Cell adherence assays were performed using human liver epithelial cell HepG2. The adherence of wild

type EDL933 to HepG2 cells in tissue culture was two-fold higher than that of rpoS and Suc++ mutants (P < 0.05) (Figure 3B), indicating that Suc++ mutants LY2835219 are impaired in cell adherence due to loss of RpoS function. This is consistent with previous results that over-expression of RpoS stimulates cell adherence [47]. Figure 3 Virulence-related traits, RDAR and cell adherence. (A) Development of RDAR morphotype is impaired in Suc++ mutants. Cells were replica-plated on CR (Congo Red) plates and incubated at 25°C for 48 h. (B) Cell adherence to epithelial cells. The adherence was expressed as the percentage of cells surviving the washing process. rpoS designates the constructed rpoS null-deletion mutant. Suc++ mutants with an intact RpoS function (rpoS +) During the screening for the Suc++ phenotype,

we found that a small proportion of Suc++ mutants Copanlisib datasheet from strains EDL933 (8%), CL106 (16%), and EPZ5676 EC6-484 (33%) were catalase-positive, a presumptive indication that RpoS was functional. To confirm this, we sequenced the rpoS region of five such Suc++ mutants (three aerobically isolated and the other two anaerobically isolated) of strain EDL933. As expected, there was no mutation in the rpoS gene in these mutant strains. However, these grew much better than wild type when grown on succinate (generation time: 240 ± 31 min) and fumarate (generation time: 306 ± 33 min) (Table 3). These data suggest that non-rpoS mutations are a minor component in the poor carbon selection process. Effect of the rpoS mutation on metabolism by Phenotype Microarray analysis RpoS

is known to negatively Hydroxychloroquine molecular weight control many genes involved in metabolism [10, 12, 48], and therefore, mutations in rpoS are likely to exert pleiotropic effects on metabolism. To test this, we compared wild type MG1655 and its derivative rpoS deletion mutants [12] using Phenotype Microarray analysis (Biolog, Hayward, CA). The rpoS mutants exhibited better respiration on 8 carbon sources and 92 nitrogen sources but less respiration on four carbon sources and one nitrogen source (Table 4). The substantial impact of rpoS mutations on nutrient utilization suggest that the beneficial effect of loss of RpoS in one selection condition may be extended to other conditions as well. Table 4 Phenotypic Microarray (PM) analyses of growth changes resulted from rpoS mutations.