The principle of the Ras Recruitment System (RRS).

<p>The system is based on a temperature sensitive GDP exchange factor (encoded by the <i>cdc25–2</i> allele) that is rendered inactive at 36°C trapping endogenous Ras1p in its inactive GDP bound form. Growth is rescued by genetic complementation with a constitutively active mutant of mammalian H-Ras (<i>RAS61</i>). To exert its function and rescue growth, Ras61p needs to be directed to the plasma membrane. This can either occur through protein-protein interactions or lipid modifications such as myristoylation or prenylation. Specifically, prenylation can either be mediated by endogenous protein prenyltransferases (wt-PPTases) that recognise naturally occurring, prenylatable CaaX-box motives or engineered protein prenyltransferases (o-PPTases) that recognise orthogonal CaaX-box motives that are not recognised by the endogenous machinery. For optimal membrane recruitment and genetic complementation in the RRS, the three most C-terminal amino acids of prenylated CaaX-box motives are removed by highly specific protein prenyl proteases located in the endoplasmic reticulum followed by carboxymethylesterification of the C-terminus.</p

Engineering FTases with altered substrate specificities.

<p>(A) CaaX-box motives with positively charged residues in the anchoring position X cannot rescue growth in the RRS and thus provide poor substrates for endogenous FTases in <i>Saccharomyces cerevisiae</i>. (B) Structural model of the αβ-FTase heterodimer derived from <i>Rattus norvegicus</i> (PDB: 1KZO). The C-terminus of the α-subunit (highlighted in blue) is separated by 40 Å from the N-terminus of the β-subunit (highlighted in red). (C) Western blot analysis of GFP-αβ-FTase fusion proteins derived from <i>R</i>. <i>norvegicus</i> expressed in <i>Leishmania tarantolae</i> cell-free expression system. The linker connecting α- and β-subunits contained a TEV protease cleavage site that is cleaved with exogenously added TEV protease. L1: Protein Ladder; L2: Uncleaved GFP-αβ-FTase; L3: GFP-αβ-FTase cleaved with TEV Protease. (D) Fluorescent scan of SDS–PAGE loaded with mCherry-K-Ras <i>in vitro</i> prenylation reaction containing single-chain GFP-αβ-FTase fusion proteins and fluorescent phosphoisoprenoid NBD-GPP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120716#pone.0120716.ref035" target="_blank">35</a>]. Addition of FPP to the reaction prevents formation of the fluorescent reaction product due to competition with the fluorescent lipid donor. L1: Protein Ladder; L2: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBP-GPP; L3: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBD-GPP, 25 μM FPP. (E) To facilitate expression and prevent cross-heterodimerisation between yeast and exogenous FTase subunits, a single-chain αβ-FTase was created based on mutant β-W102T while introducing negative charges at the bottom of the active site at β-G142D and β-G142E enabling FTase to farnesylate a CaaX-box motif with a positive charge in X and thus rescue growth in the RRS. Controls: pYES2 denotes vector control and β-W102T the unmodified, single-chain αβ-FTase^β-W102T mutant neither of which can prenylate the orthogonal CaaX-box motif.</p

Summary of constructs employed in this study.

<p>Summary of constructs employed in this study.</p

Mapping CaaX-box dependent membrane recruitment space in yeast.

<p>(A) Flow chart of the mapping experiment. The CaaX-box library was transformed into the RRS screening strain, grown for 4 days under permissive conditions at 25°C, replica plated and then grown for another 4–6 days under restrictive and permissive conditions at 37°C and 25°C. CaaX-box coding plasmid DNA was then isolated and analysed for the two different library sets by NGS with the Ion Torrent system. The enrichment was determined for each of the 8000 different CaaX-box motives by measuring the frequency of each peptide under restrictive conditions and normalising it over its frequency under permissive conditions. (B) Graphic representation of the enrichment factors of 8000 different CaaX-box motives is summarised in a 4D plot: Each axis represents the 20 different amino acids while the size of each dot is proportional to the enrichment of a specific CaaX-box motif. Only CaaX-box motives that have been enriched >3 are shown. (C) Cross-sectional views along the a₂-a₁, X-a₁ and X-a₂ axis illustrate that a₂ exerts the greatest specificity on substrate specificity with small hydrophobic residues highly preferred followed by the anchoring position X and a₁.</p

Summary of NGS counts and library coverage.

<p>¹Based on the observation that a sequence motif is detected at least twice in a particular NGS run.</p><p>²Based on the observation that a sequence motif is detected at least twice in the Naïve, 25°C or 37°C data set.</p><p>³Based on the observation that a sequence motif is detected at least twice in either the 25°C or 37°C data set.</p><p>Summary of NGS counts and library coverage.</p

β-lactam MICs and PCR results for Kp001 and Ec003.

a<p>Antibiotic abbreviations: CLA, clavulanic acid, TZB, tazobactam.</p>b<p>MICs were determined using a VITEK 2 AST-N149 card.</p

Structural features of plasmids pJEG011 and pJEG012.

<p>Comparisons to related plasmids pOXA-48a (A) and pJIE143 (B) are shown, respectively; plasmid backbones are represented by thick gray lines and areas of ≥99% sequence identity between plasmids are indicated by the light blue areas. Only the following selected genes are annotated and represented by colored arrows: plasmid replication genes, red; transposon-related genes, orange; plasmid partitioning, maintenance (<i>e.g.</i>, toxin/antitoxin systems (T/AT)), mobilization and conjugation genes, yellow; aminoglycoside resistance genes, green; β-lactam resistance genes, blue. Dashed arrows represent more than one gene or open reading frame. Insertion sequences (IS) are represented by orange pentagons with the IS number indicated within; the direction of the IS with respect to the transposase gene is indicated by the point of the pentagon. Inverted repeats associated with IS and transposons are indicated by vertical orange lines; the nucleotide sequences of the direct repeats resulting from IS and transposon insertion are indicated above or below the plasmid figures. Integron gene cassettes are represented by orange rectangles.</p

SNPs proximal to SNVs are more likely to be ASE.

<p>(A) SNPs binned by genomic distance to nearest heterozygous indel. (B) SNPs binned by nearest heterozygous un-phased <i>de novo</i> called SNP. (C) SNPs binned by the number of proximal heterozygous SNPs. (D) SNPs binned by genomic alignability. The closer a SNP is to a heterozygous SNV, the more likely that SNP is to be classified as ASE. For indels, this effect is stronger than SNPs, which are only slightly affected, however as the number of SNPs within a read length (100bp) increases, this effect is increased. In all plots, the dashed line represents the average ASE proportion for all SNPs. Numbers at the top of the bars indicate the number of SNPs in each bin (blue not ASE, red ASE).</p

Bacterial strains, plasmids and primers.

<p>Bacterial strains, plasmids and primers.</p

Antibiotic MICs for Kp001 and Kp002.

a<p>Antibiotic abbreviations: CLA, clavulanic acid, TZB, tazobactam; TMP/SXT, trimethoprim/sulfamethoxazole.</p>b<p>MICs were determined using a VITEK 2 AST-N149 card.</p