Establishment of persistent infection requires <i>arcA, fnr, frdA</i>, and <i>wrbA</i>.

<p>(A) Infection profile 42 dpi for FVB/N mice infected orally with10⁷ CFUs of wt <i>Y. pseudotuberculosis</i> (n = 20) and indicated mutant strains (each group n = 16). The infections were monitored by IVIS at certain intervals up to 42 dpi. (B) Heatmap showing differences in clearance (by <i>p</i>-value) between wt and indicated mutant strains at different time points during the 42 day infection period. Heatmap color scale, from green to yellow, was adjusted according to <i>p</i>-values from 1 to 0. <i>p</i>-values were calculated with 2×2 contingency table by Fisher’s Exact Test, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004600#ppat.1004600.s013" target="_blank">S6 Table</a>. (C) Motility profile of wt <i>Y. pseudotuberculosis</i> and indicated mutant strains under anaerobic conditions at 26°C. Images were captured by the ChemiDoc XRS System (Bio-Rad), showing the bioluminescent signal produced by <i>Y. pseudotuberculosis</i> YPIII/pIBX.</p

Hypothetical model of <i>Y. pseudotuberculosis</i> reprogramming for persistent infection in cecum.

<p>Upon initial infection, <i>Y. pseudotuberculosis</i> is still flagellated and expresses T3SS virulence genes. At the early stage of infection (2 dpi) the T3SS is important for colonization of tissue, including breaking the epithelial barrier and resisting the attack from arriving PMNs. At the persistent stage of infection (42 dpi), <i>Y. pseudotuberculosis</i> had reprogrammed its transcriptome by reducing the expression of T3SS components and increasing the expression of genes important for survival in the cecal lymphoid compartment. At this stage the bacteria are flagellated and can spread to other hosts by shedding into the feces, possibly through motility.</p

Summary of RNA-seq reads.

<p>Asterisks indicate samples without rRNA depletion.</p><p>Summary of RNA-seq reads.</p

<i>Y. pseudotuberculosis</i> infection alters the bacterial composition of the cecum.

<p>(A) Representative Bioanalyzer 2100 electrographs and associated gel pictures for replicates of <i>in vitro</i>-derived RNA samples (grown at 26°C and 37°C), <i>in vivo</i>-derived samples of early (isolated from mouse cecal tissue 2 dpi) and persistent infection (isolated from mouse cecal tissue 42 dpi), and uninfected samples (isolated from uninfected mouse cecal tissue). (B) The number of reads mapping to 16S rRNA from different bacteria in non-depleted <i>in vivo</i>-derived samples. Data represent the mean ± SD of the two replicates for each sample group. (C) Relative abundance of different bacterial phyla in samples according to reads mapped to the 16SMicrobial database. The proportions are given as the percent of bacterial phyla identified in specific samples.</p

<i>Y. pseudotuberculosis</i> undergoes transcriptional reprogramming for adaption to persistence.

<p>(A) Comparison of genes up-regulated in <i>Y. pseudotuberculosis in vitro</i> at 26°C and 37°C compared to <i>in vivo</i> during early (2 dpi) and persistent (42 dpi) stages of infection. Similarities are shown with the number of genes up-regulated in both groups. (B) Functional annotation of <i>Y. pseudotuberculosis</i> genes up-regulated during early and persistent infection (KEGG pathway mapping tool). (C) Comparison of the <i>in vivo</i> gene expression profiles and the expression profiles of bacteria grown under anaerobic conditions <i>in vitro</i>. The analysis included genes up-regulated (>1.8-fold) during anaerobic or aerobic growth in both the exponential and stationary growth phase compared to genes up-regulated during early and persistent infection. Similarities are shown with the number of genes up-regulated in both groups.</p

Up-regulated genes indicative of different environmental cues.

<p>Up-regulated genes indicative of different environmental cues.</p

Temperature-dependent reprogramming of the primary metabolism of <i>Y. pseudotuberculosis</i>.

<p>Glycolysis, acetate metabolism, β-oxidation of fatty acids, the TCA cycle and transport systems are illustrated and the genes, designated according to the annotated YPIII genome, which encode equivalent enzymes of the presented pathways are given. Genes which are differentially expressed in response to temperature are colored. The blue color shows genes induced at 25°C (≥4, dark blue, ≥2 light blue), and the red color represents genes upregulated at 37°C (≥4, dark red, ≥2 light red). The values of the expression changes are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005087#pgen.1005087.s014" target="_blank">S4 Dataset</a>.</p

CRP-dependent non-coding RNAs of <i>Y. pseudotuberculosis</i>.

<p>(A) Northern blot analyses of selected CRP-regulated non-coding RNAs. RNA samples were prepared from YPIII and YP89 (YPIII Δ<i>crp</i>) bacteria grown to stationary growth phase (stat) at 25°C and 37°C. 5S rRNA served as loading control. The size marker (nt) is indicated. (B) Interaction of CRP with the regulatory regions of selected CRP-regulated sRNA genes. Individual DNA fragments with the predicted CRP-binding site(s) (yellow boxes; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005087#pgen.1005087.s013" target="_blank">S3 Dataset</a>) used for electrophoretic mobility shift assays are illustrated. An individual sRNA promoter fragment (<i>ysr204</i>) for which no CRP-binding site was predicted was included as negative control. Respective DNA fragments were incubated with increasing concentrations of CRP and 0.2 mM cAMP. As a negative control, cAMP was omitted in samples with the highest CRP concentration (right lane). The CRP-DNA complexes were separated on 4% polyacrylamide gels. The position of specific higher molecular weight complexes is marked with an asterisk. A molecular weight standard (M) was loaded, and the corresponding molecular weights are indicated. A <i>csiD</i> PCR fragment amplified from <i>E</i>. <i>coli</i> served as an internal negative control.</p

Global identification of mRNA transcriptional start sites (TSSs).

<p>(A) Visualization of RNA-seq (+/- TAP) based cDNA sequencing reads mapped to the YPIII <i>rovA</i>, <i>hfq</i>, <i>katY</i> and <i>crp</i> gene locus using the Artemis genome browser (Release 15.0.0). (B) The 5’-UTR repertoire. The distribution and frequency of the length of 5’-UTRs is given for all mRNAs of YPIII which start upstream of the annotated translational start site. More than 40% of all 5’-UTRs are 20–60 nt in length. (Inset) Sequence conservation at the TSSs. Sequence logo computed from 1151 unaligned TSS regions (TSS is located at position +1) showing nucleotide conservation around the TSSs. The initial nucleotide of transcripts (position +1) is dominated by purines while position -1 is dominated by pyrimidines. (C) Detected conserved sequence motifs in the -35 and -10 promotor region.</p

Temperature- and growth phase-responsive protein-encoding genes and <i>trans</i>-encoded RNAs.

<p>(A) Differential expression of <i>trans</i>-encoded sRNAs of YPIII shown by Northern blot analyses. RNA samples were prepared from bacteria grown to exponential (exp) or stationary phase (stat) at 25°C and 37°C. sRNAs were detected by specific radioactive labeled probes. 5S rRNA served as loading control. The size marker (nt) is indicated. Venn diagrams illustrating temperature and growth phase-regulated (B) protein-encoding genes and (C) <i>trans</i>-encoded sRNAs in <i>Y</i>. <i>pseudotuberculosis</i> YPIII. The regulons were obtained by comparative RNA-seq using DESeq from triplicate experiments (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005087#pgen.1005087.s013" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005087#pgen.1005087.s014" target="_blank">S4</a> Datasets). Protein- and <i>trans</i>-encoded RNAs which are differentially regulated by at least 4-fold (p-value ≤0.05) are included.</p