MSB media contains high levels of divalent cations, which have been proposed to increase lateral interactions between the phosphate groups of neighboring lipid A molecules [15]. Based on Murray et al.’s finding [16] that a decrease in electrostatic repulsion between the phosphates of lipid A can help to compensate for the lack of the myristic acid residue, we investigated whether Mg2+ and Ca2+ would protect against the detrimental effects of 5% CO2. On agar plates, Mg2+ and Ca2+showed partial protection in YS873 Bortezomib clinical trial (Figure 3D). YS873, which contains the EGTA and salt resistance suppressor mutation somA

[4], grows well on LB (Figure 3A), MSB (Figure 3C), LB-0 (Figure 3E) and LB-0 sucrose (Figure 3G) agar plates in air, but not when the plates are incubated in

5% CO2 (Figures 3B, 3D, 3F, and 3H). In contrast, the strain YS873 zwf is able to grow on all of these media in CO2, indicating that the zwf mutation can compensate for the growth defect of msbB strains in CO2 (Figure 3). Subsequent experiments were performed using the YS873 (msbB somA) genetic background because unsuppressed msbB Salmonella can not grow under mammalian physiological salt conditions [4]. msbB somA Salmonella are sensitive to CO2 in LB and LB-0 broth Figure 4 shows the growth of wild type ATCC 14028, 14028 zwf, YS873, and YS873 zwf in LB and LB-0 broth, incubated in the presence or absence of 5% CO2. As shown in Figure 4, the growth of YS873 (Figure 4A), but not ATCC 14028 (Figure 4C) is greatly impaired in LB broth in the presence of 5% CO2. A significant decrease in CFU is observed Silmitasertib (Figure 4A), indicating that YS873 cells lose viability in the presence of 5% CO2 in LB broth. When a loss-of-function mutation in zwf is incorporated into YS873, no loss in viability is observed under

identical conditions, although there is a longer lag phase of growth (Figure 4A). In LB-0 broth, there are no growth defects in 14028 or 14028 zwf (Figure 4D). For YS873 and YS873 zwf, the growth defects in LB-0 in the presence of 5% CO2 are attenuated in comparison to those observed in LB broth. There is no decrease in viability in YS873 in LB-0 in 5% CO2, Dolichyl-phosphate-mannose-protein mannosyltransferase although there is impaired growth in both YS873 and YS873 zwf in LB-0 in the presence of CO2 compared to growth in the absence of CO2 (Figure 4B). Figure 4 msbB confers growth sensitivity in liquid media under CO 2 conditions containing physiological amounts of salt and this is suppressed by zwf. Two sets of Salmonella strains (YS873 and YS873 zwf; 14028 and 14028 zwf) were grown on either LB (A and C) or LB-0 (B and D) in either air or 5% CO2. YS873 has severe morphological defects in LB broth under 5% CO2 conditions that are suppressed by a loss-of-function mutation in zwf Since our results show that msbB Salmonella lose viability in the presence of 5% CO2 (Figure 4), we examined msbB mutants grown in the presence of 5% CO2 to determine if there are any defects in cell morphology or chromosome segregation.