Analysis of native gene expression of lscA in P. syringae pathovars Lack of expression of lscA had been shown before in P. syringae pv. glycinea PG4180 . However, this has not been experimentally proven for other P. syringae pathovars. Consequently, possible expression patterns of lscA variants were also analyzed in the three P. syringae pathovars pv. phaseolicola 1448A, pv. syringae B728a and pv. tomato DC3000 using cDNA synthesis and PCR. No amplicon was detected in any of the four strains as shown in Figure 6 indicating that none of the lscA variants are expressed. The specificity of the primers was demonstrated by amplifying the lscA genes from corresponding genomic DNA,
all of which gave amplicons of the expected sizes. The accuracy of reverse transcription was checked by amplifying a cDNA of a
PG4180.M6 transformant carrying a recombinant lscA gene under the control of Plac, where lscA is known to be expressed . Successful PD 332991 cDNA synthesis of total mRNA was also demonstrated by PCR amplifying the cDNA derived from the mRNA of the hexR gene, a hexose metabolism regulator Z-VAD-FMK solubility dmso . Gene hexR gave an amplicon of expected size (Figure 6) indicating correct cDNA synthesis. Figure 6 Expression of lscA in different P. syringae pathovars. The bacterial cells were harvested at OD600 of 0.5 and 2.0. Total RNA was extracted as described in the Materials and Methods followed by generation of cDNA. PCR amplification of lscA fragment on the total cDNA using strain-specific primers showed no amplicon (lscA panel) indicating no expression of lscA. Quality of the primers was checked by performing PCR amplification using genomic DNA (gDNA) as template. Amplification using an unrelated gene hexR (hexR) and artificially expressed lscA by
P lac [M6(pRA3.1)] signified correct reverse transcription. Discussion Genomic co-existence of three highly conserved genes coding for levansucrase is a feature unique to the plant APR-246 molecular weight pathogen P. syringae despite the fact that numerous other bacterial species harbor just a single copy of this gene in their genomes. Artificial expression of lscA from P. oxyclozanide syringae under the control of the Plac had been shown previously . The same study also showed that lscA could not be expressed under its own promoter. Major differences between lscA and the natively expressed genes lscB and lscC are not found in the coding sequences but in their upstream DNA regions. The upstream regions of lscB and lscC represent a possible PAPE . We previously hypothesized that this PAPE might harbor regulatory sites required for expression of levansucrase and general sugar metabolism in P. syringae. Herein, the PAPE of lscB was fused to the coding sequence of lscA and thus proven for its transcriptional activity in P. syringae. The nucleotide sequence of the predicted PAPE consists of two parts, the upstream region of lscB and the first 48-bp coding for the N-terminus of LscB.