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.

Altering textural properties of fermented milk by using surface‐engineered Lactococcus lactis

“…Interestingly, the localization effect observed for MG1363(pIL253 pil ) was not seen for strain IL1403(pIL253 pil ). This would be consistent with the fact that pilin overexpression results in much fewer pili on the surface of IL1403 as compared with MG1363 (Tarazanova et al ., ) and the limited effect on gel hardness presented above. Overall the CLSM results indicate that surface properties of L. lactis can have profound effects on the location of the cells in the fermented milk matrix.…”

“…The strains MG1365, MG1362, MG1063, MG1261 and MG1299 differ from their parent NCDO712 by carrying only one or two plasmids (Gasson, 1983). Although originally described as containing five plasmids, we recently discovered that strain NCDO712 has six plasmids (Tarazanova et al, 2016). One of these plasmids, pSH74, carries a spaCB-spaA-srtC1-srtC2 gene cluster, which encodes proteins that lead to pili formation on the cell surface upon overexpression (Tarazanova et al, 2016).…”

“…Although originally described as containing five plasmids, we recently discovered that strain NCDO712 has six plasmids (Tarazanova et al, 2016). One of these plasmids, pSH74, carries a spaCB-spaA-srtC1-srtC2 gene cluster, which encodes proteins that lead to pili formation on the cell surface upon overexpression (Tarazanova et al, 2016). The overexpression of different types of pili in L. lactis is known to cause auto-aggregation (Oxaran et al, 2012).…”

“…The biodiversity of L. lactis surface properties is very high (Tarazanova et al, 2017). Pili can significantly change the surface of L. lactis upon their overexpression (Tarazanova et al, 2016). Large surface changes can also be introduced by plasmid transfer through conjugation (Neve et al, 1987;Broadbent and Kondo, 1991;Gasson et al, 1995), or by altering the expression of genes such as those encoding autolysins (Buist et al, 1997;Visweswaran et al, 2013) or enzymes involved in galactose utilization (Grossiord et al, 2003).…”

“…Large surface changes can also be introduced by plasmid transfer through conjugation (Neve et al, 1987;Broadbent and Kondo, 1991;Gasson et al, 1995), or by altering the expression of genes such as those encoding autolysins (Buist et al, 1997;Visweswaran et al, 2013) or enzymes involved in galactose utilization (Grossiord et al, 2003). The molecular composition of the cell wall has a big impact on the roughness of the bacterial surface, on bacterial chaining and on cell aggregation (Tarazanova et al, 2016). These properties govern the interactions between LAB and the matrix components (Ly-Chatain et al, 2010;Burgain et al, 2014a).…”