Plasmids of raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 suggest a plant-based origin for the strain

Abstract

The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na(+) and K(+) transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains.

Fig. 1.

Physical and genetic maps of plasmids pVF50, pVF22, pVF21, and pVF18 of L. lactis DPC3901. Position and orientation of genes are indicated by arrows. Inner circles show the nucleotide numbering.

Fig. 1.

Physical and genetic maps of plasmids pVF50, pVF22, pVF21, and pVF18 of L. lactis DPC3901. Position and orientation of genes are indicated by arrows. Inner circles show the nucleotide numbering.

Fig. 2.

Sequence analysis of the putative polycistronic operon orf18-21. (a) Putative regulatory elements of the orf18-21 operon. The ribosome binding site (RBS), −10 and −35 promoter sequences, and rho-independent terminator signal are shown in bold and underlined. (b) Locations of the conserved domains typical of ABC transporter ATPases in Orf20: the Walker A (consensus GxxGxGKS/T, where x is any residue) and Walker B (consensus hhhhDE, where h is a hydrophobic residue) motifs involved in ATP binding and the Walker C (consensus LSGGQQ/R/KQR) motif involved in ATP hydrolysis are shown underlined. (c) TOPCONS consensus prediction of membrane topology for Orf21, showing seven transmembrane motifs (i, inside; M, transmembrane; o, outside) typical of ABC transporter permeases.

Fig. 3.

Growth curves of L. lactis DPC3901 (a), MG1363 (b), DPC3901-c1 (c), and DPC3901-c2 (d) in medium containing 0 (•), 1 (▪), 2 (▴), 3 (⋄), 4 (○), 5 (□), or 6 mM (▵) cobalt chloride. The minus or plus superscript is indicative of the absence or presence, respectively, of the orf18-21 and corA genotypes in each strain or derivative. Abs, absorbance.

Fig. 4.

(a) Plasmid DNA profiles of L. lactis DPC3901 (lane 3) and its cured derivatives DPC3901-c1 (pVF18⁻; lane 4) and DPC3901-c2 (pVF18⁻ pVF22⁻; lane 5). The plasmid profile of L. lactis DRC3 (lane 2) provides reference plasmids of large sizes (36, 51, and 65 kb) as a complement to the 2- to 16-kb molecular size ladder (lane 1). (b) PCR-based detection of specific gene features in the plasmid complement of L. lactis DPC3901, DPC3901-c1, and DPC3901-c2.