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. 2011 Jun 15;25(12):1328-43.
doi: 10.1101/gad.2038911.

A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW

Joshua W Modell  1 , Alexander C Hopkins, Michael T Laub
Affiliations

A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW

Joshua W Modell et al. Genes Dev. .

Erratum in

  • Genes Dev. 2011 Aug 1;25(15):1662

Abstract

Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.

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Figures

Figure 1.
Figure 1.
DNA damage induces global changes in gene expression and inhibits cell division. (A) Schematic of the Caulobacter cell cycle with and without DNA damage. (B) Wild-type CB15N grown to mid-exponential phase was exposed to UV light, HU, or MMC for 80 min or left untreated, and was imaged by differential interference contrast microscopy. Cells exposed to MMC were also examined after 160 min. Bar, 2 μm. (C) Gene expression profiles of DNA damage-regulated genes in Caulobacter. Profiles are shown for 74 genes significantly changed in expression level after treatment with HU, MMC, or UV light in rich (PYE) and minimal (M2G) media. The column labeled “LexA” indicates with a red box whether a gene has a LexA box upstream. The columns labeled “CtrA-direct” and “CtrA-indirect” indicate with a green or black box, respectively, whether a gene is a direct target of CtrA or is indirectly affected by CtrA. For annotation of individual genes and complete data, see Supplemental Figure S1 and Supplemental Table S1. Expression ratios are shown relative to untreated cells and are represented using the color scale shown. Gray blocks indicate missing data. (D) Graph showing induction of sidA after DNA damage, relative to other members of the SOS regulon: recA, lexA, and ssb. Response curves are the average across all six time courses in C.
Figure 2.
Figure 2.
sidA is the primary SOS-induced cell division inhibitor. (A,B) Micrographs (A) and growth curves (B) of wild-type, ΔlexA, ΔsidA, and ΔlexA ΔsidA cells grown in rich medium. Bar in the top left panel, 2 μm. (C) ΔsidA (tetR) cells were placed on agarose pads containing MMC and imaged for up to 600 min. Examples of minicell formation resulting from division near a cell pole or division at mid-cell are shown. The percentage of cells that produce minicells or divide medially are shown on the right and are compared with wild-type cells treated identically.
Figure 3.
Figure 3.
Overproducing SidA is sufficient to inhibit cell division. (A) Growth curve in rich medium of cells carrying a high-copy plasmid on which the xylose-inducible promoter drives expression of the M2 epitope only; sidA*, the originally annotated CC1927 ORF; sidA*(T2A), which harbors a mutation in the first annotated start codon; or M2-sidA, in which the M2 epitope is fused to the second start site in the originally annotated CC1927 ORF. (B) Each strain from A was grown to mid-exponential phase in rich medium supplemented with glucose to repress expression of the plasmid-encoded construct. Expression was then induced by adding xylose, and cells were imaged by differential interference contrast (DIC) microscopy after 3 and 6 h. (C) The promoter and leader region of CC1927, up through either the first or second annotated methionine, were fused to the coding region of EGFP to generate PsidA*:egfp and PsidA:egfp, respectively. Strains carrying each construct on a low-copy plasmid were grown in the presence or absence of MMC, and expression was examined by epifluorescence microscopy. Bars, 2 μm.
Figure 4.
Figure 4.
SidA does not prevent assembly of the cell division machinery. (A) Subcellular localization of FtsZ was examined in a strain expressing ftsZ-eyfp from the vanillate-inducible promoter Pvan and overexpressing M2-sidA from a xylose-inducible promoter on a high-copy plasmid. Cells were grown to mid-exponential phase in rich medium with glucose and then shifted to xylose. Vanillate was added 1.5 h before shifting to xylose. At the times indicated, samples were taken and cells were imaged by DIC and epifluorescence microscopy (top two rows). The white asterisk indicates a FtsZ ring that is no longer associated with a constriction site and so likely moved and reassembled. (Bottom two rows) Localization of FtsN was examined in a strain expressing an egfp-ftsN fusion at its native chromosomal locus and overexpressing M2-sidA from a high-copy plasmid. The percentage of cells with foci after 4.5 h in xylose is shown below the last panel. (B) Localization of FtsI was examined in a strain expressing gfp-ftsI at its native chromosomal locus and overexpressing M2-sidA from a xylose-inducible promoter carried on a high-copy plasmid. Cells were grown to mid-exponential phase in rich medium with glucose and then placed on agarose pads containing xylose to produce M2-SidA. Individual cells were followed by time-lapse microscopy, with phase and epifluorescence images captured at the time points indicated. (C) Cells expressing gfp-ftsI and xylose-inducible M2-sidA were grown in the presence of xylose for 4.5 h to inhibit cell division and induce cellular filamentation. Cells were then placed on an agarose pad containing glucose to repress M2-sidA expression. Individual cells with localized GFP-FtsI were then followed by time-lapse microscopy, with phase and epifluorescence images captured at the time points indicated. Bars, 2 μm.
Figure 5.
Figure 5.
Mutations in ftsW and ftsI suppress the SidA overproduction phenotype. (A) Summary of a sidA overexpression suppressor screen. The location of mutations identified in FtsW and FtsI are indicated on schematics representing the domain structure of each protein. (B) Each suppressor mutation was introduced into a clean wild-type background by allelic replacement, followed by transformation with the M2-sidA overexpression plasmid. Each strain was then grown to mid-exponential phase, and serial dilutions were plated on PYE supplemented with chloramphenicol to maintain the plasmid and with xylose to induce SidA. (C) Cellular morphology of strains harboring each suppressor mutation and overexpressing M2-sidA for 6 h. Bars, 2 μm. (D) Cellular morphology of strains harboring each suppressor mutation and a deletion of lexA. The doubling time of each strain in rich medium is indicated below the corresponding micrograph. (E) The location of a predicted transmembrane domain within sidA is shown schematically at the top and directly below in an alignment of the coding region of SidA orthologs. The location of a single predicted transmembrane domain within ftsI is shown schematically at the bottom and directly above in an alignment of the transmembrane domains of FtsI orthologs. The alignments are aligned to each other, using the last predicted amino acid as an anchor point. The arrow indicates a conserved phenylalanine in both alignments. Black and gray shading indicate residue conservation and similarity, respectively, found at that position in >50% of aligned sequences. (F) Subcellular fractionation of cells overexpressing M2-sidA from a xylose-inducible promoter on a high-copy plasmid for 1.5 h and expressing cckA-gfp from the chromosome. Samples from cells grown in either glucose or xylose, as indicated, were fractionated into soluble (S) and membrane (M) fractions. Samples were separated by SDS-PAGE, transferred to a PVDF membrane, cut into three pieces, and probed with antibodies specific for GFP, CtrA, or the M2 epitope.
Figure 6.
Figure 6.
SidA interacts with the late-arriving divisome components FtsW and FtsN. (A) Bacterial two-hybrid analysis of interactions between M2-SidA and cell division proteins fused to T18 and T25, as indicated. Interactions were quantified using a Miller assay and are reported relative to empty vector controls, which yielded 60 Miller units. Each interaction was measured in triplicate; error bars represent the standard error of the mean. FtsA# indicates the FtsA-MalF(TM) fusion described in the Materials and Methods. FtsIΔC was tested alone and while producing untagged FtsW, as indicated. Asterisks indicate a statistically significant difference (P < 0.05, one-sided t-test) relative to empty vector controls. (B,C) Bacterial two-hybrid analysis of interactions between M2-SidA (B) or FtsN (C) fused to T18 and mutants of FtsW fused to T25. Interactions are reported as a percentage of that measured for wild-type FtsW with M2-SidA (B) or with wild-type FtsN (C).
Figure 7.
Figure 7.
Overproducing SidA does not inhibit the translocation of septal peptidoglycan precursors. (A) Van-FL staining of wild-type, ftsZ depletion, and ftsW depletion. Wild-type cells were grown to mid-exponential phase in rich medium. The ftsZ depletion strain was grown to mid-exponential phase in the presence of xylose, washed, and then grown in the presence of xylose or glucose for 1.5 h before imaging. The ftsW depletion strain was grown to mid-exponential phase in the presence of vanillate, washed, and then grown without vanillate for 7.5 h before imaging. (B) The M2-SidA overproduction strain was synchronized, released into rich medium containing either glucose or xylose, and imaged at the times indicated. (C) A mixed population of the M2-SidA overproduction strain was imaged after growth in xylose for 4.5 h. In all panels, cells were stained with Van-FL and were imaged by DIC or phase and epifluorescence microscopy. Arrowheads indicate Van-FL staining in transverse bands and foci that are at least 1.5-fold over the cell background (see the Materials and Methods; Supplemental Fig. S4). Bars, 2 μm.
Figure 8.
Figure 8.
A DNA damage checkpoint regulating cell division in C. crescentus. Model for regulation of cell division by SidA following DNA damage. The schematic at the top shows the Caulobacter cell cycle and indicates the progression of cell division, beginning with assembly and initial stabilization of FtsZ rings in stalked cells, followed by constriction in late predivisional cells, and resulting finally in cell division. When DNA damage occurs, the FtsZ ring is still assembled, but cell division is inhibited while cells continue to elongate. SidA inhibits cell division by inserting into the membrane and binding FtsW and FtsN. The expression of sidA is under SOS control, and hence is induced following DNA damage and cleavage of the LexA repressor.
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