MINIREVIEW

crossm

Regulation of Cell Division in Bacteria by Monitoring Genome

Integrity and DNA Replication Status

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Peter E. Burby,a Lyle A. Simmonsa

a
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA

ABSTRACT All organisms regulate cell cycle progression by coordinating cell divi-
sion with DNA replication status. In eukaryotes, DNA damage or problems with repli-
cation fork progression induce the DNA damage response (DDR), causing cyclin-
dependent kinases to remain active, preventing further cell cycle progression until
replication and repair are complete. In bacteria, cell division is coordinated with
chromosome segregation, preventing cell division ring formation over the nucleoid
in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria
induce the SOS response after replication forks encounter DNA damage or impedi-
ments that slow or block their progression. During SOS induction, Escherichia coli ex-
presses a cytoplasmic protein, SulA, that inhibits cell division by directly binding
FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allow-
ing for cell division to resume. Recently, it has become clear that SulA is restricted
to bacteria closely related to E. coli and that most bacteria enforce the DNA damage
checkpoint by expressing a small integral membrane protein. Resumption of cell di-
vision is then mediated by membrane-bound proteases that cleave the cell division
inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that
are regulated independently from the canonical LexA-mediated SOS response. In this
review, we discuss several pathways used by bacteria to prevent cell division from
occurring when genome instability is detected or before the chromosome has been
fully replicated and segregated.

KEYWORDS cell cycle, cell division, checkpoint, DNA damage, SOS response

E ven though DNA is a stable macromolecule for storing genetic information, chro-
mosomes must be replicated and segregated properly before cell division can occur
(1). Further, DNA can be modified from endogenous or exogenous sources resulting in
mutagenesis or DNA damage (2, 3). Sources of DNA damage include endogenous
metabolic intermediates, such as reactive oxygen species, as well as exogenous sources,
including radiation and chemicals that react directly with DNA, causing several types of
lesions (3–5). In addition, many antibiotics produced by plants and bacteria can cause
DNA damage to competing organisms as a defense mechanism or to provide a fitness Citation Burby PE, Simmons LA. 2020.
Regulation of cell division in bacteria by
advantage (6). Therefore, DNA damage is a pervasive problem encountered by all monitoring genome integrity and DNA
organisms regardless of their natural environment. replication status. J Bacteriol 202:e00408-19.
DNA damage prevents accurate DNA replication, though the exact mechanism https://doi.org/10.1128/JB.00408-19.
Editor William Margolin, McGovern Medical
differs depending on the type of lesion encountered. For example, Escherichia coli
School
exposure to UV light results in a very rapid decrease in DNA replication due to the Copyright © 2020 American Society for
production of thymine-thymine dimers and 6-4 photoproducts (7, 8). Replicative DNA Microbiology. All Rights Reserved.
polymerases cannot utilize thymine dimers as a template because the active site only Address correspondence to Lyle A. Simmons,
accommodates a single templating base during catalysis (9–12). Similarly, alkylating lasimm@umich.edu.
Accepted manuscript posted online 23
agents, such as methyl methanesulfonate, can methylate DNA bases, preventing accu-
September 2019
rate base pairing during DNA synthesis (13). Mitomycin C is a distinct type of bifunc- Published 2 January 2020
tional alkylating agent that can react with DNA, resulting in an interstrand cross-link

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FIG 1 A model for activation of the bacterial SOS response. Activation of the SOS response begins with
accumulation of ssDNA that occurs when high levels of DNA damage are present (green polygons). The
ssDNA is subsequently coated with the protein RecA. The resulting RecA/ssDNA nucleoprotein filament
stimulates the protease activity of the transcriptional repressor LexA (yellow protein). LexA undergoes
autocleavage, resulting in derepression of the LexA regulon. Many of the genes in the LexA regulon are
involved in DNA repair, DNA damage tolerance, and regulation of cell division, a process known as a DNA
damage checkpoint. Yellow boxes represent LexA binding sites, and purple boxes represent ⫺35 and
⫺10 promoter sequences. This figure is adapted from reference 113.

(14). Interstrand DNA cross-links are particularly toxic because the two DNA strands
cannot be separated by the replicative helicase or RNA polymerase, preventing DNA
replication and transcription (15, 16). Another type of DNA damage is a break in the
phosphodiester backbone caused by agents such as ionizing radiation and the naturally
produced microbial peptides bleomycin and phleomycin (3). A break is toxic to cells
because the DNA replication machinery depends on the integrity of the template for
synthesis of the nascent strand (17, 18). For all types of DNA damage, the major
impediment is the inability to access and replicate the information stored within the
chromosome. DNA damage not only alters the coding information through mutagen-
esis or loss of information from deletions, but it can also slow chromosomal replication
and segregation. Therefore, bacteria have evolved several different methods to detect
incomplete chromosome segregation or problems with DNA integrity. Once such a
condition is detected, cells halt the progression of cell division, affording the cell time
to repair and then fully replicate its chromosome.

DNA DAMAGE ACTIVATES THE SOS RESPONSE IN BACTERIA

The SOS response is a highly conserved stress response pathway that is activated
when bacteria encounter DNA damage (19–22). Activation of the SOS response results
in increased transcription of genes important for DNA repair, DNA damage tolerance,
and regulation of cell division (23–25). In addition, many mobile genetic elements and
pathogenicity islands also sense problems with DNA replication through the SOS
response (for a review, see reference 26). The collection of genes controlled by the SOS
response is referred to as the SOS regulon. Proximal to the promoters of genes in the
SOS regulon are DNA binding sites for the transcriptional repressor LexA (27–30). When
bound to LexA binding sites, LexA prevents the transcription of genes under its control
(31–34). Thus, activation of the SOS response requires the inactivation of LexA, resulting
in activated gene transcription (Fig. 1).
Early genetic studies demonstrated that RecA is required for SOS activation (35).
RecA catalyzes the pairing of single-stranded DNA (ssDNA) to the complementary

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sequence in double-stranded DNA (dsDNA), resulting in the synapsis step of homolo-

gous recombination (36, 37). RecA is also required for LexA inactivation in vitro,
suggesting that RecA functions as a protease responsible for LexA cleavage (38).
Further characterization found that RecA bound to ssDNA acted as a coprotease
stimulating the autoproteolytic activity of LexA (39) (Fig. 1). A unique feature of the SOS
regulon is that a broad set of DNA lesions or problems with DNA replication evoke the
formation of a RecA/ssDNA nucleoprotein filament, relaying a compromise in genome
integrity activating the SOS response.
How does DNA damage lead to the accumulation of ssDNA in vivo? Treatment with

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a double-strand-break-inducing agent, such as bleomycin or ionizing radiation, leads to
activation of the SOS response (40–42). The repair of double-stranded breaks occurs
through homologous recombination (36, 43, 44), during which ssDNA is generated by
the helicase/nuclease complex RecBCD in E. coli (17), AddAB in Bacillus subtilis (45), or
AddnAB in Mycobacterium spp. (46). These enzymes bind and process double-stranded
ends (45, 47) and generate a free 3= tail onto which RecA is loaded (48–52). Thus,
double-strand breaks result in the generation of a RecA/ssDNA nucleoprotein filament
that can activate the SOS response. In B. subtilis, replication is required for the formation
of a RecA-GFP focus and DNA damage is not sufficient (53). In E. coli, replication is not
required to stimulate LexA cleavage following treatment with bleomycin (41). The
generation of ssDNA following treatments that cause other types of DNA lesions, such
as pyrimidine dimers from UV light, is less clear, though the process depends on DNA
replication (41). One prominent model is that the stalling of the replicative DNA
polymerase results in uncoupling of the replicative helicase from the replisome-
generating excess ssDNA (54). A second model is that the replication machinery stalls
at lesions such as pyrimidine dimers and will skip ahead, leaving a gap of ssDNA that
provides a suitable substrate for RecA binding (54). A primer generated from an RNA
transcript (55) or by primase (56) can allow DNA polymerase to advance beyond a DNA
lesion on the leading strand, or this can occur through template switching (57, 58). The
resulting ssDNA gap generated by DNA polymerase skipping ahead on the leading
strand can be used as the substrate for loading RecA by the RecFOR complex (59).
Indeed, activation of the SOS response following UV exposure is also dependent on the
RecFOR proteins (60). Therefore, bacteria make use of a DNA repair intermediate to
activate the expression of the genes important for DNA repair and the regulation of cell
division (Fig. 1).
DNA replication and cell division are essential processes that must be coordinated
to ensure that subsequent generations receive an accurate and complete copy of their
genetic material. Under normal conditions, DNA replication and cell division occur at
rates that allow replication and segregation of a complete chromosome to each
daughter cell. When DNA damage forms and DNA replication is blocked, bacteria make
use of multiple mechanisms to ensure that cell division is delayed, providing the cell
with enough time for DNA repair to occur. These mechanisms include nucleoid
occlusion, SOS-independent regulation of the divisome, and an SOS-dependent DNA
damage checkpoint. Below, we discuss each of these mechanisms and how they
regulate cytokinesis in bacteria.

SOS-INDEPENDENT REGULATION OF CELL DIVISION

Nucleoid occlusion. A straightforward mechanism for preventing cell division prior
to the completion of DNA replication is nucleoid occlusion (61, 62). We define the
nucleoid as the supercoiled and compacted chromosome bound by numerous pro-
teins. Nucleoid occlusion prevents the cell division machinery from operating in the
same location as the nucleoid, requiring that chromosomal replication is near complete
and chromosomes are segregated before cell division occurs (61, 62). Simply, the
nucleoid will occlude the cell division machinery and prevent FtsZ ring assembly and
constriction. In E. coli, nucleoid occlusion is accomplished by SlmA (61, 62). SlmA was
identified using a genetic screen searching for genes that were important for cell
division in the absence of the Min system (63). The Min system, composed of MinCDE,

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prevents cell division from occurring at the cell poles (64). Therefore, the logic was that
mutants defective in both pathways responsible for cell division site selection would
not survive. Indeed, mutants in slmA were isolated (63). Interestingly, the deletion of
slmA alone did not change the frequency of cell division septum formation over the
nucleoid; however, the deletion of both slmA and minCDE resulted in a drastic increase
in septa forming over the nucleoid region (63). Importantly, when DNA replication was
blocked, the deletion of slmA caused a marked increase in septum formation over
nucleoids (63). Therefore, SlmA is required to prevent cell division from occurring at the
same location as the nucleoid, thereby providing feedback inhibition to the cell division

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machinery based on the status of chromosomal replication and segregation.
Green fluorescent protein (GFP)-SlmA was shown to localize to the nucleoid, and if
the helix-turn-helix domain was removed GFP-SlmA became diffusely cytosolic (63).
SlmA, which belongs to the TetR family of transcriptional regulators, binds specific DNA
motifs around the E. coli chromosome (65, 66). In addition to DNA binding activity, SlmA
increases FtsZ protofilament breakdown (66). FtsZ is a tubulin homolog that forms a
ring structure composed of numerous protofilaments at the site of cell division (67).
SlmA bound to DNA is able to interact with the FtsZ C-terminal domain, causing
disassembly of the protofilaments (66, 68). Recent experiments have shown that SlmA
forms dynamic phase-separated droplets when bound to FtsZ (69). These results
suggest that FtsZ-SlmA condensates regulate FtsZ by forming phase-separated drop-
lets in vivo when SlmA is bound to sites on chromosomal DNA (69). By binding distinct
chromosomal loci and simultaneously inhibiting FtsZ, SlmA provides a mechanism
delaying cell division when replication and segregation of the chromosome are incom-
plete.
The bacterium B. subtilis also uses nucleoid occlusion to inhibit cell division, though
the mechanism differs from the system discovered in E. coli. The nucleoid occlusion
gene noc was also identified as a gene required in the absence of the Min system (70).
When both Noc and the Min system were absent, FtsZ failed to form a distinct band at
midcell (70). Further, when DNA replication was blocked in cells without Noc, division
septa formed over the nucleoids, providing evidence that Noc also functions as a
nucleoid occlusion factor. (70). Like SlmA, Noc is a sequence-specific DNA binding
protein that binds to several distinct chromosomal loci (71). The N terminus of Noc is
an amphipathic helix that interacts with the cell membrane (72). Noc binds the B.
subtilis chromosome localizing to the inner leaflet of the cell membrane, where it is
hypothesized to prevent the assembly of a complex of proteins necessary for cell
division called the divisome (72).
Noc is not limited to B. subtilis or other rod-shaped bacteria. Noc has also been
shown to inhibit cell division in the spherical Gram-positive bacterium Staphylococcus
aureus (73). A synthetic lethal transposon-sequencing (Tn-seq) approach identified two
genes that were lethal in a noc deletion (74). Follow-up analysis showed that mutations
in the replication initiator dnaA suppressed the synthetic lethality of noc depletion in
the double mutants (74). This analysis led to the finding that S. aureus Noc (SaNoc)
negatively regulates DNA replication initiation by demonstrating that noc-deleted cells
alone overinitiate and mutations in dnaA suppress the overinitiation phenotype (74).
Therefore, in B. subtilis and S. aureus, Noc inhibits the divisome, providing a mechanism
for the nucleoid to delay division until DNA replication is complete. In S. aureus, Noc has
an additional regulatory role in preventing precocious initiation (74). In addition, a
Noc-independent form of nucleoid occlusion following the arrest of DNA replication
has been observed in B. subtilis (75). The mechanism and cellular factors responsible for
this phenomenon are still unclear, requiring further experiments to determine the
Noc-independent process (75).
FtsL depletion. In addition to the nucleoid regulating cell division directly, the
status of DNA replication provides feedback inhibition repressing expression of the cell
division machinery. Transcriptomic studies of the SOS response in B. subtilis revealed
that many genes were regulated by DNA damage independently of the SOS-activating

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TABLE 1 DNA damage-inducible cell division inhibitorsa

Organism Inhibitor Inhibitor function Target Removal mechanism
E. coli SulA 169-amino-acid cytoplasmic protein regulated by LexA FtsZ Degraded by Lon
protease
B. subtilis YneA 105-amino-acid integral membrane protein, regulated Unknown Cleaved by CtpA and
by LexA, contains an N-terminal TM and C-terminal DdcP
LysM domain
S. aureus SosA 77-amino-acid integral membrane protein contains an Unknown Cleaved by CtpA
N-terminal TM domain
M. tuberculosis ChiZ (Rv2719c) 165-amino-acid integral membrane protein contains a Peptidoglycan hydrolysis Unknown

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TM domain toward the middle of the protein and and/or interaction
LysM domain near the C terminus; expression is with FtsI/Q
regulated by DNA damage
C. glutamicum DivS 140-amino-acid integral membrane protein with Unknown Unknown
C-terminal TM domain
C. crescentus SidA 29-amino-acid TM protein regulated by LexA FtsI/N/W Unknown
C. crescentus DidA 71-amino-acid integral membrane protein; contains FtsI/N/W Unknown
TM domain in the middle portion of the protein;
regulated by transcription factor DriD
aThistable was adapted from Fig. 1 and information from Bojer et al. (114). The information presented in this table is also from references 86, 87, 91, 107, 109, and
114 to 117. TM, transmembrane.

protein RecA (76). Further analysis showed that several operons were regulated by the
essential replication initiator protein DnaA and that a series of promoters for ⬎50 genes
(about 20 operons) had binding sites for DnaA known as DnaA boxes (76). One of the
genes that is repressed following inhibition of DNA replication is ftsL (76). FtsL is an
essential protein component of the divisome, and previous studies have shown that
FtsL is a highly unstable protein in response to DNA damage (77–79). FtsL protein levels
are normally controlled through regulated intramembrane proteolysis (RIP). FtsL con-
tains a cytoplasmic recognition motif that is cleaved by RasP, an intramembrane zinc
metalloprotease (79, 80). The DivIC protein stabilizes FtsL, preventing cleavage by RasP
(81). Inhibition of DNA replication causes an increase in cell length independent of the
SOS-dependent cell division inhibitor (see below); however, overexpression of FtsL
resulted in shorter cells on average, indicating that a decrease in FtsL is responsible for
delaying cell division in response to a block in DNA replication (76). Thus, it appears that
following a block in DNA replication, DnaA inhibits cell division indirectly by repressing
the expression of the unstable divisome protein FtsL.

SOS-DEPENDENT DNA DAMAGE CHECKPOINT

E. coli SulA. Although the above-mentioned processes contribute to the regulation
of cell division in response to DNA replication status, a prominent mechanism for cell
division control in bacteria is the SOS-dependent DNA damage checkpoint. The bac-
terial DNA damage checkpoint was originally discovered in E. coli, long before the
effects of treatment with UV light on DNA synthesis were understood (8, 82); E. coli cells
were found to filament following exposure to UV light (83). More than 3 decades later,
the existence of a specific cell division inhibitor was hypothesized (84). The hypothesis
was that expression of the gene coding for the cell division inhibitor was normally
repressed and that inhibition of DNA replication would result in derepression and
accumulation of a protein that could prevent cell division (84). Although the mecha-
nism of action varies between organisms, this hypothesis was upheld following inves-
tigation of the DNA damage checkpoint across many diverse bacterial species (a
summary is shown in Table 1).
Cell filamentation characterized by recA mutants (tif) causing SOS induction at
elevated temperatures was exacerbated in cells with a lon mutation (85). Suppressors
were isolated called sfiA because the mutations suppressed filamentation of an E. coli
tif and lon double mutant (85). A subsequent study determined that sfiA mutations
mapped to the same gene as sulA, which were so named because they suppressed DNA
damage sensitivity of lon mutants (86, 87). To test if sulA was controlled by exogenous

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FIG 2 DNA damage-dependent cell cycle checkpoint in E coli. When E. coli cells encounter DNA damage
(green polygons), the SOS response is activated, and the cell division inhibitor SulA is overexpressed
(dark green). SulA prevents cell division by inhibiting assembly of the FtsZ ring (red circles). Following
DNA repair and SOS termination, SulA is degraded by Lon protease, allowing cell division to proceed. This
figure is adapted from reference 113.

DNA damage treatments, a lacZ reporter at the sulA locus was monitored following
exposure to several types of DNA damage, which verified that sulA expression was
indeed DNA damage inducible (88). The promoter of sulA contains a binding site for the
repressor LexA (89), and binding of LexA at the promoter of sulA repressed transcription
(90). Thus, activation of the SOS response, and therefore, inactivation of LexA, result in
high levels of sulA expression.
Other methods of increasing SulA protein levels, including overexpression in the
absence of DNA damage or deletion of lon, lead to cell filamentation (91–93). One of
the early steps in cell division is the assembly of the FtsZ ring at the future division site
(67). Overexpression of SulA prevents formation of the FtsZ ring in vivo (94). One of the
original mutations that suppressed DNA damage-induced filamentation, sfiB (or sulB),
mapped to the ftsZ locus. Further studies demonstrated that mutations in ftsZ could
suppress filamentation resulting from SulA accumulation, suggesting that FtsZ could be
the direct target of SulA (95, 96). In addition, overexpression of FtsZ rescued the
inhibition of cell division following UV treatment in lon mutants, further establishing a
link between SulA function and FtsZ (97). An interaction between SulA and FtsZ was
detected using a yeast two-hybrid assay (98). Moreover, several mutations in sulA that
abolished its ability to inhibit cell division also abolished the interaction with FtsZ, and
mutations in ftsZ that are not susceptible to SulA overexpression also abolished their
interaction (98). Experiments using an FtsZ polymerization assay in vitro demonstrated
that SulA could directly inhibit FtsZ polymerization (96, 99). Mutations in SulA that
cannot block cell division failed to inhibit FtsZ polymerization, whereas mutations in
FtsZ that are refractory to SulA accumulation were insensitive to SulA in vitro (96, 99).
A detailed kinetic analysis of SulA inhibition of FtsZ polymerization revealed that SulA
increased the concentration of FtsZ required for polymerization and that SulA interac-
tion with FtsZ functions by sequestering FtsZ monomers (100). Therefore, increased
SulA production during SOS results in temporary inhibition of FtsZ ring assembly,
thereby delaying cell division until the SOS response is repressed by LexA and SulA is
degraded by Lon (Fig. 2).

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The accumulation of SulA is reversed by Lon protease activity. As mentioned above,

sulA was originally identified by isolation of a mutation that suppressed DNA damage
sensitivity in a lon mutant (86, 87). The Lon protein was later determined to be an
ATP-dependent protease (101), leading to the hypothesis that SulA could be a substrate
of Lon. Indeed, SulA was found to be highly unstable in wild-type cells; however, in a
lon mutant, SulA was stabilized, indicating that SulA stability is regulated by Lon (92).
Intriguingly, despite a significant increase in SulA stability in lon mutants, pulse-chase
labeling experiments demonstrated that SulA was still susceptible to proteolysis in vivo
(92). Subsequent studies identified a second ATP-dependent protease, ClpYQ, that

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reduces the stability of SulA in vivo and degrades SulA in vitro (102–104). Although the
evidence that Lon protease activity regulated SulA stability in vivo was convincing, it
was not yet known if SulA was a direct substrate of Lon protease. Studies using purified
Lon protease demonstrated that SulA was indeed a direct substrate (105). Thus, Lon
protease provides recovery from the DNA damage checkpoint imposed by SulA,
thereby allowing for cell division to proceed (Fig. 2).

SMALL MEMBRANE-BOUND SOS-INDUCED CELL DIVISION INHIBITORS

The identification of a DNA damage checkpoint in bacteria was an important
conceptual advance; however, extrapolation of the mechanism beyond E. coli has
proven challenging. The cell division inhibitor SulA is not well conserved among
bacteria (106, 107). Nonetheless, treatment with DNA-damaging agents does lead to
cell filamentation in B. subtilis and many other bacteria (108). The SOS response is
conserved in B. subtilis; however, sulA is not (106). Therefore, it was unclear what gene
product(s) acted as the inhibitor in B. subtilis and other Gram-positive bacteria. To
identify the gene coding for the cell division inhibitor, a microarray was performed
using wild-type and lexA mutant cells, reasoning that if there is an SOS-dependent cell
division inhibitor, it will be highly transcribed in cells lacking the LexA repressor (109).
One of the highly transcribed genes in the lexA mutant was yneA, and Northern blot
analysis confirmed that the yneA operon was induced in the absence of functional LexA
(109). The yneA operon is located adjacent to lexA in the B. subtilis genome, and
inspection of the promoter region of yneA revealed two binding sites for LexA,
providing evidence that LexA regulates yneA expression directly (109). Indeed, purified
LexA can bind to the yneA promoter region (29).
Examination of lexA mutant cells showed that they were significantly longer relative
to the wild-type strain, indicating that cell division is inhibited in the lexA mutant (109).
Deletion of the yneA operon returned the cell length to normal in the lexA mutant
strain, suggesting that yneA is necessary for inhibiting cell division (109). To test if
expression of yneA was sufficient for increased cell length, yneA was placed under the
control of an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible promoter, and cells
were found to increase in length following the addition of IPTG (109). Cell elongation
following DNA damage was mostly dependent on yneA, suggesting that YneA is the
SOS-induced cell division inhibitor (109). Thus, yneA expression is induced by DNA
damage, and inhibition of cell division is dependent on yneA (Fig. 3).
The mechanism of YneA-dependent inhibition of cell division is still not understood.
Given that SulA prevents FtsZ ring assembly (94), it was hypothesized that YneA may
also inhibit FtsZ (109). Visualization of FtsZ following YneA expression showed that FtsZ
rings still form at midcell, suggesting that YneA inhibits a different target (110). YneA
is a small protein containing an N-terminal transmembrane domain and a C-terminal
LysM domain (109, 110). LysM domains often interact with the peptidoglycan cell wall
(111). Expression of YneA causes increased cell length; however, following a shift to a
medium that does not induce YneA expression, cells rapidly divide (110). These
experiments suggest that YneA reversibly inhibits cell division. Alanine scanning mu-
tagenesis of the alpha-helix transmembrane domain demonstrated that several resi-
dues clustering to a single face of the alpha-helix were critical for YneA function in vivo,
suggesting that YneA may interact with one of the proteins with a transmembrane
domain that functions as part of the divisome or the residues are important for some

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FIG 3 DNA damage-dependent cell cycle checkpoint in B. subtilis. When B. subtilis cells encounter DNA
damage (green polygons), the SOS response is activated, and the cell division inhibitor YneA is expressed.
YneA prevents cell division through an unknown mechanism. CtpA (maroon) and DdcP (blue) cleave
YneA, deactivating the DNA damage checkpoint. This figure is adapted from reference 113.

other function of the transmembrane domain (110). The transmembrane domain is

necessary, though not sufficient, for YneA to inhibit cell division (110). Interestingly, Mo
and Burkholder found that YneA was cleaved, releasing the YneA C terminus from the
transmembrane domain into the culture medium (110). Mutation of the putative signal
peptide cleavage site did not appreciably decrease YneA processing, though mutation
to a canonical signal peptide cleavage site resulted in increased YneA processing (110).
Further, a mutation at the C terminus of YneA was found to increase YneA stability
(110). An analysis of cell elongation using these YneA variants demonstrated that YneA
was processed more quickly resulting in shorter cells, whereas the stabilizing mutations
resulted in increased cell length, suggesting that full-length YneA is the active form of
the protein (110). Together, these studies led to a model wherein YneA is the SOS-
dependent cell division inhibitor that establishes the DNA damage checkpoint, and
degradation of YneA by an unknown protease allows for cells to resume cell division
(Fig. 3).
YneA was found to be degraded by two previously uncharacterized proteases in B.
subtilis. YlbL, now named DNA damage checkpoint protease (DdcP), and C-terminal
processing protease A (CtpA) were identified through a genome-wide Tn-seq screen to
render cells sensitive to a broad range of DNA-damaging agents (112). DdcP and CtpA
have N-terminal transmembrane domains, followed by Lon and S41 peptidase domains
at their C termini, respectively (112). This work showed that expression of YneA caused
considerable growth interference in cells lacking ddcP and/or ctpA (112). Genetic
experiments showed that ectopic expression of either ddcP or ctpA could complement
the loss of the other, including the double mutant, which demonstrates overlapping

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functions for DdcP and CtpA (112). YneA protein levels accumulate in the single- or
double-protease-deletion strains, and CtpA was shown to directly cleave YneA in a
purified system (112). Because the proteases are not damage inducible, this work
invokes a model where the proteases are constitutively present in the plasma mem-
brane and cleave YneA when it is produced. Under conditions of SOS induction, YneA
levels increase and saturate the ability of the proteases to cleave YneA, causing
inhibition of cell division. After DNA repair is complete and LexA represses YneA
expression, DdcP and CtpA cleave and clear the remaining YneA, allowing cell division
to resume (Fig. 3 and Table 1) (112). The same Tn-seq screen that discovered DdcP and

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CtpA also found a cytoplasmic protein DdcA that negatively regulates YneA helping to
set the threshold of YneA necessary to induce the checkpoint (113). The discovery of
DdcA strengthens the evidence that bacterial cells have multiple mechanisms to ensure
that enough DNA damage must be detected before engaging the checkpoint.
The human pathogen Staphylococcus aureus contains the sosA gene, which, like
YneA in B. subtilis, is divergently transcribed from the lexA promoter (114). SosA is
composed of 77 amino acids containing an N-terminal transmembrane domain but
lacking a LysM domain (114). SosA expression is induced by DNA damage, and SosA is
the SOS-induced cell division inhibitor in S. aureus (114). Like YneA, SosA accumulates
in cells with a deletion in the protease gene ctpA (114). In S. aureus, SosA appears to be
cleaved by a single protease, and a cytoplasmic negative regulator similar to DdcA has
not been identified. Collectively, these results indicate that membrane-bound SOS-
induced cell division inhibitors are cleaved by membrane-bound proteases in at least
two Gram-positive bacteria.
As other investigations identified the DNA damage checkpoints in bacteria, it
became clear that the mechanism discovered in E. coli was not representative of other
species (107, 109, 114–117). In fact, the use of a small integral membrane-bound
protein similar to YneA in B. subtilis appears to be the more widespread mechanism.
Identification of additional cell division inhibitors has proved challenging because
homology searches have failed to identify homologs in distantly related bacterial
species, requiring empirical identification (116). The differences in DNA damage check-
point mechanisms likely reflect the diversity of bacterial lifestyles, given that the need
to delay cell division in response to DNA damage will depend on growth rate, the
frequency and severity of DNA damage encountered, and the DNA repair capability of
each organism.
The cell division inhibitor Rv2719c was identified by microarray as an SOS-induced
gene in Mycobacterium tuberculosis (118, 119). Rv2719c is located adjacent to the LexA
gene, providing the same gene structure as that for YneA and SosA (120). Many of the
DNA damage-inducible genes in M. tuberculosis are induced independently of RecA
through an unknown mechanism (118). Rv2719c is DNA damage inducible, and its
regulation is also independent of RecA (119). A mutational analysis identified a pro-
moter upstream of the LexA binding sites that is important for reporter expression,
indicating that Rv2719c can be regulated from the upstream promoter, allowing
transcription through the LexA binding sites even when LexA is intact (120). Further
analysis of Rv2719c expression and cell length demonstrated that increased ex-
pression is correlated with increased cell length, and specific overexpression of
Rv2719c is sufficient to cause an increase in cell length (115). Increased expression
of Rv2719c does not appear to affect FtsZ, which is similar to YneA and SosA (115).
The Rv2719c protein contains a transmembrane domain and a LysM domain similar
to YneA. Purified Rv2719c was found to possess cell wall hydrolase activity in vitro,
and expression of a GFP-Rv2719c fusion showed localization similar to nascent
peptidoglycan synthesis, suggesting that inhibitor activity may be exerted on the cell
wall (115). Overall, Rv2719c is a DNA damage-inducible cell division inhibitor that may
act on the cell wall or cell wall synthesis machinery to establish the DNA damage
checkpoint in mycobacteria.
The observation that SOS-induced cell division inhibitors are located adjacent to
lexA in the genomes of their respective organisms and that they are transcribed

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Minireview Journal of Bacteriology

divergently is a common theme that holds true for the cell division inhibitor in
Corynebacterium glutamicum (117). The divS gene in C. glutamicum is located adjacent
to lexA, and divS expression is dependent on RecA (117). Cell elongation following
treatment with DNA-damaging agents is dependent on recA and divS (117). An analysis
of the promoter uncovered LexA binding sites, and LexA binds to the promoter region
in vitro (121). Overexpression of DivS resulted in increased cell length, indicating that
increased DivS expression is sufficient to inhibit cell division (117). The DivS protein has
three domains, an N-terminal domain, a transmembrane domain, and a C-terminal
domain (Table 1). The N terminus was found to be dispensable for cell division

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inhibition, whereas nine amino acids at the C terminus were required (117). The sites
of nascent peptidoglycan synthesis and formation of FtsZ rings were found to decrease
following DNA damage, requiring DivS (117). Overexpression of DivS also resulted in
decreased sites of nascent peptidoglycan synthesis, though the exact mechanism by
which DivS activates the DNA damage checkpoint and prevents cell division is unclear.
It is also unclear how DivS inhibition is relieved, although a proteolysis-dependent
mechanism has been shown for all other bacterial systems where the mechanism is
known (Table 1) (112, 114).
The discovery of an SOS-inducible cell division inhibitor in Caulobacter crescentus
required the use of microarrays because the inhibitor is not located near lexA in the
genome (107). Expression of the gene encoding the cell division inhibitor, sidA,
increased rapidly following mitomycin C (MMC) or UV light, and inspection of the
promoter revealed putative LexA binding sites (107). The deletion of lexA resulted in
severe cell elongation that could be rescued by the deletion of sidA, and overexpression
of sidA resulted in significant cell elongation (107), demonstrating that SidA overex-
pression is sufficient to inhibit cell division (107). SidA is a 29-amino-acid protein
composed of a single transmembrane domain (Fig. 4 and Table 1) (107). A genetic
suppressor screen identified mutations in ftsW and ftsI, genes coding for two essential
components of the divisome involved in peptidoglycan synthesis at the division
septum, which suppress cell division inhibition from overexpressing SidA (107). SidA
was found to interact with FtsW in a bacterial two-hybrid assay (107). Evidence was
presented suggesting that SidA can form a complex with FtsW and FtsI, indicating that
the interaction of SidA with FtsW does not occlude binding of FtsI (107). SidA also did
not alter the localization or recruitment of these divisome proteins to midcell, and
experiments using fluorescently labeled vancomycin did not find a defect in septal
peptidoglycan synthesis following SidA overexpression (107). Together, these data
support a model wherein SidA is an SOS-induced cell division inhibitor that inhibits the
activity of the divisome to establish the DNA damage checkpoint. We suggest that the
membrane-bound SOS-induced cell division inhibitors, including SidA, are likely to be
inactivated through a protease-dependent mechanism, as demonstrated for YneA and
SosA (112, 114).

DNA DAMAGE-DEPENDENT SOS-INDEPENDENT TRANSCRIPTIONAL REGULATOR

A subsequent study in C. crescentus identified a second SOS-independent cell
division inhibitor, DidA, whose expression is DNA damage inducible, and DidA acts by
inhibiting the divisome (116). DidA is also a small protein containing a single trans-
membrane domain (Fig. 4). The authors identified a new transcription factor, DriD, that
positively regulates didA expression. Interestingly, DriD is activated via a DNA damage-
dependent mechanism, though it is still unclear how DNA damage activates DriD (116).
Therefore, in C. crescentus, there are two cell division inhibitors that interact with the
divisome to prevent cell division following DNA damage, establishing the checkpoint.
This finding is important because it suggests that other bacteria may use transcriptional
activators induced by DNA damage to upregulate the expression of cell division
inhibitors. DNA damage-activated transcription factors, like DriD, present an attractive
mechanism for bacteria that lack LexA to control the expression of their cognate cell
division inhibitor and possibly other genes canonically regulated by LexA. The identi-
fication of DriD also presents the possibility that translesion DNA polymerases involved

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Minireview Journal of Bacteriology

C
N
C
FtsI
FtsN

FtsW
Cytoplasm
N

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N
C

DNA Damage

C
N N
C
FtsI
FtsN
C
FtsW

SidA Cytoplasm
N N
N
DidA
C C

FIG 4 Model for SidA and DidA inhibition of cell division in Caulobacter spp. following DNA damage. SidA
is regulated by LexA, and DidA is regulated by the transcription factor DriD. Following DNA damage, SidA
(red) and DidA (purple) are expressed and interact with FtsW and FtsN, respectively (116). These
interactions are hypothesized to cause the FtsW/I/N complex to assume an inactive state (116). The
inhibition of FtsW/I/N prevents cytokinesis. This figure was adapted from PLoS Biology (116). The
orientation of integral membrane proteins as well as membrane-spanning regions were predicted using
Protter (122). C, C terminus; N, N terminus.

in DNA damage-dependent mutagenesis could be regulated by a DriD-type transcrip-

tional activator in bacteria that lack the classically defined LexA-dependent SOS re-
sponse. Future experiments could be directed toward understanding how the DNA
damage signal is transduced to DriD and if organisms that lack LexA regulate their DDR
through activation of a transcription factor such as DriD.

CONCLUSIONS
Bacterial cells have evolved several methods to prevent cell division when chromo-
some replication is incomplete or genome instability occurs. Although inhibition of FtsZ
by the cytoplasmic SulA protein of E. coli has served as the prototypical DNA damage-
inducible cell division inhibitor, we now know that most bacteria employ integral
membrane-bound proteins to enforce the DNA damage checkpoint (107, 109, 114–
117). Despite recent advances, the mechanism(s) of establishing the DNA damage
checkpoint by membrane-bound cell division inhibitors remains unclear. Future work
will be necessary to identify the targets for most of the inhibitors, as well as the
mechanism of inhibition exerted by SidA and DidA on FtsW/I/N. Nonetheless, it is
notable that regardless of the type of inhibitor used, deactivation of the checkpoint
involves proteolysis of the inhibitor. This suggests that even though bacterial species
have evolved different mechanisms for checkpoint enforcement, they arrived at a
common mechanism for checkpoint deactivation, ensuring rapid recovery and com-
pletion of cell division.

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Minireview
ACKNOWLEDGMENTS
We thank the anonymous referees and the editor for comments on our manuscript.
Research in the laboratory of L.A.S. is currently supported by National Institutes of
Health grant R35 GM131772. P.E.B. was supported by a predoctoral fellowship from the
National Science Foundation (grant DGE1256260) and a fellowship from the Rackham
Graduate School at the University of Michigan.
REFERENCES
1. Wang JD, Levin PA. 2009. Metabolism, cell growth and the bacterial cell
cycle. Nat Rev Microbiol 7:822–827. https://doi.org/10.1038/nrmicro2202.
2. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T.
2006. DNA repair and mutagenesis, 2nd ed. ASM Press, Washington,
DC, p 463– 497.
3. Chatterjee N, Walker GC. 2017. Mechanisms of DNA damage, repair,
and mutagenesis. Environ Mol Mutagen 58:235–263. https://doi.org/10
.1002/em.22087.
4. Ciccia A, Elledge SJ. 2010. The DNA damage response: making it safe to
play with knives. Mol Cell 40:179 –204. https://doi.org/10.1016/j.molcel
.2010.09.019.
5. Blanpain C, Mohrin M, Sotiropoulou PA, Passegue E. 2011. DNA-
damage response in tissue-specific and cancer stem cells. Cell Stem Cell
8:16 –29. https://doi.org/10.1016/j.stem.2010.12.012.
6. Demain AL, Vaishnav P. 2011. Natural products for cancer chemother-
apy. Microb Biotechnol 4:687– 699. https://doi.org/10.1111/j.1751-7915
.2010.00221.x.
7. Beukers R, Berends W. 1960. Isolation and identification of the irradia-
tion product of thymine. Biochim Biophys Acta 41:550 –551. https://doi
.org/10.1016/0006-3002(60)90063-9.
8. Setlow RB, Swenson PA, Carrier WL. 1963. Thymine dimers and inhibi-
tion of DNA synthesis by ultraviolet irradiation of cells. Science 142:
1464 –1466. https://doi.org/10.1126/science.142.3598.1464.
9. Ling H, Boudsocq F, Plosky BS, Woodgate R, Yang W. 2003. Replication
of a cis-syn thymine dimer at atomic resolution. Nature 424:1083–1087.
https://doi.org/10.1038/nature01919.
10. Doublie S, Tabor S, Long AM, Richardson CC, Ellenberger T. 1998.
Crystal structure of a bacteriophage T7 DNA replication complex at 2.2
Å resolution. Nature 391:251–258. https://doi.org/10.1038/34593.
11. Kiefer JR, Mao C, Braman JC, Beese LS. 1998. Visualizing DNA replication
in a catalytically active Bacillus DNA polymerase crystal. Nature 391:
304 –307. https://doi.org/10.1038/34693.
12. Johnson RE, Prakash L, Prakash S. 2005. Distinct mechanisms of cis-syn
thymine dimer bypass by Dpo4 and DNA polymerase eta. Proc
Natl Acad Sci U S A 102:12359 –12364. https://doi.org/10.1073/pnas
.0504380102.
13. Sedgwick B. 2004. Repairing DNA-methylation damage. Nat Rev Mol
Cell Biol 5:148 –157. https://doi.org/10.1038/nrm1312.
14. Iyer VN, Szybalski W. 1963. A molecular mechanism of mitomycin
action: linking of complementary DNA strands. Proc Natl Acad Sci
U S A 50:355–362. https://doi.org/10.1073/pnas.50.2.355.
15. Noll DM, Mason TM, Miller PS. 2006. Formation and repair of interstrand
cross-links in DNA. Chem Rev 106:277–301. https://doi.org/10.1021/
cr040478b.
16. Dronkert ML, Kanaar R. 2001. Repair of DNA interstrand cross-links. Mutat
Res 486:217–247. https://doi.org/10.1016/s0921-8777(01)00092-1.
17. Dillingham MS, Kowalczykowski SC. 2008. RecBCD enzyme and the
repair of double-stranded DNA breaks. Microbiol Mol Biol Rev 72:
642– 671, table of contents. https://doi.org/10.1128/MMBR.00020-08.
18. Michel B, Sinha AK, Leach D. 2018. Replication fork breakage and restart
in Escherichia coli. Microbiol Mol Biol Rev 82:e00013-18. https://doi.org/
10.1128/MMBR.00013-18.
19. Erill I, Campoy S, Barbe J. 2007. Aeons of distress: an evolutionary
perspective on the bacterial SOS response. FEMS Microbiol Rev 31:
637– 656. https://doi.org/10.1111/j.1574-6976.2007.00082.x.
20. Kreuzer KN. 2013. DNA damage responses in prokaryotes: regulating
gene expression, modulating growth patterns, and manipulating rep-
lication forks. Cold Spring Harb Perspect Biol 5:a012674. https://doi
.org/10.1101/cshperspect.a012674.
21. Little JW, Mount DW. 1982. The SOS regulatory system of Escherichia
coli. Cell 29:11–22. https://doi.org/10.1016/0092-8674(82)90085-x.
January 2020 Volume 202 Issue 2 e00408-19
Journal of Bacteriology
Downloaded from http://jb.asm.org/ on January 6, 2020 at Imperial College London
22. Simmons LA, Foti JJ, Cohen SE, Walker GC. 2008. The SOS regulatory
network. EcoSal Plus 2008. https://doi.org/10.1128/ecosalplus.5.4.3.
23. Kenyon CJ, Walker GC. 1980. DNA-damaging agents stimulate gene
expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A
77:2819 –2823. https://doi.org/10.1073/pnas.77.5.2819.
24. Kenyon CJ, Walker GC. 1981. Expression of the E. coli uvrA gene is
inducible. Nature 289:808 – 810. https://doi.org/10.1038/289808a0.
25. Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC. 2001.
Comparative gene expression profiles following UV exposure in wild-
type and SOS-deficient Escherichia coli. Genetics 158:41– 64.
26. Fornelos N, Browning DF, Butala M. 2016. The use and abuse of LexA by
mobile genetic elements. Trends Microbiol 24:391– 401. https://doi.org/
10.1016/j.tim.2016.02.009.
27. Schnarr M, Oertel-Buchheit P, Kazmaier M, Granger-Schnarr M. 1991.
DNA binding properties of the LexA repressor. Biochimie 73:423– 431.
https://doi.org/10.1016/0300-9084(91)90109-e.
28. Wade JT, Reppas NB, Church GM, Struhl K. 2005. Genomic analysis of
LexA binding reveals the permissive nature of the Escherichia coli
genome and identifies unconventional target sites. Genes Dev 19:
2619 –2630. https://doi.org/10.1101/gad.1355605.
29. Au N, Kuester-Schoeck E, Mandava V, Bothwell LE, Canny SP, Chachu K,
Colavito SA, Fuller SN, Groban ES, Hensley LA, O’Brien TC, Shah A,
Tierney JT, Tomm LL, O’Gara TM, Goranov AI, Grossman AD, Lovett CM.
2005. Genetic composition of the Bacillus subtilis SOS system. J Bacte-
riol 187:7655–7666. https://doi.org/10.1128/JB.187.22.7655-7666.2005.
30. Lewis LK, Harlow GR, Gregg-Jolly LA, Mount DW. 1994. Identification of
high affinity binding sites for LexA which define new DNA damage-
inducible genes in Escherichia coli. J Mol Biol 241:507–523. https://doi
.org/10.1006/jmbi.1994.1528.
31. Brent R, Ptashne M. 1981. Mechanism of action of the lexA gene
product. Proc Natl Acad Sci U S A 78:4204 – 4208. https://doi.org/10
.1073/pnas.78.7.4204.
32. Brent R, Ptashne M. 1980. The lexA gene product represses its own
promoter. Proc Natl Acad Sci U S A 77:1932–1936. https://doi.org/10
.1073/pnas.77.4.1932.
33. Little JW, Mount DW, Yanisch-Perron CR. 1981. Purified lexA protein is
a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A
78:4199 – 4203. https://doi.org/10.1073/pnas.78.7.4199.
34. Sancar A, Sancar GB, Rupp WD, Little JW, Mount DW. 1982. LexA
protein inhibits transcription of the E. coli uvrA gene in vitro. Nature
298:96 –98. https://doi.org/10.1038/298096a0.
35. Witkin EM. 1991. RecA protein in the SOS response: milestones and
mysteries. Biochimie 73:133–141. https://doi.org/10.1016/0300-9084
(91)90196-8.
36. Bell JC, Kowalczykowski SC. 2016. RecA: regulation and mechanism of
a molecular search engine. Trends Biochem Sci 41:491–507. https://doi
.org/10.1016/j.tibs.2016.04.002.
37. Moreau PL. 1985. Role of Escherichia coli RecA protein in SOS induction
and post-replication repair. Biochimie 67:353–356. https://doi.org/10
.1016/s0300-9084(85)80079-1.
38. Little JW, Edmiston SH, Pacelli LZ, Mount DW. 1980. Cleavage of the
Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci
U S A 77:3225–3229. https://doi.org/10.1073/pnas.77.6.3225.
39. Little JW. 1984. Autodigestion of lexA and phage lambda repressors.
Proc Natl Acad Sci U S A 81:1375–1379. https://doi.org/10.1073/pnas
.81.5.1375.
40. Simmons LA, Goranov AI, Kobayashi H, Davies BW, Yuan DS, Grossman
AD, Walker GC. 2009. Comparison of responses to double-strand breaks
between Escherichia coli and Bacillus subtilis reveals different require-
ments for SOS induction. J Bacteriol 191:1152–1161. https://doi.org/10
.1128/JB.01292-08.
41. Sassanfar M, Roberts JW. 1990. Nature of the SOS-inducing signal in
jb.asm.org 12
Minireview
Escherichia coli. The involvement of DNA replication. J Mol Biol 212:
79 –96. https://doi.org/10.1016/0022-2836(90)90306-7.
42. Goranov AI, Kuester-Schoeck E, Wang JD, Grossman AD. 2006. Charac-
terization of the global transcriptional responses to different types of
DNA damage and disruption of replication in Bacillus subtilis. J Bacte-
riol 188:5595–5605. https://doi.org/10.1128/JB.00342-06.
43. Ayora S, Carrasco B, Cardenas PP, Cesar CE, Canas C, Yadav T, Marchi-
sone C, Alonso JC. 2011. Double-strand break repair in bacteria: a view
from Bacillus subtilis. FEMS Microbiol Rev 35:1055–1081. https://doi
.org/10.1111/j.1574-6976.2011.00272.x.
44. Bell JC, Kowalczykowski SC. 2016. Mechanics and single-molecule in-
terrogation of DNA recombination. Annu Rev Biochem 85:193–226.
https://doi.org/10.1146/annurev-biochem-060614-034352.
45. Chédin F, Ehrlich SD, Kowalczykowski SC. 2000. The Bacillus subtilis
AddAB helicase/nuclease is regulated by its cognate Chi sequence in
vitro. J Mol Biol 298:7–20. https://doi.org/10.1006/jmbi.2000.3556.
46. Gupta R, Barkan D, Redelman-Sidi G, Shuman S, Glickman MS. 2011.
Mycobacteria exploit three genetically distinct DNA double-strand
break repair pathways. Mol Microbiol 79:316 –330. https://doi.org/10
.1111/j.1365-2958.2010.07463.x.
47. Taylor AF, Smith GR. 1985. Substrate specificity of the DNA unwinding
activity of the RecBC enzyme of Escherichia coli. J Mol Biol 185:
431– 443. https://doi.org/10.1016/0022-2836(85)90414-0.
48. Anderson DG, Kowalczykowski SC. 1997. The translocating RecBCD
enzyme stimulates recombination by directing RecA protein onto ss-
DNA in a chi-regulated manner. Cell 90:77– 86. https://doi.org/10.1016/
S0092-8674(00)80315-3.
49. Churchill JJ, Anderson DG, Kowalczykowski SC. 1999. The RecBC en-
zyme loads RecA protein onto ssDNA asymmetrically and indepen-
dently of chi, resulting in constitutive recombination activation. Genes
Dev 13:901–911. https://doi.org/10.1101/gad.13.7.901.
50. Churchill JJ, Kowalczykowski SC. 2000. Identification of the RecA
protein-loading domain of RecBCD enzyme. J Mol Biol 297:537–542.
https://doi.org/10.1006/jmbi.2000.3590.
51. Dixon DA, Kowalczykowski SC. 1993. The recombination hotspot ␹ is a
regulatory sequence that acts by attenuating the nuclease activity of
the E. coli RecBCD enzyme. Cell 73:87–96. https://doi.org/10.1016/0092
-8674(93)90162-J.
52. Taylor AF, Schultz DW, Ponticelli AS, Smith GR. 1985. RecBC enzyme
nicking at Chi sites during DNA unwinding: location and orientation-
dependence of the cutting. Cell 41:153–163. https://doi.org/10.1016/
0092-8674(85)90070-4.
53. Simmons LA, Grossman AD, Walker GC. 2007. Replication is required
for the RecA localization response to DNA damage in Bacillus
subtilis. Proc Natl Acad Sci U S A 104:1360 –1365. https://doi.org/10
.1073/pnas.0607123104.
54. Indiani C, O’Donnell M. 2013. A proposal: source of single strand DNA
that elicits the SOS response. Front Biosci 18:312–323. https://doi.org/
10.2741/4102.
55. Pomerantz RT, O’Donnell M. 2008. The replisome uses mRNA as a
primer after colliding with RNA polymerase. Nature 456:762–766.
https://doi.org/10.1038/nature07527.
56. Heller RC, Marians KJ. 2006. Replication fork reactivation downstream
of a blocked nascent leading strand. Nature 439:557–562. https://doi
.org/10.1038/nature04329.
57. Dutra BE, Lovett ST. 2006. Cis and trans-acting effects on a mutational
hotspot involving a replication template switch. J Mol Biol 356:
300 –311. https://doi.org/10.1016/j.jmb.2005.11.071.
58. Laranjo LT, Klaric JA, Pearlman LR, Lovett ST. 2018. Stimulation of
replication template-switching by DNA-protein crosslinks. Genes (Ba-
sel) 10:E14. https://doi.org/10.3390/genes10010014.
59. Morimatsu K, Kowalczykowski SC. 2003. RecFOR proteins load RecA
protein onto gapped DNA to accelerate DNA strand exchange: a
universal step of recombinational repair. Mol Cell 11:1337–1347.
https://doi.org/10.1016/s1097-2765(03)00188-6.
60. Ivancic-Bace I, Vlasic I, Salaj-Smic E, Brcic-Kostic K. 2006. Genetic evi-
dence for the requirement of RecA loading activity in SOS induction
after UV irradiation in Escherichia coli. J Bacteriol 188:5024 –5032.
https://doi.org/10.1128/JB.00130-06.
61. Adams DW, Wu LJ, Errington J. 2014. Cell cycle regulation by the
bacterial nucleoid. Curr Opin Microbiol 22:94 –101. https://doi.org/10
.1016/j.mib.2014.09.020.
62. Wu LJ, Errington J. 2011. Nucleoid occlusion and bacterial cell division.
Nat Rev Microbiol 10:8 –12. https://doi.org/10.1038/nrmicro2671.
January 2020 Volume 202 Issue 2 e00408-19
Journal of Bacteriology
63. Bernhardt TG, de Boer PA. 2005. SlmA, a nucleoid-associated, FtsZ
binding protein required for blocking septal ring assembly over Chro-
mosomes in E. coli. Mol Cell 18:555–564. https://doi.org/10.1016/j
.molcel.2005.04.012.
64. Rowlett VW, Margolin W. 2013. The bacterial Min system. Curr Biol
23:R553– 6. https://doi.org/10.1016/j.cub.2013.05.024.
65. Tonthat NK, Arold ST, Pickering BF, Van Dyke MW, Liang S, Lu Y, Beuria
TK, Margolin W, Schumacher MA. 2011. Molecular mechanism by which
the nucleoid occlusion factor, SlmA, keeps cytokinesis in check. EMBO
J 30:154 –164. https://doi.org/10.1038/emboj.2010.288.
66. Cho H, McManus HR, Dove SL, Bernhardt TG. 2011. Nucleoid occlusion
Downloaded from http://jb.asm.org/ on January 6, 2020 at Imperial College London
factor SlmA is a DNA-activated FtsZ polymerization antagonist. Proc
Natl Acad Sci U S A 108:3773–3778. https://doi.org/10.1073/pnas
.1018674108.
67. Adams DW, Errington J. 2009. Bacterial cell division: assembly, mainte-
nance and disassembly of the Z ring. Nat Rev Microbiol 7:642– 653.
https://doi.org/10.1038/nrmicro2198.
68. Schumacher MA, Zeng W. 2016. Structures of the nucleoid occlusion
protein SlmA bound to DNA and the C-terminal domain of the cyto-
skeletal protein FtsZ. Proc Natl Acad Sci U S A 113:4988 – 4993. https://
doi.org/10.1073/pnas.1602327113.
69. Monterroso B, Zorrilla S, Sobrinos᎑Sanguino M, Robles᎑Ramos MA,
López᎑Álvarez M, Margolin W, Keating CD, Rivas G. 2019. Bacterial FtsZ
protein forms phase-separated condensates with its nucleoid-
associated inhibitor SlmA. EMBO Rep 20:e45946. https://doi.org/10
.15252/embr.201845946.
70. Wu LJ, Errington J. 2004. Coordination of cell division and chromosome
segregation by a nucleoid occlusion protein in Bacillus subtilis. Cell
117:915–925. https://doi.org/10.1016/j.cell.2004.06.002.
71. Wu LJ, Ishikawa S, Kawai Y, Oshima T, Ogasawara N, Errington J. 2009.
Noc protein binds to specific DNA sequences to coordinate cell division
with chromosome segregation. EMBO J 28:1940 –1952. https://doi.org/
10.1038/emboj.2009.144.
72. Adams DW, Wu LJ, Errington J. 2015. Nucleoid occlusion protein Noc
recruits DNA to the bacterial cell membrane. EMBO J 34:491–501.
https://doi.org/10.15252/embj.201490177.
73. Veiga H, Jorge AM, Pinho MG. 2011. Absence of nucleoid occlusion
effector Noc impairs formation of orthogonal FtsZ rings during Staph-
ylococcus aureus cell division. Mol Microbiol 80:1366 –1380. https://doi
.org/10.1111/j.1365-2958.2011.07651.x.
74. Pang T, Wang X, Lim HC, Bernhardt TG, Rudner DZ. 2017. The nucleoid
occlusion factor Noc controls DNA replication initiation in Staphylococ-
cus aureus. PLoS Genet 13:e1006908. https://doi.org/10.1371/journal
.pgen.1006908.
75. Bernard R, Marquis KA, Rudner DZ. 2010. Nucleoid occlusion prevents
cell division during replication fork arrest in Bacillus subtilis. Mol Mi-
crobiol 78:866 – 882. https://doi.org/10.1111/j.1365-2958.2010.07369.x.
76. Goranov AI, Katz L, Breier AM, Burge CB, Grossman AD. 2005. A tran-
scriptional response to replication status mediated by the conserved
bacterial replication protein DnaA. Proc Natl Acad Sci U S A 102:
12932–12937. https://doi.org/10.1073/pnas.0506174102.
77. Robson SA, Michie KA, Mackay JP, Harry E, King GF. 2002. The Bacillus
subtilis cell division proteins FtsL and DivIC are intrinsically unstable
and do not interact with one another in the absence of other septa-
somal components. Mol Microbiol 44:663– 674. https://doi.org/10
.1046/j.1365-2958.2002.02920.x.
78. Daniel RA, Errington J. 2000. Intrinsic instability of the essential cell
division protein FtsL of Bacillus subtilis and a role for DivIB protein in
FtsL turnover. Mol Microbiol 36:278 –289. https://doi.org/10.1046/j
.1365-2958.2000.01857.x.
79. Bramkamp M, Weston L, Daniel RA, Errington J. 2006. Regulated in-
tramembrane proteolysis of FtsL protein and the control of cell division
in Bacillus subtilis. Mol Microbiol 62:580 –591. https://doi.org/10.1111/
j.1365-2958.2006.05402.x.
80. Parrell D, Zhang Y, Olenic S, Kroos L. 2017. Bacillus subtilis intramem-
brane protease RasP activity in Escherichia coli and in vitro. J Bacteriol
199:e00381-17. https://doi.org/10.1128/JB.00381-17.
81. Wadenpohl I, Bramkamp M. 2010. DivIC stabilizes FtsL against RasP cleav-
age. J Bacteriol 192:5260–5263. https://doi.org/10.1128/JB.00287-10.
82. Hanawalt P, Setlow R. 1960. Effect of monochromatic ultraviolet light
on macromolecular synthesis in Escherichia coli. Biochim Biophys Acta
41:283–294. https://doi.org/10.1016/0006-3002(60)90011-1.
83. Gates FL. 1933. The reaction of individual bacteria to irradiation with
jb.asm.org 13
Minireview
ultraviolet light. Science 77:350. https://doi.org/10.1126/science.77
.1997.350.
84. Witkin EM. 1967. The radiation sensitivity of Escherichia coli B: a hy-
pothesis relating filament formation and prophage induction. Proc Natl
Acad Sci U S A 57:1275–1279. https://doi.org/10.1073/pnas.57.5.1275.
85. George J, Castellazzi M, Buttin G. 1975. Prophage induction and cell
division in E. coli. III. Mutations sfiA and sfiB restore division in tif and
lon strains and permit the expression of mutator properties of tif. Mol
Gen Genet 140:309 –332.
86. Gayda RC, Yamamoto LT, Markovitz A. 1976. Second-site mutations in
capR (lon) strains of Escherichia coli K-12 that prevent radiation sensi-
tivity and allow bacteriophage lambda to lysogenize. J Bacteriol 127:
1208 –1216.
87. Huisman O, D’Ari R, George J. 1980. Further characterization of sfiA and
sfiB mutations in Escherichia coli. J Bacteriol 144:185–191.
88. Huisman O, D’Ari R. 1981. An inducible DNA replication-cell division
coupling mechanism in E. coli. Nature 290:797–799. https://doi.org/10
.1038/290797a0.
89. Cole ST. 1983. Characterisation of the promoter for the LexA regulated
sulA gene of Escherichia coli. Mol Gen Genet 189:400 – 404. https://doi
.org/10.1007/bf00325901.
90. Mizusawa S, Court D, Gottesman S. 1983. Transcription of the sulA gene
and repression by LexA. J Mol Biol 171:337–343. https://doi.org/10
.1016/0022-2836(83)90097-9.
91. Huisman O, D’Ari R, Gottesman S. 1984. Cell-division control in Esche-
richia coli: specific induction of the SOS function SfiA protein is suffi-
cient to block septation. Proc Natl Acad Sci U S A 81:4490 – 4494.
https://doi.org/10.1073/pnas.81.14.4490.
92. Mizusawa S, Gottesman S. 1983. Protein degradation in Escherichia coli:
the lon gene controls the stability of sulA protein. Proc Natl Acad Sci
U S A 80:358 –362. https://doi.org/10.1073/pnas.80.2.358.
93. Schoemaker JM, Gayda RC, Markovitz A. 1984. Regulation of cell divi-
sion in Escherichia coli: SOS induction and cellular location of the sulA
protein, a key to lon-associated filamentation and death. J Bacteriol
158:551–561.
94. Bi E, Lutkenhaus J. 1993. Cell division inhibitors SulA and MinCD
prevent formation of the FtsZ ring. J Bacteriol 175:1118 –1125. https://
doi.org/10.1128/jb.175.4.1118-1125.1993.
95. Bi E, Lutkenhaus J. 1990. Analysis of ftsZ mutations that confer resis-
tance to the cell division inhibitor SulA (SfiA). J Bacteriol 172:
5602–5609. https://doi.org/10.1128/jb.172.10.5602-5609.1990.
96. Mukherjee A, Cao C, Lutkenhaus J. 1998. Inhibition of FtsZ polymeriza-
tion by SulA, an inhibitor of septation in Escherichia coli. Proc Natl Acad
Sci U S A 95:2885–2890. https://doi.org/10.1073/pnas.95.6.2885.
97. Lutkenhaus J, Sanjanwala B, Lowe M. 1986. Overproduction of FtsZ
suppresses sensitivity of lon mutants to division inhibition. J Bacteriol
166:756 –762. https://doi.org/10.1128/jb.166.3.756-762.1986.
98. Huang J, Cao C, Lutkenhaus J. 1996. Interaction between FtsZ and
inhibitors of cell division. J Bacteriol 178:5080 –5085. https://doi.org/10
.1128/jb.178.17.5080-5085.1996.
99. Trusca D, Scott S, Thompson C, Bramhill D. 1998. Bacterial SOS check-
point protein SulA inhibits polymerization of purified FtsZ cell division
protein. J Bacteriol 180:3946 –3953.
100. Chen Y, Milam SL, Erickson HP. 2012. SulA inhibits assembly of FtsZ by a
simple sequestration mechanism. Biochemistry 51:3100 –3109. https://
doi.org/10.1021/bi201669d.
101. Chung CH, Goldberg AL. 1981. The product of the lon (capR) gene in
Escherichia coli is the ATP-dependent protease, protease La. Proc Natl
Acad Sci U S A 78:4931– 4935. https://doi.org/10.1073/pnas.78.8.4931.
102. Kanemori M, Yanagi H, Yura T. 1999. The ATP-dependent HslVU/ClpQY
protease participates in turnover of cell division inhibitor SulA in
Escherichia coli. J Bacteriol 181:3674 –3680.
103. Wu WF, Zhou Y, Gottesman S. 1999. Redundant in vivo proteolytic
activities of Escherichia coli Lon and the ClpYQ (HslUV) protease. J
Bacteriol 181:3681–3687.
104. Seong IS, Oh JY, Yoo SJ, Seol JH, Chung CH. 1999. ATP-dependent
degradation of SulA, a cell division inhibitor, by the HslVU protease in
Escherichia coli. FEBS Lett 456:211–214. https://doi.org/10.1016/s0014
-5793(99)00935-7.
January 2020 Volume 202 Issue 2 e00408-19
Journal of Bacteriology
105. Sonezaki S, Ishii Y, Okita K, Sugino T, Kondo A, Kato Y. 1995. Overpro-
duction and purification of SulA fusion protein in Escherichia coli and
its degradation by Lon protease in vitro. Appl Microbiol Biotechnol
43:304 –309. https://doi.org/10.1007/bf00172829.
106. Yasbin RE, Cheo D, Bayles KW. 1991. The SOB system of Bacillus subtilis:
a global regulon involved in DNA repair and differentiation. Res Micro-
biol 142:885– 892. https://doi.org/10.1016/0923-2508(91)90069-m.
107. Modell JW, Hopkins AC, Laub MT. 2011. A DNA damage checkpoint in
Caulobacter crescentus inhibits cell division through a direct interac-
tion with FtsW. Genes Dev 25:1328 –1343. https://doi.org/10.1101/gad
.2038911.
Downloaded from http://jb.asm.org/ on January 6, 2020 at Imperial College London
108. Love PE, Yasbin RE. 1984. Genetic characterization of the inducible
SOS-like system of Bacillus subtilis. J Bacteriol 160:910 –920.
109. Kawai Y, Moriya S, Ogasawara N. 2003. Identification of a protein, YneA,
responsible for cell division suppression during the SOS response in
Bacillus subtilis. Mol Microbiol 47:1113–1122. https://doi.org/10.1046/j
.1365-2958.2003.03360.x.
110. Mo AH, Burkholder WF. 2010. YneA, an SOS-induced inhibitor of cell
division in Bacillus subtilis, is regulated posttranslationally and requires
the transmembrane region for activity. J Bacteriol 192:3159 –3173.
https://doi.org/10.1128/JB.00027-10.
111. Buist G, Steen A, Kok J, Kuipers OP. 2008. LysM, a widely distributed
protein motif for binding to (peptido)glycans. Mol Microbiol 68:
838 – 847. https://doi.org/10.1111/j.1365-2958.2008.06211.x.
112. Burby PE, Simmons ZW, Schroeder JW, Simmons LA. 2018. Discovery of
a dual protease mechanism that promotes DNA damage checkpoint
recovery. PLoS Genet 14:e1007512. https://doi.org/10.1371/journal
.pgen.1007512.
113. Burby PE, Simmons ZW, Simmons LA. 2019. DdcA antagonizes a bac-
terial DNA damage checkpoint. Mol Microbiol 111:237–253. https://doi
.org/10.1111/mmi.14151.
114. Bojer MS, Wacnik K, Kjelgaard P, Gallay C, Bottomley AL, Cohn MT,
Lindahl G, Frees D, Veening JW, Foster SJ, Ingmer H. 2019. SosA inhibits
cell division in Staphylococcus aureus in response to DNA damage. Mol
Microbiol 112:1116 –1130. https://doi.org/10.1111/mmi.14350.
115. Chauhan A, Lofton H, Maloney E, Moore J, Fol M, Madiraju MV, Rajago-
palan M. 2006. Interference of Mycobacterium tuberculosis cell division
by Rv2719c, a cell wall hydrolase. Mol Microbiol 62:132–147. https://
doi.org/10.1111/j.1365-2958.2006.05333.x.
116. Modell JW, Kambara TK, Perchuk BS, Laub MT. 2014. A DNA damage-
induced, SOS-independent checkpoint regulates cell division in Caulo-
bacter crescentus. PLoS Biol 12:e1001977. https://doi.org/10.1371/
journal.pbio.1001977.
117. Ogino H, Teramoto H, Inui M, Yukawa H. 2008. DivS, a novel SOS-
inducible cell-division suppressor in Corynebacterium glutamicum.
Mol Microbiol 67:597– 608. https://doi.org/10.1111/j.1365-2958.2007
.06069.x.
118. Rand L, Hinds J, Springer B, Sander P, Buxton RS, Davis EO. 2003. The
majority of inducible DNA repair genes in Mycobacterium tuberculosis
are induced independently of RecA. Mol Microbiol 50:1031–1042.
https://doi.org/10.1046/j.1365-2958.2003.03765.x.
119. Brooks PC, Dawson LF, Rand L, Davis EO. 2006. The Mycobacterium-
specific gene Rv2719c is DNA damage inducible independently of
RecA. J Bacteriol 188:6034 – 6038. https://doi.org/10.1128/JB.00340-06.
120. Dullaghan EM, Brooks PC, Davis EO. 2002. The role of multiple SOS
boxes upstream of the Mycobacterium tuberculosis lexA gene—
identification of a novel DNA-damage-inducible gene. Microbiology
148:3609 –3615. https://doi.org/10.1099/00221287-148-11-3609.
121. Jochmann N, Kurze AK, Czaja LF, Brinkrolf K, Brune I, Huser AT, Hans-
meier N, Puhler A, Borovok I, Tauch A. 2009. Genetic makeup of the
Corynebacterium glutamicum LexA regulon deduced from compara-
tive transcriptomics and in vitro DNA band shift assays. Microbiology
155:1459 –1477. https://doi.org/10.1099/mic.0.025841-0.
122. Omasits U, Ahrens CH, Muller S, Wollscheid B. 2014. Protter: interactive
protein feature visualization and integration with experimental proteomic
data. Bioinformatics 30:884–886. https://doi.org/10.1093/bioinformatics/
btt607.
jb.asm.org 14