However, in silico analysis Pexidartinib research buy of all NRPS modules present in the genome of P. syringae 1448a failed to reveal any A-domains predicted to specify alanine. One possibility may be that the variant pyoverdine species was generated as an artefact of the purification process through some unexplained mechanism; however, as the additional monomer clearly seems to fall between the chromophore and lysine residue rather than being added in a peripheral fashion, this explanation seems unlikely.

An alternative explanation is that the product of P. syringae 1448a gene Pspph1923 (the single-module NRPS predicted to incorporate L-lysine; Table 2) may possess a dual activity that enables occasional incorporation of

an additional alanine residue. Unfortunately we were unable to biochemically characterize the substrate FK228 purchase specificity of this or any other of the pyoverdine NRPS modules in in vitro assays – despite obtaining soluble protein by several different strategies, none of our purified proteins appeared to retain activity. This phenomenon is not uncommon for NRPS enzymes. We note however that in ongoing work we have verified the second module of Pspph1925 is indeed a serine-activating NRPS, as predicted by our in silico analysis (Table 2); when appropriate regions of this Thiazovivin mouse gene are swapped with the equivalent regions in module 2 of P. aeruginosa else PAO1 pvdD the substrate specificity of the recombinant gene product is converted from L-threonine [19] to L-serine, and a correspondingly modified pyoverdine product is produced (MJ Calcott, JG Owen, LW Martin, IL Lamont, DF Ackerley, unpublished data). It may be that we can employ a similar ‘recombinant genetic characterization’ strategy to interrogate the substrate specificity of Pspph1923. However, for now the precise nature of the variant P. syringae 1448a pyoverdine species (peak m/z 1212, Figure 2A) remains unknown. Although an equivalent species was not previously detected in studies of other P. syringae pathovars [35, 36], it is possible that these other pathovars also produce this form. As MALDI-TOF

is not a quantitative technique the m/z 1212 peak may actually be a very minor species that happens to ionize particularly well; and as the previous studies utilized an HPLC preparative step to yield a single pure peak, this could conceivably have resulted in other minor peaks being missed. There is evidence from a previous isoelectric focusing analysis that different P. syringae pathovars produce minor variant isoforms of pyoverdine in addition to the major pyoverdine that is synthesized by all known fluorescent P. syringae isolates [45]. It is possible that the minor isoforms include variants that possess alternative side chain constituents as well as variants that have different acyl groups attached to the chromophore.