The lack of one regulatory player can deregulate the whole flagellar biosynthetic cascade and alter motility in H. pylori. Since ablation of the HP0256 gene reduced motility, we investigated #Inhibitor Library concentration randurls[1|1|,|CHEM1|]# the effect of HP0256 mutation upon the expression of the flagellar regulon using global transcript analysis. Array analysis was performed in quadruplicate, including a dye-swap. Five genes were selected to confirm the reliability of our microarray data by qRT-PCR. Transcriptional level of hpn was unchanged in the HP0256 mutant and was therefore used a control for qRT-PCR. The fold changes thus established were in good agreement
with the array data (Figure 6). The difference observed in fold-changes of flgE transcription between array data and qRT-PCR is due to the microarray analysis method used for the study. This method tends to attenuate the dispersion of the fold-changes compared to the overall signal on the slide. Figure 6 Confirmation of transcriptional Belnacasan supplier changes in selected flagellar genes in the HP0256 mutant using qRT-PCR. Fold changes and standard deviations were calculated using the era transcript abundance as reference. qRT-PCRs were performed on at least two biological replicates. A total of forty six genes had altered expression levels in the
HP0256 mutant. Nineteen genes were significantly up-regulated and twenty seven genes down-regulated in the HP0256 mutant compared to the wild-type strain (Table 1). Data for some biologically relevant genes, below the two-fold cut-off, are also included in Table 1. Among the differentially expressed genes, seventeen encode proteins associated with the membrane. Table 1 Gene list of significantly up- and down-regulated
genes in the HP0256 mutant based on the array experiment. TIGR orf no. Putative gene product (gene) Expression ratio p-value Down-regulated genes: Hp26695-0092 type II restriction enzyme M protein (hsdM) 0.15 0.02 HpJ99-1132 dimethyladenosine transferase 0.17 0.00 Hp26695-0093 click here alpha-2-fucosyltransferase 0.22 0.01 Hp26695-0229 outer membrane protein (omp6) ( hopA ) 0.24 0.01 Hp26695-0492 flagellar sheath associated protein ( hpaA3 ) 0.26 0.00 Hp26695-1210 serine acetyltransferase (cysE) 0.26 0.00 Hp26695-1587 conserved hypothetical protein 0.27 0.00 Hp26695-1208 ulcer associated adenine specific DNA methyltransferase 0.27 0.00 Hp26695-0610 toxin-like outer membrane protein 0.32 0.03 Hp26695-1207 hypothetical protein 0.34 0.01 HpJ99-0055 putative 0.35 0.03 Hp26695-1211 hypothetical protein 0.37 0.00 Hp26695-0430 hypothetical protein 0.40 0.04 Hp26695-1492 conserved hypothetical nifU-like protein 0.41 0.01 Hp26695-0805 lipooligosaccharide 5G8 epitope biosynthesis-associated protein 0.42 0.02 Hp26695-1203a Preprotein translocase subunit SecE 0.43 0.01 Hp26695-1219 hypothetical protein 0.43 0.04 Hp26695-0711 hypothetical protein 0.45 0.01 Hp26695-1180 pyrimidine nucleoside transport protein (nupC) 0.46 0.