The induction level of nanE in the presence of sialic acid and cAMP was similar to the expression observed when sialic acid alone was added. The 5 bp insertion eliminated the cAMP-dependent activation of nanE that was observed in the 2019ΔcyaA ΔnagB strain. In both the 2019ΔcyaA and 2019ΔcyaA ΔnagB backgrounds, altered helical phasing also resulted in the induction of siaP when cAMP was added (Figures 5A and 5C). In the 2019ΔcyaA+5 strain, the 5 bp insertion led to a 43-fold increase in siaP expression in the presence of cAMP (from 6-fold
in 2019ΔcyaA) and a 29-fold increase (from 2-fold in 2019ΔcyaA) when both cAMP and sialic acid were present. Taken together, these results indicate that altering the helical phasing succeeded in uncoupling SiaR- and CRP-mediated regulation of the nan and siaPT operons. It resulted in nanE expression becoming unresponsive Crenigacestat order to cAMP, much like it is in the 2019ΔcyaA ΔsiaR mutant. Altered helical phasing also prevented SiaR from exerting a negative influence on
the expression of siaP. We conclude that the insertion eliminated the ability of SiaR and CRP to interact to regulate both the nan and siaPT operons. SiaR and CRP bind to their respective operators simultaneously Mocetinostat chemical structure Binding of SiaR to an operator in the intergenic region between nanE and siaP was demonstrated previously . The putative operator of CRP was identified in silico and was found to overlap the region protected by SiaR in a DNase I protection assay by three base pairs. The ability of both proteins to bind to their operators was examined using the electrophoretic mobility shift assay (EMSA). Both proteins were able to bind to a probe comprising the region between the G protein-coupled receptor kinase two operons and CRP binding was dependent on the addition of cAMP (Figure 6A). When both proteins were included in the binding reaction, the DNA probe was shifted slightly higher than the SiaR-bound probe. This indicates that both proteins bind to their operators simultaneously, further supporting the hypothesis that the two regulators interact to regulate the adjacent nan and siaPT operons. Figure 6 Electrophoretic mobility
shift assay. A. Binding of both SiaR and CRP to the nan-siaPT intergenic region. Both SiaR and CRP bind to the probe individually and CRP binding is dependent on the presence of cAMP. Both proteins bind the probe simultaneously as indicated by the higher shift of the probe when both proteins are added. B. GlcN-6P enhances binding of SiaR. Two-fold serial dilutions of SiaR were added to binding reactions in the absence and presence of 100 μM GlcN-6P. More probe was shifted when GlcN-6P was present. GlcN-6P alters binding of SiaR to its operator Many transcriptional regulators exhibit altered binding selleck inhibitor affinity for their operator sequences when a co-regulator is bound. To determine the effect of GlcN-6P on SiaR binding, EMSA was used.