2019ΔcyaA and derived double mutants were grown in sRPMI. Sialic acid and cAMP were added 30 min prior to RNA extraction. Expression of nanE and siaP were measured by qRT-PCR. Results are presented as fold change relative to a culture that received

neither sialic acid nor cAMP. SiaR and CRP interact to regulate the adjacent nan and siaPT operons Previous work demonstrated that in a siaR mutant, CRP and cAMP are unable to influence nan operon expression [14]. Since the current studies were performed using different mutant constructs, the experiments were repeated with the double deletion mutant to confirm the previously observed phenotype. The 2019ΔcyaA ΔsiaR mutant was examined by qRT-PCR (Figure 4) and regardless of Salubrinal whether sialic acid or cAMP was added, expression of nanE did not change relative to the control. In the absence of SiaR, cAMP activated the expression of the siaPT operon, while the nan operon was unaffected. Figure 4 Expression of nanE and siaP in 2019Δ cyaA ΔsiaR. Cultures grown with sialic acid (open bars), cAMP (gray 5-Fluoracil bars), and both sialic acid and cAMP (black bars) were compared to a reference culture that received neither. Examination of the results obtained from 2019ΔcyaA

ΔnagB revealed a large change in the expression of nanE that was cAMP-dependent (Figure 5B). The addition of sialic acid alone (which would be converted to GlcN-6P) led to a 16-fold induction of nanE while the addition of cAMP alone had no effect. The addition of both sialic acid and cAMP resulted in an 83-fold induction of nanE, indicating that the combination of GlcN-6P and cAMP significantly increase the induction of the nan operon. Epothilone B (EPO906, Patupilone) These results provide evidence of cAMP-dependent activation of both the nan and siaPT operons. Since cAMP does not induce

nanE expression in a siaR mutant, this suggests that cAMP-dependent activation of nanE requires SiaR. SiaR and CRP may physically interact to activate nan operon expression. Figure 5 Expression of nanE and siaP is altered by helical phasing. Expression of nanE and siaP in 2019ΔcyaA (A), 2019ΔcyaA ΔnagB (B), 2019ΔcyaA+5 bp (C), and 2019ΔcyaA ΔnagB+5 bp (D). Cultures grown with sialic acid (open bars), cAMP (gray bars), and both sialic acid and cAMP (black bars) were compared to a reference culture that received neither. To demonstrate that SiaR and CRP interact to regulate the nan and siaPT operons, alteration of helical phasing was used. Alteration of helical phasing is accomplished by the insertion of one half turn to the helix BV-6 between the SiaR and CRP operators. Briefly, 5 bp was inserted between the SiaR and CRP binding sites in strains 2019ΔcyaA and 2019ΔcyaA ΔnagB, resulting in strains 2019ΔcyaA+5 and 2019ΔcyaA ΔnagB+5, respectively. These strains were examined by qRT-PCR and the results were compared with those obtained from the parent strains.