SiaR was found to repress the expression of both the siaPT and nan operons, thus regulating both transport and catabolism. Binding of SiaR to the intergenic region between these two operons was demonstrated and the region of DNA protected by SiaR was identified. As expected, it was found Sotrastaurin mw that inactivation of siaR lead to a reduction in surface sialylation, demonstrating the need to control the expression of sialic acid catabolism. In addition to SiaR, the cAMP receptor protein (CRP) was STAT inhibitor identified as a
regulator of the siaPT operon, however a role in the regulation of the nan operon was not observed [12, 14]. This is in part consistent with the observation that sialic acid is a cAMP-independent sugar [15]. In H. influenzae, CRP has been shown to regulate utilization of galactose, ribose, xylose, and fucose [15], in addition to regulating the development of competence [16]. We now report on the role of intermediates in the Neu5Ac catabolic pathway in SiaR-mediated TSA HDAC price regulation. Also, the potential interaction between SiaR and CRP was investigated. SiaR was found to utilize glucosamine-6-phosphate (GlcN-6P) as a co-activator in the presence of the CRP-cAMP complex.
SiaR and CRP were found to act in a cooperative manner to regulate the expression of the divergent transporter and catabolic operons. Our results reveal a unique mechanism of regulation of two divergent operons regulated by two transcription factors from a single location. Results Promoter structure of the nan and siaPT operons The transcriptional start sites of the nan and siaPT operons were identified using primer extension analysis. Primers that bound in the nanE and siaP open reading frames were used. Two major start sites were identified for the nan operon, 104 (TS-1 nan ) and 20 (TS-2 nan
) bp from the start codon of nanE (Figure 2A). The presence of additional minor bands may be the result of addional start sites or RNA degradation or processing. The analysis identified a single transcriptional start site (TS-1 siaPT ) 107 bp upstream of the start codon of siaP (Figure 2B). SPTLC1 This organization leaves 140 bp in between TS-1 nan and TS-1 siaPT . The putative CRP binding site is located at -59 to -80 relative to TS-1 siaPT and at -59 to -80 relative to TS-1 nan (Figure 2C). This organization suggests that the siaPT promoter falls into the class I group of CRP-dependent promoters [17]. A consensus -10 sequence was identified for TS-1 nan and was found to partially overlap the SiaR binding site, consistent with the role SiaR plays in repression of the nan operon. The relative location of TS-1 nan to the SiaR operator, in addition to the identification of a consensus -10 box, suggests that this start site would be primarily involved in SiaR-mediated regulation, however, the relative contribution of the two nan promoters will need to be examined in more detail. Figure 2 Primer extension analysis of the nan and siaPT operons.