Defects in flagellar function were identified by the absence of outward migration on the media; this was then confirmed by direct observation under a light microscope.
An inverse PCR method was used to amplify the sequence flanking the inserted mini-Tn5 transposon in the chromosome of the swarming-defective Navitoclax mutants. Genomic DNA from each mutant was isolated according to the cetyltrimethylammonium bromide protocol and completely digested with TaqI. The DNA fragments were self-ligated with T4 DNA ligase and then used as templates for inverse PCR with the primers P904 (5′-GGAGAGGCTATTCGGCTATG-3′) and P194c (5′-GTAAGGTGATCCGGTGGATG-3′), which were designed according to the motile sequence of Mini-Tn5-Km plasmid. The PCR products were separated by agarose gel electrophoresis and then purified using a gel extraction kit (Watson Biotechnologies Inc.). The PCR products were directly sequenced at Shanghai GeneCore BioTechnologies.
If sequencing failed, the PCR products were ligated to PMD18-T vector (Takara Co. Ltd, Dalian, China) and the sequencing was attempted again. To identify the mutant genes, nucleotide sequence databases were searched with the blastn and blastx programs developed by the National Center for Biotechnology Information (NCBI). Flagellin was isolated from bacterial cells according to the method described by DePamphilis & Adler (1971). The bacterial cells were suspended in 0.1 M Tris-HCl find more buffer (pH 7.5). Flagellar filaments were sheared with a tissue homogenizer at maximum speed for 30 s. The bacteria were observed microscopically to ascertain loss of motility. Cell debris was removed from the flagella by centrifugation at 15 000 g for 15 min, and flagellar filaments were then pelleted from
the supernate by ultracentrifugation and then suspended in 0.1 M Tris-HCl buffer (pH 7.5). Sodium dodecyl sulfate-polyacrylamide Evodiamine gel electrophoresis (SDS-PAGE) was used to analyze the purity of samples. Flagellin protein dissolved in Tris-HCl buffer was emulsified in Freund’s incomplete adjuvant (1 : 3). One rabbit was immunized three times at intervals of 2 weeks. Serum was collected 1 week after the final injection and stored at −20 °C. Bacteria were suspended in Tris-HCl buffer and then adjusted to 1 OD600 nm. All the cell lysates were subjected to SDS-PAGE electrophoresis and then transferred onto a nitrocellulose membrane. The flagellin was visualized via Western blotting with enhanced chemiluminescence detection (Pierce). Rabbit polyclonal antiflagellin serum was used as the primary antibody. The secondary antibody was a goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. Detection was performed according to the protocol of the supplier. The surface hydrophilicity of bacterial cells was quantified using bacterial adherence to hydrocarbon (BATH) test, originally described by Rosenberg et al. (1980).