Abstract
Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.
Highlights
Lactococcus lactis is a gram-positive, non-pathogenic, non-spore forming lactic acid bacterium (LAB) that is often isolated from plant material or a dairy environment [1,2]
Sequencing of the total DNA of NCDO712 allowed detecting 11 Single Nucleotide Polymorphisms (SNPs) between the chromosomes of L. lactis NCDO712 and its plasmid-cured derivative MG1363 [66]. The latter was isolated in 1983 following multiple rounds of chemical- and protoplast-induced plasmid curing [26]
The nucleotide sequencing data suggests the occurrence of genome re-arrangements mainly due to mobile genetic elements, but their verification was outside the scope of this study
Summary
Lactococcus lactis is a gram-positive, non-pathogenic, non-spore forming lactic acid bacterium (LAB) that is often isolated from plant material or a dairy environment [1,2]. It is widely used in the dairy industry as a starter culture for the production of cheese, buttermilk and quark. Other important plasmid-encoded functions include bacteriocin production [11,12] and resistance [13,14], metal ion resistance [15], antibiotic resistance [16,17] and bacteriophage resistance [18,19]. Several genes related to lactococcal surface properties are carried on plasmids [21,22,23], such as aggL, a gene responsible for cell auto-aggregation, or genes responsible for adhesion to mucus [24]
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