coli and Salmonella[17].

The periplasmic chaperone CpxP binds to both the CpxA periplasmic domain and to certain misfolded proteins, which are degraded by the periplasmic protease DegP, therefore integrating information about their turnover status to the kinase activity of CpxA [18–20]. The outer membrane lipoprotein NlpE activates the CpxA protein upon its overexpression [21] and is required for CpxA protein activation BAY 11-7082 datasheet after adhering to hydrophobic surfaces [22]. Additional upstream components have been proposed to integrate other stresses in a process that is independent of the CpxP and NlpE pathways [17, 23]. For example, the CpxR/CpxA system confers a copper resistance phenotype even in CpxP and NlpE mutants [24]. Notably, nlpE (cutF or STM0241) is a pseudogene in Salmonella[25]. Here, we aimed to identify candidate connector genes that may integrate the signals of other systems. We identified a small protein as a novel connector-like factor from screening high copy plasmid https://www.selleckchem.com/products/mi-503.html clones that could affect the CpxR/CpxA system status. Results Identification of a plasmid clone that activates cpxP transcription To

conduct a genetic screen for novel connector proteins acting on the CpxR/CpxA system, we constructed a strain harboring a cpxP lac transcriptional fusion in Salmonella. The cpxP gene was chosen as a readout of the activation status of the CpxR/CpxA system because it is likely directly regulated exclusively by this system, unlike other CpxR-activated genes that are also controlled by envelope stress-responsive systems [26–28]. The lacZY genes were inserted after the cpxP stop codon to ensure that the CpxP protein retained the ability to repress the CpxR/CpxA system. Then, Salmonella chromosomal DNA was partially digested with Sau3AI and ligated with the high-copy-number plasmid pUC19 (digested with BamHI) to generate a DNA fragment library. Of approximately 10,000 cpxP-lac Salmonella

transformants, a plasmid clone termed pWN1 yielded stable blue colonies on LB plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) and ampicillin and RG7420 chemical structure was isolated four times. The blue color of the pWN1 strain was due to elevated cpxP-lac fusion expression. We demonstrated that this strain exhibit ~8-fold higher β-galactosidase activity than the same strain harboring the vector control or the plasmid clone pUC19-R1 that was randomly selected during the screening as a negative control (Figure 1A). Sequence analysis revealed that pWN1 harbors only the intact STM1852 open reading frame (ORF), which appeared to encode a 63-amino acid protein with no homology to any protein of known function, as well as the 3’ region of STM1851 and the 5’ region of pphA (Figure 1B).