The Type IV Pilin Pila Couples Surface Attachment and Cell-Cycle Initiation

Downloaded by guest on October 1, 2021 albce crescentus Caulobacter persistent control and formation sur- biofilm colonization infections. inhibit prevent host to strategies for sensing, offer pathogens face mimetics in peptide CIP importance type that the their suggests of and simultaneously conservation pili and broad IV The progression attachment. synthesized cell-cycle surface chemically inhibits “cell-cycle initiates the for peptide of CIP Addition CIP sequence cell peptide. signaling the pilin” PilA of initiating the onset the termed of signal We system, and helix cycle. signaling levels, transmembrane two-component c-di-GMP acid PleC-PleD cellular mem- increase the 17-amino inner activate the the to by in PilA reservoir sensed of pilin is retraction monomeric brane Upon the the signaling via (c-di-GMP). filaments, primary initiation di-GMP pili the cell-cycle cyclic as to messenger contact PilA second surface protein couples pilin that IV input type the in identify initiation surface how cell-cycle for fun- drives mechanism (FRET) sensing molecular in of the transfer sequencing uncover is energy to transposon resonance microscopy adhesion fluorescence use cellular with we conjunction to Here, response importance. cell- damental in 2019) regulate 18, progression November and review surfaces for cycle (received colonize 2020 bacteria 13, March how approved Understanding and CT, Haven, New University, Yale Jacobs-Wagner, Christine by Edited a Medico Del Luca in attachment initiation surface cell-cycle couples and PilA pilin IV type The aigdnmc niesnl el n n ht eie its besides that, find www.pnas.org/cgi/doi/10.1073/pnas.1920143117 and cells sig- single c-di-GMP inside quantify genetically we dynamics a (12), naling with biosensor conjunction c-di-GMP in encoded microscopy pleiotropic ini- (FRET) cell-cycle exerts transfer to that attachment in PilA surface protein tiation bacterial links pilin and IVc control type remained the has within initiation cell-cycle to attachment elusive. the how surface understanding of rial However, involved mechanism (9–11). determinants sensing flagella molecular key cells surface and as tactile pili implicated in Both stalked previously 8) mechanisms. been swarmer sensing have (7, replication-competent motile tactile from CtrA on state depends surface-attached regulator cell-cycle in master change into The cell-cycle 1A). (c-di-GMP)–dependent by the (Fig. di-GMP driven of phase cyclic S degradation messenger replicative the second the into in the entering remains cell, and pili, cell polar swarmer stalked of and motile flagellum round polar G new the a a 1A). with whereas initiates equipped (Fig. immediately replication, programs cell cycle, chromosome development stalked life specialized sessile and biphasic The with the differentiation types During cell cellular 6). with (5, Caulobacter progression interlinking mediated In cell-cycle attachment intimately (1–4). irreversible is flagella, to polysaccharides and transition reversible pili by as a a such with by structures begins followed surface Attachment by cycle. mediated life phase, biphasic a into T microscopy FRET nttt fMlclrSsesBooy eateto ilg,Eidgen Biology, of Department Biology, Systems Molecular of Institute 1 nti ok erpr nasotppiesga encoded signal peptide short a on report we work, this In ecl-yl oe bacterium model (Caulobacter cell-cycle he hs o enditra eoedfeetaigit a into differentiating before interval defined a for phase sn ursec eoac energy resonance fluorescence Using Caulobacter. iie smerclyadpoue w distinct two produces and asymmetrically divides a eefe)itgae ufc colonization surface integrates hereafter) ai Cerletti Dario , | c-di-GMP | yeI pilin IV type ufc sensing surface Caulobacter, Caulobacter a hlp Sch Philipp , We crescentus . Caulobacter albce crescentus Caulobacter | TnSeq nelnsbacte- interlinks | achle ¨ a atisChristen Matthias , siceTcnsh ohcueZ Hochschule Technische ossische ¨ albce crescentus Caulobacter doi:10.1073/pnas.1920143117/-/DCSupplemental at online information supporting contains article This 1 oeiaie ies . C BY-NC-ND) (CC 4.0 NoDerivatives License distributed under is article access open This Submission.y Direct PNAS a is article This B.C. interest.y competing and no M.C. declare P.S. and authors authors.y The data; and all analyzed from D.C., input B.C. L.D.M., with and paper research; M.C., the P.S., wrote designed D.C., B.C. L.D.M., and research; performed M.C., L.D.M., contributions: Author growth reduced during initiation cell-cycle check- cell-cycle conditions. delays specific a that of presence point the suggests finding This of Appendix). culturing (SI the (13) that strain found reporter affects We cell-cycle or length described slowdown relative ously G the uniform the determined 1,092 a we of to phases, in cell-cycle time resulted particular replication growth the ( reduced extending min cycle, 14.4 cell the of 10 to labora- ture standard the 30 Under freshwater 84 of ). S1 natural temperature growth Table its tory Appendix , in (SI encountered the habitat range of temperature time organism the replication reduced model to the cell-cycle cycle profiled proteobacterial cell Initiation. we the Cell-Cycle conditions, adjust growth cells Delays bacterial Checkpoint how understand Cell-Cycle Specific A signal Results input G specific monomeric the a at filaments, as signaling functions c-di-GMP pili triggers membrane that IVc inner type the in forming PilA in role structural oeta .-odices nterltv uaino the of duration relative the in increase 1.4-fold G than more a owo orsodnemyb drse.Eal [email protected] or [email protected] Email: addressed. [email protected]. be may correspondence whom To hmtpst oto ifimfrainadtetbacterial treat and formation infections. biofilm promising control represent to pilin of chemotypes mimetics peptide attachment. synthetic surface the Thus, inhibits to and initiation, sensing, corresponding surface cell-cycle mimics activates peptide subunit pilin synthetic the of acid portion membrane trigger 17-amino provision the and a that di-GMP show of we stimulus cyclic Furthermore, a messenger initiation. as second cell-cycle function the membrane activate inner to pilin in the increase depoly- within concomitant induces The subunits filaments. attachment pili initi- surface of cell-cycle that merization with propose sensing We surface ation. interlinks transduction that signal pro- molecular pathway we a Here, on adhesion. findings promote systems-level vide to bacteria many the of on found surface appendages proteinaceous long dynamic, are Pili Significance 1 ± hs,idctn ea ncl-yl ntain(i.1B). (Fig. initiation cell-cycle in delay a indicating phase, . i.Hwvr hnrsrcigtegot tempera- growth the restricting when However, min. 1.2 1 ◦ a,1 ,w bevda1-odices nteduration the in increase 13-fold a observed we C, .T netgt whether investigate To S1). Table Appendix, SI hs yfloecnemcocp sn previ- a using microscopy fluorescence by phase n etChristen Beat and , rc,Z urich, ¨ rc 03 Switzerland 8093, urich y ¨ . y raieCmosAttribution-NonCommercial- Commons Creative ◦ C, Caulobacter . y Caulobacter a,1 https://www.pnas.org/lookup/suppl/ NSLts Articles Latest PNAS 1 Spaetransition. phase –S Caulobacter t10 at elctsevery replicates ◦ caused C | across f8 of 1 To α- ±

MICROBIOLOGY forward genetic screen based on quantitative selection analy- AB40 Non-replicative Replicative sis coupled to transposon sequencing (TnSeq) (14, 15). TnSeq (G1) (S) measures genome-wide changes in transposon insertion abun- 30 * dance upon subjecting large mutant populations to different selection regimes and enables genome-wide identification of 20 essential genes. We hypothesized that the profiling of growth rate-dependent changes in gene essentiality will elucidate the Swarmer Stalked 10 components of the cell-cycle machinery fundamental for cell- cycle initiation under reduced growth conditions. We selected

c-di-GMP Cells in G1 phase [%] 0 Caulobacter transposon mutant libraries for prolonged growth 10 30 ◦ ◦ CtrA at low temperatures (5 C and 10 C) and under standard lab- oratory cultivation conditions (25◦C and 30 ◦C). Cumulatively, we mapped, for each condition, between 397,377 and 502,774 C shkA rcdA unique transposon insertion sites across the 4.0-million base Caulobacter nepR pairs (Mbp) genome corresponding to a transposon insertion density of 4 to 5 bp (SI Appendix, Table S2). °C To identify the factors required for cell-cycle progression, we 5 10 25 focused our analysis on essential genes (Dataset S1) that are pilA 30 cpdR expressed in a cell-cycle–dependent manner (SI Appendix and C. crescentus refs. 16–18). Among 373 cell-cycle–controlled genes, we found genome fliH 45 genes that were essential under all growth conditions (Fig. 1C 4.0 Mbp and Dataset S1), including five master regulators (ctrA, gcrA, sciP, ccrM, and dnaA), 11 divisome and cell wall components (ftsABILQYZ, fzlA, and murDEF), and six DNA replication and pleC segregation factors (dnaB, ssb, gyrA, mipZ, parB, and ftsK), as pleD well as 23 additional genes encoding key signaling factors and cro cellular components required for cell-cycle progression (Dataset S1). Collectively, these 45 genes form the core components of the xseB lon bacterial cell-cycle machinery. core cell cycle genes essential at slow growth (12) (45) Components of the c-di-GMP Signaling Network Are Conditionally Essential for Slow Growth. During reduced growth conditions, we D found 12 genes that specifically became essential (Fig. 1C). To TnSeq selection [°C] gain insights into the underlying genetic modules, we performed 5102530 a hierarchical clustering analysis and grouped these 12 genes

lon CCNA_02037 Endopeptidase according to their growth rate-dependent fitness profile into

pleD CCNA_02546 Diguanylate cyclase three functional clusters, A, B, and C (Fig. 1D and SI Appendix). Cluster A contained four conditionally essential genes that rcdA CCNA_03404 CtrA proteolysis regulator exhibited a large decrease in fitness during slow-growth condi- cpdR CCNA_00781 Proteolytic ClpXP adapter tions (Fig. 1D and SI Appendix, Fig. S1). Among them were flbE CCNA_00952 Flagellar assembly protein pleD, cpdR, rcdA, and lon that all comprise important regula- pilA CCNA_03043 Type IVc pilin tors for cell-cycle–controlled proteolysis. The diguanylate cyclase pleC CCNA_02567 Histidine kinase PleD produces the bacterial second messenger c-di-GMP, which shkA CCNA_00137 Histidine kinase

cluster B cluster A becomes restricted to the stalked cell progeny upon asymmet- CCNA_02103 Hypothetical protein ric cell division and is absent in the newly born swarmer cell

nepR CCNA_03590 Anti-sigma factor (Fig. 1A and ref. 12). During the G1–S phase transition, c-di-

xseB CCNA_02151 Exodeoxyribonuclease GMP levels rise again and trigger the proteolytic clearance of the cell-cycle master regulator CtrA (Fig. 1A), which is mediated CCNA_02315 Cro/CI repressor cluster C cro/CI by ClpXP and the proteolytic adaptor proteins RcdA, CpdR, Tn5 hits [log2 fold] and PopA (19–22). Similarly, the ATP-dependent endopeptidase -4 -2 0 Lon is responsible for the degradation of the cell-cycle master regulators CcrM, DnaA, and SciP (23–25). Taken together, these Fig. 1. TnSeq identifies conditionally essential genes required during findings underscore the importance of controlling proteolysis of reduced growth conditions. (A) Caulobacter divides asymmetrically into a replication-competent stalked cell and a swarmer cell. The master regulator CtrA and other cell-cycle regulators to maintain cell-cycle pro- CtrA inhibits DNA replication in swarmer cells and is proteolytically cleared gression at low growth rates when intrinsic protein turnover rates upon an increase in cellular levels of the second messenger c-di-GMP at are marginal (26). the G1–S phase transition. (B) Cellular replication under slow-growth con- Cluster B contained five genes, including the two histidine ditions increases the relative duration of the G1 phase as determined by a kinase genes pleC and shkA, a gene of unknown function encod- fluorescently tagged cell-cycle reporter strain. (C) TnSeq across the 4.0-Mbp ing a conserved hypothetical protein (CCNA 02103), and the Caulobacter genome defines 45 core-essential cell-cycle genes (gray marks) type IV pilin gene pilA and the flagellar assembly ATPase ◦ ◦ ◦ ◦ to sustain growth at 5 C, 10 C, 25 C, and 30 C (outer to inner track) and flbE/fliH (Fig. 1D and SI Appendix, Fig. S2). Multiple genes of 12 conditionally essential genes required for slow-growth conditions (blue cluster B participate in c-di-GMP signaling. Among them, we marks). (D) Hierarchical clustering analysis of the 12 conditionally essential genes required during reduced growth conditions. found the sensor kinase PleC that functions upstream and acti- vates the diguanylate cyclase PleD over phosphorylation. The hybrid kinase ShkA comprises a downstream effector protein of TnSeq Identifies Conditionally Essential Cell-Cycle Genes. To iden- PleD, which binds c-di-GMP and phosphorylates the TacA tran- tify the complete set of genes required for cell-cycle progres- scription factor responsible for the initiation of the stalked cell- sion at different growth rates, we designed a systems-wide specific transcription program (27, 28). The flagellum assembly

2 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1920143117 Del Medico et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 ufc.Tecl tahsa iepit0sadplsisl vradsac f1.3 al. of et distance Medico a Del over itself pulls and s 0 point time at attaches cell The surface. orsodn lt fteqatfidcd-M eesoe ouaino 0samrclsaesonfrwl ye(ry and (gray) type wild for shown are cells swarmer 50 of population a G the over in (C levels resting to levels. arrows) c-di-GMP similar by c-di-GMP Scale quantified (highlighted cellular intervals. cells the low Swarmer bar. of exhibit color plots cycle the cell by corresponding indicated of as initiation concentration prior c-di-GMP cytoplasmic phase the to wild-type corresponding of nm) populations (527/480 of swarmer copy synchronized plasmid-born of images a (FRET) by microscopic ratio Dual-emission (B) (S). Caulobacter 2. Fig. con- is activity PleC that suggests by which integrated amino-terminal domain, signal an comprises periplasmic external PleC of unknown. in remained type resolved has been PleC the has (30), PleD detail target molecular diguanylate downstream resides the its PleC activating of G mechanism hierarchy kinase the signaling sensor at the PleD the cyclase of B, top and the A at clusters Progression. iden- within network Cell-Cycle signaling tified c-di-GMP Initiate the of to components the Signaling Among c-di-GMP Induces PilA slow-growth under progression conditions. cell-cycle with interfere may programs that transcriptional alternative likely and genes responses these stress of induces linked disruption directly However, signaling. not c-di-GMP components to outer-circle form 1D which (Fig. S3), VII Fig. exodeoxyribonuclease the of unit (CCNA factor scription cell-cycle coordinate c-di-GMP conditions. to slow-growth that importance under progression indicate fundamental effector in of data c-di-GMP which, is these a signaling sol- apparatus, Collectively, as the identified (29). export form been complex flagellar also FliJ the has and Pseudomonas, of FliI component with uble together FlbE/FliH ATPase lse opie h niim factor antisigma the comprised C Cluster ouaindsrbto fitaellrcd-M ocnrtosi synchronized in concentrations c-di-GMP intracellular of distribution Population (A) signal. initiating cell-cycle as functions PilA pilin IV type The el.Tepplto hw ioa -iGPdsrbto orsodn osamrcls(G cells swarmer to corresponding distribution c-di-GMP bimodal a shows population The cells. A cells [%] cells [%] cells [%] cells [%] 12 15 12 15 12 15 12 15 3 6 9 0 0 3 6 9 3 6 9 0 3 6 9 50 8% G1 11% G1 12% G1 n =1216 ielpeFE irsoyadpaecnrs mgso a of images phase-contrast and microscopy FRET Time-lapse (D) D. c-di-GMP [nM] 1 52% G1 200 and pilA, Spaetasto.Wieteactivation the While transition. phase –S 21) and 02315), S S wt 350 S S n =1570 n =1325 500 n =1026 ∆pilA 200 350 500 650 50 650 xseB opeetdb h xgnu diino h I etd (100 peptide CIP the of addition exogenous the by complemented c-di-GMP [nM] B noigtesalsub- small the encoding r/Itran- Cro/CI a nepR, wt and FRET +CIP + IAppendix, SI pilA ielpeFE irsoyfloigcl-yl rgeso n ( Bottom) and progression cell-cycle following microscopy FRET Time-lapse (Top) ) D C wiecnor ro nraigteclua -iGPlevel. c-di-GMP cellular the increasing prior contour) (white µm c-di-GMP [nM] in remaining cells) 1,216 of out into G (143 the transitioned 11.8% only quickly with cells phase, S synchronized the of majority the microscopy that FRET by cells 2A swarmer (Fig. individual and in wild-type levels synchronized di-GMP We (12). cycle c-di-GMP ∆pilA cell fluctuating the the along that of pre- levels biosensor monitoring have FRET time-resolved encoded we permits genetically cells, a single engineered inside func- viously G dynamics kinase the PleC signaling prolong the c-di-GMP to of expected activation is impairs tion that c- mutation the in Any G increase the strong at a levels to di-GMP leads PleD, subsequently and PleC, and input wild-type a external in dynamics unknown hitherto PleC. kinase the sensor the PilA for by that perceived candidate speculated signal we likely Thus, 31). a (13, is swarmer- PleC the as to pole PilA pattern cell Furthermore, specific localization periplasm. subcellular pro- same the pilin the into IV shares type translocated the is of with PilA portion together tein C-terminal coclustered The 1D). PilA (Fig. B, PleC conditionally cluster identified within the genes Among essential signal. external an by trolled wt 200 350 500 ots hshptei,w oioe h -iGPsignaling c-di-GMP the monitored we hypothesis, this test To FRET phase FRET phase 50 0 1 0 0 2 4 6 180 160 140 120 100 el xrsigti RTboesradqatfidc- quantified and biosensor FRET this expressing cells 2 04 80 40 20 0 -20 hs,a niae ylwcd-M ees(i.2 (Fig. levels c-di-GMP low by indicated as phase, and Caulobacter 04 08 0 2 4 160 140 120 100 80 60 40 20 .I h idtp oto,w observed we control, wild-type the In Appendix). SI cell cycleprogression[min] 010150 100 50 Caulobacter, wre el(ht ros tahn oa agar an to attaching arrows) (white cell swarmer 1 Spaetasto Fg 1A (Fig. transition phase –S time [s] 1 n el htudretG underwent that cells and ) ) suooosso RTeiso ratios emission FRET show Pseudocolors µM). ∆pilA ∆pilA 1 akrud h ciainof activation The background. wre hs.T monitor To phase. swarmer n =50 60 mutants, NSLts Articles Latest PNAS wt 200 350 500 650 50 ∆pilA ∆pilA

1 c-di-GMP [nM] rd t10-min at (red) complemented oStransition S to n e.12). ref. and | A f8 of 3 and 1

MICROBIOLOGY B). In contrast, we observed that more than 52.2% (821 out of (pilA1–14) did not restore the cell-cycle timing defects of a ∆pilA 1,570 cells) of all of the synchronized ∆pilA mutant cells exhib- mutant (Fig. 3A and SI Appendix, Fig. S6 and Table S6). We ited a delay in the G1–S transition and maintained low c-di-GMP concluded that the translocation of PilA into the periplasm is a levels for a prolonged interval (Fig. 2 A and B). Consistent with prerequisite to signal cell-cycle initiation. an increase in the duration of the G1 phase, we also observed Upon translocation and cleavage of the N-terminal leader that ∆pilA swarmer cells exhibited a 7.7% increase in cell length sequence, the mature form of PilA resides as a monomeric compared to wild-type swarmer cells (SI Appendix, Table S3). protein anchored in the inner membrane (38). Through the Similarly, using time-lapse studies to follow signaling trajecto- action of a dedicated type IV pilus assembly machinery, the ries of individual cells, we found that ∆pilA mutants exhibited inner membrane-bound reservoir of PilA polymerizes into polar a more than 1.7-fold increase in the duration of the G1 phase pili filaments that mediate initial attachment to surfaces (39). as compared to wild-type cells (65 ± 3 min versus 38 ± 1 min However, only pilA, but none of the other components of the for ∆pilA and wild type, respectively; Fig. 2C and SI Appendix, pilus assembly machinery, became essential in our TnSeq screen Table S4). Providing an episomal copy of pilA restored the cell- under slow-growth conditions (Fig. 1C, SI Appendix, Table S7, cycle timing defect of a ∆pilA mutant strain (Fig. 2 A and B). and Dataset S1). Furthermore, unlike ∆pilA mutants, deletion When following the formation of duplicated replication origins mutants of the pilus assembly machinery genes cpaA, cpaD, and in synchronized cells using a fluorescent CFP-ParB reporter (32, cpaE did not prolong the G1 phase but, in contrast, shortened 33), we observed that a ∆pilA mutant showed a 1.4-fold lower the duration of the G1 phase by twofold as compared to the wild- proportion of cells that entered S phase as compared to wild type control (4%, 3%, and 5% G1 cells; SI Appendix, Fig. S5). type (18.9 ± 1.2% versus 26.5 ± 1.2% at 37 min after syn- The observation that the translocation of PilA monomers across chrony; SI Appendix, Fig. S4B and Table S5). Furthermore, when the inner membrane is necessary but that the subsequent poly- monitoring c-di-GMP signaling dynamics in attaching swarmer merization of PilA monomers into pilin filaments is dispensable cells through time-lapse FRET microscopy, we observed that, for cell-cycle initiation suggested that the inner membrane reser- upon surface contact, swarmer cells showed, within seconds, a voir of the monomeric form of PilA functions as an input signal cell body displacement, consistent with pili retraction in the for c-di-GMP–mediated cell-cycle signaling. micrometer range, which preceded a rapid rise in cellular c-di- GMP levels (Fig. 2D and Movie S1). Collectively, these findings The Transmembrane Helix of PilA Comprises a 17-Amino Acid Pep- establish the type IV pilin PilA as an input signal interlinking tide Signal That Mediates Cell-Cycle Initiation. The mature form surface attachment with cell-cycle progression and initiation of of PilA is a small 45-amino acid protein comprising a highly chromosome replication. hydrophobic N-terminal alpha helix, which anchors PilA in the inner membrane (36, 38), and an adjacent variable alpha- PilA Controls Cell-Cycle Initiation via the Sensor Kinase PleC helical domain protruding into the periplasm. To identify the and the Diguanylate Cyclase PleD. PleC is a bifunctional phos- portion of the matured PilA protein responsible for trigger- phatase/kinase that switches activity in a cell-cycle–dependent ing cell-cycle initiation, we constructed a panel of C-terminally manner (34). In the newborn swarmer cell, PleC first assumes truncated pilA variants and assessed their ability to comple- phosphatase activity to inactivate the diguanylate cyclase PleD ment the cell-cycle defect of a chromosomal ∆pilA mutant and establish low cellular c-di-GMP levels. Subsequently, PleC by quantifying c-di-GMP dynamics in single cells using FRET switches to a kinase and activates PleD at the G1–S phase tran- microscopy (SI Appendix). The expression of a truncated PilA sition. To test whether PilA functions as an input signal for variant that includes the leader sequence and the first five the PleC-PleD signaling cascade, we measured c-di-GMP sig- N-terminal amino acids of the matured PilA protein (PilA 5aa) naling dynamics and compared the duration of the G1 phase did not complement the cell-cycle initiation defect of a ∆pilA in single- and double-deletion mutants using FRET microscopy mutant (Fig. 3A and SI Appendix, Fig. S6 and Table S6). How- (SI Appendix, Fig. S5). Similar to ∆pilA mutants, ∆pleD mutants ever, increasing the N-terminal portion of the matured PilA also prolonged the G1 phase more than twofold as compared to protein to 17, 25, and 37 amino acids (PilA 17aa, PilA 25aa, and the wild-type control (18% and 22% versus 9% G1 cells in wild PilA 37aa) restored the cell-cycle initiation defects of a ∆pilA type; SI Appendix, Fig. S5). However, a ∆pilA, ∆pleD double mutant (Fig. 3A and SI Appendix, Fig. S6 and Table S6). Based mutant only marginally increased the duration of the G1 phase on these findings, we concluded that a small N-terminal peptide and showed similar frequencies of swarmer cells with low-c-di- sequence covering only 17 amino acids from the mature PilA GMP levels as compared to a strain lacking solely pleD (28% and (Fig. 3A) is sufficient to initiate c-di-GMP–dependent cell-cycle 22% G1 cells; SI Appendix, Fig. S5). This genetic evidence sug- progression. Accordingly, we annotated this peptide sequence as gests that PilA resides upstream of PleD. Thus, besides its role cell-cycle initiating pilin (CIP) sequence (Fig. 3A). as a structural component of the type IVc pilus, PilA likely com- prises an input signal for the sensor kinase PleC, which serves as Chemically Synthesized CIP Peptide Initiates Cell-Cycle Progression. the cognate kinase of PleD, to increase c-di-GMP levels at the Next, we asked whether the translocation of PilA from the cyto- G1–S phase transition. plasm or the presence of a PilA reservoir in the periplasm is sensed. If signaling depends solely on the presence of The Inner Membrane Reservoir of the Type IV Pilin PilA Signals Cell- membrane-inserted PilA, we speculated that exogenous provi- Cycle Initiation. PilA harbors a short 14-amino acid N-terminal sion of chemical-synthesized CIP peptide should restore the leader sequence required for the translocation across the inner cell-cycle defects in a ∆pilA mutant. To test this hypothesis, membrane that is cleaved off by the peptidase CpaA (35–37). we incubated synchronized swarmer cells of a ∆pilA mutant in To test whether the translocation of PilA is a prerequisite for the presence of 100 µM of chemically synthesized CIP peptide signaling the cell-cycle initiation, we constructed a cytosolic ver- and assayed c-di-GMP signaling dynamics by FRET microscopy sion of PilA (pilA15–59) lacking the N-terminal leader sequence (Fig. 2 A and B). Indeed, we found that addition of the CIP pep- needed for the translocation of the matured PilA across the tide induced cell-cycle transition into the S phase as indicated membrane. Unlike the full-length PilA, the episomal expression by a more than twofold lower abundance of G1 swarmer cells of the translocation-deficient version of PilA did not comple- with low c-di-GMP levels as compared to an untreated ∆pilA ment the cell-cycle timing defect of a ∆pilA mutant (Fig. 3A cell population (8% vs. 17% G1 cells, upon 15 min incubation and SI Appendix, Fig. S6 and Table S6). Similarly, we found of synchronized swarmer cells with CIP or the mock control, that solely expressing the N-terminal leader sequence of PilA Fig. 2 A and B). These findings suggest that the addition of

4 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1920143117 Del Medico et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 ∆pilA 3. Fig. e eioe al. et Medico Del interacted (T18-PilA2-31) (T18- sequence PilA CIP of T18 the fusions the only T18-domain from or (40). cyclase PilA2-59) domains split adenylate T25 the and of in reconstitution interactions (BACTH) through assay protein–protein two-hybrid bacterial measured a and used we periplasmic PleC, the with of interacts PleC domain directly effector PilA modulates downstream whether PilA test the To of of PleD. phosphorylation sequence promote CIP to the activity G the that shortens suggests peptide CIP phase the of S5 addition Fig. the that Appendix, observation (SI levels c-di-GMP low with Peptide. CIP kinase the sensor Senses the PleC of Domain Periplasmic The CIP hydrophobic the progression. via cell-cycle sensed initiate to is sequence PilA inner of the solely reservoir but membrane polymerization nor translocation that the demonstrated neither results these Collectively, G activator. the cell-cycle shortens peptide CIP the region of SEM; transmembrane attachment are surface the bars relative in Error the bundles of (D) helical chart domain. four bar kinase dimerized cytoplasmic corresponding the the between in domains residues of rearrangement chemoreceptor histidine chart structural conserved bar of induce the family will at The domain the autophosphorylation region. induce sensor with membrane to PleC similarity the the suggests highlight of (41) lines (green) server Dashed portion SWISS-MODEL from TM2). (TM1, pilin the helices IV with in transmembrane type (40) PleC fusion BACTH two of T18-PilA the possess by of domain detected processing that as periplasmic prevent (42) PilA2-31) to (CIP, the PilA used of of were portion (PilA∆DE11,12) modeling peptide variants CIP homology PilA the Cleavage-deficient or plates. (PilA2-59) reporter PilA X-Gal matured the using with interact PleC2-50) (TM1, helix transmembrane h eilsi esrdmi fPe Pe235 rafamn oeigol h first the only covering fragment a or (PleC2-315) PleC of domain sensor periplasmic The (B) variants. PilA truncated C-terminal and N- of panel a with opeetto ftecl-yl ntaindfc of defect initiation cell-cycle the of Complementation (A) peptide. CIP the of action of mode proposed and effects, pleiotropic mapping, Genetic + control A B PilA 17aa PilA 5aa No leader Leader PilA 37aa PilA 25aa PilA PleC Caulobacter o Type IVpilinPilAprotein Neisseria l Cell-cycle initiatingpeptide(CIP) ∗∗∗∗ ATAIEYGLIVALIAVVI pleC N - control P B5i h (Right the in CB15 leader PilA PilA assduhe el ooi h G the omit to cells daughter causes << 1Y) h M gen neat ihteCPppieprino iA(le.(Right (blue). PilA of portion peptide CIP the with interacts (green) TM1 The (1AY2). 10 − 4 CIP CIP CIP CIP CIP . 1 hs n,tu,fntosa a as functions thus, and, phase rsnead(Left and presence )

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P PleC-TM1

PilA P shrci coli Escherichia eeinof Deletion Caulobacter hmclysnhszdCPppie( peptide CIP synthesized chemically 100-µM of absence ) 1 phase 1 D C E - B5uo ficbto ihu ( without incubation of h 1 upon CB15

nareetwt idn oeivligmmrn helix membrane involving (44). mode domains binding output a other with the and agreement regulate kinase transmit In to chemore- cytoplasmic that membrane of helices the dimeric activity TM across from two of signaling harbor Mlp37 ligand-induced domains family 3C; receptor the [Fig. These domains with ceptor predicted exhibits similarity PleC of structural domain periplasmic the that revealed 3B (Fig. (T18-PilA2-31) (T18- peptide PilA S8). Table CIP with Appendix, the interaction or for PilA2-59) sufficient TM1 the was solely We covering (T25-PleC2-50) fragment we domain. PleC peptide, PleC N-terminal an periplasmic CIP that the the found of 305). of to variants site 283 truncated binding and assayed the 50 to characterize 28 further residues To at positioned helices TM2, transmembrane of and two (TM1 domain comprises domain periplasmic PleC intact periplasmic 3B the (Fig. (T25-PleC2-315) of PleC fusions T25-domain with Attachment Plaques assay tutrlhmlg oeig(WS-OE)(41) (SWISS-MODEL) modeling homology Structural T1 M control control 2 T M PleC PilA C I Φ P b hg ucpiiiyasyadcorresponding and assay susceptibility phage Cbk binding Caulobacter idn fPl i le oteteTM1 the the to blue) (in PilA of Binding ) n a )o ih(n with or 9) = c and t i v n a ) (E 5). = t .The S8). Table Appendix, SI e ii iAwsmdldot the onto modeled was PilA pilin d h NSLts Articles Latest PNAS i s rsa iltsann and staining violet Crystal ) Surface attachment Phage susceptibility (C coli. E. t .cholera V. 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 i I peptide. CIP 100-µM 9) = d i n e 0 0100 k i n (Left ) a 100 **** **** s e 4,43)]. (42, Structural ) and | f8 of 5 SI

MICROBIOLOGY interactions, the PilA structural model consists of a single Typeyp IV ppilus transmembrane-spanning alpha helix (Fig. 3C). How could retractionretraction binding of the CIP peptide to TM1 of the periplasmic domain of PleC lead to activation of the output kinase domain? One surface sensinging possibility is that binding of the CIP peptide to the PleC receptor domain induces a structural rearrangement between the dimerized four helical bundles in the transmembrane region OMO to activate autophosphorylation of the cytoplasmic kinase domain (Fig. 3C). In support of a ligand-induced conformational PleC rearrangement in the receptor complex, we observed that T18 PilAPilA and T25 fusions to the N-terminal portion of PleC spanning the TM1, the periplasmic domain, and the TM2 (T18-PleC2-315 and T25-PleC2-315) showed strong interaction, but detected IM a more than 39% reduction in adenylate cyclase activity when CpaF coexpressing also the CIP peptide (SI Appendix, Table S9). HK HK Collectively, these findings suggest that the histidine kinase PleC senses the membrane-inserted PilA pool through direct inter- activated kinase action between the CIP sequence and the first transmembrane helix (TM1) of the periplasmic receptor domain.

The CIP Peptide Reduces Surface Attachment and ΦCbk Phage Sus- DivK-P PleD-P Pili retraction ceptibility. In addition to omitting the G1 phase, deletion of the sensor kinase pleC also results in pleiotropic defects including a lack of polar pili and defects in initial attachment to surfaces (45). CckA c-di-GMP To gain further evidence that PilA, upon binding, modulates the activity of the sensor kinase PleC, we investigated whether the addition of the chemically synthesized CIP peptide also impairs Inactivation Proteolysis additional PleC-specific output functions. Indeed, when assaying CtrA for the presence of functional pili using the pili-specific bacterio- phage ΦCbK, we found that incubating synchronized wild-type Phage Caulobacter for 10 min with the CIP peptide resulted in a 77.2 ± Cell cycle initiation resistance Attachment 2.1% decrease in bacteriophage CbK susceptibility (Fig. 3D), Fig. 4. Model of the type IV pilin-mediated signaling network coupling suggesting that binding of the CIP peptide impairs pili function. surface sensing with cell-cycle initiation. Surface-induced retraction of type Furthermore, we also found that the addition of the CIP peptide IV pili leads to accumulation of monomeric PilA in the inner membrane, to wild-type Caulobacter CB15 cells reduced initial attachment where PilA interacts with the transmembrane portion of the sensor kinase by 71.3 ± 1.6% (Fig. 3E), with a half-effective peptide concentra- PleC. Binding of PilA activates the kinase function of PleC to promote phos- tion (EC50) of 8.9 µM (SI Appendix, Fig. S7) and a Hill coefficient phorylation of the downstream effectors PleD and DivK, and activates pili of 1.6, suggesting positive cooperativity in the binding of CIP to retraction as part of a positive feedback loop. DivK and PleD synergisti- a multimeric PleC receptor complex. Altogether, these findings cally promote inactivation and degradation of the master regulator CtrA support a model in which the CIP sequence of PilA functions as to initiate cell-cycle progression. a pleiotropic small-peptide modulator of the sensor kinase PleC, leading to premature cell-cycle initiation, retraction of type IV sequence for translocation, the CIP sequence for cell-cycle sig- pili, and impairment of surfaces attachment. naling functions, and structural determinants required for pili polymerization. Discussion Caulobacter exhibits a biphasic life cycle where newborn Understanding how bacteria regulate cell-cycle progression in swarmer progeny undergo an obligate differentiate into surface- response to external signaling cues is of fundamental impor- attached stalked cells to initiate DNA replication. How could tance. In this study, we used a TnSeq approach to identify genes PilA couple cell-cycle initiation to surface sensing? Type IV required for cell-cycle initiation. Comparing deviations in gene pili have been shown to serve as adhesion filament as well as essentiality between growth at low temperatures (5◦C and 10◦C) mechanosensors (38, 46, 47), that, upon surface contact, induce and under standard laboratory cultivation conditions (25◦C and the retraction of pili filaments with concomitant accumulation of 30 ◦C) allowed us to pinpoint genes required exclusively for cell- monomeric PilA in the inner membrane (9). Our results from cycle initiation. We identified the pilin protein PilA together with genetic dissection of PilA in conjunction with FRET microscopy six additional components of a multilayered c-di-GMP signal- to monitor c-di-GMP signaling dynamics upon surface contact ing network (Fig. 1) as key determinants that control cell-cycle (Fig. 2D and Movie S1) suggest a model where high levels of PilA initiation. Using FRET microscopy studies, we quantified c- monomers in the inner membrane are sensed via the N-terminal di-GMP signaling dynamics inside single cells and found that, CIP sequence that binds and activates the sensor kinase PleC besides its structural role in forming type IVc pili filaments, to promote phosphorylation of the diguanylate cyclase PleD PilA comprises a specific input signal for activation of c-di-GMP (Fig. 4). In such a model, surface sensing by pili induces a sharp signaling at the G1–S phase transition (Fig. 4). Furthermore, increase in intracellular c-di-GMP levels and activates the prote- we show genetic evidence that PilA functions upstream of the olytic clearance (19, 21) of the master regulator CtrA to initiate PleC-PleD two-component signaling system and present data cell-cycle progression (Fig. 4). that the monomeric PilA reservoir is sensed through a short In support of this model, parallel studies conducted by the 17-amino-acid-long N-terminal peptide sequence (CIP), which laboratory of Yves Brun and colleagues show that chemical directly interacts with the first transmembrane helix anchoring or genetic obstruction of pili retraction leads to an increase the periplasmic domain of the sensor kinase PleC (Fig. 4). It is in the cellular c-di-GMP pool, premature replication initiation, remarkable that the Caulobacter PilA is a multifunctional pro- polar remodeling, and stalked-cell differentiation (48). However, tein that encodes, within a 59-amino acid polypeptide, a leader the precise structural mechanism of how initial surface contact

6 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1920143117 Del Medico et al. Downloaded by guest on October 1, 2021 Downloaded by guest on October 1, 2021 n lohspeorpcefcson effects pleiotropic has pro- also chemically and a 2A) of (Fig. addition cells (52–54). the in signaling treat c-di-GMP that behavior the modulates to show specifically peptide tolerance, difficult we CIP duced work, infections antibiotic our chronic resultant In make its can with initiation, which cell-cycle formation motility, biofilm cellular of and regulator key a is GMP) attachment surface G the permanent at differentiation induce surface cellular to and adhesive (28) of programs transcrip- cell-specific secretion tional stalk of (51), activation and motility (9), polysaccharides flagellar cell-cycle of couple inhi- including bition second- to functions, output c-di-GMP mechanism orthogonal multiple a to a initiation to provides reservoir cascade PilA messenger membrane inner the via mechanism. PleC sensing c-di-GMP surface in of increase robust activity permanent a a kinase and to levels the leading war, state, of retraction tug the con- the perma- wins surface in where model a pili a locks In to phosphatase. tact a again to lead extended reset are is pili not PleC activity when and kinase reversed do dynamic PleC is retraction as state to upon levels, induced c-di-GMP planktonic due cellular in the concentrations increase nent in fluctuations PilA cycling Small membrane surfaces. pili inner sense the to in integration provides signal PleC kinase/phosphatase robust bifunctional the through voir including biogenesis. dif- pili functions, in and output initiation evolved the cell-cycle distinct have surface-induced regulate into pilins to kinase of organism pool the ferent membrane switch mechanisms inner to signaling the convergent reported sensing PleC, Thus, was (50). to bind- PilS state contrast and to phosphatase domain, In PilA sensor (50). of N-terminal ing PilS different inter- a kinase possesses intramembrane sensor PilS direct the via with expression actions own their interactions in regulate transmembrane reported via been kinases also histidine has to pilins the of kinase/phosphatese of ing sensor bundle 3 the (Fig. transmembrane of PleC the domain receptor to periplasmic portion CIP sensing. of surface embedded robust retraction to induce leading pole, to cell loop the at feedback pili positive additional pilus individual a con- an induces peak of at likely retraction pili blocking of Thus, retraction (49). and centrations levels increased intermediate promoting at dynamics, activity pilus affects second-messenger also the c-di- itself that of c-di-GMP showed activation PilA work subsequent elevated Recent and signaling. to exten- membrane GMP inner leading pilus the prevents subunits, in turn, of levels in reincorporation which, and state, sion retraction generation locks that the force filament in contact, the pili within surface rearrangements structural upon (9). to that, leads activities is high- dynamic of possibility their attachment One lose covalent upon conjugates, pili, molecular-weight that showed In studies question. ter, open an remains retraction pili stimulates e eioe al. et Medico Del .C en,C .Elsn .Dce,Y .Bu,Bceilahso ttesnl-ellevel. single-cell the Li at adhesion G. Bacterial Brun, 2. V. Y. Ducret, A. Ellison, K. C. Berne, C. 1. .J amr .Fit .Bok,Bceilcl tahet h einn fabiofilm. a of beginning the attachment, cell Bacterial Brooks, J. Flint, S. Palmer, J. 3. .H .MAas .Saio h rhtcueadcnevto atr fwhole-cell of pattern conservation and architecture The Shapiro, L. McAdams, H. in H. development of Regulation 7. loop: the in Getting Brun, V. Y. Curtis, D. P. 6. the in genes Differentiation of Shapiro, Identification L. Brun, V. 5. Y. Larson, E. D. Bodenmiller, D. Hinz, A. Smith, S. C. 4. h atra eodmsegrcci iunlt (c-di- diguanylate cyclic second-messenger bacterial The sensing surface coupling perspective, engineering an From reser- PilA the sensing integration, signal of level the On membrane- the via binds PilA that find we BACTHs, Using a.Rv Microbiol. Rev. Nat. adhesins. oto circuitry. control crescentus. (1976). 377–407 (2003). in 1432–1442 holdfast 185, adhesive the of synthesis for required Biotechnol. Microbiol. Ind. tal. et o.Microbiol. Mol. irbo.Ml il Rev. Biol. Mol. Microbiol. ufc otc tmltstejs-ntm elyeto bacterial of deployment just-in-time the stimulates contact Surface , B .Ml Biol. Mol. J. and 1–2 (2018). 616–627 16, C 15 (2012). 41–51 83, and 7–8 (2007). 577–588 34, 83 (2011). 28–35 409, .Bind- S9). and S8 Tables Appendix, SI hr pilins where aeruginosa, Pseudomonas Caulobacter 34 (2010). 13–41 74, 1 Spaetransition. phase –S elcycle. cell albce crescentus. Caulobacter nu e.Microbiol. Rev. Annu. Caulobac- Caulobacter .Bacteriol. J. 30, J. 5 .Christen M. 15. Christen B. 14. Christen B. 13. in synthesis pilus reduced Christen have M. mutants Flagellar 12. Brun, V. Y. Rusch, messenger–mediated B. Second D. Ellison, Jenal, K. U. C. Pfohl, T. 11. Sprecher, S. K. Deshpande, S. Hug, I. 10. in Generation. Library TnSeq measurements. TnSeq to according temperatures classifi- growth essentiality cell-cycle–controlled the each contains of S1 cation Dataset assays. susceptibility phage and levels c-di-GMP cellular in quantify to procedures microscopy FRET sequencing, Tn5 conditions, growth dard Appendix SI Methods and Materials infections. bacterial surface treat block to to pathogens and in networks sensing signaling c-di-GMP genetic dissect chemical to a tool into developable promising potentially col- a is their host represents and and identified chemotype and peptide infection pili CIP deter- drive the IV to (55), be type onization pathogens human to of in conservation remains role the broad central porins via chronic the up other taken of Given or is mined. part or channel own secretion is its can on CpaC that peptide membrane outer CIP formation the chemical-synthesized penetrate biofilm the of Whether infections. control target drug the attractive an for be might c-di-GMP, of production poral in susceptibility D phage and adhesion surface oB.C. to of temperature environmental an maintain to unit 25 heating a with Apo Plan bined Hamamatsu a a and source, camera, light CCD CoolLED ORCA-ERA precisExcite a with microscope inverted Microscopy. FRET at format 96-well dimethyl a vol/vol use. in 10% stored with and supplemented (DMSO) plates, sulfoxide the off pooled 5 separately of the temperatures library, selection 30 respective the or at the incubated on were (PYE) Depending plates medium gentamicin. rich extract with peptone-yeast supplemented on selected were libraries mutant .CliradM hnihe o eeosypoiigsris n S. and strains, Technology providing of work Institute This Federal generously Z thank characterization. Swiss (ETH) the peptide We for from with support manuscript. help W.-D. institutional Thanbichler the for received Wennemers and on M. H. comments and support, and helpful Loosli for sequencing Collier Miller for I. J. S. Center and Genomics Hardt Functional Zurich ACKNOWLEDGMENTS. S1–S9 Tables Appendix, SI was Statement. peptide Availability CIP Data the uM. and 100 salts, of concentration M2 final a in to resuspended synchronized administered to was was 4% fraction peptide of swarmer the concentration and DMSO DMSO, final ter 100% a in in kept applied were Stocks GENEART. entific Assay. Peptide CIP 12. ref. to according settings channel and acquisition the neFudto rns31003A Grants Foundation ence .C .Ellison K. C. 9. Brilli M. 8. Caulobacter Caulobacter and t in ity networks. division. cell bacterial crescentus . Caulobacter motor. rotary bacterial a by response tactile Science analysis. genomic comparative A proteobacteria: ◦ A00wl-yeand wild-type NA1000 uigteiaig igetm on custoswr ae under taken were acquisitions point time Single imaging. the during C ◦ rc,EHRsac rn T-81- oBC,adSisNtoa Sci- National Swiss and B.C., to 16-1 ETH-08 Grant Research ETH urich, .A ona ooisapae,teslce uatlbaiswere libraries mutant selected the appeared, colonies as soon As C. ¨ albce crescentus. Caulobacter .Teeoe h I etd,rgltn h spatiotem- the regulating peptide, CIP the Therefore, E). 3–3 (2017). 535–538 358, h iest n vlto fcl yl euaini alpha- in regulation cycle cell of evolution and diversity The al., et rc al cd c.U.S.A. Sci. Acad. Natl. Proc. h seta eoeo bacterium. a of genome essential The al., et ihtruhu dnicto fpoenlclzto dependency localization protein of identification High-throughput al., et smercldsrbto ftescn esne -iGPupon c-di-GMP messenger second the of distribution Asymmetrical al., et btuto fplsrtato tmltsbceilsraesensing. surface bacterial stimulates retraction pilus of Obstruction al., et uniaieslcinaayi fbacteriophage of analysis selection Quantitative al., et nldsdtie ecitoso tan,mdaadstan- and media strains, of descriptions detailed includes el,CPppieasy unicto fsraeattachment, surface of quantification assay, peptide CIP cells, eegnrtda rvosydsrbd(4 5.Transposon 15). (14, described previously as generated were RTiaigwspromdo io cis Ti-E Eclipse Nikon a on performed was imaging FRET h I etd a ree rmTem ihrSci- Fisher Thermo from ordered was peptide CIP The Science .Bacteriol. J. etakC qioadR clpahfo the from Schlapbach R. and Aquino C. thank We and ∆pilA Tn5 .Ml Biol. Mol. J. 2519 (2010). 1295–1297 328, l aadsusdi h ae r vial in available are paper the in discussed data All rnpsnmtgnss rwhslcinand selection growth mutagenesis, transposon aae S1. Dataset yestrtdtasoo uatlibraries mutant transposon hypersaturated 646 310030 166476, ouain ro oFE irsoy The microscopy. FRET to prior populations 0011 (2019). e00031-19 201, 6148 (2010). 4681–4686 107, Caulobacter 1–3 (2016). 419–430 428, λ Science 100× M yt Biol. Syst. BMC NSLts Articles Latest PNAS 3–3 (2017). 531–534 358, i h Mojcie com- objective, DM Ph3 Oil 864 n CRSII5 and 184664, eepolda different at profiled gene o.Ss.Biol. Syst. Mol. −80 Caulobacter ◦ 2(2010). 52 4, ◦ o subsequent for C ,10 C, b susceptibil- φCbK 2 (2011). 528 7, ◦ Caulobac- ,25 C, | Fg 3 (Fig. 177164 f8 of 7 ◦ C,

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