For Southern hybridization, digoxigenin-11-dUTP-labeled pnxIIIA probes were generated using the primer-pair pnx3A-probe-f and pnx3A-probe-r and the genomic DNA of P. pneumotropica ATCC 35149. The genomic DNAs of the reference strains SCH772984 in vitro were digested with HindIII and loaded on agarose gels. The hybridization and detection protocol used has
been described previously . Immunoelectron microscopy Bacterial cells were fixed with 4% (w/v) paraformaldehyde, 0.25% (v/v) glutaraldehyde, and 5% sucrose in 1.5 ml of 0.1 M phosphate buffer (pH 7.4) for 2 h at 4°C. The cells were harvested at 1000 × g for 10 min. The pellets were then rinsed with the same buffer and dehydrated by passing them through an ethanol series. Samples were embedded in LR-white resin. Thin sections were placed in PBS with 5% bovine serum albumin (BSA) for 30 min at RT ABT-263 cell line and then incubated with rabbit anti-PnxIIIA IgG diluted
to 1:100 with 1% BSA in PBS for 4 h at RT. The sections were washed 3 times in PBS and incubated with goat anti-rabbit IgG conjugated with 10-nm immunogold particles (BBInternational, Cardiff, UK) diluted to 1:50 with 5% BSA in PBS for 1 h. The sections were subsequently stained with uranyl acetate and lead citrate and viewed under a JEOL JEM-1200EX electron microscope (JEOL, Tokyo, Japan) at 80 kV. Nucleic acid accession numbers The nucleotide sequences of pnxIIIE, pnxIIIA, pnxIIIB, pnxIIID, and tolC were deposited in GenBank through DNA Data Bank of Japan, and the assigned accession numbers were AB568084, AB568085, AB568086, AB568087, and AB568088, respectively. Acknowledgements This study was partially supported by a grant-in-aid Dimethyl sulfoxide (20700369) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. Electronic supplementary material Additional file 1: Multiple alignments of the 3 regions with repeat sequences in PnxIIIA. The numbers at the terminus represent the position of each protein. Identical residues and similarity substitutions
are highlighted in black and gray, respectively. Each organism and protein are represented by abbreviations as follows: PN, PnxIIIA from P. pneumotropica ATCC 35149; PR, RTX family exoprotein A from Proteus mirabilis ATCC 29906 (accession no., EEI46927); EC, putative RTX family exoprotein from E. coli CFT073 (AAN78844); CA, cell wall surface anchor family protein from Cardiobacterium hominis ATCC 15826 (EEV87836); AN, possible LPXTG anchored adhesin from Anaerococcus tetradius ATCC 35098 (EEI83830); MH, hemolysin-type calcium-binding protein from Marinomonas sp. strain MWYL1 (ABR70778); VC, RTX toxin from V. cholerae M66-2 (ACP05873); PS, putative outer membrane adhesin-like protein from Psychrobacter sp. PRwf-1 (ABQ94037); FL, probable aggregation factor core protein MAFp3 from Dokdonia donghaensis MED134 (EAQ39910); ST, putative RTX family exoprotein from Streptococcus suis 98HAH33 (ABP91341).