Vibrational Alchemy of DNA: Exploring the Mysteries of Hybridization under Cooperative Strong Coupling with Water

Vibrational strong coupling (VSC) as an emerging technology can modify molecular reactivities. Previously, we found that VSC can drive deoxyribonucleic acid (DNA) coassembly in the dark; however, the underlying mechanism still lacks sufficient exploration. Here, we take the melting temperature (Tm) as a measure to systematically study how VSC affects DNA hybridization in the optical resonator. The results indicate a strong correlation between Tm and coupling strength; i.e., VSC with O–H stretching vibration can significantly reduce Tm and favor nonspecific hybridization of DNA molecules. The mechanism is ascribed to the cooperative VSC between water and base pairs, since the number of hydrogen bonds is much higher in solvent water than in DNA molecules. More interestingly, concentrations of Mg2+ also play key roles in changing Tm due to the alteration of the hydrogen-bond network. The hybridization landscape of DNA molecules is significantly reduced due to the increased active energy contributed by Rabi splitting, which is proven by fluorescence quenching-based Tm measurement. Gel electrophoresis further demonstrates that VSC can induce DNA to form unstable nanoassemblies. This work sheds light on how the reactant-solvent cooperative VSC regulates bioreactions, which is expected to find wide applications in the biomedicine field

Cell Density- and Quorum Sensing-Dependent Expression of Type VI Secretion System 2 in <i>Vibrio parahaemolyticus</i>

<div><p>Background</p><p><i>Vibrio parahaemolyticus</i> AphA and OpaR are the two master quorum sensing (QS) regulators that are abundantly expressed at low cell density (LCD) and high cell density (HCD), respectively, with a feature of reciprocally gradient production of them with transition between LCD and HCD. The type VI secretion system 2 (T6SS2) gene cluster can be assigned into three putative operons, namely VPA1027-1024, VPA1043-1028, and VPA1044-1046. T6SS2 contributes to adhesion of <i>V. parahaemolyticus</i> to host cells.</p> <p>Methodology/Principal Findings</p><p>OpaR box-like sequences were found within the upstream promoter regions of all the above three operons, while none of AphA box-like elements could be identified for them. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that OpaR bound to the promoter regions of these three operons to stimulate their transcription, while AphA negatively regulated their transcription most likely through acting on OpaR. This regulation led to a gradient increase of T6SS2 transcription with transition from LCD to HCD.</p> <p>Conclusions/Significance</p><p><i>V. parahaemolyticus</i> OpaR and AphA positively and negatively regulate T6SS2 expression, respectively, leading to a gradient elevation of T6SS2 expression with transition from LCD to HCD. T6SS2 genes are thus assigned as the QS regulon members in <i>V. parahaemolyticus</i>.</p> </div

Regulation of T6SS2 genes by OpaR.

<p>The primer extension (a), LacZ fusion (b), EMSA (c), DNase I footprinting (d) assays were performed to characterize the regulation of VPA1027-1024, VPA1043-1028, and VPA1044-1046 operons by OpaR. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#pone-0073363-g002" target="_blank">Figure 2</a> for detail annotations.</p

Regulation of T6SS2 genes by AphA.

<p>Lanes C, T, A, and G represent the Sanger sequencing reactions. The minus and positive numbers indicated the nucleotide positions upstream and downstream of indicated genes. <b>a</b>) <b>Primer extension</b>. An oligonucleotide primer was designed to be complementary to the RNA transcript of each gene tested. The primer extension products were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcriptional start sites were indicated by arrows with nucleotides and positions. <b>b</b>) <b>LacZ fusion</b>. The target promoter-proximal DNA region was cloned into the <i>lacZ</i> transcriptional fusion vector pHRP309 and then transformed into WT or <i>ΔAphA</i> to determine the promoter activity, i.e., the β-galactosidase activity (miller units) in the cellular extracts. <b>c</b>) <b>EMSA</b>. The radioactively labeled promoter-proximal DNA fragments were incubated with increasing amounts of purified His-AphA protein and then subjected to 4% (w/v) polyacrylamide gel electrophoresis. If there was the association of His-AphA and target DNA, the band of free DNA disappeared with increasing amounts of His-AphA, resulting in a retarded DNA band with decreased mobility, which presumably represented the DNA-AphA complex. Shown also was the schematic representation of the EMSA design. <b>d</b>) <b>DNase I footprinting</b>. Labeled coding or non-coding DNA probes were incubated with increasing amounts of purified His-AphA (Lanes 1, 2, 3, and 4 containing 0, 35.1, 46.8, and 58.5 pmol, respectively) and then subjected to DNase I footprinting assay. The footprint regions were indicated by vertical bars with positions.</p

Organization of promoter-proximal DNA regions.

<p>The promoter-proximal DNA regions of indicated genes were derived from RIMD 221063.</p

Action of <i>V. parahaemolyticus</i> QS systems.

<p>The regulatory associations between LuxO, Qrr sRNAs, AphA, and OpaR were summarized with the integration of relevant observations in <i>V. parahaemolyticus</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B4" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B5" target="_blank">5</a>] and closely related <i>V. harveyi</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B3" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B32" target="_blank">32</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B40" target="_blank">40</a>]. The grey fonts denoted the inhibited production of relevant proteins or the cease of relevant regulatory cascades. At LCD, low concentrations of autoinducers lead to phosphorylation of LuxO (LuxO-P), and LuxO-P activates expression of Qrr sRNA genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B32" target="_blank">32</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B33" target="_blank">33</a>]. Redundant Qrr sRNAs promote AphA translation and, meanwhile, inhibit OpaR translation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B34" target="_blank">34</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B36" target="_blank">36</a>]. AphA further represses <i>opaR</i> transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B3" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B5" target="_blank">5</a>]. Overproduced AphA feeds back to inhibit transcription of <i>qrr2-3</i> and its own gene [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B3" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B5" target="_blank">5</a>]. In addition, over-production of Qrr sRNAs and LuxO-P triggers three additional feedback regulatory loops: i) LuxO-P represses transcription of its own gene, ii) Qrr sRNAs inhibits <i>luxO</i> translation, and iii) Qrr sRNAs repress translation of <i>luxMN</i> encoding the membrane-anchoring autoinducer-binding receptor protein LuxM and its cognate receptor LuxN [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B37" target="_blank">37</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B39" target="_blank">39</a>]. The above feedbacks will contribute to control the LuxO-P, Qrr and AphA levels within the physiological states. At HCD, high concentrations of autoinducers reverse the phosphate flow in the circuit, leading to dephosphorylation of LuxO. Dephosphorylated LuxO is inactive as a regulator, leading to cessation of Qrr sRNA production; thus, there is no production of AphA but OpaR translation occurs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B34" target="_blank">34</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B35" target="_blank">35</a>]. OpaR in turns represses <i>aphA</i> transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B4" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B40" target="_blank">40</a>] but stimulates T6SS2 transcription (this study), and it also feeds back to inhibit its own expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B4" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B40" target="_blank">40</a>]. OpaR is also able to activate the <i>qrr2-4</i> transcription, leading to rapid down-regulation of <i>opaR</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B4" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B40" target="_blank">40</a>]; this OpaR-<i>qrr</i> feedback dramatically accelerates transition from HCD to LCD, but it has no effect on QS behaviors at steady-state LCD or HCD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073363#B41" target="_blank">41</a>]. Taken together, there is the reciprocal gradients of cellular AphA and OpaR levels during transition between LCD and HCD, and AphA and OpaR act as the master QS regulators at LCD and HCD, respectively. In addition, T6SS2 transcription enhances in a gradient manner with transition from LCD to HCD, which is coordinately controlled by AphA and OpaR.</p