PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

Background: Protein folding in the envelope is a crucial limiting step of protein export and secretion. In order to better understand this process in Lactococcus lactis, a lactic acid bacterium, genes encoding putative exported folding factors like Peptidyl Prolyl Isomerases (PPIases) were searched for in lactococcal genomes. Results: In L. lactis, a new putative membrane PPIase of the cyclophilin subfamily, PpiA, was identified and characterized. ppiA gene was found to be constitutively expressed under normal and stress (heat shock, H2O2) conditions. Under normal conditions, PpiA protein was synthesized and released from intact cells by an exogenously added protease, showing that it was exposed at the cell surface. No obvious phenotype could be associated to a ppiA mutant strain under several laboratory conditions including stress conditions, except a very low sensitivity to H2O2. Induction of a ppiA copy provided in trans had no effect i) on the thermosensitivity of an mutant strain deficient for the lactococcal surface protease HtrA and ii) on the secretion and stability on four exported proteins (a highly degraded hybrid protein and three heterologous secreted proteins) in an otherwise wild-type strain background. However, a recombinant soluble form of PpiA that had been produced and secreted in L. lactis and purified from a culture supernatant displayed both PPIase and chaperone activities. Conclusions: Although L. lactis PpiA, a protein produced and exposed at the cell surface under normal conditions, displaye

Cholate-Stimulated Biofilm Formation by Lactococcus lactis Cells ▿ †

Bile acid resistance by Lactococcus lactis depends on the ABC-type multidrug transporter LmrCD. Upon deletion of the lmrCD genes, cells can reacquire bile acid resistance upon prolonged exposure to cholate, yielding the ΔlmrCDr strain. The resistance mechanism in this strain is non-transporter based. Instead, cells show a high tendency to flocculate, suggesting cell surface alterations. Contact angle measurements demonstrate that the ΔlmrCDr cells are equipped with an increased cell surface hydrophilicity compared to those of the parental and wild-type strains, while the surface hydrophilicity is reduced in the presence of cholate. ΔlmrCDr cells are poor in biofilm formation on a hydrophobic polystyrene surface, but in the presence of subinhibitory concentrations of cholate, biofilm formation is strongly stimulated. Biofilm cells show an enhanced extracellular polymeric substance production and are highly resistant to bile acids. These data suggest that non-transporter-based cholate resistance in L. lactis is due to alterations in the cell surface that stimulate cells to form resistant biofilms