Overview of the design of synonymous <i>tuf</i> alleles.

<p>The first forty codons in <i>tuf</i> were left unchanged to reduce the possible impact of N-terminal codon usage bias (hatched region). Black bars represent codons that have been changed to synonymous codons. (<b>A</b>) Wild-type <i>tufA</i> gene. (<b>B</b>) Leucine codons (N = 25) were changed to UUA, UUG, CUU, CUC or CUA, respectively. (<b>C</b>) The leucine codons in the first half of the <i>tuf</i> gene (N = 13) were changed to UUA, CUC or CUA, respectively. (<b>D</b>) The leucine codons in the second half of the <i>tuf</i> gene (N = 12) were changed to UUA, CUC or CUA, respectively. (<b>E</b>) Proline codons (N = 19) were changed to CCU, CCC or CCA. (<b>F</b>) Both leucine and proline codons (N = 44) were changed: in one <i>tuf</i> allele all leucine codons were changed to UUG and all proline codons to CCU; in the other <i>tuf</i> allele all leucine codons were changed to UUA and all proline codons to CCA. (<b>G</b>) All arginine CGU codons (N = 17) were changed to CGG. (<b>H</b>) All valine GUU codons (N = 21) were changed to GUC.</p

Selective disadvantage per codon per generation of synonymous <i>tuf</i> alleles.

<p>Selective disadvantage as a function of <b>(A)</b> logarithm of the relative adaptiveness and <b>(B)</b> increase in translational time for the <i>tufA</i> gene (filled circle) and synonymous <i>tuf</i> alleles with changes affecting codons for leucine (filled squares), proline (filled triangles), arginine (open squares), valine (open circles) and combinations of leucine and proline (filled diamonds). Results are shown as means ± 95% confidence interval. Codons are indicated next to the marker. Black line indicates regression fit.</p

The selective disadvantage of synonymous <i>tuf</i> alleles.

<p>The selective disadvantage of synonymous <i>tuf</i> alleles.</p

Analysis of <i>tuf</i> mRNA.

<p>Relative <i>tuf</i> mRNA content (dark grey) and relative <i>tuf</i> mRNA half-life (light grey) for wild-type <i>tufA</i> and synonymous <i>tuf</i> alleles with all leucine codons changed to UUA, UUG, CUU, CUC or CUA, respectively. Results are shown as means ± standard deviation.</p

Selective disadvantage per generation of synonymous <i>tuf</i> alleles.

<p>Selective disadvantage as a function of <b>(A)</b> absolute changes in predicted mRNA free energy, and number of hexamers with a binding affinity to the anti-SD sequence in 16S rRNA of <b>(B)</b> < -6 kcal/mol and <b>(C)</b> < -4 kcal/mol. Values are shown for the <i>tufA</i> gene (filled circle) and synonymous <i>tuf</i> alleles with changes affecting codons for leucine (filled squares), proline (filled triangles), arginine (open squares), valine (open circles) and combinations of leucine and proline (filled diamonds). Results are shown as means ± 95% confidence interval. Black line indicates regression fit.</p

RpoB and RpoS mutants have a growth advantage on aging colonies.

<p>Fold increase in wild-type and mutant cells added to 24 h wild-type colonies and allowed to age for a further 7 days. The box plots show the first quartile, median, and third quartile values. Outlier indicated by a triangle. Statistical significance of differences in the distribution of values between strains, compared to the wild-type, is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255.s002" target="_blank">Table S2</a> and indicated in the figure by asterisks (*  = 95% confidence interval, ***  = 99.9% confidence interval).</p

Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies

<div><p>When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in <i>rpoB</i> (RNA polymerase β-subunit) or <i>rpoS</i> (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of <i>acs</i> (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of <i>rpoB</i> and <i>rpoS</i> mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the <i>rpoB</i> mutant, and significantly increased in the aging <i>rpoS</i> mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of <i>rpoB</i> and <i>rpoS</i> mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.</p></div

Inactivating <i>relA</i> does not alter step-time of the <i>tufA499</i> mutant.

a<p>All strains carried the F-factor <i>F′128 pro</i>⁺<i>lac</i>⁺<i>zzf-1831</i>::Tn<i>10d-spc</i>.</p>b<p><b>Step time ± standard deviation (sec).</b></p>c<p>p-values calculated by unpaired t-tests, comparing the step-time of the mutant strains to the TH7480 <i>tufA</i> wild-type.</p>d<p>p-values calculated by unpaired t-tests, comparing <i>relA21</i>::Tn<i>10</i> strains to the corresponding <i>relA</i>+ strain.</p

Acetate uptake rate in young and aged colonies.

<p>Colonies of 24 h (open squares) and 7 days (closed diamonds) were suspended in ¹⁴C-acetate. Samples were taken every 10 seconds for one minute. <b>A</b>. Wild-type colonies. <b>B</b>. <i>rpoB</i> P564L colonies. <b>C</b>. Δ<i>rpoS</i> colonies. Each data point represents the average of three independent measurements, with standard deviations as error bars.</p

Protein quantification in aging colonies.

<p>Total protein was prepared from colonies of either wild-type, <i>rpoB</i> P564L mutant or Δ<i>rpoS</i> mutant, all grown for 1, 3, 5 and 7 days. Concentrations of the proteins of interest were quantified by Single Ion Monitoring mass spectrometry. Bars represent averages of two or three peptides per protein, measured in biological duplicates, each measured twice, with error bars representing standard deviation between the runs. Concentration values are in arbitrary units, normalized to total protein in the samples, to enable direct comparison between different days and different samples. The day 5 sample from the Δ<i>rpoS</i> mutant could not be analyzed. Statistical significance of differences, compared to wild-type samples of the same age, are indicated in the figure (-  =  not significant, *  = 95% confidence interval, **  = 99% conficence interval, ***  = 99.9% confidence interval). <b>A</b>. Concentration of Acs, acetyl-CoA synthase. <b>B</b>. Concentration of AceA, isocitrate lyase. <b>C</b>. Concentration of AceB, malate synthase. <b>D</b>. Concentration of AckA, acetate kinase. <b>E</b>. Concentration of Pta, phosphotransacetylase.</p