Bacterial strains, media, growth conditions and transformations
Lactococcus lactis subsp. lactis BGKP1 (Agg+) was isolated from semi-hard homemade cheese using standard microbiological procedures. Preliminary strain classification was done according to its fermentation ability using API 50CHL (Api System SA; Bio-Merieux, Montelieu-Vercieu, France), temperature of growth (30°C, 37°C, and 45°C), growth in the presence of salt (4% and 6.5%) and pH tolerance. Further taxonomic classification of BGKP1 involved repPCR with (GTG)5 primer , and sequencing of amplified 16S rDNA . A non-aggregating derivative BGKP1-20 (Agg-), L. lactis subsp. lactis BGMN1-596 (9), L. lactis subsp. cremoris MG1363  and Enterococcus faecalis BGZLS10-27  were used for homologous and heterologous expression of the aggregation phenotype. Lactococcal and enterococcal strains were grown at 30°C in M17 medium  supplemented with 0.5% glucose (GM17) and stored in the same medium containing 15% (w/v) glycerol (Sigma Chemie GmbH, Deisenhofen, Germany) at -80°C. L. lactis and E. faecalis electrocompetent cells were prepared and transformed as previously described  using an Eppendorf Electroporator (Eppendorf, Hamburg, Germany). E. coli strain DH5α  was used for cloning experiments and plasmid propagation. DH5α was grown at 37°C in Luria-Bertani (LB) medium . Agar plates were prepared by addition of agar (1.5% w/v) to the corresponding broth. E. coli competent cells were prepared using chemical treatment and subjected to heat shock transformation. Transformants were selected using the antibiotic erythromycin (5 μg ml-1 for lactococci and enterococci or 250 μg ml-1 for E. coli). Bacteriocin and proteinase activity were determined as described previously .
Cultures of BGKP1 and BGKP1-20 were grown in 10 ml of GM17 to a density of 109 cells ml-1. Approximately 106 cells of each strain were added to 10 ml of GM17 and cultures were incubated at 30°C. Aliquots from each culture were taken every hour and plated on solid GM17 medium to calculate generation time for each strain. Experiments were done in triplicate.
Molecular cloning techniques like end filling of DNA fragments with the Klenow fragment of DNA polymerase, dephosphorylation, ligation, PCR amplification and agarose gel DNA electrophoresis were carried out essentially as described previously . The mini-prep method  was used for isolation of large plasmids from lactococci. Plasmids from E. coli were isolated using the QIAprep Spin Miniprep kit according to the manufacturer's recommendations (QIAGEN, Hilden, Germany). The DNA fragments from agarose gels were purified using a QIAqick Gel extraction kit as described by the manufacturer (QIAGEN). Digestion with restriction enzymes was conducted according to the supplier's instructions (Fermentas, Vilnius, Lithuania).
Determination of the effect of ions, pH and proteinase K on aggregation ability of L. lactis subsp. lactis BGKP1
The effect of different ions and pH values on the BGKP1 aggregation phenotype was tested using cells that were three times washed in bi-distilled water until the aggregation phenotype was lost. Cells were then resuspended in buffers of different pH and solutions of various ions. 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.
The ability of BGKP1 and BGKP1-20 to form biofilms was tested as previously described by Christensen and coauthors . Pseudomonas aeruginosa PAO1 and Escherichia coli DH5α were used as the positive and negative control strains respectively. The experiments were done in 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 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, 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 (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. coli DH5α by the blue/white color method on LB plates containing erythromycin (250 μg ml-1), IPTG (0.1 mM) and X-Gal (40 μg ml-1). The obtained constructs carrying fragments of the largest plasmid pKP1 were designed as pAZIL-KPSl8 (16181 bp pKP1 plasmid linearized in SalI at position 10784 resulting in a disrupted aggL gene), pAZIL-KPE6 (9151 bp EcoRI fragment of pKP1, position 2198-11349), pAZIL-KPBg1 (10572 bp BglII fragment of pKP1, position 4953-15525), pAZIL-KPSc1 (8873 bp SacI fragment of pKP1, 6289-15162), pAZIL-KPPvBg2 (6322 bp PvuI-BglII fragment of pKP1, position 9303-15525), and pAZIL-KPPvSc1 (5859 bp PvuI-SacI fragment of pKP1, 9303-15162). Restriction enzyme digestion and sequencing of the constructs were performed to show that the anticipated final plasmid constructs had been obtained. The constructs were isolated from E. coli and then transferred to L. lactis subsp. lactis BGKP1-20 (Agg-), L. lactis subsp. lactis BGMN1-596 and L. lactis subsp. cremoris MG1363 by electroporation. The obtained Emr transformants were tested for expression of the aggregation phenotype.
DNA sequencing and analysis
For DNA sequencing, pAZIL-KPSl8 and the other constructs aforementioned were isolated from E. coli using a QIAprep Spin Miniprep Kit (QIAGEN) as recommended by the manufacturer. Plasmids were sequenced by primer-walking of both strands in Macrogen's sequencing service (Seoul, Korea). Sequence annotation and the database search for similar sequences were performed using BLAST site programs at the National Center for Biotechnology Information . The DNA Strider program was used for open reading frame (ORF) prediction.
Nucleotide sequence accession number
The nucleotide sequences of the partial 16S rDNA sequence of L. lactis subsp. lactis BGKP1, plasmids pAZILcos and pKP1 were submitted to the EMBL GenBank under accession numbers FR873574, FR872379 and FR872378, respectively.