Sequence and structural homology of essential genes and their high copy suppressors.

<p>The Protein Information Resource (<a href="http://pir.georgetown.edu/pirwww/search/pairwise.shtml" target="_blank">http://pir.georgetown.edu/pirwww/search/pairwise.shtml</a>) was used to generate Smith-Waterman amino acid alignments and the corresponding e-values; lower e-values imply higher homology between sequences <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002803#pgen.1002803-Smith1" target="_blank">[28]</a>. MAMMOTH (Matching Molecular Models from Theory) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002803#pgen.1002803-Ortiz1" target="_blank">[29]</a> was used to generate pairwise structural alignments; a value above 4.5 (p = 0.01) indicates significant structural homology <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002803#pgen.1002803-Ortiz1" target="_blank">[29]</a>; if no structure was available, a structure model from <a href="http://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi" target="_blank">http://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi</a> was used. Essential genes that could be knocked out in the presence of their HCS are indicated with an asterisk.</p

Patterns of Evolutionary Conservation of Essential Genes Correlate with Their Compensability

<div><p>Essential genes code for fundamental cellular functions required for the viability of an organism. For this reason, essential genes are often highly conserved across organisms. However, this is not always the case: orthologues of genes that are essential in one organism are sometimes not essential in other organisms or are absent from their genomes. This suggests that, in the course of evolution, essential genes can be rendered nonessential. How can a gene become non-essential? Here we used genetic manipulation to deplete the products of 26 different essential genes in <em>Escherichia coli</em>. This depletion results in a lethal phenotype, which could often be rescued by the overexpression of a non-homologous, non-essential gene, most likely through replacement of the essential function. We also show that, in a smaller number of cases, the essential genes can be fully deleted from the genome, suggesting that complete functional replacement is possible. Finally, we show that essential genes whose function can be replaced in the laboratory are more likely to be non-essential or not present in other taxa. These results are consistent with the notion that patterns of evolutionary conservation of essential genes are influenced by their compensability—that is, by how easily they can be functionally replaced, for example through increased expression of other genes.</p> </div

Few high copy suppressors are found for conserved and consistently essential genes.

<p>We gathered information on orthologous genes in nine additional taxa for which essentiality has been experimentally investigated. All genes listed are essential in <i>E.</i> coli; for each gene, the orthologue in other taxa is indicated as essential (E; white background), non-essential (N; light grey), associated with large fitness reduction (N*), or unknown (U). Cases in which there is no orthologue are indicated with an A (absent; dark grey background). For example, <i>spoT</i>, which is essential in <i>E. coli</i>, has been found to be essential in only four out of eight other taxa and is absent from one. The second column indicates the high copy suppressors that were isolated (genes for which suppressors were isolated are highlighted in light grey). In parentheses are high copy suppressors that were recovered from the screen but which were not analyzed further. A cladogram showing the evolutionary relationships of these taxa is shown at the top of the table. Abbreviations: <i>S.ty, Salmonella typhi Ty2; A.ba, Acinetobacter baylyi; C.cr, Caulobacter crescentus NA100; F.tu, Francisella tularensis U112; S.pn, Streptococcus pneumonia TIGR4; B.su, Bacillus subtilis 168; S.au, Staphylococcus aureus 8325; M.ge, Mycoplasma genitalium G37; M.pu, Mycoplasma pulmonis UAB CTIP</i>. a) No reciprocal best hit orthologue of <i>ftsK</i> exists in <i>B. subtilis</i> due to an apparent duplication (genes BSU16800 and BSU29805); neither gene is essential. b) No reciprocal best hit orthologue of <i>plsC</i> exists in <i>F. tularensis</i> due to an apparent duplication (FTN1749 and FTN1750); only FTN1749 is essential.</p

Differential colony formation of conditional lethal mutant strains with suppressive plasmids.

<p>In six cases we isolated HCS, but were unable to knockout the corresponding essential gene even in the presence of the HCS. For each essential gene, the top row illustrates the growth that is observed when the native promoter is replaced by P_ara, under permissive (0.1% arabinose) or restrictive (0.4% glucose) conditions. The bottom row indicates the growth observed for the same strain containing the plasmid with the corresponding HCS, under permissive (0.1% arabinose) or restrictive (0.4% glucose) conditions. Expression of the HCS was induced with 50 µM IPTG. When expression of the essential gene is repressed, robust growth is only observed in the presence of the high copy suppressor. To grow the spot plates, cultures were grown overnight in 0.1% arabinose with 15 µg/ml chloramphenicol, diluted into LB medium, and 5 µl of the indicated dilution were spotted onto plates containing either 0.1% arabinose, or 0.4% glucose with 50 µM IPTG. The arabinose plates were incubated for 24 hours, while the glucose IPTG plates were incubated for 48 hours at 37°, except for P_ara-<i>degS</i>, which was incubated for 14 hours.</p

Possible functional similarities between non-complementing high copy suppressors and essential genes.

<p>We derived depletion phenotypes of conditional lethal mutants and the original function of high copy suppressor genes from literature. Proposed modes of action of the high copy suppressor genes are also based on evidence from literature.</p

Differential colony formation of conditional lethal mutant strains on arabinose or glucose-containing LB agar plates.

<p>The native promoters of 23 genes or operon pairs were replaced with the arabinose-inducible <i>araBAD</i> promoter (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002803#s3" target="_blank">Methods</a>). By shifting these mutant strains from medium with 0.1% L-arabinose to medium without L-arabinose and supplemented with 0.4% D-glucose, expression of the essential gene was repressed. In all cases this resulted in growth inhibition or severe growth defects. The gene whose native promoter was replaced is indicated on the right hand side of each row; the ancestral strain TB741 is shown at the top. In the case of <i>degS</i>, substantial growth occurs even after repression, suggesting that growth only becomes inhibited once the gene products are sufficiently depleted; this has been observed previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002803#pgen.1002803-Alba1" target="_blank">[19]</a>. This is also true, although to a lesser extent, for <i>spoT</i>, <i>ftsK</i>, and <i>ygjD</i>. To grow the spot plates, cultures were grown overnight in 0.1% arabinose, diluted into LB medium, and 5 µl of the indicated dilution were spotted onto plates and incubated at 37° for 10 hours. Those strains indicated with a cross were incubated for 24 hours.</p

Predicted equilibrium fitness as a function of phenotypic complexity (n_e).

<p>Results are shown for populations of size 100 (black), ten (grey), and three (white). An exponential fitness decline in which Q = 1 was used (yielding a fitness function of f(d) = exp(−d)). Circles indicate the average fitness reached in the simulation model; curves indicate the analytical results.</p

Noise in gene expression is dependent on the functional importance of the downstream gene.

<p>Conserved non-essential genes exhibit less noise. Conservation is calculated as the number of gamma-proteobacterial taxa in which an orthologous gene copy is present. Promoters were binned according to the number of taxa in which an orthologue was found; the relationship is highly significant (for an unbinned analysis, Spearman's rho = −0.19, p = 7.2e-12, n = 1350 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002443#pgen.1002443.s005" target="_blank">Figure S5</a>)). A nonparametric linear fit using Thiel's method <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002443#pgen.1002443-Thiel1" target="_blank">[71]</a> is shown in black.</p

Dependence of variation in mRNA expression on mean mRNA expression level and derivation of a noise metric.

<p>A. The observed variance in mRNA expression increases with increasing mean expression level. Shown are five promoters with various levels of mean and variance in expression (from left to right: rho transcription termination factor; <i>prpR</i> transcriptional dual regulator, <i>bolA</i> transcriptional dual regulator; tyrosyl-tRNA synthetase; and <i>dps</i>, an iron sequestration and DNA damage protein). B. The expression level and observed standard deviation for all 1522 promoters used in the analysis. The genes shown in panel A are highlighted in red (the left-most red dot is <i>rho</i>, the right-most dot is <i>dps</i>). C. The coefficient of variation decreases initially with increasing expression, but plateaus at higher expression levels. D–F. Analogous histograms and graphs to those shown in panels A–C), but calculated from log-transformed data. As discussed in the text, our focus is on variation in expression; we thus derived a measure of variation in mRNA expression that is independent of the mean level of mRNA expression, and any measurement artifacts associated with changes in the mean. This allows us to test whether mean and variation in expression can be decoupled due to selection or changes in the promoter sequence. The noise metric is the vertical deviation from a smooth spline (blue) calculated from the running median (orange) of mean log expression level versus the CV of log expression. The slight decrease in CV at low expression levels (panels C and E) is because fluorescence values lower than one cannot occur. Thus, for weakly expressed genes, the distribution specifying the variation in expression levels is truncated at one, decreasing the CV.</p

Noise in gene expression is dependent on the functional importance of the downstream gene.

<p>Conserved non-essential genes exhibit less noise. Conservation is calculated as the number of gamma-proteobacterial taxa in which an orthologous gene copy is present. Promoters were binned according to the number of taxa in which an orthologue was found; the relationship is highly significant (for an unbinned analysis, Spearman's rho = −0.19, p = 7.2e-12, n = 1350 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002443#pgen.1002443.s005" target="_blank">Figure S5</a>)). A nonparametric linear fit using Thiel's method <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002443#pgen.1002443-Thiel1" target="_blank">[71]</a> is shown in black.</p