Growth rates in batch cultures are similar for the autoregulated WT and constitutive strains.

<p>(A) Western blot analyses of PhoB expression. Wild type strains BW25113 (WT) and RU1622 (WT-<i>yfp</i>), IPTG-inducible strains RU1616 (LAC) and RU1618 (TRC), and the constitutive strain RU1617 (KON) were grown to Pi-depletion under different IPTG concentrations and assayed for PhoB expression. Growth curves (B) and doubling times (C) of strains with different PhoB levels. Indicated strains were grown in MOPS media with an initial Pi concentration of 50 µM. Depletion of Pi accompanies the change of the growth rate and cells quickly enter into the stationary phase (shaded). Numbers after the “+” sign indicate IPTG concentrations. Relative doubling times were determined by fitting the exponential growth curves with an exponential function and compared to the doubling time of the WT strain. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#s2" target="_blank">Results</a> are shown as the mean and SD of at least four independent experiments.</p

Evolutionary Tuning of Protein Expression Levels of a Positively Autoregulated Two-Component System

<div><p>Cellular adaptation relies on the development of proper regulatory schemes for accurate control of gene expression levels in response to environmental cues. Over- or under-expression can lead to diminished cell fitness due to increased costs or insufficient benefits. Positive autoregulation is a common regulatory scheme that controls protein expression levels and gives rise to essential features in diverse signaling systems, yet its roles in cell fitness are less understood. It remains largely unknown how much protein expression is ‘appropriate’ for optimal cell fitness under specific extracellular conditions and how the dynamic environment shapes the regulatory scheme to reach appropriate expression levels. Here, we investigate the correlation of cell fitness and output response with protein expression levels of the <i>E. coli</i> PhoB/PhoR two-component system (TCS). In response to phosphate (Pi)-depletion, the PhoB/PhoR system activates genes involved in phosphorus assimilation as well as genes encoding themselves, similarly to many other positively autoregulated TCSs. We developed a bacteria competition assay in continuous cultures and discovered that different Pi conditions have conflicting requirements of protein expression levels for optimal cell fitness. Pi-replete conditions favored cells with low levels of PhoB/PhoR while Pi-deplete conditions selected for cells with high levels of PhoB/PhoR. These two levels matched PhoB/PhoR concentrations achieved via positive autoregulation in wild-type cells under Pi-replete and -deplete conditions, respectively. The fitness optimum correlates with the wild-type expression level, above which the phosphorylation output saturates, thus further increase in expression presumably provides no additional benefits. Laboratory evolution experiments further indicate that cells with non-ideal protein levels can evolve toward the optimal levels with diverse mutational strategies. Our results suggest that the natural protein expression levels and feedback regulatory schemes of TCSs are evolved to match the phosphorylation output of the system, which is determined by intrinsic activities of TCS proteins.</p></div

Intermediate PhoB levels lead to a slower adaptation pace and distinct adaptive genotypes.

<p>(A) PhoB levels of the indicated adapting cultures. IPTG concentrations of 0 µM and 40 µM were included in the medium to give low and intermediate initial PhoB levels, both of which were lower than the optimal concentration. (B) PhoB expression in batch cultures from random colonies isolated from above cultures. No IPTG was added to batch cultures and PhoB levels were used for phenotype classification of individual colonies. (C) Population of individual colonies with different PhoB expression phenotypes. Colonies isolated from adapting cultures were classified into three groups by PhoB expression levels (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#pgen.1003927.s003" target="_blank">Figure S3</a>). High level corresponds to the autoregulated WT PhoB concentration seen under Pi-deplete conditions and low level is comparable to the basal expression of the original LAC strain. Colonies with intermediate levels substantially different from the above two levels were classified as the medium PhoB expression phenotype. (D) Mapping of adaptive mutations. Colonies with elevated PhoB levels were selected for sequencing of the <i>lac</i> promoter and <i>lacI</i>. Mutations in <i>lacO</i>1 and <i>lacI</i> as well as numbers of colonies carrying the particular mutation are indicated. Dots represent identical nucleotides and dashes represent absence of nucleotides. For genotype α, PCR with multiple <i>lacI</i>-specific primers failed to yield any products, suggesting the loss of the entire <i>lacI</i>, possibly due to deletion. (E) IPTG-induced PhoB levels of selected colonies with different genotypes.</p

Adapted LAC₀ cells carry promoter regulation mutations for optimal expression of PhoB.

<p>AP activities (A) and PhoB levels (B) of individual colonies isolated from LAC₀ and LAC₁₅₀ cultures. Indicated strains were grown in batch cultures with 50 µM (Lo) or 2 mM Pi (Hi) for 3 h. PhoB levels are shown for cells grown in Pi-replete medium (2 mM) except for WT. Error bars are SDs of at least four independent experiments. (C) IPTG-induced PhoB expression of indicated strains under Pi-deplete conditions. (D) Schematic representation of adaptive mutations. In the LAC strain, LacI binds to <i>lacO</i> regions at the <i>lac</i> promoter before <i>phoBR</i>. The presence or absence of <i>lacI</i> is indicated by “+” or “−” symbols. Sequences of the <i>lacO</i>1 site from adaptive mutants are shown with dots representing identical sequences. (E) Repression of PhoB expression by additional copies of <i>lacI</i>. Indicated colonies were transformed with the <i>lacI_q</i>-containing plasmid pRG177 and probed for PhoB expression in the absence of IPTG.</p

Cells expressing low levels of PhoB adapt to Pi-deplete environments by increasing PhoB expression.

<p>Time-dependent AP activities (A) and PhoB levels (B) are shown for continuous cultures with indicated strains, BW25113 (WT) and RU1616 (LAC). Pi concentrations of 300 µM (Hi) and 12 µM (Lo) in the inlet fresh medium were used for Pi-replete and -deplete environments, respectively. Numbers after the “+” sign indicate IPTG concentrations. 0.3 OD₆₀₀*ml of cells collected from the chemostat outlet flow were used for analyses of AP activities and PhoB expression. Error bars are SDs of three independent experiments and unseen error bars are smaller than symbols.</p

Cells overexpressing PhoB adapt to Pi-replete environments by abolishing PhoB expression.

<p>(A) PhoB levels of the adapting culture under the Pi-replete condition. RU1618 (TRC) was induced with 15 µM IPTG to achieve overexpression of PhoB and allowed for adaptation in continuous cultures. (B) PhoB expression in batch cultures from random colonies isolated at different times. Lower case letters indicate genotypes identified in selected colonies that were subjected to sequencing analyses. (C) List of mutations for adapted cells with altered PhoB expression.</p

Autoregulation of PhoB/PhoR amplifies the graded output response.

<p>(A) Positive autoregulation of the PhoB/PhoR system. (B) PhoB-regulated transcription output of autoregulated (WT) and non-autoregulated (LAC) strains. RU1465 (WT) and RU1653 (LAC) carrying a <i>phoA-yfp</i> reporter were grown in MOPs medium with indicated initial Pi concentrations. Depletion of Pi led to activation of the <i>phoA-yfp</i> reporter and mean fluorescence of ∼20000 cells was determined. IPTG concentrations of 0 and 150 µM were used to induce PhoB expression in the LAC strain to achieve two different constant levels, corresponding to basal and autoregulated WT PhoB concentrations, respectively (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#pgen.1003927.s004" target="_blank">Table S1</a>). Error bars are SDs of three independent experiments and unseen error bars are smaller than symbols. (C) Population distribution of single-cell output responses of RU1465 (WT) at indicated initial Pi concentrations. Dashed lines represent lognormal fit of cell population distribution.</p

PhoB expression levels correlate with cell fitness in continuous competition assays.

<p>(A) Non-fluorescent bacteria population in continuous cultures when competed against WT-<i>yfp</i> (RU1622). A 50∶50 mixture of WT-<i>yfp</i> with indicated strains were grown in continuous cultures. Pi-replete and -deplete conditions were achieved with different Pi concentrations, 300 µM (Hi) and 12 µM (Lo), in the fresh medium supplied through the chemostat inlet. Bacteria population at 24 h was used to evaluate cell fitness under Pi-replete (B) and -deplete (C) conditions. Different PhoB levels were achieved with different IPTG concentrations using the following strains: BW25113 (WT, solid square), RU1631 (Δ<i>phoB</i>, open square), RU1616 (LAC, open circle), RU1617 (KON, open diamond), RU1618 (TRC, open triangle) and RU1619 (KON <i>phoB</i>^D53A, solid diamond). PhoB amount was determined previously from 0.3 OD₆₀₀*ml of cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#pgen.1003927-Gao2" target="_blank">[19]</a>. A value of 3 pmol in these cells is estimated to correspond to a PhoB concentration of ∼10 µM. Dotted lines represent linear and lognormal trend lines under respective conditions. The solid line represents the saturating dependence of output PhoB∼P levels on total amount of PhoB measured previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#pgen.1003927-Gao2" target="_blank">[19]</a>. The star marks the PhoB∼P level at the autoregulated WT concentration of PhoB. Error bars are SDs and the number of independent experiments is documented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003927#pgen.1003927.s004" target="_blank">Table S1</a>.</p

Serum Lipids and Breast Cancer Risk: A Meta-Analysis of Prospective Cohort Studies

<div><p>Purpose</p><p>Epidemiologic studies exploring causal associations between serum lipids and breast cancer risk have reported contradictory results. We conducted a meta-analysis of prospective cohort studies to evaluate these associations.</p><p>Methods</p><p>Relevant studies were identified by searching PubMed and EMBASE through April 2015. We included prospective cohort studies that reported relative risk (RR) estimates with 95% confidence intervals (CIs) for the associations of specific lipid components (i.e., total cholesterol [TC], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and triglycerides [TG]) with breast cancer risk. Either a fixed- or a random-effects model was used to calculate pooled RRs.</p><p>Results</p><p>Fifteen prospective cohort studies involving 1,189,635 participants and 23,369 breast cancer cases were included in the meta-analysis. The pooled RRs of breast cancer for the highest versus lowest categories were 0.96 (95% CI: 0.86–1.07) for TC, 0.92 (95% CI: 0.73–1.16) for HDL-C, 0.90 (95% CI: 0.77–1.06) for LDL-C, and 0.93 (95% CI: 0.86–1.00) for TG. Notably, for HDL-C, a significant reduction of breast cancer risk was observed among postmenopausal women (RR = 0.77, 95% CI: 0.64–0.93) but not among premenopausal women. Similar trends of the associations were observed in the dose-response analysis.</p><p>Conclusions</p><p>Our findings suggest that serum levels of TG but not TC and LDL-C may be inversely associated with breast cancer risk. Serum HDL-C may also protect against breast carcinogenesis among postmenopausal women.</p></div

Stratified meta-analyses of three lipid components and breast cancer risk.

<p>Stratified meta-analyses of three lipid components and breast cancer risk.</p