Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

SulA does not sequester FtsZ in Escherichia coli cells during the SOS response.

In Escherichia coli, the SulA protein is synthesized during the SOS response to arrest cell division. Two possible models of SulA action were proposed: the sequestration and the capping. In current paper, to clarify which model better reflects the SulA effect on cell division upon the SOS response, the FtsZ/SulA ratio was estimated inside cells based on fusion of both FtsZ and SulA to fluorescent protein mNeonGreen. This allowed to quantify this ratio by fluorescence microscopy as well as western blotting; moreover, the effect of SulA on FtsZ distribution patterns in cells was analyzed based on fluorescence microscopy images. The SulA concentration in cells under the SOS response was shown to be several times (about 10) lower than that of FtsZ. The effect of SulA was unequal to corresponding decrease in FtsZ concentration. These results are supported by uneven FtsZ distribution in cells under the SOS response. Together the results of current work indicate that the division arrest by SulA protein in E. coli cells could not be explained by the sequestration model.

The division protein FtsZ interacts with the small heat shock protein IbpA in Acholeplasma laidlawii.

Small heat shock proteins (sHSPs) control the proteins stability in the cell preventing their irreversible denaturation. While many mycoplasmas possess the sHSP gene in the genome, Acholeplasma laidlawii is the only mycoplasma capable of surviving in the environment. Here we report that the sHSP IbpA directly interacts with the key division protein FtsZ in A. laidlawii, representing the first example of such interaction in prokaryotes. FtsZ co-immunoprecipitates with IbpA from A. laidlawii crude extract and in vitro binds IbpA with KD ~ 1 µM. Proteins co-localize in the soluble fraction of the cell at 30-37 °C and in the non-soluble fraction after 1 h exposition to cold stress (4 °C). Under heat shock conditions (42 °C) the amount of FtsZ decreases and the protein remains in both soluble and non-soluble fractions. Furthermore, in vitro, FtsZ co-elutes with IbpAHis6 from A. laidlawii crude extract at any temperatures from 4 to 42 °C, with highest yield at 42 °C. Moreover, in vitro FtsZ retains its GTPase activity in presence of IbpA, and the filaments and bundles formation seems to be even improved by sHSP at 30-37 °C. At extreme temperatures, either 4 or 42 °C, IbpA facilitates FtsZ polymerization, although filaments under 4 °C appears shorter and with lower density, while at 42 °C IbpA sticks around the bundles, preventing their destruction by heat. Taken together, these data suggest that sHSP IbpA in A. laidlawii contributes to the FtsZ stability control and may be assisting appropriate cell division under unfavorable conditions.

New insights into FtsZ rearrangements during the cell division of Escherichia coli from single-molecule localization microscopy of fixed cells.

FtsZ - a prokaryotic tubulin homolog - is one of the central components of bacterial division machinery. At the early stage of cytokinesis FtsZ forms the so-called Z-ring at mid-cell that guides septum formation. Many approaches were used to resolve the structure of the Z-ring, however, researchers are still far from consensus on this question. We utilized single-molecule localization microscopy (SMLM) in combination with immunofluorescence staining to visualize FtsZ in Esherichia coli fixed cells that were grown under slow and fast growth conditions. This approach allowed us to obtain images of FtsZ structures at different stages of cell division and accurately measure Z-ring dimensions. Analysis of these images demonstrated that Z-ring thickness increases during constriction, starting at about 70 nm at the beginning of division and increasing by approximately 25% half-way through constriction.