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The Type IV Pilin Pila Couples Surface Attachment and Cell Cycle Initiation in Caulobacter Crescentus

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The Type IV Pilin Pila Couples Surface Attachment and Cell Cycle Initiation in Caulobacter Crescentus bioRxiv preprint doi: https://doi.org/10.1101/766329; this version posted September 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The type IV pilin PilA couples surface attachment and cell cycle initiation in Caulobacter crescentus Luca Del Medicoa, Dario Cerlettia, Matthias Christena,1, and Beat Christena,1 aInstitute of Molecular Systems Biology, Department of Biology, ETH Zürich, Zurich, Switzerland This manuscript was compiled on September 27, 2019 1 Understanding how bacteria colonize surfaces and regulate cell cy- quantify c-di-GMP signalling dynamics inside single cells and 30 2 cle progression in response to cellular adhesion is of fundamental found that, besides its structural role in forming type IVb pili 31 3 importance. Here, we used transposon sequencing in conjunction filaments, monomeric PilA in the inner membrane functions 32 4 with FRET microscopy to uncover the molecular mechanism how as a specific input signal that triggers c-di-GMP signalling at 33 5 surface sensing drives cell cycle initiation in Caulobacter crescen- the G1-S phase transition. 34 6 tus. We identified the type IV pilin protein PilA as the primary signal- 7 ing input that couples surface contact to cell cycle initiation via the Results 35 8 second messenger c-di-GMP. Upon retraction of pili filaments, the 9 monomeric pilin reservoir in the inner membrane is sensed by the A specific cell cycle checkpoint delays cell cycle initiation. To 36 10 17 amino-acid transmembrane helix of PilA to activate the PleC-PleD understand how bacterial cells adjust the cell cycle to reduced 37 11 two component signaling system, increase cellular c-di-GMP levels growth conditions, we profiled the replication time of the a- 38 12 and signal the onset of the cell cycle. We termed the PilA signaling proteobacterial cell cycle model organism Caulobacter across 39 13 sequence CIP for cell cycle initiating pilin peptide. Addition of the the temperature range encountered in its natural freshwater 40 14 chemically synthesized CIP peptide initiates cell cycle progression habitat (Table S1). Under the standard laboratory growth 41 15 and simultaneously inhibits surface attachment. The broad conser- temperature of 30°C, Caulobacter replicates every 84 ± 1.2 42 16 vation of the type IV pili and their importance in pathogens for host min. However, when restricting the growth temperature to 43 17 colonization suggests that CIP peptide mimetics offer new strategies 10°C, we observed a 13-fold increase in the duration of the 44 18 to inhibit surface-sensing, prevent biofilm formation and control per- cell cycle, extending the replication time to 1092 ± 14.4 min 45 19 sistent infections. (Table S1). To investigate whether reduced growth resulted in 46 a uniform slow-down or affects particular cell cycle phases, we 47 Caulobacter crescentus | c-di-GMP | Type IV pilin | TnSeq | FRET determined the relative length of the G1 phase by fluorescence 48 microscopy microscopy using a previously described cell cycle reporter 49 strain (11) (Materials and Methods). We found that the 50 1 he cell cycle model bacterium Caulobacter crescentus culturing of Caulobacter at 10°C caused a more than 1.4-fold 51 2 T(Caulobacter thereafter) integrates surface colonization increase in the relative duration of the G1 phase indicating a 52 3 into a bi-phasic life-cycle. Attachment begins with a reversible delay in cell cycle initiation (Fig. 1b). This finding suggested 53 4 phase, mediated by surface structures such as pili and flagella, the presence of a specific cell cycle checkpoint that delays cell 54 5 followed by a transition to irreversible attachment mediated cycle initiation during reduced growth conditions. 55 6 by polysaccharides (1–4). In Caulobacter surface sensing is in- 7 timately interlinking with cellular differentiation and cell cycle 8 progression (5, 6). During the bi-phasic life cycle, Caulobacter Significance Statement 9 divides asymmetrically and produces two distinct cell types Pili are hair-like appendages found on the surface of many 10 with specialized development programs (Fig. 1a). The sessile bacteria to promote adhesion. Here, we provide systems-level 11 stalked cell immediately initiates a new round of chromosome findings on a molecular signal transduction pathway that in- 12 replication, whereas the motile swarmer cell, equipped with terlinks surface sensing with cell cycle initiation. We propose 13 a polar flagellum and polar pili, remains in the G1 phase that surface attachment induces depolymerization of pili fila- 14 for a defined interval before differentiating into a stalked cell ments. The concomitant increase in pilin sub-units within the 15 and entering into the replicative S phase driven by the sec- inner membrane function as a stimulus to activate the second 16 ond messenger c-di-GMP dependent degradation of the cell messenger c-di-GMP and trigger cell cycle initiation. Further- 17 cycle master regulator CtrA (7, 8) (Fig. 1a). The change more, we show that the provision of a 17 amino acid synthetic 18 in cell cycle state from motile swarmer into surface attached peptide corresponding to the membrane portion of the pilin 19 replication-competent stalked cells depends on tactile sensing sub-unit mimics surface sensing, activates cell cycle initiation 20 mechanisms. Both pili and flagella have been previously impli- and inhibits surface attachment. Thus, synthetic peptide mimet- 21 cated as key determinants involved in tactile surface sensing ics of pilin may represent new chemotypes to control biofilm 22 (9, 10). However, understanding the molecular mechanism of formation and treat bacterial infections. 23 how Caulobacter interlinks bacterial surface attachment to cell 24 cycle initiation has remained elusive. LDM, MC, and BC conceived the research; LDM performed transposon mutagenesis experiments, 25 In this work, we report on a short peptide signal encoded LDM perfomed FRET microscopy, DC performed time-lapse FRET microscopy; LDM, MC, and BC analyzed data; LDM, MC, and BC wrote the manuscript. 26 within the type IVb pilin protein PilA that exerts pleiotropic No conflict of interest declared. 27 control and links bacterial surface attachment to cell cycle 28 Caulobacter initiation in . Using FRET microscopy in conjunc- 1To whom correspondence should be addressed. E-mail: [email protected]; 29 tion with a genetically encoded c-di-GMP biosensor (11), we [email protected] bioRxiv | September 27, 2019 | 1–17 bioRxiv preprint doi: https://doi.org/10.1101/766329; this version posted September 27, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. a b forward genetic screen based on quantitative selection analysis 59 40 coupled to transposon sequencing (TnSeq) (12, 13). TnSeq 60 Non-replicative Replicative measures genome-wide changes in transposon insertion abun- 61 (G1) (S) 30 * dance upon subjecting large mutant populations to different 62 selection regimes and enables genome-wide identification of 63 20 essential genes. We hypothesised that the profiling of growth- 64 rate dependent changes in gene essentiality will elucidate the 65 10 Swarmer Stalked components of the cell cycle machinery fundamental for cell 66 67 c-di-GMP cells in G1 phase [%] cycle initiation under reduced growth conditions. We selected 0 10 30 Caulobacter transposon mutant libraries for prolonged growth 68 CtrA Growth [ºC] at low temperatures (5°C and 10°C) and under standard labo- 69 ratory cultivation conditions (25°C and 30°C). Cumulatively, 70 c shkA rcdA we mapped for each condition between 397’377 and 502’774 71 unique transposon insertion sites across the 4.0 Mbp Caulobac- 72 nepR ter genome corresponding to a transposon insertion densities 73 °C 74 5 of 4-5 bp (Table S2). 10 25 To identify the factors required for cell cycle progression, 75 30 cpdR pilA we focused our analysis on essential genes (Data SI) that are 76 C. crescentus expressed in a cell cycle-dependent manner (Materials and 77 genome fliH Methods, (14–16)). Among 373 cell cycle-controlled genes, 78 4.0 Mbp we found 45 genes that were essential under all growth con- 79 ditions (Fig. 1c, Data SI), including five master regulators 80 pleC (ctrA, gcrA, sciP, ccrM and DnaA), eleven divisome and cell 81 pleD wall components (ftsABILQYZ, fzlA and murDEF), six DNA 82 cro replication and segregation factors (dnaB, ssb, gyrA, mipZ, 83 xseB parB and ftsK ) as well as 23 genes encoding for key signalling 84 lon factors and cellular components required for cell cycle pro- 85 core cell cycle genes essential at slow growth (12) gression (Data SI). Collectively, these 45 genes form the core 86 (45) components of the bacterial cell cycle machinery. 87 d TnSeq selection [°C] Components of the c-di-GMP signalling network are condi- 88 5 10 25 30 tionally essential for slow growth. During reduced growth con- 89 lon CCNA_02037 Endopeptidase ditions, we found 12 genes that specifically became essential 90 pleD CCNA_02546 Diguanylate cyclase (Fig. 1c). To gain insights into the underlying genetic modules, 91 rcdA CCNA_03404 CtrA proteolysis regulator we performed a hierarchical clustering analysis and grouped 92 cluster A _ cpdR CCNA 00781 Proteolytic ClpXP adapter these 12 genes according to their growth-rate dependent fit- 93 flbE CCNA_00952 Flagellar assembly protein ness profile into three functional clusters A, B and C (Fig. 1d, 94 pilA CCNA_03043 Type IVb pilin Materials and Methods). 95 CCNA_02567 Histidine kinase pleC Cluster A contained four conditionally essential genes that 96 _ cluster B shkA CCNA 00137 Histidine kinase exhibited a large decrease in fitness during slow-growth con- 97 CCNA_02103 Hypothetical protein ditions (Fig 1d, Fig.
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