Oxidation of methionine, which was chosen as a variable modification parameter, added another 16 Da to the peptide mass which subsequently increased the mass of the NSPLASMSNINYAPTIWSR fragment to 2,138 Da. This mass was exactly the same as the mass of a recovered peptide which did not find a match during the NCBI search since the respective fusion peptide SB202190 cell line is not present in the database. Thus, the synthesis of the LscBUpNA fusion protein could also be proven. The majority of previous LscA-related studies have been performed with P. syringae pv. glycinea PG4180 [9, 10, 23, 24]. However, thus far, there was no evidence for a lack of lscA expression in other pathovars of P. syringae. Since the genomes

of P. syringae pv. phaseolicola 1448A, pv. syringae B728a and pv. tomato DC3000 are fully sequenced [19–21], template-specific oligonucleotide primers for cDNA-based mRNA detection could be designed. Although mRNA samples were extracted during different growth stages, namely, early-logarithmic and late-logarithmic phase, no amplicons could be detected in

any of the strains suggesting that lscA variants were not expressed. PCR amplification, using respective genomic DNA as template, proved that the primers were binding correctly. An independent gene, hexR, coding for a conserved hexose metabolism regulator protein HexR, was chosen to see if the total mRNA had been reverse transcribed correctly [25]. This PCR amplification gave correct sized amplicon of 880-bp for all the four strains demonstrating the accuracy of the used method. PCR amplification was also performed on the cDNA obtained from mRNA samples of PG4180.M6 containing Selleck AZD3965 lscA under the control of P lac . This experiment gave the same-sized amplicon as for genomic DNA again proving the accuracy of the method. In summary, for we propose that lscA could be an ancestral Lsc variant in P. syringae as suggested by high throughput screening Srivastava et al. [24]. During evolution, the inactive promoter perhaps did not allow expression

of lscA after this gene had potentially been introduced to an ancestral P. syringae. An evolutionary gene duplication of lscA followed by an insertion of a prophage-borne PAPE might have led to a new lsc variant, i.e. lscB which in turn got duplicated yielding lscC or vice-versa. As a result of this evolutionary process, two functional and expressed lsc genes emerged in the plant pathogen, for which utilization of sucrose, and perhaps levan formation, might be particularly important. The advantage of an additional in planta fitness-increasing and possibly virulence-promoting factor [29] could have helped this organism to selectively establish itself as a potent plant pathogen. As a consequence of this hypothesis, one could speculate on a loss of the supposedly non-expressed lscA during further evolutionary steps, a phenomenon also previously hypothesized by Smits et al. [30]. Conclusions The differential expression of levansucrases in P.