title: Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.
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.
authors.affiliation: NIZO food research B.V., Ede, The Netherlands.|TI Food and Nutrition, Wageningen, The Netherlands.|Molecular Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Mariya
authors.initials: M
authors.lastname: Tarazanova
authors.affiliation: NIZO food research B.V., Ede, The Netherlands.|TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Marke
authors.initials: M
authors.lastname: Beerthuyzen
authors.affiliation: TI Food and Nutrition, Wageningen, The Netherlands.|Centre for Molecular and Biomolecular Informatics, Radboud UMC, Nijmegen, The Netherlands.|Microbial Bioinformatics, Ede, The Netherlands.
authors.forename: Roland
authors.initials: R
authors.lastname: Siezen
authors.affiliation: TI Food and Nutrition, Wageningen, The Netherlands.|Host-Microbe Interactomics Group, Animal Sciences, Wageningen University, Wageningen, The Netherlands.
authors.forename: Marcela M
authors.initials: MM
authors.lastname: Fernandez-Gutierrez
authors.affiliation: TI Food and Nutrition, Wageningen, The Netherlands.|Molecular Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Anne
authors.initials: A
authors.lastname: de Jong
authors.affiliation: TI Food and Nutrition, Wageningen, The Netherlands.|Molecular Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: van der Meulen
authors.affiliation: TI Food and Nutrition, Wageningen, The Netherlands.|Molecular Genetics, University of Groningen, Groningen, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Kok
authors.affiliation: NIZO food research B.V., Ede, The Netherlands.|TI Food and Nutrition, Wageningen, The Netherlands.
authors.forename: Herwig
authors.initials: H
authors.lastname: Bachmann
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D003227:Conjugation, Genetic; D003300:Copper / Q000494:pharmacology; D004269:DNA, Bacterial; D024881:Drug Resistance, Bacterial / Q000235:genetics; D010861:Fimbriae, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000187:drug effects / Q000235:genetics* / Q000648:ultrastructure; D005810:Multigene Family; D009561:Nisin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009561:Nisin; D003300:Copper