DNA Translocation through Low-Noise Glass Nanopores

The effect of electron irradiation-induced shrinking on glass nanocapillaries with diameters ranging from 75 to 14 nm was analyzed by measuring the conductance characteristics with and without DNA translocation. We have investigated nanocapillary shrinking with a scanning electron microscope from several perspectives to understand the geometry of the shrunken nanocapillary. On the basis of this observation, the conductance was modeled with respect to the nanocapillary diameter, which allowed reproducing the experimental results. We then translocated DNA through the shrunken nanocapillaries and measured higher conductance drops for smaller diameters, reaching 1.7 nS for the 14 nm diameter nanocapillary. A model taking into account the conical shape of the shrunken nanocapillaries also supported this dependence. Next, we calculated the noise in the form of the standard deviation of the ionic conductance (between 0.04 and 0.15 nS) to calculate a signal-to-noise ratio (SNR) and compared it with nanopores embedded in 20 nm thick silicon nitride membranes. This shows that although nanocapillaries have smaller signal amplitudes due to their conical shape, they benefit from a lower noise. The glass nanocapillaries have a good SNR of about 25 compared with the SNR of 15 for smaller sized nanopores in silicon nitride membranes. The ability to use a modified model of nanopores to mimic the block conductance by DNA translocation provides a theoretical framework to support experimental results from translocating polymers such as DNA

Time-lapse microscopy series of bacteria exposed to exogenous DNA.

<p>The images were captured in the red channel to visualize ComEA-mCherry at intervals of 3 sec (A) or 2 min (B). Matlab-computed maximal fluorescence intensity-plots are shown in A (corresponding fluorescent image in inset). Heat-maps showing the fluorescence intensities of the mCherry signal are depicted in the lower row of panel B. (C) Time-lapse microscopy series as in (B), but in a <i>comEC</i> minus background. The corresponding movies are available online (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s012" target="_blank">movies S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s013" target="_blank">S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s014" target="_blank">S4</a>).</p

ComEA binds to DNA <i>in vivo</i>.

<p>(A) Plasmid-encoded <i>gfp</i> (tat-GFP) or <i>comEA-gfp</i> (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in <i>E. coli</i>. The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the <i>tat-comEA-gfp</i> expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent <i>V. cholerae</i> cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a <i>comEC</i> positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.</p

Working model of how DNA translocation across the outer membrane might occur in <i>V. cholerae</i>

<p>(based on the current study and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Chen2" target="_blank">[2]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Burton1" target="_blank">[4]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Claverys2" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Krger1" target="_blank">[14]</a>). The key components addressed in this study are indicated. It has been suggested that a (pseudo)pilus <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Chen1" target="_blank">[1]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Chen2" target="_blank">[2]</a>, which is similar to type 2 secretion systems (T2SS) and type IV pili (Tfp), represents a core element of the DNA uptake machinery <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Seitz2" target="_blank">[11]</a>. It is assumed that the Tfp crosses the outer membrane through a secretin pore formed by PilQ <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Chen2" target="_blank">[2]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Burton1" target="_blank">[4]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Wolfgang1" target="_blank">[74]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Korotkov1" target="_blank">[75]</a>. This secretin could also provide the point of entry for incoming DNA. A single pilus retraction event might open the secretin pore so that short stretches of the tDNA can enter the periplasm by Brownian motion (or through partial binding to the pilus structure). ComEA would then bind to the tDNA via its HhH-associated lysine residues favoring translocation via a Brownian ratchet mechanism, which is modulated by the effective ComEA DNA-binding kinetics, binding spacing, and concentration. The ComEA-loaded tDNA might eventually interact with the inner membrane transporter ComEC <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Draskovic1" target="_blank">[76]</a>, which would transport the DNA into the cytoplasm. Similar to Gram-positive bacteria, it is assumed that incoming DNA enters the cytoplasm of Gram-negative bacteria single-stranded.</p

<i>In silico</i>-prediction of the ComEA-DNA complex.

<p>(A) Protein sequence alignment of the two helix-hairpin-helix motifs (HhH1-2) of ComEA/ComE homologs from the indicated organisms (using the ComEA residue numbering). The sequence conservation is shown in tones of blue (dark blue = highly conserved). (B) 3D model of ComEA and its predicted DNA binding mode based on comparative modeling using the ComEA-related protein of <i>T. thermophilus</i> HB8 (PDB ID: 2DUY) as a template (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066.s011" target="_blank">movie S1</a>). The non-sequence-specific DNA backbone phosphate interactions with K62 and K63 are shown (inset). (C) The electrostatic potential at the molecular surface of ComEA is reported within a ±160 k_BT/e range (negative values in red, positive values in blue).</p

Localization of the ComEA protein in naturally competent <i>V. cholerae</i> cells.

<p>(A) Expression and distribution of ComEA-mCherry (upper row) or signal sequence[ComEA] (amino acids 1–25)-mCherry fusion proteins (lower row) within competent <i>V. cholerae</i> cells. Fluorescent signals for mCherry or DAPI-stained genomic DNA were visualized and compared with each other (merge) and the corresponding phase contrast image (Ph). (B) Representative fluorescence loss in photobleaching (FLIP) experiment to demonstrate the degree of mobility of ComEA-mCherry in live bacteria. Bleaching of the region-of-interest (ROI) 1 (indicated as 1 in the images on the right) was initiated after the acquisition of 20 frames and repeated after every frame. The fluorescence intensities of ROIs 1–3 were measured for a total of 20 sec and normalized to the average fluorescence intensity of the first 10 frames. The moving averages (period n = 5) are indicated with black lines. The average fluorescence intensity projections before (pre-bleach) and after bleaching (post-bleach) are shown on the right. Scale bars, 2 µm.</p

ComEA but not ComEA^K62/63A binds to DNA <i>in vitro</i>.

<p>EMSA using the 200<i>comEA</i> gene as a probe (panels A and B). A total of 0.4 pmol of DNA was incubated without or with increasing amounts of the ComEA-mCherry-Strep (A) and ComEA^K62/63A-mCherry-Strep (B) protein (lanes 2 to 10: 0, 2, 4, 6, 8, 10, 12, 16, and 20 pmol of protein). Free DNA, free protein, and the DNA/protein complex are indicated by the arrows. L: DNA ladder (representative bp are indicated on the left). Panel C: AFM images of DNA, proteins, and DNA/protein complexes absorbed on mica. AFM images from left to right: bare DNA fragments (DNA to protein ratio 1∶0); DNA/protein complex at a molecular ratio of 1∶2.5; DNA/protein complex at a molecular ratio of 1∶10. The proteins bound to the DNA are marked with black arrows; unbound proteins are labeled by white arrows. The height or Z scale is shown on the right and is the same for all three panels displaying 270 nm×270 nm scan areas.</p

ComEA homologs from different naturally competent bacteria compensate for the absence of ComEA in <i>V. cholerae</i>.

<p>First row: Schematic representation of the ComEA protein of <i>V. cholerae</i> from amino acid 1 to the end. Domains such as the helix-hairpin-helix motifs (HhH) and the signal sequence (signal) are also depicted. The signal sequence cleavage site (scissor) was predicted through the SignalP 4.1 server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Petersen1" target="_blank">[73]</a> for ComEA<i>^V.c.</i> and for its homologs. Designed constructs replacing wild-type ComEA on the chromosome of the respective <i>V. cholerae</i> strains are indicated below the WT ComEA scheme. First, the two ComEA mutants lacking either of both HhH motifs are indicated. In gray: the ComEA protein of <i>B. subtilis</i> (<i>B.s.</i>; not to scale compared to the other constructs and as indicated by the two diagonal lines). The transmembrane domain of ComEA<i>^B.s.</i> was removed to avoid toxicity/insolubility problems <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Provvedi1" target="_blank">[21]</a>. ComEA(C-term) of <i>B. subtilis</i> refers to the C-terminal part of the protein, which still allowed DNA binding <i>in vitro </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004066#pgen.1004066-Provvedi1" target="_blank">[21]</a>. Both ComEA<i>^B.s.</i> and ComEA(C-term)<i>^B.s.</i> were tested without and with mCherry fused to the C-terminus (− mCherry/ + mCherry). In white: ComEA homologs (ComE1) of the Gram-negative bacteria <i>Neisseria gonorrhoeae</i> (<i>N.g.</i>), <i>Haemophilus influenzae</i> (<i>H.i.</i>), and <i>Pasteurella multocida</i> (<i>P.m.</i>). The original signal sequence of each of those proteins was removed. All constructs were expressed from the native <i>comEA</i> promoter and encoded the <i>V. cholerae</i> ComEA-specific signal sequence (residues 1–25) to allow proper translocation across the inner membrane. Natural transformation was tested for all strains and the transformation frequencies (TF) are indicated on the right. The average of at least three independent biological replicates is shown (± SD). </p