We performed qRT-PCR reactions on RNA preparations
extracted from strain 2787 at different points during growth in LB broth at 37 °C with shaking. We used primers specific for the aah gene, for the aidA gene and a pair of primers amplifying a region encompassing the 3′-end of aah and the 5′-end of aidA (Fig. 1a). Primers specific for the rpoD genes were used to normalize Pembrolizumab and compare the amounts of transcripts that could be amplified (Fig. 2a). The amplification with the aah-aidA primers shows that the two genes can be transcribed from a single bicistronic message. The levels of mRNA detected with the three pairs of primers varied significantly during growth. The pattern of variation was similar for the three primer pairs: there was an initial decrease during the log phase, most likely because of dilutions of existing
RNA pools from the overnight culture, and then an abrupt increase in the early-stationary phase. This has been observed with RpoS-controlled genes (Gordia & Gutierrez, 1996; Fomenko et al., 2001), and is therefore in agreement with our identification of RpoS-specific consensus sequences for the P149 promoter. Averaging three different experiments, the only statistical CP-690550 datasheet difference was between the amounts of transcripts detected with the aah and aidA primers at the mid-log phase. This suggests that there is a promoter allowing the transcription of the aidA gene alone, despite our failure to identify it by RACE. This is consistent with previous results, however, because residual AIDA-I expression was seen in constructs lacking the 5′-end of aah (Benz & Schmidt, 2001). A weak promoter upstream of aidA could account for these previous results STK38 that used a cloned fragment in a multicopy plasmid and explain why, in a wild-type context, we could not readily identify this promoter. To confirm the
qRT-PCR results, we performed a Western blot on total extracts of 2787 using anti-AIDA antibodies (Fig. 2b). The antibodies are specific for the glycosylated form of AIDA-I (Charbonneau et al., 2007), and therefore report the expression of Aah and AIDA-I. Glycosylated AIDA-I is expressed as a 150 kDa pro-protein that is self-cleaved into a 100 kDa mature protein (Suhr et al., 1996; Charbonneau et al., 2009). We observed a slight decrease in the amounts of AIDA-I between the early-log phase and the mid-log phase and a marked increase at the early-stationary phase, in agreement with the qRT-PCR experiments. We cloned the 426 nucleotides upstream of the start codon of aah in a multicopy vector bearing a promoterless lacZ gene. We transformed 2787 with this construct or with a promoterless control construct. As shown in Fig. 3a, the amount of LacZ initially decreased during the log phase and increased sharply at the early-stationary phase. There was no activity with the control plasmid.