Changes in gene expression due to the deletion of tRNA^Other.

<p>Expression of genes in <i>B. cereus</i> Δ<i>tRNA^Other</i> reletive to wt grown in rich media (light green) and iron depleted media (dark green) was determined by qRT-PCR. Data presented in log₂.</p

Predicted interactions between tRNA^Other-containing transcripts and putative mRNA targets.

<p>Target prediction parameters for <i>nos</i> (<b>A</b> and <b>B</b>) and <i>sod</i> (<b>C</b> and <b>D</b>) included terminator removal, a hybridization seed of 9, G:U basepairs included, and alignment score determined with a P-value set to 0.05 (<b>A</b> and <b>C</b>) or thermodynamic energy (<b>B</b> and <b>D</b>). Vertical lines (|) indicate a Watson-Crick base pair, and dots (<b>:</b>) indicate a G-U wobble base pair. The numbered nucleotide positions are relative to the start and stop codons for <i>sod</i> and <i>nos</i> mRNAs, respectively. Target prediction parameters for <i>cymR</i> included terminator removal, a hybridization seed of 8, and G:U basepairs included. Alignment was focused on the start codon (<b>E</b>), the stop codon (<b>F</b>), or the coding sequence (<b>G</b> and <b>H</b>). Alignment score was determined with a P-value set to 0.01 (<b>C</b>) or thermodynamic energy (<b>E</b>, <b>F</b> and <b>H</b>). Vertical lines (|) indicate a Watson-Crick base pair, and dots (<b>:</b>) indicate a G-U wobble base pair. The numbered nucleotide positions are relative to the start and stop codons for <i>cymR</i> mRNA. Ribosome-binding sites and start codons are underlined (note that <i>sod</i> and <i>cymR</i> homologue sequences are written 3′ to 5′. Targets were predicted using TargetRNA and sRNATarget.</p

Proposed role for tRNA^Other in <i>B. cereus</i> antibiotic resistance.

<p>Low levels of ferrous iron (Fe²⁺) limit DNA binding by Fur, which de-represses expression of tRNA^Other. tRNA^Other induces expression of <i>pbp1a</i>, <i>trpS1</i>, <i>nos</i>, <i>cymR</i>, <i>spx</i>, and <i>sod</i> by undetermined mechanisms. An increase in <i>pbp1a</i> expression leads to an increase in cell wall thickness, which can reduce susceptibility to certain antibiotics. TrpRS1 interacts with and induces activity of NOS, which, together with SOD, reduces endogenous oxidative stress. CymR and Spx are both involved in regulation of gene expression in the response to, and in order to combat, oxidative stress.</p

Deletion of tRNA^Other alters resistance to vancomycin and hydrogen peroxide in <i>B. cereus</i>.

<p><b>A</b>, effect of tRNA^Other on vancomycin resistance. Wt (black) and Δ<i>tRNA^Other</i> (white) <i>B. cereus</i> strains were grown in LB + DIP (□), LB + vancomycin (⋄), or LB + vancomycin + DIP (○) at 37°C with shaking at 250 rpm. Averages of three growth curves are shown, and error bars represent standard deviations. <b>B</b>, effect of tRNA^Other on hydrogen peroxide resistance. Wt (black) and Δ<i>tRNA^Other</i> (white) <i>B. cereus</i> strains were grown in LB (□) or LB +400 μM H₂O₂ (○) at 37°C with shaking at 250 rpm. <b>C</b>, effect of tRNA^Other on nitric oxide production. Comparison of NO levels in wt (black bars) and Δ<i>tRNA^Other</i> (white bars) cells in the presence of vancomycin or DIP. Average fluorescence is presented in arbitrary units (a.u.). When used, vancomycin was added at a final concentration of 2 μg/ml.</p

Changes in transcript levels between wt and <i>B. cereus</i> Δ<i>tRNA^Other</i> during growth under moderate and low iron conditions.

*<p>Δ, <i>B. cereus</i> Δ<i>tRNA^Other</i>; wt, wild-type <i>B. cereus</i> ATCC14579.</p>‡<p>Negative control transcript levels were determined with a standard curve.</p

Mapping of tRNA^Other.

<p><b>A</b>, Putative promoter and Fur binding sites (DBTBS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041248#pone.0041248-Sierro1" target="_blank">[28]</a>). Thioesterase indicates the ORF immediately 5′ of tRNA^Other. <b>B</b>, 5′ and 3′ mapping by RACE. Circle with a vertical line at the bottom indicates an intrinsic terminator. Numbers indicate 5′ and 3′ nt relative to the originally predicted 5′-and 3′- ends of <i>tRNA^Other</i>.</p

Transcript profiles of wt and <i>B. cereus</i> Δ<i>tRNA^Other</i>.

<p>Transcript level data is presented as the average of two microarrays. Each dot represents the transcript level for one gene. The three green lines, from top left to bottom right, indicate 2-fold higher, equal, and 2-fold lower transcript levels for Δ<i>tRNA^Other</i> relative to wt <i>B. cereus</i>. White dots represent transcripts with a significant 2-fold change or greater.</p

Morphology of wt and Δ<i>tRNA^Other B. cereus.</i>

<p><b>A</b>, TEM of wt and Δ<i>tRNA^Other B. cereus</i> cell walls. Average cell wall thickness: wt, 60.4±4.6 nm; Δ<i>tRNA^Other</i>, 45.0±3.9 nm. <b>B</b>, Biofilm formation. Wt (black bars) and Δ<i>tRNA^Other</i> (white bars) strains were grown under high and low (+DIP) iron conditions. Data was normalized to wt <i>B. cereus</i> grown in LB.</p

Phenotypes¹ of <i>B. cereus</i> Δ<i>tRNA^Other</i> relative to wt determined by BIOLOG phenotypic array analyses.

1<p>Only a subset of phenotype changes is shown corresponding to intensity losses above an arbitrary threshold of 100 units or greater. Intensity corresponds to the area under the curve divided by number of reads. The array was conducted in duplicate after incubation of the strains at 37°C for 24 h.</p