Furthermore, our data showing that a loss-of-function mutation in gnd (which produces the second enzyme of the PPP pathway, Figure 2) does not suppress sensitivity to CO2 suggests that the production of 6-phosphogluconate, by either Zwf or gluconate kinase, contributes to CO2 sensitivity in msbB Salmonella. MsbB as a virulence factor? Several publications Dinaciclib cost cite MsbB as a virulence factor that is necessary for both septic shock and the ability to invade and persist in mammalian cells [5, 17, 29]. However, owing to the fact that msbB Salmonella were tested under 5% CO2 conditions,
the lack of virulence may be partially or fully due to the inability of msbB Salmonella to grow in the presence of the 5% CO2. Further experimentation with msbB zwf Salmonella will be necessary to determine which virulence defects are attributable to msbB lipid A and those
that arise from sensitivity to 5% CO2. Based upon this study and earlier studies on the sensitivity of zwf mutant to superoxides, zwf may both reduce virulence on one hand, yet potentiate growth under CO2 conditions on the other, further complicating virulence analyses. Conclusion Here, we report new growth defects in msbB Salmonella: sensitivity to gluconate Danusertib ic50 and growth in hypertonic, acidic or 5% CO2 conditions. These characteristics are in addition to the previously reported growth defects in the presence of salt, EGTA, polymyxin, or MacConkey media. Previous studies showing that MsbB is a virulence factor require further evaluation of the role that CO2 sensitivity plays. The potential for cryptic, spontaneous mutations remains a possibility that should be addressed by re-transduction under non-selective conditions followed by plating independently under CO2 and ambient
air. We have created an msbB somA zwf Salmonella strain that is resistant to growth under acidic or 5% CO2 conditions. This strain contains a loss-of-function mutation Thalidomide in zwf, an enzyme in the pentose phosphate pathway that produces CO2 as it converts a 6 carbon sugar to a 5 carbon sugar. The study of the virulence of msbB zwf Salmonella will allow the determination of what types of virulence are attributable to cells having an MsbB lipid A independent of sensitivity to 5% CO2, which is required for in vitro and in vivo virulence assays. Methods Bacterial strains, plasmids, phage and media The bacterial strains and plasmids used in this study are listed in Table 1. The Salmonella msbB insertion/deletion for tetracycline resistance was described by Low et al. . P22 mutant HT105/1int201 (obtained from the Salmonella Genetic Stock Center, Calgary, Canada) was used for Salmonella transductions. Salmonella enterica serovar Typhimurium strains were grown on LB-0 or MSB agar or in LB, LB-0, buffered LB or MSB broth. MSB media consists of LB (Luria-Bertani media, ) with no NaCl and supplemented with 2 mM MgSO4 and 2 mM CaCl2. LB-0 is LB media with no NaCl. Buffered LB pH 7.5 and pH 6.