Complementation of induced <i>yhcS</i> antisense RNA by P<i>spac</i>-driven <i>lacABC.</i>

<p>Growth curves of control strain, YJ107 (A), <i>lacABC</i> complemented strain, YJ307 (B), in TSB containing 5 µg/ml of erythromycin and various concentrations of ATc (in nanograms/milliliter). (C) Represents the growth curves of the above strains in the absence of ATc. The overnight cultures of <i>S. aureus</i> strains were diluted to ∼10⁴ CFU/ml with TSB containing appropriate antibiotics and different concentrations of an inducer [anhydrotetracycline, (ATc), at concentrations of 0, 50, 250, 500, or 750 ng/ml]. Cell growth was monitored at 37°C by measuring the optical density at 600 nm (OD₆₀₀) every 15 min, with 1 min of mixing before each reading in a SpectraMax plus Spectrophotometer. The growth curves represent one of three repeated experiments.</p

The Essential <em>yhcSR</em> Two-Component Signal Transduction System Directly Regulates the <em>lac</em> and <em>opuCABCD</em> Operons of <em>Staphylococcus aureus</em>

<div><p>Our previous studies suggested that the essential two-component signal transduction system, YhcSR, regulates the <em>opuCABCD</em> operon at the transcriptional level, and the <em>Pspac</em>-driven <em>opuCABCD</em> partially complements the lethal effects of <em>yhcS</em> antisense RNA expression in <em>Staphylococcus aureus</em>. However, the reason why <em>yhcSR</em> regulon is required for growth is still unclear. In this report, we present that the <em>lac</em> and <em>opuC</em> operons are directly transcriptionally regulated by YhcSR. Using real-time RT-PCR we showed that the down-regulation of <em>yhcSR</em> expression affected the transcription of <em>lacA</em> encoding galactose-6-phosphotase isomerase subunit LacA, and <em>opuCA</em> encoding a subunit of a glycine betaine/carnitine/choline ABC transporter. Promoter-<em>lux</em> reporter fusion studies further confirmed the transcriptional regulation of <em>lac</em> by YhcSR. Gel shift assays revealed that YhcR binds to the promoter regions of the <em>lac</em> and <em>opuC</em> operons. Moreover, the <em>Pspac</em>-driven <em>lacABC</em> expression <em>in trans</em> was able to partially complement the lethal effect of induced <em>yhcS</em> antisense RNA. Likewise, the <em>Pspac</em>-driven <em>opuCABCD</em> expression <em>in trans</em> complemented the growth defect of <em>S. aureus</em> in a high osmotic strength medium during the depletion of YhcSR. Taken together, the above data indicate that the <em>yhcSR</em> system directly regulates the expression of <em>lac</em> and <em>opuC</em> operons, which, in turn, may be partially associated with the essentiality of <em>yhcSR</em> in <em>S. aureus</em>. These results provide a new insight into the biological functions of the <em>yhcSR</em>, a global regulator.</p> </div

Gel shift mobility analysis of genes regulated by YhcR.

<p>Promoter regions for each gene were obtained as described. The mobility of labeled promoter fragments without addition of YhcR is shown in lane 1 (0). Different amounts of YhcR (0.5, 1, 2, 3 µg) were incubated with each DIG-labeled promoter probes: <i>P_lac</i> (A) and <i>P_opuC</i> (B) in 20 µl reaction volume. –represents without unlabeled specific competitor;+represents in the presence of 100-fold extra unlabeled specific competitor. Both BSA (bovine serum albumin) and SaeR (an unrelated response regulator of <i>S. aureus</i>) were used as nonspecific binding controls. Approximate 0.2 pmol of DIG-labeled promoter DNA fragment was used in each reaction.</p

Primers used in this study.

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

Effect of the complementation of <i>opuC</i> operon on bacterial growth in high osmotic medium conditions during the depletion of YhcSR.

<p>Growth curves of control strain, YJ2002 (A), <i>yhcSR</i> antisense RNA strain, YJ107 (B), and <i>opuCABCD</i> complemented strain, YJ207 (C) in CDM containing 5 µg/ml erythromycin and where indicated 750 ng/µl ATc, 1 M NaCl, and 1 µM choline chloride were added. The overnight cultures of <i>S. aureus</i> strains were diluted with CDM containing appropriate antibiotics and additives as indicated. Cell growth was monitors at 37°C by measuring the optical density at 600 nm (OD₆₀₀) every 15 min, with 1 min of mixing before each reading in a BioTek Synergy II microplate reader. The growth curves represent one of three repeated experiments.</p

Real-time RT-PCR (qPCR) and microarray analysis of gene expression in mid-log phase of growth, using the <i>yhcS</i> antisense RNA strain.

<p>qPCR* (control): The number inside parentheses is the fold change of specific genes in control strain YJ2002 with versus without ATc. –represents down-regulated gene expression during inactivating <i>yhcSR</i> system; +represents up-regulated gene expression during inactivating <i>yhcSR</i> system.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Analysis of transcriptional regulation of <i>lac</i> operon by the YhcSR system using promoter-<i>lux</i> reporter fusion.

<p>The impact of the down-regulation of <i>yhcSR</i> on the expression of <i>lac</i> operon was determined by monitoring the bioluminescence intensity during growth. The overnight cultures of <i>S. aureus</i> strains were diluted to ∼10⁴ CFU/ml with TSB containing appropriate antibiotics and in the absence (solid dots ) or presence of 200 ng/ml of inducer, ATc (solid dashes). Both bioluminescence signals and cell growth were monitored at 37°C by measuring the light intensity with a Chiron luminometer and optical density at 600 nm (OD₆₀₀) with a SpectraMax plus Spectrophotometer every 30 min. To eliminate the effect of bacterial growth, the relative light units (RLU) were calculated (light intensity/OD₆₀₀) from triplicate readings at different times during growth.</p

Effect of the deletion of the <i>ilv</i>-<i>leu</i> operon on the essentiality of Gcp for growth.

<p>The growth curves of wild-type control, WCUH29 carrying pYH4-lacI, in nutrient complete CDM (A) and in ILV dropout CDM (B). The growth curves of the <i>ilv</i>-<i>leu</i> operon deletion mutant (JW290211) in nutrient complete CDM (C) and in ILV dropout CDM (D); the growth curves of the <i>ilv</i>-<i>leu</i> operon deletion and defined <i>Pspac</i>-regulated <i>gcp</i> expression strain (JW290311) in nutrient complete CDM (E) and in ILV dropout CDM (F). The growth curves are monitored by kinetically measuring the optical density at OD600 nm in the corresponding culture medium in the presence of different concentrations of inducer, IPTG (0. 10, 25 and 100 µM), in every 15 min at 37°C using a BioTek Synergy plate reader. These figures are one representative of three independent experiments.</p

Determine the effect of depletion of Gcp on t6A content of tRNA isolated from staphylococcal cells using LC-MS/MS.

<p>tRNA isolated and purified from the defined <i>Pspac</i>-regulated <i>gcp</i> expression mutant with (Gcp+) and without inducer IPTG (200 μM) (Gcp-) at exponential phase of growth (OD600nm ∼0.5) and processed for LC-MS/MS analysis of t6A as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046836#s4" target="_blank">Materials and Methods</a>. The t6A content was normalized to the respective adenosine content and represented to % t6A/A ratio (the numerical values).</p