CodY is involved in a positive regulation of <i>prfA</i> transcription.

<p>A. Schematic representation of <i>hly</i>, <i>plcA</i> and <i>prfA</i> gene organization and respective promoter regions. B. Luminescence measurements of <i>ΔcodY</i>, <i>ΔprfA</i> and WT <i>L. monocytogenes</i> bacteria harboring a pPL2-P<i>_hlylux</i> plasmid indicating P<i>_hly</i> promotor activity during growth in MDM with low BCAA concentrations. C. Luminescence measurements of <i>ΔcodY</i>, <i>ΔprfA</i> and WT <i>L. monocytogenes</i> bacteria harboring pPL2-P3<i>_plcA/prfAlux</i> plasmid indicating P3<i>_plcA/prfA</i> promoter activity during growth in MDM with low BCAAs concentrations D. Luminescence measurements of <i>ΔcodY</i>, <i>ΔprfA</i> and WT <i>L. monocytogenes</i> bacteria harboring pPL2-P1P2<i>_prfAlux</i> plasmid indicating P1P2<i>_prfA</i> promoter activity during growth in MDM with low BCAAs concentrations. Overnight precultures were grown in MDM. The data represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean.</p

Rli60 mediates <i>Lm’s</i> BCAA partial-auxotrophy.

<p><b>(A)</b> Presentation of maximal OD measurements (600 nm) of WT bacteria and indicated mutants grown in minimal defined medium containing decreasing concentrations of BCAA (800 μM, 80 μM, 20 μM and without a supplement of BCAA). Maximal OD values were extracted from growth analyses presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007283#pgen.1007283.g004" target="_blank">Fig 4B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007283#pgen.1007283.s004" target="_blank">S4 Fig</a>. The data represent 3 biological replicates (N = 3). Error bars represent standard deviation. <b>(B)</b> Growth of WT <i>Lm</i> and indicated mutants in minimal defined medium, which is completely lack of BCAA. Growth was measured by a Synergy HT BioTek plate reader at 37°C for 55 h. The data represent 3 biological replicates (N = 3). Error bars represent standard deviation.</p

Sequential regulation of the <i>ilv-leu</i> operon.

<p>A schematic model of the <i>ilv-leu</i> operon regulation. In <i>Lm</i> the BCAA biosynthesis pathway is regulated by two sequential mechanisms, the first using the classical CodY repression under rich BCAA conditions and the second using Rli60-ribosome-mediated attenuation under poor BCAA conditions. Upon a drop in BCAA levels, CodY repression is alleviated, <i>rli60</i> is transcribed, forming two alternative RNA structures that terminate or anti-terminate the transcription of the downstream genes. Transcription attenuation is dictated by the leader peptide translating ribosome as classically shown for translation-coupled ribosome-mediated attenuation. This mechanism relies on two types of regulators, a global <i>i</i>.<i>e</i>., CodY and a specific <i>i</i>.<i>e</i>., Rli60. RNAP, RNA polymerase.</p

<i>rli60</i> and <i>ilvD</i> transcription pattern under varying BCAA concentrations.

<p><b>(A)</b> Schematic representation of <i>rli60</i> and <i>ilvD (LMRG_01131</i>) genomic region. A single promoter is indicated upstream to <i>rli60</i> (-10 and -35 are marked as white boxes). Two putative CodY binding sites are indicated upstream to <i>rli60</i> and to <i>ilvD</i> (grey boxes). The transcription start site (TSS), based on a 5’-RACE analysis (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007283#pgen.1007283.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007283#pgen.1007283.s002" target="_blank">S2</a> Figs), is marked with an arrow. <b>(B)</b> Comparative qRT-PCR analysis of <i>rli60</i> and <i>ilvD</i> mRNA levels in WT bacteria grown in BHI, MM and LBMM media. <i>rli60</i> and <i>ilvD</i> mRNA levels were normalized to the levels of <i>rpoD</i> mRNA and to the transcription level in BHI. The data represent 3 biological replicates (N = 3). Error bars indicate standard deviation. represent <i>P</i>-values (* = P<0.05, n.s. = non-significant), calculated using Student’s <i>t</i>-test. <b>(C)</b> Transcription analysis of <i>rli60</i> and <i>ilvD</i> mRNA in WT bacteria grown in BHI and in bone marrow derived macrophages (BMDM) using a Nanostring analysis. The data represent 2 biological replicates (N = 2). Error bars indicate standard deviation. <i>P</i>-values (n.s. = non-significant) were calculated using Student’s <i>t</i>-test. <b>(D)</b> Northern blot analysis of <i>rli60</i> and <i>ilvD</i> mRNA transcripts using ³²P-labeled specific probes. Total RNA was extracted from WT <i>Lm</i> and <i>ΔcodY</i> bacteria grown in BHI, MM and LBMM and hybridized with Rli60 (left panel) or <i>ilvD</i> (right panel) probe. 23S and 16S rRNA were used as a loading control, and <i>Δrli60</i> RNA extracts as a negative control. All samples were tested on the same membrane, and were separated only for visualization.</p

CodY regulates the transcription of virulence and metabolic genes under limiting concentrations of BCAAs.

<p>A. Relative luminescence measurements (RLU) indicating activation of <i>hly</i> promoter under growth of WT <i>L. monocytogenes</i> (harboring pPL2-P<i>_hlylux</i> plasmid) in BHI, MDM and MDM with low concentration of isoleucine. B. Optical density measurements of the same cultures of WT <i>L. monocytogenes</i> containing pPL2-P<i>_hlylux</i> plasmid, growing in BHI, MDM and MDM with low concentration of isoleucine. The results represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean. C. Relative luminescence measurements indicating activation of <i>hly</i> promoter in <i>ΔcodY</i> mutant and WT bacteria during growth in MDM and MDM with low concentration of isoleucine. The results represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean. D. RT-qPCR analysis of <i>prfA</i> and <i>actA</i> transcription levels during growth in MDM with low levels of isoleucine in the <i>ΔcodY</i> mutant, WT bacteria and <i>ΔcodY</i> complemented strain harboring pPL2-<i>codY</i> plasmid with and without IPTG. Levels are represented as RQ, relative to BHI and normalized to <i>rpoD</i> mRNA. The data represent 3 independent experiments (N = 3). Error bars indicate a 95% confidence interval. E. RT-qPCR analysis of <i>ilvC</i> and <i>purH</i> transcription levels in <i>ΔcodY</i> mutant and WT bacteria during growth in BHI and in MDM media with reduced concentrations of BCAAs: 100 µg/ml and 10 µg/ml. Represented as RQ, relative to BHI and normalized to <i>rpoD</i> mRNA. The data represent 3 independent experiments (N = 3). Error bars indicate a 95% confidence interval. In all experiments (A–E) overnight precultures were grown in MDM. F. Intracellular growth curve of the <i>ΔcodY</i> mutant, WT bacteria and the <i>ΔcodY</i> complemented strain (with and without IPTG) in primary BMD macrophage cells. The data represent 3 independent experiments (N = 3). Error bars indicate a standard error of the mean.</p

<i>L. monocytogenes</i> metabolic mutants grow less efficiently in macrophage cells.

<p>A. Intracellular growth curves of WT <i>L. monocytogenes</i> and metabolic mutants. Left panel: Δ<i>ilvC</i>, Δ<i>argD</i>, Δ<i>purH</i>, Δ<i>aroE</i> mutants (for complemented strains, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002887#pgen.1002887.s003" target="_blank">Figure S3</a>). Right panel: the newly identified metabolic mutants Δ<i>rhaB</i> and Δ<i>hisC</i>, and complemented strains harboring a copy of <i>rhaB</i> and <i>hisC</i> genes on the pPL2 plasmid. The experiment was performed 3 times and representative growth curves are shown. Error bars indicate standard error. B. Percentage of bacteria that escaped macrophage phagosomes at 2.5 h.p.i. as determined by microscope fluorescence assays. The data represents 3 independent experiments (N = 3).</p

The genes selected for knock out encode key enzymes in the metabolic pathways predicted to be active during intracellular growth.

<p>The genes selected for knock out encode key enzymes in the metabolic pathways predicted to be active during intracellular growth.</p

Restriction of BCAA biosynthesis promotes <i>Lm</i> virulence.

<p><b>(A)</b> qRT-PCR analysis of <i>hly</i>, <i>actA</i> and <i>prfA</i> transcription levels in WT <i>Lm</i> and indicated mutants grown in LBMM. mRNA levels are represented as relative quantity (RQ) and normalized to <i>rpoD</i>. The data represent 4 biological replicates (N = 4). Error bars indicate standard deviation. Asterisks represent <i>P</i>-values (* = P<0.05, ** = P<0.01, *** = P<0.001, n.s. = non-significant), calculated using Student’s <i>t</i>-test. <b>(B)</b> <i>Δrli60</i> and <i>codY-R61A/Δrli60</i> bacteria are attenuated for virulence compared to WT <i>Lm</i> bacteria during <i>in-vivo</i> competitive infection of mice. Bacterial loads in the spleens and livers of C57BL/6 mice after 2 days of infection with a 1:1 ratio of WT <i>Lm</i> and mutant bacteria (either <i>Δrli60</i> or <i>codY-R61A/Δrli60</i>). pPL2 containing a Kanamycin (Km) or Spectinomycin (Spec) resistance cassette were used to identify each bacterial strain. The experiment was done reciprocally, switching the antibiotic cassete between the two strains. Colony forming units (CFU) of mutant bacteria in each organ are presented as relative to the CFU of WT bacteria and are significantly different from CFU (WT/Mutant) = 1, calculated using Student’s <i>t</i>-test (* = p<0.05). Mutant Km/WT Spec CFU is not significantly different from the reciprocal Mutant Spec/WT Km CFU.</p

Metabolic pathways predicted to be differentially active during <i>L. monocytogenes</i> intracellular growth by standard bioinformatics analysis and iMAT analysis.

a<p>- Based on hypergeometric enrichment test.</p><p>NP-Not predicted.</p

Nutrient availability influences transcription of <i>L. monocytogenes</i> metabolic and virulence genes.

<p>A. Transcription levels of key metabolic genes during growth of WT <i>L. monocytogenes</i> in BHI medium, minimal defined medium (MDM) and MDM with low concentrations (10-fold less) of BCAAs, histidine (His), arginine (Arg) and adenine (Ade). Transcriptional levels are presented as relative quantity (RQ), relative to BHI medium. B. Transcription levels of <i>prfA</i>, <i>hly</i>, and <i>actA</i> virulence genes during growth of WT <i>L. monocytogenes</i> in BHI medium, MDM, MDM with low concentrations of BCAAs, His, Arg and Ade and in MDM with low concentrations of phenylalanine (Phe) and tryptophan (Trp) (10 µg ml⁻¹). Transcriptional levels are presented as RQ, relative to BHI medium. C. Transcription levels of <i>prfA</i>, <i>hly</i>, and <i>actA</i> virulence genes during growth of WT <i>L. monocytogenes</i> in MDM and MDM with low concentrations of the designated nutrients. Transcriptional levels are presented as RQ, relative to MDM. D. Transcription levels of <i>ilvC</i>, <i>hly</i>, and <i>actA</i> genes during growth of WT <i>L. monocytogenes</i> in MDM and MDM with low concentrations of the designated nutrients. Transcriptional levels are presented as RQ, relative to MDM. Overnight precultures were grown in MDM media and diluted for growth under the indicated conditions. Bacteria were harvested at O.D._{600 nm} of 0.3, representing exponential growth. mRNA levels were normalized to <i>rpoD</i> mRNA. The results represent 3 independent experiments (N = 3). Error bars indicate a 95% confidence interval.</p