Transformants carrying either of the two fusion constructs produced levan similar to the PG4180.M6 mutant complemented with lscB. Western blotting, zymographic detection, and qRT-PCR analyses confirmed these results but also allowed a more detailed view; native lscB and the lscB UpN A fusion had similar mRNA expression levels while that of the fusion lscB Up A, which lacked the 48-bp of N-terminal LscB-coding region, had less. Consequently, one might speculate that although

the -450 bp upstream DNA region of lscB, which includes the TSS as determined Rucaparib clinical trial in this study, is sufficient for expression of lscA, the first 48-bp of the lscB ORF increase the level of its expression. Since our respective results of Western blotting and zymographic detection of Lsc activity were indistinguishable from each other, it could be concluded that the N-terminus of LscB might not be involved in altering check details of enzymatic activities. A peculiar observation was the electrophoretic migration of the individual proteins or fusion proteins in polyacrylamide gels. The observed faster migration of LscBUpNA as compared to LscB under denaturing conditions could potentially be attributed to the apparent mass shift for two proteins with nearly identical

molecular masses as described earlier [26]. Interestingly, the migration of LscBUpNA was significantly slower than that of LscB under native conditions. This finding might demonstrate that modest changes in the protein’s surface charge might result in significant find more alterations of electrophoretic mobility [22, 27, 28]. Although the different migration rates of the proteins or fusion proteins under native or denaturing conditions suggested that the synthesized

proteins were indeed different from each other, a MALDI-TOF analysis of each of the proteins was conducted using protein samples from zymograms. The produced levan surrounding the proteins did not seem to impact mass spectrometric analysis. The MASCOT score for each of the identified proteins was above the significance threshold of 100. The sample from the PG4180.M6(lscB) sample gave LscB from P. syringae pv. phaseolicola 1448A as the first significant match which was in line with the high homology of the respective genes in the close relatives pv. glycinea and pv. phaseolicola [24]. The sample from PG4180.M6(lscBUpA) which should synthesize only LscA gave the first significant match as LscA from P. syringae pv. glycinea race 4 strain. This proved that the lscB Up A fusion actually synthesized an active LscA and confirmed earlier findings that artificial expression of LscA of PG4180 leads to levan formation [10]. Although the majority of obtained peptides for the sample representing LscBUpNA were LscA-borne as expected, the unique N-terminal 2,122-Da peptide NSPLASMSNINYAPTIWSR could be detected.