A Student’s t-test was used to determine if the difference in fold change was significant between LVS and the ΔpdpC mutant. Since PdpC was found to localize to the bacterial inner membrane, it would be possible that its absence affected the integrity of the bacterial membrane and, therefore, we investigated whether ΔpdpC may be defective for membrane integrity and/or sensitive to stress stimuli. We found this particularly pertinent in view
of the recent finding that so called hypercytotoxic F. tularensis mutants, often deficient for membrane-associated proteins or LPS, are prone to intracellular lysis, which leads to increased levels of pyroptosis . The LPS profile of ΔpdpC, as judged by use of an LPS antibody, was indistinguishable from that of LVS (data not shown) and, moreover, it did not Selleckchem Staurosporine show increased SIS3 research buy susceptibility to a detergent, SDS, a cell-permeable dye, EtBr, or an antibiotic
that penetrates deficient Gram-negative membrane, Vancomycin, nor to stress-related stimuli such as low pH, temperature, or H2O2 (Additional file 1: Table S1). Additionally, since it was shown that growth of hypercytotoxic mutants was delayed in Chamberlain’s medium, but not in TSB , in vitro growth of the ΔpdpC mutant was investigated. However, the mutant grew as well as LVS in both Chamberlain’s medium and TSB as well as on solid media. Therefore, we conclude that the ΔpdpC mutant showed intact membrane integrity and thereby none of the features typical of hypercytotoxic mutants. By performing PCR using primers specific for pdpC and other FPI genes, we found that pdpC was part of a large transcript including the 12 FPI genes from pdpA to pdpE (data not shown). To investigate the possibility of polar effects in the mutant, we measured the expression of FPI genes using RT-qPCR. The transcription
of genes directly upstream of pdpC was not affected, nor was there any effect on the pdpE gene immediately downstream, indicating cAMP a lack of polar effects of the gene deletion, while, surprisingly, the genes in the iglA D operon were downregulated, although only two of them to a significant extent (Table 1). The downregulation also included the corresponding proteins, IglA, B, C, and D, but also the levels of VgrG and IglH were lower in the mutant (Figure 3). Thus, there appear to be both transcriptional and translational effects resulting from the absence of PdpC. The absence of pdpC did not affect expression of any of mglA, sspA, pmrA genes (data not shown), all of which encode proteins that positively check details regulate FPI expression . We also used a bacterial two-hybrid (B2H) assay to determine the possibility that PdpC may form a regulatory complex together with the FPI regulatory proteins SspA, MglA, FevR, and PmrA , but none of these were found to interact with PdpC, although a novel PmrA-PmrA interaction was determined, nor did PdpC interact with any of the other members of the FPI (data not shown).