The in vitro PDE activity of the purified STM0551-His fusion protein was determined using the specific substrate, RXDX-106 bis (pNPP). The purified FimY-His fusion protein was used as a control since FimY amino acids exhibited no domain related to PDE

activity. STM0551 possesses “EVL” conserved residues that may form the putative active site that varies from the consensus “EAL” sequence. We constructed a substitution mutation in which the glutamic acid (E) at position 49 was replaced by alanine (A) in the stm0551 allele. A fusion protein of this construct was prepared with the same procedure described for STM0551 and FimY and was designed as STM0551E49A-His. The reactions that contained STM0551 exhibited a statistically significant 1.75-fold increase in the release of p-nitrophenol compared to that containing FimY and STM0551E49A (both reaction mixtures contained the same amount of protein [10 μg]) (Figure 6). This result suggests that STM0551 could function as a PDE. Figure 6 Phosphodiesterase activity. In vitro phosphodiesterase activity assays compared the abilities of the purified STM0551-6xHis, FimY-6xHis, and STM0551E49A-6xHis proteins to cleave the specific substrate, bis (pNPP). Release of p-nitrophenol was determined at 410 nm. * p<0.05. Discussion

The regulatory pathway of type 1 fimbriae in S. Typhimurium involves several genes including find more the fim gene cluster and other genes such as lrp[8–14]. The Salmonella pathogenicity island 1 (SPI1) and flagellar systems also crosstalk with type 1 fimbriae [23]. Several studies have indicated that the mechanism controlling the intracellular c-di-GMP concentration plays a critical role in regulating fimbrial

production. For example, MrkJ, a PDE, regulates type-3 fimbrial production in Klebsiella pneumoniae[19]. Deletion of mrkJ resulted in an increase in type-3 fimbrial production [19]. In Escherichia coli S fimbriae are regulated by a PDE, SfaY [24]. Production of CupA fimbriae of Pseudomonas aeruginosa is controlled by both the GGDEF domain in protein, PA1120, and PvrR that contains an EAL domain [25]. The FimK of Klebsiella pneumoniae contains the EAL domain and deletion of fimK conferred hyperpiliation of type 1 fimbriae Racecadotril in this bacterium [26]. Our present finding may add one more example to this fimbrial regulation/c-di-GMP concentration circuit. The stm0551 gene of S. Typhimurium is located within the fim gene cluster but has not previously been investigated. The predicted amino acids of STM0551 showed similarity to those of proteins with PDE activity, so it was interesting to further dissect the function of stm0551 in terms of type 1 fimbrial regulation. The parental strain S. Typhimurium LB5010, is an LT2 derivative and displays a variable fimbrial phase [21]. A static broth culture favors S.