Cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. The photochemical mechanism by which switching occurs has yet to be elucidated. In this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. The rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution pH in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. Mass spectrometry suggests that the photoconversion product is a thiol−cyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated π-electron system of the dye.

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide͞polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived ␤-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a ⌬ 2 -isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon ␤-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.mass spectrometry ͉ orphan gene cluster ͉ hybrid nonribosomal peptide͞polyketide ͉ polyketide methylation P olyketides and nonribosomal peptides are classes of secondary metabolites that are synthesized by the iterative coupling of malonyl (Mal) derivatives and amino acids, respectively. The catalytic machinery responsible for the biosynthesis of these natural products comprises modular synthases that incorporate, and often subsequently tailor, monomer units into the growing small molecule by a thiotemplated mechanism. Often, the sequence of protein domains and modules is colinear with respect to the sequence of biosynthetic reactions catalyzed by the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machinery (1-3). Because of their highly modular nature, the small-molecule products of PKSs, NRPSs, and hybrid NRPS-PKS systems can often be predicted by bioinformatic approaches, and there have been recent experimental validations of such predictions (4-6). However, for many cases in which the biosynthetic product encoded by a given gene cluster is unknown, functional characterization of the synthase by bioinformatic methods alone is difficult or impossible.One such orphan cluster for which the product structure is unknown is the hybrid NRPS-PKS pksX cluster from Bacillus subtilis (7-9). Although the pksX cluster has been proposed to encode the biosynthesis of difficidin (7,9,10), recent work has demonstrated that the NRPS portion of the gene cluster is active, disqualifying difficidin, which does not contain any amines, as a candidate for the biosynthetic product of this cluster (11). Long thought to be cryptic, there is now evidence that it is responsible for the biosynthesis of a product that kills streptomycetes (P. D. Straight, M. A. Fischbach, C.T.W., and R. Kolter, unpublished results). Additionally, this cluster does not follow the usual NRPS-PKS colinearity rules because it has only three trans-acting acyltransferases that may load up to 17 of the 20 predicted thiolation domains of the cluster.We were intrigued by a series of predicted ORFs withi...

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule, revealing NAD-linked RNA in E. coli and S. venezuelae. Subsequent characterization shows NAD is a 5’ modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs, and is present at a surprisingly high abundance of ~3000 copies per cell.