SummaryThe F plasmid is actively partitioned to daughter cells by the sopABC gene. To elucidate the partitioning mechanisms, we simultaneously analysed movements of the plasmid and the SopA ATPase in single living cells. SopA, which is a putative motor protein assembled densely near nucleoid borders and formed a single discrete focus associated with less dense filamentous distribution along the long axis of the cell. The dense SopA focus oscillates between cell poles. The direction of the plasmid motion switches as the SopA focus switches its position. The velocity of the plasmid motion stays constant while it oscillates moving towards the SopA focus. The low density filamentous distribution of SopA persisted throughout the SopA oscillation. The focus associated with filamentous distribution of SopA was also observed in a cell without nucleoid. The SopA filament may guide the movement of the plasmid as a railway track and lead it to cell quarters.

To date, solid-state nanopores have been fabricated primarily through a focused-electronic beam via TEM. For mass production, however, a TEM beam is not suitable and an alternative fabrication method is required. Recently, a simple method for fabricating solid-state nanopores was reported by Kwok, H. et al. and used to fabricate a nanopore (down to 2 nm in size) in a membrane via dielectric breakdown. In the present study, to fabricate smaller nanopores stably—specifically with a diameter of 1 to 2 nm (which is an essential size for identifying each nucleotide)—via dielectric breakdown, a technique called “multilevel pulse-voltage injection” (MPVI) is proposed and evaluated. MPVI can generate nanopores with diameters of sub-1 nm in a 10-nm-thick Si3N4 membrane with a probability of 90%. The generated nanopores can be widened to the desired size (as high as 3 nm in diameter) with sub-nanometre precision, and the mean effective thickness of the fabricated nanopores was 3.7 nm.

SummaryRecently, it has been reported that prokaryotes also have a mitotic-like apparatus in which polymerized fibres govern the bipolar movement of chromosomes and plasmids. Here, we show evidence that a nonmitotic-like apparatus that does not form polymerized filaments carries out plasmid partitioning. P1 ParA, which is a DNA-binding ATPase protein, was found to be distributed through the whole nucleoid and formed a dense spot at the centre of the nucleoid. The fluorescent intensity of the ParA spot blinked, and then the spot gradually migrated from the midcell to a cell quarter position. Such distribution was not observed in anucleate cells, suggesting that the nucleoid could be a matrix for gradual distribution of ParA. Plasmid DNA constantly colocalized at the spot of ParA and migrated according to spot migration and separation. Thus, the gradient distribution of ParA determines the destination of partitioning plasmids and may direct plasmids to the cell quarters.

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 μs base(-1)) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 μs base(-1)). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

Optical chromatography, a new separation technique involving the use of a radiation force and a medium flow, is used for trace analysis of protein. Two polystyrene beads, coated with antibody (anti-mouse IgG), are combined in the presence of an antigen (mouse IgG). The bound (B) and free (F) beads are readily separated by optical chromatography, and the B/F ratio can be correlated with the concentration of antigen (protein). Nanomolar concentrations of protein can be measured by this technique. The rates of the forward and reverse immunological reactions were independently determined by measuring the time of formation and dissociation, respectively, of the immunobeads.