One microliter of EZ-Tn5 Tnp was mixed with 50 μL of the competent cells of YS-11. The mixture was placed in an ice-cold 2 mm-gapped cuvette (BioRad Laboratories Inc., Hercules, CA). The cells were transformed by electroporation
using Gene Pulser II (BioRad) at 2.5 kV, 25 μF, and 200 Ω. After electroporation, 1 mL of SOC medium (Invitrogen, Carlsbad, CA) was immediately added to the cell suspension, and the culture was incubated at 37 °C for 1 h. One hundred microliters of the cell suspension was plated on TSAY containing 50 μg mL−1 of kanamycin (Nacarai Tesque, Kyoto, Japan). Four hundred and eighty-six colonies grown on selection plates were transferred into TSBY containing 50 μg mL−1 of kanamycin EPZ-6438 order for screening mutants deficient in exopolysaccharide production. The viscosity of spent culture media of 486 mutants was measured using a rotary viscometer (Tokimec Inc.) as described above. Mutants showing lower viscosity than that of the parent strain YS-11 were further investigated by means of SEM to observe selleck compound cell surface-associated structures as described previously. Mutants that had completely lost the meshwork-like structures around cells were selected as putative knockout mutants
for genes involved in the formation of biofilm-like structures. Southern hybridization was carried out to confirm a single insertion of transposon on genomic DNA. The genomic DNA from a mutant strain without exopolysaccharide production was purified using the GNOME Kit (Qbiogene Inc., Morgan Irvine, CA) and digested with a restriction enzyme PstI (Takara Bio, Ohtsu, Japan). The DNA fragments
Protein kinase N1 were electrophoresed on a 0.8% SeaKem agarose gel (Takara Bio), transferred to a positively charged nylon membrane (Hybond-N+, Amersham Biosciences Corp., Piscataway, NJ), and fixed on the membrane by UV light irradiation (HL-2000 HybriLinker, UVP Inc., Upland, CA). To detect an insertion of EZ-Tn5 Tnp, a digoxigenin (DIG)-labeled probe designed from the sequence of EZ-Tn5 Tnp was generated using the PCR DIG probe synthesis Kit (Roche Applied Science, Mannheim, Germany) with a primer pair (Table 1) to amplify a kanamycin-resistant gene in EZ-Tn5 Tnp (EZ-Tn5 Tnp sequence is available at http://www.epibio.com/pdftechlit/techlit_eztn.asp). The membrane was prehybridized (30 min, 65 °C) in a hybridization solution (DIG Easy Hyb Granules, Roche Applied Science) and subsequently hybridized overnight at 65 °C with 2 μL mL−1 of DIG-labeled probe in a hybridization solution. The detection of DIG-labeled probes was carried out according to the manufacturer’s instruction in a DIG Luminescent Detection Kit (Roche Applied Science). Alignments of flanking regions of the inserted EZ-Tn5 Tnp were analyzed using a DNA Walking SpeedUp Premix Kit (Seegene Inc., Seoul, Korea) according to the instruction of the kit.