce1-Ce3-Ce5-Ce4-Ce6. (B) PFGE patterns of genomic DNA from L. lactis strains.

FIG. 4.—

Alignment of the chromosomes of Lactococcus lactis KF147, IL1403, MG1363, and SK11. Colored blocks surround a section of the genome sequence that aligns to part of another genome. Inverted regions are depicted as blocks below the genome's center line. Inside each block, Mauve draws a similarity profile of the genome sequence. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Regions outside the blocks, or shown as white space, lack detectable homology with the other genomes and contain sequence elements specific to that strain. The locations of the six I-CeuI cut sites that indicate the locations of the 23S rRNA genes are shown above each strain.

FIG. 5.—

maximum representation with parsimony (MRP) supertree for Lactococcus lactis and other lactic acid bacteria derived from 1,160 single gene families. Listeria species were selected as an outgroup. Bootstrap scores for all nodes are displayed.

FIG. 6.—

(A) Relationship between the lengths of the Ce1 and Ce2 chromosomal regions of Lactococcus lactis. (B) Relationship between variances of different I-CeuI fragments standardized by their average size and the average size of the corresponding fragments.

FIG. 7.—

Alignment of the Ce6 region of the chromosomes of Lactococcus lactis IL1403, KF147, MG1363, and SK11 and identification of the genes present. Insertions common to the cremoris strains MG1363 and SK11 are shown in red, and the genes found only in SK11 are shown in blue. The fusA pseudogene in SK11 is shown in mauve.

FIG. 8.—

(A) Plasmid profile of Cit⁺ Lactococcus lactis strains. Lane 1, Invitrogen supercoiled DNA ladder; lane 2, Invitrogen 1 kb DNA ladder. (B) Southern blot of (A) hybridized with a PCR-amplified product of the citP gene. Where two bands are present, they represent open and closed circular forms of the same plasmid.