DNA-Membrane Anchor Facilitates Efficient Chromosome Translocation at a Distance in Bacillus subtilis

Abstract

Chromosome segregation in sporulating Bacillus subtilis involves the tethering of sister chromosomes at opposite cell poles. RacA is known to mediate chromosome tethering by interacting with both centromere-like elements in the DNA and with DivIVA, a membrane protein which localizes to the cell poles. RacA has a secondary function in which it assists in nucleoid condensation. Here we demonstrate that, in addition to positioning and condensing the chromosome, RacA contributes to efficient transport of DNA by the chromosome segregation motor SpoIIIE. When RacA is deleted, one-quarter of cells fail to capture DNA in the nascent spore, yet 70% of cells fail to form viable spores without RacA. This discrepancy indicates that RacA possesses a role in sporulation beyond DNA capture and condensation. We observed that the mutant cells had reduced chromosome translocation into the forespore across the entire length of the chromosome, requiring nearly twice as much time to move a given DNA locus. Additionally, functional abolition of the RacA-DivIVA interaction reduced translocation to a similar degree as in a racA deletion strain, demonstrating the importance of the RacA-mediated tether in translocation and chromosome packaging during sporulation. We propose that the DNA-membrane anchor facilitates efficient translocation by SpoIIIE, not through direct protein-protein contacts but by virtue of physical effects on the chromosome that arise from anchoring DNA at a distance.IMPORTANCE To properly segregate their chromosomes, organisms tightly regulate the organization and dynamics of their DNA. Aspects of the process by which DNA is translocated during sporulation are not yet fully understood, such as what factors indirectly influence the activity of the motor protein SpoIIIE. In this work, we have shown that a DNA-membrane tether mediated by RacA contributes to the activity of SpoIIIE. Loss of RacA nearly doubles the time of translocation, despite the physically distinct locations these proteins and their activities occupy within the cell. This is a rare example of an explicit effect that DNA-membrane connections can have on cell physiology and demonstrates that distant changes to the state of the chromosome can influence motor proteins which act upon it.

Keywords: Bacillus; chromosome organization; chromosome segregation; sporulation.

FIG 1

RacA contributes to sporulation by assisting SpoIIIE activity. (A) Sporulation efficiency of wild-type and ΔracA cells. Sporulation was induced by resuspension in minimal medium for 24 h. The 30% ± 5% efficiency for ΔracA cells was calculated as the number of spores as a fraction of wild-type CFU, where normalization of cell density was conducted at resuspension. Error bars are the standard deviations between dilutions; 100 to 300 colonies were counted for each condition. An efficiency of 31% ± 4% was observed by a different technique, sporulation by exhaustion. (B) Distribution of translocation times measured by live-cell time-lapse imaging of YFP and CFP signals from the forespores of sporulating cells. One hundred cells were acquired for wild type (WT), SpoIIIE^D586A (D586A), and ΔracA cells. The fraction is the proportion of the cells which saw a complete translocation event (YFP signal followed by CFP) with a given duration between the appearances of each signal. The vertical lines denote the mean translocation time in a given background, where for wild-type cells it was 11 min, SpoIIIE^D586A cells it was 25 min, and ΔracA cells it was 19 min. The cfp gene was at the 90° locus in each strain. (C) Translocation efficiency of wild-type (black) and ΔracA (gray) cells in three SpoIIIE backgrounds. DNA translocation by wild type SpoIIIE (WT), SpoIIIE^D586A (D586A), and SpoIIIE^Δγ (Δγ) of the 90° locus is shown after sporulation by resuspension; 500 to 1,000 forespores were included across 3 to 10 fields of view in technical replicates. Time points early in sporulation or with slowly sporulating strains may include 100 to 200 forespores.

FIG 2

RacA contributes to DNA transport throughout the process of translocation. (A) DNA translocation of wild-type (black) and ΔracA (gray) cells, transporting either the 117° (left) or 138° (right) locus and either wild-type SpoIIIE (WT), SpoIIIE^D586A (D586A), or SpoIIIE^Δγ (Δγ); 500 to 1,000 forespores were included across 3 to 10 fields of view in technical replicates at each time point. Time points early in sporulation or with slowly sporulating strains may include 100 to 200 forespores. (B) Efficiency of transport for ΔracA cells with either wild-type SpoIIIE (WT), SpoIIIE^D586A (D586A), or SpoIIIE^Δγ (Δγ) and the CFP reporter at either the 90° (black), 117° (dark gray), or 138° (light gray) locus. Efficiency of transport is the CFP/YFP ratio of ΔracA normalized to wild type 225 min after the initiation of sporulation by resuspension. Data are from Fig. 2A and error bars are the standard deviations between fields of view. Fold change is relative to wild-type efficiency of transport.

FIG 3

Deletion of 21 residues from the C terminus of DivIVA abolishes its interaction with RacA. (A) Sample images of RacA-GFP in wild-type cells stained with FM4-64. Bar, 1 μm. White arrowheads point to forespores empty of DNA (left) and containing DNA (right). (B) Fractions of forespores with (gray) and without (black) detectable RacA-GFP fluorescence 180 min after sporulation by resuspension. At least 500 forespores were counted for each strain. (C) Sample images of TetR-mCherry in wild-type cells stained with TMA-DPH and used for line scans. Bar, 1 μm. The white arrowhead denotes a TetR-mCherry focus. (D) Diagram of line scan (black line) through a forespore, intersecting a TetR-mCherry focus (black circle) and the closest region of TMA-DPH-stained membrane. These line scans were used for calculating the distance d between the locus and the membrane. (E) Distributions of distances calculated between each focus and the nearest portion of the membrane. For wild-type cells, the mean distance was 83 nm (upper black vertical line) and for DivIVAΔ11 cells, the mean was 107 nm, while for DivIVAΔ21 cells, the mean was 162 nm (lower black vertical line). One hundred line scans were performed for each strain.

FIG 4

Abolition of anchoring by RacA nullifies its contribution to DNA translocation activity by SpoIIIE. (A) DNA translocation efficiency in wild-type and DivIVAΔ21 cells after sporulation by resuspension. (Top) Wild-type SpoIIIE cells (WT); (middle) SpoIIIE^D586A cells (D586A); (bottom) SpoIIIE^Δγ cells (Δγ). Wild type (black), divIVAΔ11 (dark gray), and divIVAΔ21 (light gray) are each depicted. The cfp reporter gene is at the 90° locus; 500 to 1,000 forespores were included across 3 to 10 fields of view in technical replicates at each time point. Time points early in sporulation or with slowly sporulating strains may include 100 to 200 forespores. (B) The efficiency of transport of ΔracA cells (black) compared to that of divIVAΔ21 cells (gray) with wild-type SpoIIIE, SpoIIIE^D586A, and SpoIIIE^Δγ. The efficiencies for ΔracA and divIVAΔ21 were standardized to wild-type RacA and wild-type DivIVA, respectively, 180 min after sporulation by resuspension. The data depicted are the same as in the translocation efficiency plots in Fig. 1B and 4A, and error bars are the standard deviations between fields of view. The cfp gene was at the 90° locus in each strain.