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

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 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

Influence of mutations affecting acetate metabolism on the growth advantage of <i>rpoB</i> P564L and Δ<i>rpoS</i> mutants.

<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, as a function of acetate metabolism activity. <b>A</b>. Relative to the <i>rpoB</i> 564L mutant. <b>B</b>. Relative to the Δ<i>rpoS</i> mutant. The box plots show the first quartile, median, and third quartile values. Outliers are indicated as triangles. Δ<i>apa</i> indicates deletion of the three genes, <i>ackA-pta</i> and <i>acs</i>. Statistical significance of differences, compared to the <i>rpoB</i> or <i>rpoS</i> mutants, 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 is indicated in the figure by asterisks (**  = 99% confidence interval, ***  = 99.9% confidence interval).</p

Outline of central metabolism indicating acetate production and utilization pathways.

<p>Outline of central metabolism showing the acetate synthesis and utilization pathways <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255-Wolfe1" target="_blank">[24]</a>. Gene names are shown in italics, substrates and products are referred to in the text.</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

Influence of the <i>acs</i> status of the background colony on the growth advantage of Rif^R and RpoS mutants.

<p>Fold increase in wild-type and mutant cells added to 24 h wild-type or Δ<i>acs</i> colonies and allowed to age for a further 7 days. The box plots show the first quartile, median, and third quartile values. Outliers are indicated as triangles. Statistical significance of differences between strains 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 is indicated in the figure by asterisks (**  = 99% confidence interval, ***  = 99.9% confidence interval).</p

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

Proline tRNAs are less acylated in the slow-growing <i>tufA499</i> mutant.

<p>Northern blot measurements of aminoacylation levels of the proline isoacceptors, in wild-type (TH7507) and <i>tufA499</i> mutant (TH7509). Values are averages of four or five independent measurements and normalized to the wild-type, standard deviation shown as error bars. The differences between the wild-type and the <i>tufA499</i> strains are statistically significant according to an unpaired t-test, proK: p = 0.0007, proM: p = 0.0010, proV: p = 0.0027.</p

Inactivating <i>dksA</i> does not compensate for <i>tufA499</i>.

a<p>Dt is doubling time of the bacterial cultures, ± standard deviation.</p