In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

GO biological process enrichment for the 882 deletion strains below the threshold of detection by microarray in the BCprot relative to the gene universe of strains present in at least one deletion collectio

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Induction of plasmid-encoded <i>sxy</i> and <i>pilF2</i> genes in strain ZK126.

<p>PCR reactions on cDNA (lanes 1 to 4) confirm that both <i>sxy</i> (515 bp; top) and <i>pilF2</i> (265 bp; bottom) are strongly expressed when IPTG is added. As a control for DNA contamination of RNA preparations, PCR reactions were also performed on the RNA samples before reverse transcription. As expected, these reactions generated no product (lanes 4 to 8). Lanes 1 and 5: p<i>Ecsxy</i>_low; Lanes 2 and 6: p<i>Ecsxy</i>_low with IPTG; Lanes 3 and 7: p<i>Ecsxy</i>; Lanes 4 and 8: p<i>Ecsxy</i> with IPTG.</p

Gene expression upon induction of plasmid-encoded <i>sxy</i> genes.

<p>qRT-PCR quantification of changes in the expression of <i>sxy</i>, <i>ppdD</i> and <i>yrfD</i> upon <i>sxy</i> induction in strain BW25113 after 60 mins expression time. Each bar represents the average of two independent biological replicates for each gene, with error bars indicating standard deviation from the mean. Expression levels shown were normalised for each mRNA sample using 23S rRNA levels.</p

Expression of PpdD pilin protein upon induction of plasmid-encoded <i>sxy</i> genes.

<p>Western blotting with PpdD antiserum was performed on whole-cell extracts from broth-grown BW25113 60 mins after IPTG induction of <i>sxy</i> expression. Lane 1: p<i>EcppdD</i> (positive control, His-tagged ppdD); Lane 2: p<i>Ecsxy</i>; Lane 3: p<i>Ecsxy</i>_low. The positions of native processed (15 kDa) and His-tagged (17 kDa) PpdD proteins are indicated with arrows.</p

Mapping of the <i>dfr1</i> mutations onto the yeast DFR1 model.

<p>(A) Cartoon representation of the structural model for the yeast DFR1, colored by sequence conservation (red is conserved, blue is divergent), with surface shown in transparency. The identified resistance point mutations are indicated with a sphere. MTX is highlighted in yellow, and NADPH in green. Mutations largely cluster around conserved residues near the active site of the enzyme. The <i>dfr1</i> mutations of residues interacting with MTX (yellow, B) or NADPH (green, C) are mapped onto the yeast DFR1 model. The represented protein is colored by sequence conservation as in (A).</p

Expression of hDHFR and hDHFRL1 in <i>DFR1</i>/<i>dfr1</i> heterozygote yeast.

<p>(A) The hDHFR and hDHFRL1 active sites in complex with folate (in red; PDB ID 4M6K). The conserved W25 residue in hDHFR forms a hydrophobic interaction stabilized by a hydrogen bond with folate (left panel). In contrast, R25 in hDHFRL1 cannot form hydrophobic interactions with folate, but the stabilizing hydrogen bond is still present (right panel). The side chains of W25 and R25 residues are shown in sticks. (B) The fitness of yeast mutants upon exposure to MTX (1 mM sublethal dose) or DMSO solvent (2% v/v) were evaluated over 24 hours in a Tecan shaker-reader at 30°C. Error bars indicate standard error, n = 3.</p

Validation of methotrexate resistant dfr1 mutations.

<p>The average fitness of the candidate <i>dfr1</i> haploid (A) and diploid (B) mutants upon exposure to MTX (1 mM sublethal dose) or DMSO solvent (2% v/v) were evaluated over 24 hours in a Tecan shaker-reader at 30°C. Mutant alleles expressed in the presence of a wild-type <i>DFR1</i> copy are listed with (+). Mutants are colored according to their relative doubling time in comparison to the wild-type strains: BY4742 (WT, A) and <i>DFR1</i>/<i>dfr1</i>Δ (WT, B) with: blue for longer doubling time (Slower than WT), grey for comparable doubling time (Fit as WT), and red for shorter doubling time (Faster than WT). The average growth under MTX conditions of the control strains are indicated with a dashed line. Error bars indicate standard error, n = 3.</p

Single nucleotide variant allele frequency estimates for DFR1 loci associated with methotrexate resistance.

<p>Estimated variant allele frequencies (VAFs) are shown for DFR1 loci that have a significantly different VAF compared to the starting T0 time point (as described in Methods). Each VAF estimate is based on a sample size of n = 3 following the sample collection design in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006275#pgen.1006275.s002" target="_blank">S2 Fig</a>. Loci are depicted along the rows and time points along the columns. The heatmap shows the relative change in the VAF at each time point compared to T0, in the diploid (left panel) and haploid (right panel) pools. The estimated VAF at T0 is shown along the left side of the heatmap. The promoter and terminator are delineated from the coding sequence by black and blue bars respectively to the right of the heatmap. Non-synonymous mutations are indicated by the wild-type and variant residue. A bullet beside the residue indicates that the locus is conserved with hDHFR.</p

Workflow for methotrexate resistance screen.

<p>The plasmid-borne <i>dfr1</i> variomics library contains ~2 x 10⁵ independent <i>dfr1</i> alleles maintained in a diploid <i>DFR1</i>/<i>dfr1</i>Δ heterozygote strain. The diploid pool was sporulated and cultured in haploid selective media to generate a <i>dfr1</i> pool in a <i>dfr1</i>Δ haploid background. Both diploid and haploid strains were grown in the presence of MTX (2 mM) or DMSO solvent control (2% v/v) over a 6-day timecourse (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006275#pgen.1006275.s002" target="_blank">S2 Fig</a>). MTX-treated cultures were subsequently harvested for plasmid extraction followed by PCR amplification of the dfr1 alleles. Nextera XT libraries were prepared for DFR1-amplicon sequencing, and non-reference <i>dfr1</i> variant alleles were identified using the RVD analysis tool. Candidate <i>dfr1</i> point mutations were validated by constructing the individual <i>dfr1</i> mutants and their MTX resistance confirmed in growth assays.</p