Effect of <i>yidC</i> antisense expression on corresponding gene transcript.

<p>Relative Real-time quantitative PCR (qPCR) was carried out to compare <i>yidC</i> mRNA expression in uninduced and induced pHN678-yidC harbouring <i>E. coli</i> DH5α cells. The CT values were averaged from triplicate samples and the expression ratio was calculated. Comparison of expression ratio of <i>yidC</i> gene upon its antisense induction in <i>E. coli</i> with different IPTG concentrations showed that <i>yidC</i> mRNA expression decreased with increasing IPTG concentration. However, the expression is almost same in cells treated with 80 and 160 µM IPTG.</p

Fold sensitization of <i>yidC</i> antisense expressing <i>E. coli</i> DH5α cells to various antibacterial agents.

<p>Various antibiotics belonging to six different classes based on their mode of action namely cell wall synthesis inhibition (vancomycin and phosphomycin), protein synthesis inhibition (kanamycin and hygromycin), nucleic acid synthesis inhibition (mitomycin and novobiocin), cell membrane disruption (polymyxin B and colistin), metabolic antagonism (trimethoprim) and fatty acid synthesis inhibition (triclosan) as well as some plant natural products such as membrane bound ATPase activity inhibiting essential oils eugenol and carvacrol, cell division protein FtsZ inhibiting alkaloid berberine as well as some other plant natural products named in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057370#s2" target="_blank">materials and methods</a> (data not shown) were screened for sensitization of YidC depleted strain of <i>E. coli</i> DH5α as compared to control cells (<i>E. coli</i> DH5α harbouring only vector or expressing antisense of <i>murA</i> essential gene). Fold sensitization represents the change in antibiotic IC50 values upon antisense induction. <i>E. coli</i> DH5α cells expressing <i>yidC</i> antisense showed considerable sensitization to eugenol (16 fold) and carvacrol (8 fold) as compared to control cells. However other natural products and antibiotics with known mode of action did not result in fold sensitization equal to or greater than that of <i>murA</i> antisense expressing E. coli cells to phosphomycin (positive control).</p

Primers used in this study.

<p>Primers used in this study.</p

Plasmids used in the study.

*<p>antisense target locations are specified relative to the start codon.</p

Effect of antisense RNA mediated <i>yidC</i> silencing on <i>E. coli</i> susceptibility to eugenol and carvacrol.

<p>Growth curves for <i>E. coli</i> DH5α carrying the control (pHN678 and pHN678-murA) and pHN678-yidC plasmids. (A) In absence of any antibacterial agent. (B) In presence of sub lethal concentration of eugenol. (C) In presence of sub lethal concentration of carvacrol. The growth curve experiment was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057370#s2" target="_blank">materials and methods</a>. Antisense expression was induced by 70 µM IPTG. Data are the means ± standard deviations of the experiment performed in triplicate.</p

Effect of IPTG concentration on growth of <i>E. coli</i> DH5α harbouring <i>yidC</i> antisense expressing vector (pHN678-yidC).

<p>Graph on left represents growth of <i>E. coli</i> DH5α harbouring <i>yidC</i> antisense expressing vector (pHN678-yidC) while graph on right represents growth of <i>E. coli</i> DH5α harbouring <i>murA</i> antisense expressing vector (pHN678-murA) used as positive control. Antisense of the essential genes was induced by IPTG at different concentrations (40, 80, 160 and 320 µM). Addition of IPTG had a clear inhibitory effect on the growth of <i>E. coli</i> DH5α cells harbouring antisense inserts which increased with increasing IPTG concentration.</p

Selected target genes for expression analysis by qRT-PCR.

<p>Selected target genes for expression analysis by qRT-PCR.</p

List of putative 31 sRNAs and their features.

<p>Up ORF: Upstream Open Reading frame; dist Up ORF: distance of sRNA from Upstream Open Reading frame; UpORF dir: Upstream Open Reading frame direction; dir sRNA: direction of small RNA expression; len sRNA: length of sRNA; dist Dn ORF: distance of sRNA from downstream open reading frame.</p

Effect of AbsR25 sRNA overexpression.

<p>Comparison of expression of predicted putative target genes in <i>A. baumannii</i> cells harbouring recombinant plasmid over-expressing AbsR25 sRNA. Expression of target genes was studied under arabinose induced and uninduced conditions. The experiments were performed in triplicates.</p

Real time expression of selected target genes.

<p>Relative expression of <i>in silico</i> predicted putative target genes [A1S_3401 (hypothetical protein), A1S_3251 (amino acid transporter LysE), A1S_1791 (putative tartrate symporter MFS superfamily protein), A1S_2269 (RtcR - Regulator of RNA terminal phosphate cyclase), A1S_1505 (hypothetical protein), A1S_0229 (hypothetical protein), A1S_3342 (putative arsenate reductase), A1S_1627 (hypothetical protein), A1S_2584 (major facilitator superfamily family drug transporter), A1S_2660 (RND efflux transporter), A1S_1331 (major facilitator superfamily transporter)] of AbsR25 sRNA under different concentrations of EtBr.</p