Lactococcus Lactis NZ9000 Research Articles

SdrF, a Staphylococcus epidermidis Surface Protein, Binds Type I Collagen

Staphylococcus epidermidis is the leading cause of device-related infections. These infections require an initial colonization step in which S. epidermidis adheres to the implanted material. This process is usually mediated by specific bacterial surface proteins and host factors coating the foreign device. Some of these surface proteins belong to the serine-aspartate repeat (Sdr) family, which includes adhesins from Staphyloccus aureus and S. epidermidis. Using a heterologous expression system in Lactococcus lactis to overcome possible staphylococcal adherence redundancy we observed that one of these Sdr proteins, SdrF, mediates binding to type I collagen when present on the lactococcal cell surface. We used lactococcal recombinant strains, a protein-protein interaction assay and Western ligand blot analysis to demonstrate that this process occurs via the B domain of SdrF and both the alpha1 and alpha2 chains of type I collagen. It was also found that a single B domain repeat of S. epidermidis 9491 retains the capacity to bind to type I collagen. We demonstrated that the putative ligand binding N-terminal A domain does not bind to collagen which suggests that SdrF might be a multiligand adhesin. Antibodies directed against the B domain significantly reduce in vitro adherence of S. epidermidis to immobilized collagen.

Bioluminescence-based bioassays for rapid detection of nisin in food

We have developed a method for determining ultralow amounts of nisin in food samples that is based on luminescent biosensor bacteria. Modified bacterial luciferase operon luxABCDE was placed under control of the nisin-inducible nisA promoter in plasmid pNZ8048, and the construct was transformed into Lactococcus lactis strains NZ9800 and NZ9000. The nisRK genes of these strains allow them to sense nisin and relay the signal to initiate transcription from nisA promoter. The resulting luminescence can be directly measured from living bacteria without the addition of exogenous substrates. Induction leads to detectable luminescence within ten minutes. Lyophilization of the biosensor cells produced viable and inducible sensor elements that can be utilized as freshly cultivated cells for rapid detection of nisin. The linear dose–response relationship perceived in the assay facilitates quantification of nisin in samples. The sensitivity of the nisin bioassay was 0.1 pg/ml in pure solution and 3 pg/ml in milk, exceeding the performance of all previously reported methods.

Gene cloning, functional expression and secretion of the S-layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a in Lactococcus lactis

The ∼93-kDa surface layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a forms a regular crystalline array providing a nanopatterned matrix for the future display of biologically relevant molecules. Lactococcus lactis NZ9000 was established as a safe expression host for the controlled targeted production of SgsE based on the broad host-range plasmid pNZ124 Sph, into which the nisA promoter was introduced. SgsE devoid of its signal peptide-encoding sequence was cloned into the new vector and purified from the cytoplasm at a yield of 220 mg l − of expression culture. Secretion constructs were based on the signal peptide of the Lactobacillus brevis SlpA protein or the L. lactis Usp45 protein, allowing isolation of 95 mg of secreted rSgsE l −1. N-terminal sequencing confirmed correct processing of SgsE in L. lactis NZ9000. The ability of rSgsE to self-assemble in suspension and to recrystallize on solid supports was demonstrated by electron and atomic force microscopy.