galU gene expression in Streptococcus pneumoniae

BONOFIGLIO L; GARCÍA E; MOLLERACH M

Simposio; 8th International Symposium on Pneumococci and Pneumococcal Diseases; 2012

ISPPD Association

The galU gene of Streptococcus pneumoniae (Spn) encodes an UDP-glucose pyrophosphorylase absolutely required for capsule biosynthesis. Since eukaryotic GalU appear to be completely unrelated to their prokaryotic counterparts, we postulate that GalU may be an appropriate target for the search of new drugs to control pathogenicity of Spn. Comparison of completely Spn sequenced genomes showed that galU and gpdA are adjacent and in the same orientation. Using OperonDB software we found that both genes are cotranscribed in the same unit in Spn and 60 genomes belonging to firmicutes. Transcriptional terminator prediction using TranstermHP showed that no terminator is present between gpdA and galU; however, a terminator was detected downstream galU gene. A promoter prediction program identified 4 putative promoter regions, upstream of gdpA. DNA fragments containing these putative promoters were cloned in the promoter probe vector pLSE4. Promoter activity was detected in one of these constructions. The cloned fragment was screened for the presence of Shine Dalgarno (SD) like sequence, -35 and -10 boxes consensus sequences. An extended −10 site T-TG-TATAAT that lacks a −35 site was detected, the putative transcription start site is a purine and the SD sequence found is exactly the consensus. The predicted promoter region is highly conserved in 23 Spn completely sequenced genomes. Expression of galU gene was explored by semiquantitative real time RT-PCR at differents points of the growth curve, indicating that galU gene is expressed in exponential phase. These results contribute to the knowledge of the expression mechanism of GalU enzyme.