Conclusions The c-di-GMP pathway is used by most bacteria (but not eukaryotes or Archaea) to regulate numerous biological processes [36]. Several lines of evidence have indicated the concentration of c-di-GMP, balanced by diguanylate cyclase (DGC) and phosphodiesterase (PDE), account for the fimbrial regulatory network in some microorganisms. In S. Typhimurium, it has been demonstrated that production of curli fimbriae was inhibited by a PDE STM3611[18]. However, no other type of fimbrial expression in this microorganism has thus far been shown to be controlled by DGC or PDE. The present study revealed that a previously uncharacterized
stm0551 gene, which could encode a PDE, contributes to the down-regulation of type 1 fimbrial expression in S. Typhimurium. Our finding may provide valuable information that may LDE225 in vivo help to further elucidate the complicated type 1 fimbrial regulatory circuit in this pathogen. Methods Bacterial strains, plasmids, and culture media The bacterial strains, plasmids, and primers used in the present study are listed in Table 1 and Table 2. The S. Typhimurium strain used was LB5010, an LT2 derivative [21]. This strain produces type 1 fimbriae and has a variable fimbrial phase. Bacteria were cultured in Luria-Bertani (LB) broth (Difco/Becton AT9283 concentration Dickinson, Franklin Lakes, NJ) or plated on LB agar. When required, media
were supplemented with antibiotics at the following concentrations: 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 20 μg/ml chloramphenicol. Antibiotics were obtained from Sigma (St. Louis, MO). To detect gene expression, 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was used (MDbio, Taipei, Taiwan). Construction of a S. Typhimurium stm0551 mutant A stm0551 mutant was created by one-step gene inactivation method as described previously [20]. Briefly, a kanamycin-resistance Protein kinase N1 gene from pKD13 tagged with a flanking sequence of the stm0551 gene was generated by a polymerase chain reaction (PCR) technique. The designed nucleotide sequence was generated
with Pfu polymerase (Fermentas, St. Leon-Rot, Germany) on a GeneAmp PCR system 2700 thermal cycler (Applied Biosystems, Foster City, CA) and initially incubated at 94 ° C for 3 min, followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min. Primers used in this approach are listed in Table 3. Then, the PCR product was introduced by electroporation into S. enterica serotype Typhimurium LB5010 possessing the pKD46 plasmid which expressed λ Red recombinase [20]. All transformants were grown on LB agar containing kanamycin. The constructed mutants were verified by PCR with primers located in the flanking sequence of the stm0551 gene. Yeast agglutination and guinea pig erythrocyte hemagglutination test for type 1 fimbriae Tested bacteria were cultured in static LB broth at 37°C for 48 h or on LB agar at 37°C overnight.