doi: 10.1371/journal.pone.0041248. Epub 2012 Jul 18.
A pseudo-tRNA modulates antibiotic resistance in Bacillus cereus
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- PMID:22815980
- PMCID: PMC3399842
- DOI: 10.1371/journal.pone.0041248
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A pseudo-tRNA modulates antibiotic resistance in Bacillus cereus
Theresa E Rogers et al. PLoS One.2012.
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Abstract
Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.
Conflict of interest statement
Figures
A, effect of tRNAOther on vancomycin resistance. Wt (black) and ΔtRNAOther (white)B. cereus 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, effect of tRNAOther on hydrogen peroxide resistance. Wt (black) and ΔtRNAOther (white)B. cereus strains were grown in LB (□) or LB +400 μM H2O2 (○) at 37°C with shaking at 250 rpm.C, effect of tRNAOther on nitric oxide production. Comparison of NO levels in wt (black bars) and ΔtRNAOther (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.
A, TEM of wt and ΔtRNAOther B. cereus cell walls. Average cell wall thickness: wt, 60.4±4.6 nm; ΔtRNAOther, 45.0±3.9 nm.B, Biofilm formation. Wt (black bars) and ΔtRNAOther (white bars) strains were grown under high and low (+DIP) iron conditions. Data was normalized to wtB. cereus grown in LB.
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 ΔtRNAOther relative to wtB. cereus. White dots represent transcripts with a significant 2-fold change or greater.
Expression of genes inB. cereus ΔtRNAOther reletive to wt grown in rich media (light green) and iron depleted media (dark green) was determined by qRT-PCR. Data presented in log2.
A, Putative promoter and Fur binding sites (DBTBS [28]). Thioesterase indicates the ORF immediately 5′ of tRNAOther.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 oftRNAOther.
Low iron concentration was achieved by addition of the iron chelator DIP (250 μM). High iron concentration was achieved by the addition of FeSO4 (250 μM). Green and red asterisks indicate presence and absence of a PCR signal, respectively. DNA marker is HyperLadder V (Bioline). PCR of 2-fold dilutions of tRNAOther cDNA from cells grown for 30 (A) and 120 min (B), in LB with low (Lanes to the left of the DNA marker) or high (Lanes to the right of the DNA marker) iron concentrations. Arrows indicate RT-PCR product for tRNAOther at 74 bp.
Target prediction parameters fornos (A andB) andsod (C andD) included terminator removal, a hybridization seed of 9, G:U basepairs included, and alignment score determined with a P-value set to 0.05 (A andC) or thermodynamic energy (B andD). Vertical lines (|) indicate a Watson-Crick base pair, and dots (:) indicate a G-U wobble base pair. The numbered nucleotide positions are relative to the start and stop codons forsod andnos mRNAs, respectively. Target prediction parameters forcymR included terminator removal, a hybridization seed of 8, and G:U basepairs included. Alignment was focused on the start codon (E), the stop codon (F), or the coding sequence (G andH). Alignment score was determined with a P-value set to 0.01 (C) or thermodynamic energy (E,F andH). Vertical lines (|) indicate a Watson-Crick base pair, and dots (:) indicate a G-U wobble base pair. The numbered nucleotide positions are relative to the start and stop codons forcymR mRNA. Ribosome-binding sites and start codons are underlined (note thatsod andcymR homologue sequences are written 3′ to 5′. Targets were predicted using TargetRNA and sRNATarget.
Low levels of ferrous iron (Fe2+) limit DNA binding by Fur, which de-represses expression of tRNAOther. tRNAOther induces expression ofpbp1a,trpS1,nos,cymR,spx, andsod by undetermined mechanisms. An increase inpbp1a 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.
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