A DNA Damage-Induced, SOS-Independent Checkpoint Regulates Cell Division in Caulobacter crescentus

Cells must coordinate DNA replication with cell division, especially during episodes of DNA damage. The paradigm for cell division control following DNA damage in bacteria involves the SOS response where cleavage of the transcriptional repressor LexA induces a division inhibitor. However, in Caulobacter crescentus, cells lacking the primary SOS-regulated inhibitor, sidA, can often still delay division post-damage. Here we identify didA, a second cell division inhibitor that is induced by DNA damage, but in an SOS-independent manner. Together, DidA and SidA inhibit division, such that cells lacking both inhibitors divide prematurely following DNA damage, with lethal consequences. We show that DidA does not disrupt assembly of the division machinery and instead binds the essential division protein FtsN to block cytokinesis. Intriguingly, mutations in FtsW and FtsI, which drive the synthesis of septal cell wall material, can suppress the activity of both SidA and DidA, likely by causing the FtsW/I/N complex to hyperactively initiate cell division. Finally, we identify a transcription factor, DriD, that drives the SOS-independent transcription of didA following DNA damage.National Institutes of Health (U.S.) (Grant R01GM082899)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Control of a Programmed Cell Death Pathway in Pseudomonas aeruginosa by an Antiterminator

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream. AlpA thus functions in a mechanistically unusual manner to control the expression of virulence genes in this opportunistic pathogen

A Self-Lysis Pathway that Enhances the Virulence of a Pathogenic Bacterium

In mammalian cells, programmed cell death (PCD) plays important roles in development, in the removal of damaged cells, and in fighting bacterial infections. Although widespread among multicellular organisms, there are relatively few documented instances of PCD in bacteria. Here we describe a potential PCD pathway in Pseudomonas aeruginosa that enhances the ability of the bacterium to cause disease in a lung infection model. Activation of the system can occur in a subset of cells in response to DNA damage through cleavage of an essential transcription regulator we call AlpR. Cleavage of AlpR triggers a cell lysis program through de-repression of the alpA gene, which encodes a positive regulator that activates expression of the alpBCDE lysis cassette. Although this is lethal to the individual cell in which it occurs, we find it benefits the population as a whole during infection of a mammalian host. Thus, host and pathogen each may use PCD as a survival-promoting strategy. We suggest that activation of the Alp cell lysis pathway is a disease-enhancing response to bacterial DNA damage inflicted by the host immune system

Pervasive Targeting of Nascent Transcripts by Hfq

SUMMARY Hfq is an RNA chaperone and an important posttranscriptional regulator in bacteria. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), we show that Hfq associates with hundreds of different regions of the Pseudomonas aeruginosa chromosome. These associations are abolished when transcription is inhibited, indicating that they reflect Hfq binding to transcripts during their synthesis. Analogous ChIP-seq analyses with the post-transcriptional regulator Crc reveal that it associates with many of the same nascent transcripts as Hfq, an activity we show is Hfq dependent. Our findings indicate that Hfq binds many transcripts co-transcriptionally in P. aeruginosa, often in concert with Crc, and uncover direct regulatory targets of these proteins. They also highlight a general approach for studying the interactions of RNA-binding proteins with nascent transcripts in bacteria. The binding of post-transcriptional regulators to nascent mRNAs may represent a prevalent means of controlling translation in bacteria where transcription and translation are coupled

DidA is a small, inner membrane protein that interacts with FtsN.

<p>(A) The subcellular localization of DidA was examined in a strain expressing <i>M2-yfp-didA</i> from the xylose-inducible promoter P<i>_xyl</i> on a low-copy plasmid. Cells were grown to mid-exponential phase in rich media with glucose and then shifted to xylose. At the times indicated, cells were imaged by phase and epifluorescent microscopy. In the fluorescent micrographs, cell boundaries were added after imaging. (B) Subcellular fractionation of cells overexpressing <i>3×M2-didA</i> from the P<i>_van</i> promoter on a medium-copy plasmid for 1.5 hours and expressing the transmembrane protein <i>cckA-gfp</i> from P<i>_cckA</i> on the chromosome. Samples were fractionated into soluble (S) and membrane (M) fractions and analyzed by Western blot. The membrane was cut into three pieces, indicated by dashed lines, and probed with antibodies specific for the GFP, CtrA, or M2 epitope. (C) Bacterial two-hybrid analysis of interactions between T25-DidA and cell division proteins fused to T18, as indicated. The FtsIΔC construct lacking the C-terminal catalytic domain previously showed interactions with FtsW and FtsN as expected, unlike the full-length version of FtsI <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977-Modell1" target="_blank">[13]</a>. The interacting pair T18-M2-SidA and T25-FtsN was included for comparison. <i>E. coli</i> strains harboring each pair of fusions were plated on LB, and colonies were restruck on MacConkey plates containing maltose. Red streaks indicate positive interactions. −/− indicates empty vectors negative control, +/+ indicates the zip/zip fusions used as a positive control. (D) Subcellular localization of FtsZ, FtsW, FtsI, and FtsN were examined in strains expressing <i>ftsZ-yfp</i> from the chromosomal P<i>_van</i> promoter, or <i>venus-ftsW</i>, <i>gfp-ftsI</i> or <i>gfp-ftsN</i> from its native chromosomal locus. Each strain was transformed with a medium-copy plasmid expressing <i>didA</i> from the P<i>_van</i> promoter. Strains were grown to mid-exponential phase and samples imaged by phase and epifluorescent microscopy after addition of vanillate for 4.5 hours. In the fluorescent images, cell outlines were drawn based on the phase micrographs. Bar, 2 µm. (E) Strains from (D) were grown to mid-exponential phase and 10-fold serial dilutions were plated on rich media supplemented with vanillate to induce <i>didA</i> expression.</p

Two independent pathways regulate cell division in <i>Caulobacter</i> following DNA damage.

<p>(A–B) Two cell division inhibitors are induced following DNA damage in <i>Caulobacter</i>. <i>sidA</i> is induced by cleavage of the SOS repressor LexA while <i>didA</i> is induced by DriD. SidA and DidA are small transmembrane proteins that can block cell division by preventing the divisome subcomplex FtsW/I/N from assuming an active state, designated FtsW/I/N*. FtsW/I/N* could promote division by enhancing peptidoglycan synthesis and remodeling, by triggering FtsZ constriction, or by coordinating these activities.</p

Mutations that suppress <i>sidA</i> and <i>didA</i> overexpression likely hyperactivate cell division.

<p>(A) The strains indicated were grown to mid-exponential phase in rich media and imaged by phase microscopy. Bar, 2 µm. (B) Each strain indicated was grown to mid-exponential phase and average cell length, relative to wild-type, was calculated (all <i>n</i>>440). Error bars represent standard error of the mean (SEM), and asterisks indicate <i>p</i><0.01 (*) or <i>p</i><0.0001 (**). The strain denoted <i>ftsW**I*</i> combines the mutations <i>ftsW(F145L, A246T)</i> and <i>ftsI(I45V)</i>. Separate graphs are shown for cell length measurements made on different days. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>. (C) Wild-type, <i>ΔsidAΔdidA</i>, <i>ftsW(A246T)</i>, and <i>ftsW**I*</i> cells were grown to mid-exponential phase and plated in 10-fold dilutions on rich media containing no additives, 0.35 µg/ml MMC or 6 µg/ml cephalexin. (D) The strains from (C) were grown to mid-exponential phase in rich media and treated with MMC or cephalexin at the concentrations in (C) for 6 hours. PI at 5 µM was added 1.5 hours before imaging. Cells were imaged by phase and fluorescence microscopy; cell lengths and percentage of PI+ cells are shown by bar graphs. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>.</p

DriD directly activates <i>didA</i>.

<p>(A) Wild-type cells harboring low-copy plasmids expressing <i>egfp</i> from the <i>sidA</i> or <i>didA</i> promoters were treated with MMC (0.5 and 3 µg/ml), hydroxyurea (HU; 0.5 and 3 mg/ml) or zeocin (2.5 and 15 µg/ml) and then analyzed by Western blot using an α-GFP antibody. (B) Diagram of <i>driD</i> indicating the predicted helix-turn-helix (HTH) and WYL domains. Arrows indicate transposon insertion sites in the genetic screen that identified <i>driD</i>. (C) Wild-type, <i>ΔdriD</i>, and <i>ΔrecA</i> cells were transformed with the P<i>_sidA</i> and P<i>_didA</i> reporter plasmids from (A) and treated with 3 µg/ml MMC or 15 µg/ml zeocin for 1 hour. Samples were analyzed by Western blot using an α-GFP antibody. (D) 10-fold serial dilutions of the strains indicated were grown on plates containing 0.35 µg/ml MMC. (E) <i>ΔdriD</i> cells carrying a low-copy plasmid producing a control construct (P<i>_xyl-ftsW-egfp</i>), untagged DidA, or DidA fused at either its N- or C-terminal end to a 3×M2 tag and expressed from the <i>didA</i> promoter were treated with 15 µg/ml zeocin for 45 minutes. Samples were analyzed by Western blot using an α-FLAG/M2 antibody. (F) <i>ΔdriD</i> cells carrying a low-copy plasmid expressing either <i>driD</i> or <i>driD-3×M2</i> from the <i>driD</i> promoter were treated with 15 µg/ml zeocin for 45 minutes. DriD was immunoprecipitated with an α-FLAG/M2 antibody and promoter occupancy was analyzed by quantitative PCR using primers specific for P<i>_didA</i>. Fold-enrichment values were normalized relative to the enrichment of a region within the coding sequence of <i>ruvA</i>. For raw data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s014" target="_blank">Data S3</a>.</p

DidA is sufficient to inhibit cell division.

<p>Growth curves (A) and micrographs (B) of strains overexpressing <i>didA</i>. Cells harboring a low- or medium-copy plasmid that expresses <i>didA</i> from the vanillate-inducible promoter P<i>_van</i> were grown in rich medium with or without vanillate for the times indicated. Bar, 2 µm.</p

<i>didA</i> is induced by DNA damage and is not SOS regulated.

<p>(A) Wild-type and Δ<i>recA</i> cells were grown in rich medium to mid-exponential phase and treated with 1 µg/ml MMC for 30 minutes. Expression values, the average of two biological replicates, are shown for the 50 most upregulated genes in wild-type cells with fold-change ratios calculated in comparison to mock treated cells. The dashed line corresponds to fold-change values that are identical in wild-type and <i>ΔrecA</i> cells. For complete data, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s002" target="_blank">Figure S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001977#pbio.1001977.s012" target="_blank">Data S1A</a>. (B) CC3114 and CCNA03212 (<i>didA</i>) are shown schematically in their genomic context. Nucleotide positions relative to the annotated CC3114 start site are shown below. The gray shaded region represents a predicted transmembrane domain. (C) Western blot of cells producing DidA fused to a C-terminal 3×M2 epitope from the chromosomal <i>didA</i> locus. Cells were grown to mid-exponential phase and treated with 1 µg/ml MMC for the times indicated. (D) Western blot of wild-type, Δ<i>recA</i> and <i>lexA(K203A)</i> cells expressing <i>didA-3×M2</i> from its native locus treated with 1 µg/ml MMC for 1 hour. Membranes (C–D) were blotted with the α-FLAG/M2 antibody.</p