PMC

Spider plot of mean significant attribute intensities for (a) R1, R1‐W and R1‐E and (b) R2, R2‐W and R2‐E rum 1. “R1” is rum 1, “R1‐W” is 1:2 (v/v) dilution of rum 1 with water to achieve 20% ABV, “R1‐E” is 1:2 (v/v) dilution of rum 1 with 40% ethanol to achieve 40% ABV. “R2” is rum 2, “R2‐W” is 1:2 dilution (v/v) of rum 2 with water to achieve 20% ABV, “R2‐E” is 1:2 (v/v) dilution of rum 1 with 40% ethanol to achieve 40% ABV. “A” is aroma, “ABM” is aroma‐by‐mouth, “AT” is aftertaste, “MF” is mouthfeel, “Ta” is taste

Rum Mut is an activated form of pathogen-encoded Rum polymerase. (A) Sketch showing the conversion of inactive Rum pol (RumA′₂B) to an activated mutasome by the transfer of a RecA monomer from the 3′-tip of RecA* followed by the binding of a molecule of ATP to form Rum Mut = RumA′₂B-RecA-ATP. Rum Mut is catalytically active in the absence of RecA*. (B, C) Rum Mut integrity analysis. To show that Rum pol and RecA assemble as a physical complex, Rum Mut (and Rum pol), containing a His tag on RumB, was bound to a Ni²⁺-NTA resin and washed extensively to remove proteins not associated with the complex. An SDS-page analysis of the eluted complex showed 3 gel protein bands for Rum Mut corresponding to RumB, RumA′ and RecA, thus confirming the integrity of the mutasomal complex. (D, E) Activity of Rum pol and Rum Mut measured on p/t DNA in the presence and absence of ATP or ATPγS. The ‘RecA*’ lane shows transactivation of Rum pol in the presence of RecA*. % PE refers to percent of p/t extended, Ec RecA is E. coli RecA and Rum Mut-Ec refers to Rum Mut complex assembled with Ec RecA. The reactions (1 h, saturating dNTP concentrations) were carried out on hairpin (HP) p/t DNA with a 3 nt template overhang (oh), radioactively-labelled with ³²P at the 5′-template-end (see Materials and Methods).

RecA homologs from variety of bacterial pathogens form Rum Mut complexes that require activation by ATP or ATPγS for DNA synthesis. (A) A ClustalW multiple sequence alignment of RecA homologs encoded by different bacterial pathogens. Protein sequences were obtained from Genbank sequence files: NP_417179 (Ec RecA); YP_005228402 (Kp RecA); WP_048608224 (Pr RecA); AAF93711 (Vc RecA); NP_252307 (Pa RecA); NP_217253 (Mt RecA); YP_499795 (Sa RecA). Identical or highly conserved amino acid residues are shaded and blocked together. Amino acid differences present in both Mt RecA and Sa RecA, which form weakly active Rum Mut-Mt and inactive Rum Mut-Sa, are highlighted in red. (B) An unrooted phylogenetic tree indicating the evolutionary relatedness of RecA homologs. The scale bar corresponds to an estimated amino acid substitutions per site of 0,01 (the number of substitutions divided by the length of the sequence). (C) Activity of Rum Mut assembled with RecA homologs measured for 1 h on 3 nt oh HP DNA in the absence and presence of activating ATP/ATPγS and with saturating concentration of dNTPs. All RecA homologs, with the exception of Sa RecA, form active Rum Mut complexes that require activation with ATP/ATPγS for DNA synthesis. The ‘RecA*’ lane shows transactivation of Rum pol in the presence of RecA*. Rum Mut-Pr, Rum Mut-Vc, Rum Mut-Kp, Rum Mut-Pa, Rum Mut-Mt and Rum-Sa abbreviations refer to the identity of bound RecA homologs.

a. Map of Scotland and location of the Isle of Rum (red box). b Simplified geological map of the Isle of Rum. c Geological map of the northern limb of the ELI with simplified geological map of the peridotites and allivalites in this study. After Emeleus