, 2009) to obtain pKT-cra, which was then transformed into the Δcra strain. Stationary-phase overnight cultures grown in YLB medium at pH 7.0 were diluted to 106 CFU mL−1 in PBS at pH 4.5 and incubated at 37 °C for 2 h. The cultures were serially diluted and plated onto YLB agar plates and colonies were counted after 20 h growth at 37 °C. Percent survival was calculated as described previously (Hu et al., 2009). All assays were repeated at least three check details times and the data were analyzed by Student’s
t-test. We applied 2D gel to screen proteins whose expression was induced or repressed at pH 4.5, which is a sublethal pH for YpIII (Hu et al., 2009); 21 proteins showed more than twofold changes in all three replicate experiments (Fig. 1). These proteins were identified by MALDI-TOF MS and are summarized in Table 1. Among these proteins, eight proteins involved in carbohydrate metabolism were up- or downregulated over twofold at pH 4.5 (Fig. 2a). It is worth noting that the three proteins that were involved in the beginning step of
fructose metabolism selleck screening library (FruB-1, FruB-2, FruK) (Ow et al., 2007) were all upregulated by acid challenge (Fig. 2a and b). To further confirm the increased expression of fruBKA at acidic pH, we constructed translational lacZ fusions of fruB∷lacZ and fruA∷lacZ, which are located at the beginning and end of the fruBKA transcription unit (Fig. 3a). As seen in Fig. 3b, in accordance with our 2D gel results, higher β-galactosidase activities of both fruB∷lacZ and fruA∷lacZ fusions were observed at pH 4.5 than at pH 7.0, suggesting that expression of fruBKA is acid induced. Expression of the fruBKA operon encoding FruB, FruK and FruA was reported to be negatively controlled by a transcription factor Cra at physiological pH in several bacteria (Saier & Ramseier, 1996). This raised the question of whether the acid-induced fruBKA expression is mediated by Cra. To address this question, we constructed translational cra∷lacZ fusion and compared the β-galactosidase activities with or without acid challenge. β-Galactosidase
activities of cells challenged with acid were obviously lower than those without challenge, suggesting cra expression is repressed by acid (Fig. 4a). Furthermore, we constructed the cra deletion strain named Δcra and compared fruB and fruA Regorafenib in vitro expressions in Δcra and YpIII wild-type strains. Expressions of fruB and fruA were both acid induced in YpIII wild-type strain (Fig. 4b and c). But there was no significant difference of β-galactosidase activities at pH 7.0 and at pH 4.5 in Δcra, although the values in Δcra were obviously higher than in YpIII, which confirmed the Cra regulates fruBKA expression in YpIII. Together, these results suggested that the acid induction of fruBKA expression is mediated by repressed expression of Cra at acidic pH. It was established that Cra acts as a global regulatory protein (Crasnier-Mednansky et al.