Of the identified proteins, CpxR and Dps (Additional File 4) are those commonly associated with stress resistance. CpxR is part of the two-component regulatory system CpxAR which controls gene Proteasome inhibitor expression in response to numerous external stimuli, including those responsible for alterations in the cell envelope [22–25]. The DNA-binding protein (Dps) has shown an ability to protect several pathogenic bacteria during acid stress, as well as when subjected to various oxidative stresses [26–30]. It is produced primarily throughout stationary phase and its expression
is regulated by the stationary phase sigma factor RpoS (σ38), OxyR, and IHF . Dps sequesters iron, thereby limiting Fenton-catalyzed JNK inhibitor oxyradical formation, and also physically protects DNA against environmental assaults by sequestering it into a highly stable biocrystal complex . Quantitative Real-time PCR Quantitative real-time PCR was performed to determine if the proteins upregulated in PA cultures (Dps, CpxR, SodA, RplE, and RplF) were overexpressed at the transcriptional level as well.
check details A relative quantification experiment was performed; therefore, the level of expression of each target in the PA adapted culture was compared to the level of gene expression of the identical target gene in the unadapted culture. The expression of each gene in unadapted cultures was taken to be the
basal level of expression for that particular gene (for the growth conditions used in this study) to which the expression in PA adapted cultures was compared. This method allowed the changes in gene expression of our selected targets to be carefully quantified. The relative quantification of each target gene was calculated from the data obtained using the comparative CT (ΔΔCT) method. Interestingly, qRT-PCR results did not fully coincided with all of the previously obtained proteomic results from 2 D electrophoresis (Figure 3). When compared to unadapted cultures, only two of the five targets overexpressed at the proteomic level (Dps and Fludarabine nmr CpxR) showed increased expression at the transcriptional level (p < 0.05). cpxR showed a 20.8% increase in expression in PA adapted cultures, while dps from PA adapted cultures showed a 50.7% increase in expression over that from unadapted cultures. Expression of rplE and rplF in PA adapted cultures was only 82.1% and 99.5% respectively, of those from unadapted cultures. This difference in gene expression was not statistically significant (p > 0.05). Finally, sodA showed a significant decrease in expression after exposure to PA (p < 0.01). Its expression in PA adapted cultures was only 52.2% of that in unadapted cultures.