The following buffers were used: KCl (pH 3.0), HCl-glycine (pH 3.0), Na-citrate (pH 4.0 to 6.0), Tris-HCl (pH 7.0 to 10.0) and Tris-NaOH (pH 11.0 to 12.0). The following ions were examined: K+, Na+, Ca++, Mg++ and Fe+++ in concentrations of 0.1, 1, and 10 mM. Proteinase K (1 μg ml-1) treatment was done in TE (10 mM Tris, 1mM EDTA, pH8) buffer for 1 h at 37°C. Determination of aggregation phenotype was based on absorption at 600 nm. Biofilm formation The ability of BGKP1 and BGKP1-20 to form biofilms was tested as previously described by Christensen and coauthors [43]. Pseudomonas aeruginosa PAO1 and Escherichia coli DH5α were used as the positive and negative control strains

respectively. The experiments were done in Tanespimycin clinical trial triplicate. Analysis of cell surface proteins of L. lactis subsp. lactis BGKP1 and its non-aggregating derivative Cells from overnight culture (250 ml) of strain BGKP1 and its Agg- derivative find more BGKP1-20

were harvested by centrifugation and washed in 50 ml bi-distilled water. Proteins from the wash were precipitated with ammonium sulphate (25% saturation). Precipitated proteins were resuspended in 10 mM Tris-HCl, pH 8.5, and applied on SDS-PAGE (10%). The obtained bands were visualized by Coomassie blue staining. Construction of shuttle-cloning vectors The pAZIL shuttle-cloning vector and pAZILcos cosmid vector were constructed in order to perform the molecular analysis of BGKP1 plasmid pKP1 [see Additional File 1]. The tetracycline resistance gene of pACYC184 was replaced with the lacZ gene from the replicative form of M13 mp18 phage using ClaI/NarI and HincII/AvaII restriction enzymes, Selleck CH5183284 resulting in cloning vector pAZ1. In the next step, the chloramphenicol resistance gene from pAZ1 was removed using ScaI and XmnI restriction enzymes and the vector was fused with lactococcal cloning vector pIL253,

previously digested with EcoRI-XbaI restriction enzymes and blunted with Klenow enzyme, resulting in shuttle cloning vector pAZIL. To obtain a cosmid vector for the construction of cosmid libraries of lactococcal genomes, the cos site was introduced into the unique SacII (7697) restriction site of the pAZIL vector. The DNA fragment containing the cos site was obtained by PCR amplification with primers cosF-CATGTTTGACCGCGGATCATCG and cosR-CTAGACACCGCGGAAGCTAGC Morin Hydrate (SacII restriction sites are underlined). Afterwards, the PCR amplicon was digested with SacII and ligated with SacII-digested pAZIL resulting in the pAZILcos cosmid vector. Construction of various plasmid pKP1 derivatives Strain BGKP1 harbors at least three plasmids. Total plasmids isolated from strain BGKP1 were digested with different restriction enzymes (SalI, EcoRI, BglII, SacI, PvuI and BglII, SacI and PvuI). The resulting fragments were cloned into pAZIL vector digested with the same restriction enzymes (except for BglII, which was cloned into BamHI) and selected in E.