bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 Sensory domain of the cell cycle kinase CckA in Caulobacter crescentus
2 regulates the differential DNA binding activity of the master regulator CtrA
3
4 Sharath Narayanan, Lokesh Kumar and Sunish Kumar Radhakrishnan*
5
6 School of Biology, Indian Institute of Science Education and Research,
7 Thiruvananthapuram 695016, Kerala, India.
8
9 *Correspondence to: [email protected]
10
11
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13
14
15
16
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21 Abstract
22 Sophisticated signaling mechanisms allow bacterial cells to cope with environmental
23 and intracellular challenges. Activation of specific pathways facilitates the cells to
24 overcome cellular damage and thereby warrant integrity. Here we demonstrate the
25 pliability of the CckA-CtrA two component signaling system in the freshwater bacterium,
26 Caulobacter crescentus. Our forward genetic screen to analyse suppressor mutations
27 that can negate the chromosome segregation block induced by the topoisomerase IV
28 inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA.
29 Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in
30 increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator.
31 Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide
32 increase in the DNA occupancy of CtrA, but specifically enriched its affinity to the
33 chromosomal origin of replication, Cori, and a very small sub-set of CtrA regulated
34 promoters. We show that through this enhanced binding of CtrA to the Cori, cells are
35 able to overcome the toxic defects rendered by stable NstA through a possible slow
36 down in the chromosome cycle. Taken together, our work opens up an unexplored and
37 intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA
38 binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis
39 because of the highly conserved nature of CckA-CtrA pathway in alphaproteobacteria.
40
41
42
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43 Introduction
44 Bacteria harbor robust signaling mechanisms, to respond to numerous
45 environmental challenges both inside and outside the cell. Exquisitely fine tuned
46 regulatory cascades in bacteria impart their effect at a precise spatio-temporal scale to
47 bring about specific morphological and functional programs, in response to the changes
48 in the internal or external milieu. The aquatic α-proteobacterium, Caulobacter
49 crescentus (henceforth Caulobacter), has emerged as a powerful model organism for
50 studying the complex signaling mechanisms that control cell cycle and development in
51 response to environmental cues. During its cell cycle, Caulobacter undergoes
52 asymmetric division to produce progenies with distinct developmental fates. One of the
53 daughter cells, the swarmer cell, acquires a dispersal fate wherein its motility is assisted
54 by the polar flagellum and a tuft of pili (1,2). In contrast, the stalked daughter cell
55 acquires a sedentary fate and is in an S-phase-like state capable of replicating its
56 chromosome and proliferating by cytokinesis (Figure 1A) (3,4). The G1-like swarmer cell
57 has to terminally differentiate into a stalked cell to enter into the proliferative phase. This
58 G1 to S-like transition is marked by the shedding of the flagellum, retraction of the pili,
59 and production of a stalk at the same cell pole (Figure 1A).
60 In the swarmer cells, the master transcriptional regulator, CtrA, inhibits the DNA
61 replication. The Caulobacter origin of replication, Cori, is bound by CtrA, which prevents
62 replisome formation in the swarmer cells (5). Concurrent with the swarmer to stalked
63 cell transition, CtrA is degraded by proteolysis and thus facilitating the binding of DnaA,
64 the replication initiator, to the Cori triggering chromosome replication.(6). Apart from
65 blocking DNA replication initiation, CtrA also serve as a transcription factor to drive the
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66 expression of numerous developmentally important genes in a cell cycle dependent
67 manner (7).
68 The differential activity of CtrA in swarmer and stalked cells is of paramount
69 significance for generating different cell fates. Multiple levels of regulation involving
70 control at the level of synthesis, stability, and activity exist for the regulation of CtrA
71 during cell cycle (8,9). The phosphorylated form of CtrA (CtrA~P) represents the active
72 form that binds to DNA (10). The phosphorylation of CtrA is catalyzed by an essential
73 hybrid cell cycle histidine kinase/phosphatase, CckA, which phosphorylates CtrA
74 through the single domain histidine phosphotransferase, ChpT (11-14). In the swarmer,
75 and pre-divisional cells, the kinase activity of CckA ensures the abundance of active
76 CtrA~P, while in the stalked cell compartment, the phosphatase activity of CckA is
77 predominant ensuring the dephosphorylation, and degradation, of CtrA (Figures 1A and
78 1B) (15).The bifunctional nature of CckA is governed by the second messenger, cyclic-
79 di-guanylate (c-di-GMP). Binding of c-di-GMP to CckA causes an inhibition of its kinase
80 activity and trigger the phosphatase activity. The swarmer to stalked cell transition is
81 accompanied by an increase in the levels of c-di-GMP, which causes the transition of
82 CckA from the kinase to the phosphatase mode (16,17).
83 Recent evidences have shown that in addition to developmental regulatory
84 proteins such as CtrA, the cell cycle progression in Caulobacter is controlled by a
85 cytoplasmic redox fluctuation (18). We have shown that a redox dependent regulator,
86 NstA, whose activation is coupled to the cytoplasmic redox state, inhibits the DNA
87 decatenation activity of topoisomerase IV (Topo IV) during the early stages of cell cycle
88 (18). Apart from the cytoplasmic redox control of NstA activity, additional layers of
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89 regulation for NstA exist at the level of transcription by the transcription factors, GcrA
90 and CcrM, and at the level of protein abundance by the ClpXP protease. A stable
91 version of NstA, NstADD, is resistant to protein degradation by ClpXP. Overproduction
92 of NstADD from an inducible promoter induces lethality in Caulobacter (18). In this
93 study, we wished to investigate the regulatory networks that possibly fine-tune NstA
94 activity in vivo. Towards this, we exploited the lethality induced by NstADD, to conduct
95 an unbiased forward genetic screen to analyze extragenic suppressor mutation(s) that
96 can negate NstADD toxicity. Strikingly, through this screen we have identified
97 suppressor mutations in cckA that influences the DNA binding activity of CtrA in a
98 distinctive manner. We show that the CckA(L228P) mutation though enhances the
99 CtrA~P levels, does not universally increase the binding of CtrA on a genome-wide
100 scale. Rather, we found that the CckA(L228P) mutation specifically increases the
101 binding of CtrA~P at the Cori and a very small sub-set of CtrA dependent promoters.
102 Finally, we show that the enhanced binding of CtrA to the Cori rescues the toxicity
103 caused by NstADD by possibly slowing down the chromosome replication process to
104 compensate for the slowed down segregation caused by the inhibitory effects of
105 NstADD on the Topo IV.
106
107 Results
108 Suppressor mutations in CckA alleviate NstADD toxicity
109 Overproduction of the cell-cycle stable form of the Topo IV inhibitor, NstADD,
110 leads to fitness defect and impaired chromosome segregation in Caulobacter (18). To
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111 unearth the signaling network that regulate NstA, we exploited the lethality induced by
112 NstADD overproduction in a genetic screen to identify extragenic suppressors that could
113 tolerate NstADD toxicity (See Materials and Methods). Strikingly, whole-genome
114 sequencing revealed that all nine extragenic suppressors harbored a mutation in the
115 gene encoding the cell cycle histidine kinase, cckA (Supplementary Figure S1C). The
116 suppressor mutations were CckA-(L228P), (A317V), (D364G), (R356C), (F392L),
117 (F493C) and (F496C) (Figure 1C, Supplementary Figures S1B, S2A and S2B).
118 Interestingly, all the point mutations in cckA, except the L228P substitution, were
119 located either in the histidine kinase domain or the ATP binding domain of the CckA
120 protein (Supplementary Figure S1C). Remarkably the L228P mutation, which mapped
121 to the PAS-B domain in CckA, rendered developmental defects such as cell
122 filamentation in the WT and the ∆nstA mutant (Figure 1D, Supplementary Figure S2A).
123 To confirm that it is the L228P mutation in cckA that conferred resistance to NstADD
124 toxicity, we backcrossed the cckA(L228P) mutation into WT and ∆nstA. The
125 backcrossed cells were indeed able to tolerate the NstADD overexpression
126 (Supplementary Figure S1A). The fact that the CckA(L228P) mutation was not in the
127 kinase or the ATP binding domain of CckA, prompted us to investigate further the
128 mechanism by which the cckA(L228P) mutant induced the developmental defects and
129 negated the chromosome segregation defect attributed by NstADD in Caulobacter.
130 CckA(L228P) mutation leads to increased CtrA~P levels but not increase in CtrA
131 binding or activity
132 The cell cycle histidine kinase, CckA, is the primary kinase that activates the
133 master cell cycle transcriptional regulator, CtrA, by phosphorylation (10). The
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134 phosphorylated form of CtrA (CtrA~P) binds efficiently to its target promoters on the
135 chromosome regulating transcription (10,11,19). Therefore, we decided to investigate if
136 the L228P mutation in the PAS-B domain of CckA could affect CtrA~P levels.
137 Interestingly, in vivo phosphorylation analysis revealed that the relative levels of CtrA~P,
138 compared to total CtrA, was two fold higher in the cckA(L228P) mutant than the wild
139 type cells (Figure 2A).
140 It has been shown that increase in CtrA~P levels could result in elevated levels of
141 CtrA binding to its target promoters (20,21). Therefore, we decided to analyze the
142 binding of CtrA~P on well-established CtrA-dependent promoters such as pilA, tacA and
143 kidO (22-24), by quantitative chromatin immunoprecipitation (qChIP) analysis using
144 CtrA specific antibodies. Surprisingly, our qChIP experiments revealed that the binding
145 of CtrA to the PpilA, PkidO and PtacA promoters were not significantly different in the
146 cckA(L228P) mutant when compared to wild-type (Figure 2D, Supplementary Figure
147 S3A, B). Further, β-galactosidase (LacZ)-based promoter-probe assays using the
148 promoters of pilA and tacA in WT, ∆nstA and ∆nstA cckA(L228P) mutant backgrounds,
149 revealed that there was no measurable differences in the PpilA and PtacA promoter
150 activities in the cckA(L228P) mutant (Figures 2B and 2C).
151 Collectively, these results indicated that the cckA(L228P) mutation increased
152 CtrA~P levels. Nevertheless, this surge in CtrA~P levels did not result in increased
153 binding of CtrA, or elevated promoter activity, on at least the G1-specific promoters of
154 CtrA such as PkidO, PpilA and PtacA. Furthermore, these observations opened up the
155 possibility that CckA, apart from its kinase activity, may be influencing the DNA-binding
156 activity of CtrA, only at specific promoter regions in the Caulobacter genome.
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157 CckA influences the promoter specific binding of CtrA
158 Next we decided to investigate if the absence of difference in binding of CtrA,
159 despite increased CtrA~P levels in the cckA(L228P) mutant, is specific to a subset of
160 CtrA dependent promoters. Towards this, we performed chromatin immunoprecipitation
161 followed by deep sequencing (ChIP-seq) to analyze the CtrA occupancy on its target
162 promoters in the cckA and cckA(L228P) mutant backgrounds on a genome-wide scale.
163 From the ChIP-seq analyses it was evident that the cckA(L228P) mutation enhanced
164 the CtrA occupancy at the promoter regions of target genes whose transcripts peaked
165 at late S-phase, including the Class II flagellar genes such as pleA, fliQ, fliL, fliM, fliJ
166 and fliI (25), pilus secretion genes, cpaA and cpaB (26), flagellar regulatory genes, flbT
167 and flbA (27), and the chemotaxis genes, motA and motB (28) (Figures 3 A, C ,D and
168 Supplementary Dataset 1). To corroborate if this increase in binding of CtrA to these
169 promoters resulted in an increased promoter activity, we analyzed the activity of the fliM
170 promoter (PfliM) and the flbT promoter (PflbT) using LacZ reporter fusions to these
171 promoters (PfliM-lacZ and PflbT-lacZ). Our analyses showed that indeed the activities of
172 PfliM and PflbT were increased in the cckA(L228P) mutant background commensurate
173 with the increase in binding of CtrA to these promoters (Supplementary Figure S4A, B).
174 Further, the qChIP experiments confirmed the enhanced binding of CtrA at the promoter
175 region of flbT, in the ∆nstA cckA (L228P) background when compared to the WT or
176 ∆nstA (Supplementary Figure S3C).
177 Interestingly, in addition to the S-phase specific promoters, the ChIP-seq data
178 also revealed a significant increase in binding of CtrA to the chromosomal origin of
179 replication, Cori (Figure 3B). The, qChIP experiment also confirmed the increase in CtrA
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180 occupancy at the Cori, in the cckA(L228P) mutant. In comparison to the WT or ∆nstA, a
181 three-fold increase in the binding of CtrA at Cori was evident in the cckA(L228P) mutant
182 background (Supplementary Figure S3D). Together, these data pointed towards the
183 differential DNA binding of CtrA, as a result of the cckA(L228P) point mutation to S-
184 phase specific target promoters, and Cori, possibly regulated through the PAS-B domain
185 of CckA.
186
187 CtrA mediated repression of replication initiation mitigate NstA induced lethality
188 Next, we wondered if the increase in the CtrA binding to the CorI is what
189 contributes to the rescue of the toxicity induced by the overproduction of stable NstADD.
190 We hypothesized that the specific modulation of CtrA activity by the CckA(L228P)
191 mutation leading to enhanced binding of CtrA to the Cori may be delaying replication
192 initiation. Further, this delay in replication initiation may be slowing down the
193 chromosome cycle to compensate for a reduced activity of a downstream event in the
194 chromosome cycle. For example, the slow down in segregation process by the
195 reduction in the decatenation activity of Topo IV by NstADD (18). To test this
196 hypothesis, we decided to monitor the appearance or movement of the newly replicated
197 chromosomal origin in the cckA(L228P) mutant. In Caulobacter, it has been well
198 demonstrated that the newly formed origin of replication is immediately tethered to the
199 opposite pole upon initiation of replication (29). The chromosome partitioning protein,
200 ParB, specifically binds to the regions near Cori and moves along with the Cori upon
201 initiation of replication (30,31). Therefore, the movement of fluorescently tagged ParB,
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202 GFP-ParB, can be used as a proxy to monitor the movement of the newly formed Cori to
203 the opposite cell pole (31,32). Localization experiments using GFP-ParB showed that
204 the ΔnstA cckA(L228P) mutant had stalked cells with either single GFP-ParB foci
205 (22.4%; Figure 4A red arrow heads, Figure 4C), or with two GFP-ParB foci, partially
206 segregated, with the second foci still migrating to the opposite pole (33.8%; Figure 4A
207 white arrow heads, Figure 4C). This was unlike in ΔnstA cells wherein the newly
208 replicated Cori along with GFP-ParB was immediately tethered to the opposite pole upon
209 initiation of replication (86.9%; Figures 4B and C). From this observation we inferred
210 that in the cckA(L228P) mutant, the initiation of replication and the elongation processes
211 of the chromosome was slowed. This slow down may well be due to the increase in
212 CtrA binding to the Cori. We also observed multiple GFP-ParB foci in Caulobacter cells,
213 overproducing NstADD (Figure 4D), unlike the control samples with pMT335 vector
214 alone, wherein, bipolar GFP-parB foci were predominant (Figure 4D). Thus we surmise
215 that in the cells overproducing NstADD, multiple rounds of DNA replication are initiated
216 and the chromosome decatenation is hampered (18). To counter this effect,
217 cckA(L228P) point mutation enhances the CtrA binding at the Cori, which can
218 significantly slow down the replication cycle. Our hypothesis was further corroborated by
219 the observation that the increase in the binding of CtrA to the Cori is still retained after
220 the overexpression of NstADD. In comparison to the WT or ΔnstA, overexpressing
221 NstADD, the cckA(L228P) mutant cells overproducing NstADD, had a significant
222 increase in the occupancy of CtrA at the Cori (Figure 5B).
223 The CtrA binding boxes at the origin overlaps with the DnaA binding sites (6,33).
224 Thus when CtrA~P is abundant in the system, the DnaA binding to the origin is inhibited
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225 leading to inhibition of chromosome replication initiation (34). Therefore, we speculated
226 that, if it is the increase in CtrA binding that is leading to slow down in replication, then
227 such an inhibition should be relieved by titrating out CtrA at the Cori by the
228 overexpression of DnaA. Indeed, the overproduction of DnaA or its constitutively active
229 ATP bound form, DnaA(R357A), from the xylose inducible promoter (Pxyl) on a medium
230 copy plasmid (35) caused a considerable decrease in cell filamentation of SN208 cells
231 (Figure 5A). In addition, the qChIP experiments, also confirmed that the CtrA occupancy
232 at Cori was greatly reduced by about 70%, post the DnaA/DnaA(R357A) overexpression
233 in the SN208 mutant (Figure 5C). Altogether, these results indicated at the increased
234 binding of CtrA to Cori facilitated by the cckA(L228P) mutation can alleviate toxicity
235 attributed to NstADD overproduction.
236
237 Discussion
238 The highly conserved CckA-CtrA signal transduction pathway in α-proteobacteria
239 has several implications in development and pathogenesis. For example, during the
240 early stages of symbiosis, in the nitrogen fixing bacteria, Sinorhizobium meliloti (S.
241 meliloti), the role of CckA and its regulation has been shown to be essential (36).
242 Likewise, the viability of the intracellular pathogen, Brucella abortus (B. abortus), in the
243 human macrophages is dependent on the CckA-ChpT-CtrA pathway (37). The
244 regulatory networks involving CtrA can be related to the specific lifestyle of the
245 bacterium. For instance, while in Caulobacter CtrA is involved in cell-fate control, and
246 cell cycle, by fine-tuning the stalked and swarmer cell programs, the control of cell
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247 envelope composition by CtrA is prevalent in B. abortus and Rhizobium leguminosarum
248 (38,39), reiterating the plasticity of CckA-CtrA pathway.
249 In this study, we shown that the L228P mutation in the PAS-B domain of CckA
250 not only increases the CtrA~P levels but also rewires the preferential binding of CtrA to
251 its target promoters (Figure 2A, 3 and Supplementary Dataset 1). The PAS-B domain in
252 CckA has been shown to be necessary for regulation of its auto kinase activity, and for
253 the switching of CckA between the kinase and the phosphatase modes (40). The CckA
254 phosphatase activity during the swarmer to stalked cell transition is triggered by the
255 binding of the effector molecule, c-di-GMP, to the PAS-B domain (16,17,40). Therefore,
256 it is conceivable that the cckA(L228P) mutation possibly perturbs the binding of c-di-
257 GMP to CckA thereby locking CckA in a kinase active form leading to increased CtrA~P
258 levels in the cckA(L228P) mutant (Figure 6).
259 Surprisingly, the above-mentioned increase in the CtrA~P levels does not
260 translate into a uniform increase in the binding of CtrA on all its target sites on the
261 chromosome. The increased binding happens only at the Cori and a sub-set of S-phase
262 specific CtrA promoters. Previous studies have shed light on the additional components,
263 SciP and MucR, which modulate the activity of the CtrA-dependent promoters during
264 cell cycle (41-43). While MucR specifically represses G1-phase promoters of CtrA, SciP
265 has been shown to negatively regulate the CtrA-dependent promoters whose activity
266 are known to peak at the S-phase of the cell cycle (42). Interestingly, our comparative
267 ChIP-Seq analysis, revealed that the CckA(L228P) substitution contributes to specific
268 enhancement of CtrA binding at S-phase promoters, which are also bound by SciP. The
269 mechanistic mode of repressing the CtrA transcription by SciP involves a direct
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270 interaction between SciP and CtrA (43). Interestingly, SciP does not perturb the DNA
271 binding activity of CtrA and it blocks the RNA polymerase recruitment to the CtrA
272 activated promoters (43). Moreover, SciP itself is under the direct transcriptional control
273 of CtrA (41). Nevertheless, our ChIP-Seq analysis shows that in the cckA(L228P)
274 mutant the binding of CtrA to the sciP promoter is not significantly altered
275 (Supplementary Dataset 1) indicating that sciP transcription might not be altered in
276 CckA(L228P). Therefore, it is tempting to speculate that the CckA(L228P) substitution
277 possibly facilitates CtrA to overcome the inhibition imparted by SciP either by directly
278 acting on SciP or by making CtrA more potent to compete for the RNA polymerase. It
279 may also be possible that the CckA kinase could be regulating the interaction between
280 SciP and CtrA, in a direct or indirect manner. However, this hypothesis, remains to be
281 investigated further and our results pave way for exploring this intriguing aspect of the
282 CckA-CtrA pathway.
283
284 Materials and methods
285 Growth Conditions and Media
286 Caulobacter strains were grown on rich PYE media (0.2% peptone, 0.1% yeast
287 extract, 1 mM MgSO4, 0.5 mM CaCl2) or minimal M2G media (M2 -1X salt solution
288 [Na2HPO4 0.87gm/L, KH2PO4 0.53gm/L, NH4Cl 0.25gm/L] supplemented with 0.5 mM
289 MgSO4, 0.2 % Glucose, 10 µM FeSO4.EDTA, 0.5 mM CaCl2) (44), and incubated at
290 29°C, unless specifically mentioned. The Caulobacter strains were subjected to
291 electroporation, øCr30 mediated transductions, and intergeneric conjugations (using
292 E.coli S17-1) as previously described (24,45,46). E. coli strains, EC100D (Epicentre,
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293 WI, USA), and S17-1 were grown on LB media and incubated at 37°C, unless
294 specifically mentioned.
295
296 In vivo phosphorylation
297 In vivo phosphorylation experiments were performed as described previously (47). A
298 single colony of cells picked from a PYE agarose plate was washed with M5G medium
299 lacking phosphate and was grown overnight in M5G with 0.05 mM phosphate to an
300 optical density of 0.3 at 660 nm. One milliliter of culture was labeled for 4 min at 28°C
32 301 using 30 µCi of γ-[ P]ATP. Upon lysis, proteins were immunoprecipitated with 3 ml of
302 anti-CtrA antiserum and Protein A agarose (Roche, CH) and the precipitates were
303 resolved by SDS–polyacrylamide gel electrophoresis and radiolabelled CtrA was
304 quantified and were normalized to the relative cellular content as determined by
305 immunoblotting of lysates.
306
307 Extragenic suppressor Screen
308 ∆nstA or WT cells were UV irradiated with 700 and 900×100 µJ/cm2 energy using
309 CL 1000 UV Cross linker (UVP, Cambridge, UK). The irradiated cells were diluted into
310 PYE media followed by 6 hr incubation. The cells were then electroporated with plasmid
311 pMT335-Pvan-nstADD and plated on PYE supplemented with gentamycin and vanillate
312 inducer. Individual colonies were then grown in liquid PYE containing gentamycin, and
313 vanillate inducer for overnight. Two criteria were used for confirming the extragenic
314 mutation: (i) plasmids from the selected mutants were again transformed into WT
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315 Caulobacter and checked for nstA toxicity, to avoid the possibility that the suppression
316 is due to mutation on the plasmid, and (ii) plasmid cured mutants were retransformed
317 with fresh pMT335-Pvan-nstADD, to confirm that the toxicity suppression is indeed due to
318 a mutation in the chromosome. Mutations were mapped by next generation sequencing
319 on an Illumina platform at Fasteris, Switzerland.
320
321 Chromatin immunoprecipitation (ChIP)
322 ChIP experiments were carried out as described earlier (24). Mid-log phase cells
323 were cross-linked in 10 mM sodium phosphate (pH 7.6) and 1% formaldehyde at room
324 temperature for 10 minutes and on ice for 30 min thereafter, washed thrice in phosphate
325 buffered saline (pH 7.4) and lysed in 5000 Units of Ready-Lyse lysozyme solution
326 (Epicentre Technologies, WI, USA). Lysates were sonicated on ice using 7 bursts of 30
327 sec to shear DNA fragments to an average length of 0.3-0.5 kbp. The cell debris were
328 cleared by centrifugation at 14,000 rpm for 2 min at 4°C. Lysates were normalized by
329 protein content, diluted to 1 mL using ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2
330 mM EDTA, 16.7 mM Tris-HCl [pH 8.1], 167 mM NaCl plus protease inhibitors
331 [CompleteTM EDTA-free, Roche, Switzerland]), and pre-cleared with 80 µl of protein-
332 A agarose (Roche, Switzerland) saturated with 100 µg BSA and 300 µg Salmon sperm
333 DNA. Ten % of the supernatant was removed and used as total chromatin input
334 DNA. To the remaining supernatant, anti-CtrA (20) antibody was added (1:500 dilution),
335 and incubated overnight at 4°C. Immuno complexes were trapped with 80 µL of protein-
336 A agarose beads pre-saturated with BSA-salmon sperm DNA. The beads were then
337 washed once each with low salt buffer (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20
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338 mM Tris-HCl [pH 8.1], 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2
339 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl) and LiCl buffer (25 mM LiCl,
340 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]), and twice
341 with TE buffer (10 mM Tris-HCl[pH 8.1], 1 mM EDTA). The protein•DNA complexes
342 were eluted in 500 µL freshly prepared elution reagent (1% SDS, 1 mM NaHCO3). This
343 was supplemented with NaCl to a final concentration of 300 mM and incubated
344 overnight at 65°C to reverse the crosslinks. The samples were treated with 2 µg of
345 Proteinase K (Roche, Switzerland) for 2 hr at 45°C in after addition of 40 mM EDTA and
346 40 mMTris-HCl (pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol
347 (25:24:1), ethanol-precipitated using 20 µg of glycogen as carrier, and resuspended in
348 50 µL of sterile deionized water. The comparative ChIP-followed by deep Sequencing
349 (ChIP-Seq), was done using the next generation sequencing on an Illumina platform at
350 Fasteris, Switzerland.
351
352 Quantitative PCR (qPCR) analyses
353 qPCR was performed on a CFX96 Real Time PCR System (Bio-Rad, CA, USA) using
354 10% of each ChIP sample, 12.5 µL of SYBR® green PCR master mix (Bio-Rad, CA,
355 USA), 200 nM of primers and 6.5 µl of water per reaction. Standard curve generated
356 from the cycle threshold (Ct) value of the serially diluted chromatin input was used to
357 calculate the % input value of each sample. Average values are from triplicate
358 measurements done per culture. The final data was generated from three independent
359 cultures. The SEM shown in the figures was derived with Origin 7.5 software (OriginLab
360 Corporation, Northhampton, MA, USA). Cori_Fwd (5’-CGCGGAACGACCCACAAACT-3’)
16 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
361 and Cori_Rev (5’-CAGCCGACCGACCAGAGCCA-3’) primer pairs as described earlier
362 (35) were used to amplify the region near ori precipitated by anti-CtrA antibody. To
363 check CtrA binding on PpilA, the DNA region analysed by real time PCR was from
364 nucleotide -287 to -91 relative to the start codon of pilA (24). A PkidO fragment
365 comprising nt 3,857,810–3,858,141 of the NA1000 genome sequence was quantified
366 (23) to monitor CtrA binding on PkidO. To quantify CtrA occupancy at the promoter of
367 flbT, the DNA region, -280 to +30 relative to the start codon of flbT , was used. The DNA
368 region from -226 to +30 relative to the start codon of tacA was analysed for quantifying
369 CtrA occupancy on PtacA (24).
370
371 ChIP-seq data analysis
372 The FASTQ files were checked for quality of sequencing using FastQC software,
373 version 0.11.5. The first ten bases showed distortion, due to which it was decided to trim
374 the first ten bases from all short reads. The reads were trimmed at the 5' end for 10
375 bases using fastx_trimmer tool from Fastx-toolkit version 0.0.14. The preprocessed
376 reads were mapped to the Caulobacter Cresentus NA1000 reference genome
377 (CP001340.1) using aligner Bowtie version 1.0.0 using the following parameter: -m 1, -
378 S, -v 2. Around 36.9 million reads mapped uniquely to the reference genome for the wild
379 type cckA and 21 million reads for the mutant cckA(L228P) strains.
380 Further, the aligned reads were imported onto Seqmonk (version 1.38.1) to build the
381 sequence read profiles. The genome was subdivided into 50bp probes and a value
382 representing the number of reads mapping to the genome within a probe was calculated
383 using the Read Count Quantitation option. The probe list with the quantified value for
17 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
384 each probe was exported. Custom Perl scripts were used to compute the relative
385 abundance of each probe as a percent with respect to the total uniquely mapped reads
386 for each dataset. A cutoff was determined as average reads plus twice the standard
387 deviation of the sample to differentiate between candidate peaks and background noise.
388 The candidate peaks were annotated using custom Perl scripts. A probe was annotated
389 with a gene if the centre of the probe was within a distance of -500 and +100 bases
390 from the transcription start site of the gene, taking into account the orientation of the
391 gene as well. If a probe is found to satisfy the condition for two genes (each on either
392 strand), then both the genes are reported. A list of nearby RNA genes are also reported
393 separately. Probes without an annotation are labeled as 'NO ANNO'.
394
395 Microscopy
396 Differntial interference contrast (DIC) and fluorescence microscopy were
397 performed on a Nikon Eclipse 90i microscope equipped with 100X oil TIRF (1.49
398 numerical aperture) objective and a coolSNAP HQ-2 (Photometrics, USA) CCD camera.
399 Cells were placed on a 1% agarose solidified pads for imaging. Images were processed
400 and analyzed with the Metamorph software (Molecular Devices, USA).
401
402 β-Galactosidase Assay
403 The cultures harbouring were incubated at 29°C till it reached 0.1-0.4 OD@660
404 nm (A660). 50 µl of the cells were treated with a 10 µl of chloroform followed by the
18 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
405 addition of 750 µ l of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1mM
406 MgSO4.7H2O, pH 7.0) followed by 200 µl of Ortho Nitro Phenyl-β-D-Galactoside (from
407 stock concentration of 4 mg/ml dissolved in 100 mM potassium phosphate buffer [pH
408 7.0]). The reaction mixture was incubated at 30°C till yellow color was developed.
409 Finally 500µl of 1 M Na2CO3 solution was added to stop the reaction and absorbance at
410 420nm (A420) of the supernatant was noted using Z-buffer as the blank. The miller units
411 (U) were calculated using the equation U= (A420 X 1000) / (A660 X t X v), where ‘t’ is the
412 incubation time (min), ‘v’ is the volume of culture taken (ml). Experimental values were
413 average of three independent experiments. The SEM shown in the figures was derived
414 with Origin 7.5 software (OriginLab Corporation, Northhampton, MA, USA).
415
416 Acknowlegdements
417 We thank Justine Collier and Patrick Viollier for materials, Rashmi Sukumaran for
418 her assistance with the ChIP-seq analysis, and Srinivasa Murty Srinivasula for
419 discussions and critical comments on the manuscript. S.N. is supported by a graduate
420 fellowship from IISER Thiruvananthapuram. L.K is supported by a Junior Research
421 Fellowship from the Council of Scientific & Industrial Research (CSIR) India. This work
422 was supported by funds from the Wellcome Trust-DBT India Alliance (500140/Z/09/Z)
423 through an Intermediate fellowship to S.K.R., and funds from the SwarnaJayanti
424 Fellowship (DST/SJF/LSA-01) from Department of Science and Technology, India, to
425 S.K.R.
426
19 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
427 References
428 1. Skerker, J.M. and Berg, H.C. (2001) Direct observation of extension and 429 retraction of type IV pili. Proc Natl Acad Sci U S A, 98, 6901-6904. 430 2. Curtis, P.D. and Brun, Y.V. (2010) Getting in the loop: regulation of development 431 in Caulobacter crescentus. Microbiol Mol Biol Rev, 74, 13-41. 432 3. Skerker, J.M. and Laub, M.T. (2004) Cell-cycle progression and the generation of 433 asymmetry in Caulobacter crescentus. Nat Rev Microbiol, 2, 325-337. 434 4. Jenal, U. (2000) Signal transduction mechanisms in Caulobacter crescentus 435 development and cell cycle control. FEMS Microbiol Rev, 24, 177-191. 436 5. Taylor, J.A., Ouimet, M.C., Wargachuk, R. and Marczynski, G.T. (2011) The 437 Caulobacter crescentus chromosome replication origin evolved two classes of 438 weak DnaA binding sites. Mol Microbiol, 82, 312-326. 439 6. Marczynski, G.T. and Shapiro, L. (2002) Control of chromosome replication in 440 caulobacter crescentus. Annu Rev Microbiol, 56, 625-656. 441 7. Laub, M.T., Chen, S.L., Shapiro, L. and McAdams, H.H. (2002) Genes directly 442 controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc Natl 443 Acad Sci U S A, 99, 4632-4637. 444 8. Ryan, K.R., Judd, E.M. and Shapiro, L. (2002) The CtrA response regulator 445 essential for Caulobacter crescentus cell-cycle progression requires a bipartite 446 degradation signal for temporally controlled proteolysis. J Mol Biol, 324, 443-455. 447 9. Ausmees, N. and Jacobs-Wagner, C. (2003) Spatial and temporal control of 448 differentiation and cell cycle progression in Caulobacter crescentus. Annu Rev 449 Microbiol, 57, 225-247. 450 10. Jacobs, C., Ausmees, N., Cordwell, S.J., Shapiro, L. and Laub, M.T. (2003) 451 Functions of the CckA histidine kinase in Caulobacter cell cycle control. Mol 452 Microbiol, 47, 1279-1290. 453 11. Biondi, E.G., Reisinger, S.J., Skerker, J.M., Arif, M., Perchuk, B.S., Ryan, K.R. 454 and Laub, M.T. (2006) Regulation of the bacterial cell cycle by an integrated 455 genetic circuit. Nature, 444, 899-904. 456 12. Angelastro, P.S., Sliusarenko, O. and Jacobs-Wagner, C. (2010) Polar 457 localization of the CckA histidine kinase and cell cycle periodicity of the essential 458 master regulator CtrA in Caulobacter crescentus. J Bacteriol, 192, 539-552. 459 13. Iniesta, A.A. and Shapiro, L. (2008) A bacterial control circuit integrates polar 460 localization and proteolysis of key regulatory proteins with a phospho-signaling 461 cascade. Proc Natl Acad Sci U S A, 105, 16602-16607. 462 14. Jacobs, C., Domian, I.J., Maddock, J.R. and Shapiro, L. (1999) Cell cycle- 463 dependent polar localization of an essential bacterial histidine kinase that 464 controls DNA replication and cell division. Cell, 97, 111-120. 465 15. Chen, Y.E., Tsokos, C.G., Biondi, E.G., Perchuk, B.S. and Laub, M.T. (2009) 466 Dynamics of two Phosphorelays controlling cell cycle progression in Caulobacter 467 crescentus. J Bacteriol, 191, 7417-7429. 468 16. Lori, C., Ozaki, S., Steiner, S., Bohm, R., Abel, S., Dubey, B.N., Schirmer, T., 469 Hiller, S. and Jenal, U. (2015) Cyclic di-GMP acts as a cell cycle oscillator to 470 drive chromosome replication. Nature, 523, 236-239.
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471 17. Dubey, B.N., Lori, C., Ozaki, S., Fucile, G., Plaza-Menacho, I., Jenal, U. and 472 Schirmer, T. (2016) Cyclic di-GMP mediates a histidine kinase/phosphatase 473 switch by noncovalent domain cross-linking. Science Advances, 2, e1600823. 474 18. Narayanan, S., Janakiraman, B., Kumar, L. and Radhakrishnan, S.K. (2015) A 475 cell cycle-controlled redox switch regulates the topoisomerase IV activity. Genes 476 Dev, 29, 1175-1187. 477 19. Pierce, D.L., O'Donnol, D.S., Allen, R.C., Javens, J.W., Quardokus, E.M. and 478 Brun, Y.V. (2006) Mutations in DivL and CckA rescue a divJ null mutant of 479 Caulobacter crescentus by reducing the activity of CtrA. J Bacteriol, 188, 2473- 480 2482. 481 20. Domian, I.J., Quon, K.C. and Shapiro, L. (1997) Cell type-specific 482 phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S 483 transition in a bacterial cell cycle. Cell, 90, 415-424. 484 21. Spencer, W., Siam, R., Ouimet, M.C., Bastedo, D.P. and Marczynski, G.T. (2009) 485 CtrA, a global response regulator, uses a distinct second category of weak DNA 486 binding sites for cell cycle transcription control in Caulobacter crescentus. J 487 Bacteriol, 191, 5458-5470. 488 22. Chen, J.C., Hottes, A.K., McAdams, H.H., McGrath, P.T., Viollier, P.H. and 489 Shapiro, L. (2006) Cytokinesis signals truncation of the PodJ polarity factor by a 490 cell cycle-regulated protease. Embo J, 25, 377-386. 491 23. Radhakrishnan, S.K., Pritchard, S. and Viollier, P.H. (2010) Coupling prokaryotic 492 cell fate and division control with a bifunctional and oscillating oxidoreductase 493 homolog. Dev Cell, 18, 90-101. 494 24. Radhakrishnan, S.K., Thanbichler, M. and Viollier, P.H. (2008) The dynamic 495 interplay between a cell fate determinant and a lysozyme homolog drives the 496 asymmetric division cycle of Caulobacter crescentus. Genes Dev, 22, 212-225. 497 25. Boyd, C.H. and Gober, J.W. (2001) Temporal regulation of genes encoding the 498 flagellar proximal rod in Caulobacter crescentus. J Bacteriol, 183, 725-735. 499 26. Skerker, J.M. and Shapiro, L. (2000) Identification and cell cycle control of a 500 novel pilus system in Caulobacter crescentus. Embo J, 19, 3223-3234. 501 27. Schoenlein, P.V., Gallman, L.S. and Ely, B. (1989) Organization of the flaFG 502 gene cluster and identification of two additional genes involved in flagellum 503 biogenesis in Caulobacter crescentus. J Bacteriol, 171, 1544-1553. 504 28. Johnson, R.C. and Ely, B. (1979) Analysis of nonmotile mutants of the dimorphic 505 bacterium Caulobacter crescentus. J Bacteriol, 137, 627-634. 506 29. Shebelut, C.W., Guberman, J.M., van Teeffelen, S., Yakhnina, A.A. and Gitai, Z. 507 (2010) Caulobacter chromosome segregation is an ordered multistep process. 508 Proceedings of the National Academy of Sciences of the United States of 509 America, 107, 14194-14198. 510 30. Mohl, D.A. and Gober, J.W. (1997) Cell cycle-dependent polar localization of 511 chromosome partitioning proteins in Caulobacter crescentus. Cell, 88, 675-684. 512 31. Thanbichler, M. and Shapiro, L. (2008) Getting organized--how bacterial cells 513 move proteins and DNA. Nat Rev Microbiol, 6, 28-40. 514 32. Mohl, D.A., Easter, J., Jr. and Gober, J.W. (2001) The chromosome partitioning 515 protein, ParB, is required for cytokinesis in Caulobacter crescentus. Mol 516 Microbiol, 42, 741-755.
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517 33. Siam, R., Brassinga, A.K. and Marczynski, G.T. (2003) A dual binding site for 518 integration host factor and the response regulator CtrA inside the Caulobacter 519 crescentus replication origin. J Bacteriol, 185, 5563-5572. 520 34. Shaheen, S.M., Ouimet, M.C. and Marczynski, G.T. (2009) Comparative analysis 521 of Caulobacter chromosome replication origins. Microbiology, 155, 1215-1225. 522 35. Fernandez-Fernandez, C., Gonzalez, D. and Collier, J. (2011) Regulation of the 523 activity of the dual-function DnaA protein in Caulobacter crescentus. PLoS One, 524 6, e26028. 525 36. Pini, F., Frage, B., Ferri, L., De Nisco, N.J., Mohapatra, S.S., Taddei, L., 526 Fioravanti, A., Dewitte, F., Galardini, M., Brilli, M. et al. (2013) The DivJ, CbrA 527 and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium 528 meliloti. Mol Microbiol, 90, 54-71. 529 37. Willett, J.W., Herrou, J., Briegel, A., Rotskoff, G. and Crosson, S. (2015) 530 Structural asymmetry in a conserved signaling system that regulates division, 531 replication, and virulence of an intracellular pathogen. Proc Natl Acad Sci U S A, 532 112, E3709-3718. 533 38. Francis, N., Poncin, K., Fioravanti, A., Vassen, V., Willemart, K., Ong, T.A., 534 Rappez, L., Letesson, J.J., Biondi, E.G. and De Bolle, X. (2017) CtrA controls cell 535 division and outer membrane composition of the pathogen Brucella abortus. Mol 536 Microbiol, 103, 780-797. 537 39. Vanderlinde, E.M. and Yost, C.K. (2012) Mutation of the sensor kinase chvG in 538 Rhizobium leguminosarum negatively impacts cellular metabolism, outer 539 membrane stability, and symbiosis. J Bacteriol, 194, 768-777. 540 40. Mann, T.H., Seth Childers, W., Blair, J.A., Eckart, M.R. and Shapiro, L. (2016) A 541 cell cycle kinase with tandem sensory PAS domains integrates cell fate cues. Nat 542 Commun, 7, 11454. 543 41. Tan, M.H., Kozdon, J.B., Shen, X., Shapiro, L. and McAdams, H.H. (2010) An 544 essential transcription factor, SciP, enhances robustness of Caulobacter cell 545 cycle regulation. Proc Natl Acad Sci U S A, 107, 18985-18990. 546 42. Fumeaux, C., Radhakrishnan, S.K., Ardissone, S., Theraulaz, L., Frandi, A., 547 Martins, D., Nesper, J., Abel, S., Jenal, U. and Viollier, P.H. (2014) Cell cycle 548 transition from S-phase to G1 in Caulobacter is mediated by ancestral virulence 549 regulators. Nat Commun, 5, 4081. 550 43. Gora, K.G., Tsokos, C.G., Chen, Y.E., Srinivasan, B.S., Perchuk, B.S. and Laub, 551 M.T. (2010) A cell-type-specific protein-protein interaction modulates 552 transcriptional activity of a master regulator in Caulobacter crescentus. Mol Cell, 553 39, 455-467. 554 44. Ely, B. (1991) Genetics of Caulobacter crescentus. Methods Enzymol, 204, 372- 555 384. 556 45. Chen, J.C., Viollier, P.H. and Shapiro, L. (2005) A membrane metalloprotease 557 participates in the sequential degradation of a Caulobacter polarity determinant. 558 Mol Microbiol, 55, 1085-1103. 559 46. Poindexter, J.S. (1964) Biological Properties and Classification of the 560 Caulobacter Group. Bacteriol Rev, 28, 231-295.
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561 47. Janakiraman, B., Mignolet, J., Narayanan, S., Viollier, P.H. and Radhakrishnan, 562 S.K. (2016) In-phase oscillation of global regulons is orchestrated by a pole- 563 specific organizer. 113, 12550-12555.
564
565 Figure legends
566 Figure 1. Cell cycle regulation in Caulobacter crescentus by the CckA-CtrA
567 pathway. (A) Schematic representation of the dual switching of CckA between kinase
568 mode (blue) and phosphatase mode (orange) in the swarmer and stalked cell
569 compartments, respectively. The graded bars indicate the time during which CtrA
570 (black) is present during the cell cycle. (B) The bidirectional flow of phosphate between
571 CckA, ChpT and CtrA. In the swarmer cells, CckA transfers the phosphate group to the
572 phosphotranferase, ChpT, which further donate phosphate group to CtrA. Thus, the
573 phosphorylated form of CtrA (CtrA~P) represent the active form, wherein CtrA can bind
574 to various target promoters of several cell regulated genes, as well as repress the
575 initiation of chromosome replication. (C) Growth of ΔnstA, and the suppressor mutant,
576 ΔnstA cckA(L228P), upon nstADD overexpression. Cells, as indicated, were diluted five
577 folds and spotted on media containing 0.5 mM vanillate. (D) Differential interference
578 contrast (DIC) image of the suppressor mutant, ΔnstA cckA(L228P). Scale bar: 2µm.
579
580 Figure 2. Effect of CckA(L228P) mutation on CtrA. (A) In vivo phosphorylation
581 experiment denoting CtrA~P/CtrA levels in WT and ΔnstA cckA(L228P) mutants. The
582 relative β -galactosidase activity (in percentage) of (B) the PtacA-lacZ and (C) the PpilA-
583 lacZ reporters in WT, ΔnstA and ΔnstA cckA(L228P) cells. (D) Data of qChIP analysis
584 showing the CtrA occupancy at the promoter region of pilA (PpilA) in WT, ΔnstA and
23 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
585 ΔnstA cckA(L228P) cells. The values±SE, represented in A, B, C and D are the average
586 of at least three independent experiments.
587
588 Figure 3. The CckA(L228P) mutation leads to differential CtrA binding. (A)
589 Genome-wide comparative ChIP-Seq using polyclonal antibodies to CtrA, denoting the
590 occupancy of CtrA on the chromatin of cckA vs ΔnstA cckA(L228P) mutant cells. The
591 color key indicate the degree by which the occupancy of CtrA is varied in selected
592 targets, as a result of the CckA(L228P) substitution. The color key at the bottom is
593 expressed as log2 ratio (see the Supplementary data set for the complete list of target
594 genes). Traces of the occupancy of CtrA at (B) the chromosomal origin of replication,
595 Cori (C) the promoter of fliQ (D) the promoter of flbT. The Figures 3B-D were derived
596 from the ChIP-Seq data and the traces of CtrA in WT are denoted in green, and in
597 ΔnstA cckA(L228P) mutant is denoted in orange.
598
599 Figure 4. Suppression of NstADD toxicity by the cckA(L228P) mutant. (A)
600 DIC and fluorescent micrographs of the extragenic suppressor mutant, ΔnstA
601 cckA(L228P), harboring gfp-parB at the chromosomal xylX locus (xylX::Pxyl-gfp-parB).
602 Red arrow-heads: cells with one GFP-ParB foci; white arrow-heads: cells with two
603 partially segregated GFP-ParB foci. (B) DIC and fluorescent micrographs of ΔnstA cells
604 expressing gfp-parB from xylX::Pxyl-gfp-parB. The cells were treated in the same
605 manner, as described in A. (C) Data representing the stalked cells with one (blue), two
606 partially segregated (orange), normal bipolar (yellow), and multiple (grey) GFP-ParB foci
24 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
607 in ΔnstA cckA(L228P) (data from 1032 stalked cells) or ΔnstA (data from 944 stalked
608 cells). (D) DIC and fluorescent micrographs of WT cells harboring xylX::Pxyl-gfp-parB,
609 and overexpressing NstADD from Pvan on pMT335 or carrying the vector alone. The
610 cells in (A), (B) and (D) were treated with 0.3% xylose to induce the production of GFP-
611 ParB. Cells in (D) were additionally treated with 0.5 mM vanillate for 3h to induce
612 NstADD production. Scale bar in A,B and D: 2µm.
613
614 Figure 5. DnaA overexpression can alleviate the filamentation phenotype
615 induced by the CckA(L228P). (A) DIC images showing ΔnstA cckA(L228P) harboring
616 the vector or expressing dnaA or dnaA(R357A) from xylose inducible promoter on the
617 medium copy vector, pJS 14. The cells were grown to exponential phase in PYE
618 supplemented with 0.2% glucose, prior to the addition of 0.3% xylose. The xylose
619 induction was done for 6h. (B) qChIP data depicting the CtrA occupancy at Cori in WT,
620 ΔnstA, and ΔnstA cckA(L228P) genetic backgrounds, after the overexpression of
621 NstADD for three hours with 0.5mM vanillate inducer. (C) The qChIP data showing the
622 CtrA occupancy at Cori in ΔnstA cckA(L228P) cells, after the overexpression of DnaA or
623 DnaA(R357A) from pJS14. The cells were treated in the same manner, as described in
624 (A). The values±SE, represented in (B) and (C) are the average of at least three
625 independent experiments.
626
627 Figure 6. Model for CckA(L228P) function. (A) The membrane bound bi-
628 functional kinase/phosphatase CckA (yellow), in its kinase form, phosphorylates CtrA
25 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
629 (orange), via the intermediate phosphotransferase, ChpT (green). The active
630 phosphorylated form of CtrA (CtrA~P) binds to the DNA and triggers the transcription
631 during G1- and late S-phase of the cell cycle, and inhibits chromosome replication in the
632 G1 cells by binding to Cori. When the phosphatase activity of CckA is predominant (See
633 Fig. 1A), the phosphate flow reverses dephosphorylating and inactivating CtrA. (B) The
634 CckA(L228P) mutation possibly leads to a predominant CckA kinase acitivity thereby
635 increasing CtrA~P levels. In addition, the CckA(L228P) mutation, through an yet to be
636 understood mechanism, specifically increases the CtrA~P binding at Cori and S-phase
637 specific promoters.
638
26 Narayanan et al. Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B
G1 S Division CckA~P
ChpT~P CckA kinase CtrA~P CckA phosphatase Chromosome Target replication promoters
CtrA~P CtrA~PCtrA~P
C D ΔnstA cckA(L228P) + pPvan-nstADD
ΔnstA
ΔnstA cckA(L228P)
+ vanillate
27 A B
Relative β - galactosidase Relative CtrA~P/CtrA (%) bioRxiv preprint 100 100 200 400 300 500 600 50 0
0 activity (%)
WT P doi: WT certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/300178 pilA ΔnstA -lacZ
cckA (L228P)ΔnstA cckA (L228P)ΔnstA ; this versionpostedApril14,2018. 28 D C Relative β - galactosidase % input DNA activity (%) 0.14 0.12 0.00 0.02 0.10 100 0.04 0.06 0.08 50 0 The copyrightholderforthispreprint(whichwasnot Narayanan etal.Figure2 CtrA occupancyatP
WT P WT tacA ΔnstA
ΔnstA -lacZ
cckA (L228P)ΔnstA cckA (L228P)ΔnstA pilA A bioRxiv preprint CCNA_02816 CCNA_00425 CCNA_03263 CCNA_02286 CCNA_03308 CCNA_00999 (mopJ) CCNA_03689 CCNA_00977 CCNA_00210 CCNA_01779 CCNA_00470 CCNA_03906 CCNA_03200 CCNA_03119 CCNA_00467 CCNA_00982 CCNA_02782 CCNA_02114 CCNA_03248 CCNA_00203 CCNA_01059 (rsaA) CCNA_01297 CCNA_02726 CCNA_00504 CCNA_02820 CCNA_00505 CCNA_01305 (rpsJ) CCNA_00045 CCNA_02935 CCNA_02436 CCNA_00165 (pssY) CCNA_02061 CCNA_03404 (rcdA) CCNA_03607 CCNA_00126 CCNA_01426 CCNA_01421 CCNA_01966 CCNA_03268 (dgrB) CCNA_01451 (cspD) CCNA_00166 CCNA_02402 CCNA_00318 CCNA_00353 CCNA_02723 CCNA_00803 CCNA_02246 (mipZ) CCNA_01163 CCNA_00044 CCNA_02922 CCNA_00352 CCNA_01328 (rpsM) CCNA_01087 CCNA_02343 CCNA_03395 CCNA_01532 (flaY) CCNA_01140 CCNA_02664 (fliX) CCNA_02342 CCNA_03890 CCNA_03863 CCNA_00244 CCNA_00533 (hfsI) CCNA_00875 CCNA_01281 (glpK) CCNA_01644 CCNA_03713 CCNA_00519 CCNA_02665 (flgI) CCNA_01268 CCNA_03246 CCNA_00232 CCNA_03552 CCNA_01004 (flgB) CCNA_00257 CCNA_01129 (fliQ) CCNA_02412 CCNA_03993 CCNA_02523 CCNA_00847 CCNA_02243 CCNA_03135 (fliI) CCNA_03790 CCNA_01530 (fljJ) CCNA_01269 CCNA_00001 (Cori) CCNA_00729 CCNA_02625 (ftsQ) CCNA_01926 (dgcB) CCNA_03605 CCNA_00163 CCNA_00354 CCNA_00439 CCNA_03879 (hemE) CCNA_00348 CCNA_02534 CCNA_03198 CCNA_00351 CCNA_03692 CCNA_01465 CCNA_02105 CCNA_03803 CCNA_00350 CCNA_03325 CCNA_03326 doi: certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/300178 0.5 1 1.5 2 2.5 ; this versionpostedApril14,2018.
Relative abundance Relative abundance Relative abundance 29 C B (%) in ChIP−Seq D (%) in ChIP−Seq (%) in ChIP−Seq
0.00 0.01 0.02 0.03 0.04 0.000 0.005 0.010 0.015 0.020 0.025 0.00 0.05 0.10 0.15 020 020 0 001500 1000 500 1 4042500 4042000 CA080CN_12 fliQ CCNA_01128 CCNA_03890 1633500 hemE flbA 230 230 1234000 1233500 1233000 The copyrightholderforthispreprint(whichwasnot flbA nt positionongenome nt positionongenome nt position on genome Narayanan etal.Figure3 640 640 1635000 1634500 1634000 WT cckA(L228P) WT C WT ori cckA(L228P) cckA(L228P) lTflaF flbT fliR CCNA_0002 Narayanan et al. Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
A B ΔnstA cckA(L228P) xylX::Pxyl-gfp-parB ΔnstA; xylX::Pxyl-gfp-parB DIC GFP DIC GFP
WT + xylX::Pxyl-gfp-parB C D DIC GFP 100 4.3 90 13.7 80 + vector 70 30.1 60 50 86.9
% cells 40 33.8 30 20 + nstADD 10 22.4 5.3 0 3.5
ΔnstA ΔnstA
cckA(L228P)
30 Narayanan et al. Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not A certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ΔnstA cckA(L228P) + vector pPxyl-dnaA pPxyl-dnaA(R357A)
B CtrA occupancy at Cori C CtrA occupancy at Cori 0.040 0.07 0.035 0.06 0.030 0.05 0.025 0.04 0.020 0.03 0.015 % input DNA % input DNA 0.02 0.010
0.01 0.005
0.00 0.000
WT ΔnstA ΔnstA + vector + dnaA
cckA(L228P) WT + vector + dnaA(R357A) + Pvan-nstADD ΔnstA cckA(L228P)
31 Narayanan et al. Figure 6
bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A B
CckA IM CckA IM L228P
↓↓ P ↓P
ChpT ChpT ↓ influences DNA ↓P ↓P ↓ ? binding of CtrA CtrA ↔CtrA~P CtrA CtrA~P ↓ ↓ G1-phase promoters ↓ Replication G1-phase promoters S-phase promoters ↓ Replication S-phase promoters
32 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
1 SUPPLEMENTARY INFORMATION
2
3
4 Supplementary Figure S1. (A) Growth of ΔnstA cells overproducing NstADD, and 5 harboring either wild-type cckA or cckA(L228P) or cckA(L228P)-back-cross (bc). (B) 6 Growth of Caulobacter harboring the wild-type cckA, or NstADD toxicity suppressor 7 mutants of cckA, upon nstADD overexpression. In (A) and (B), cells were diluted five- 8 fold and spotted on media containing 0.5 mM vanillate. The mutant cckA(F496C) is in 9 the ΔnstA genetic background, the rest were in WT background. (C) Schematic 10 denoting the CckA suppressor mutations. The CckA(L228P) mutation resides in the 11 PAS-B domain, whereas the other point mutations are harbored either in the histidine 12 kinase domain or the ATP binding domain of CckA.
33 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
13
14
15
16 Supplementary Figure S2. (A) DIC images of the various point mutants of cckA in WT 17 genetic background. (B) DIC image of ∆nstA cckA(F496C) mutant.
34 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
18
19 20 Supplementary Figure S3. The qChIP data indicating the CtrA occupancy at (A) the 21 promoter of kidO (PkidO), (B) the promoter of tacA (PtacA), (C) the promoter of flbT (PflbT) 22 and (D) at the chromosomal origin of replication, Cori, in WT, ∆nstA, and ∆ nstA 23 cckA(L228P) cells. The data represented are the average of 3 independent experiments 24 ±SE.
25
35 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
26
27
28 Supplementary Figure S4. Relative β-galactosidase activities of (A) PfliM-lacZ reporter, 29 and (B) PflbT-lacZ reporter, in WT, ∆nstA, and ∆ nstA cckA(L228P) cells. The data 30 represented in (A) and (B) are the average of 3 independent experiments ±SE.
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36 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
39 Supplementary Methods
40 Strain construction
41 The various cckA point mutant strains namely SN208 [∆nstA cckA (L228P)],
42 LK98 [WT cckA(R356C)], LK100 [∆nstA cckA(F496C)], LK109 [WT cckA(L228P)],
43 LK122 [WT cckA(A317V)], LK124 [WT cckA(F493C)], LK128 [WT cckA(F392L)] and
44 LK135 [WT cckA(D364G)] were generated by Ultraviolet (UV) radiation based
45 mutagenesis using WT or SKR1797 (∆nstA) (1).
46 The strains namely SN227 [∆nstA cckA (L228P) + pMT 335-nstADD], SN1140
47 [WT cckA(L228P) + pMT335-Pvan-nstADD], SN1141 [WT cckA(A317V) + pMT335-Pvan-
48 nstADD], SN1142 [WT cckA(F493C) + pMT335-Pvan-nstADD], SN1144 [WT
49 cckA(F392L) + pMT335-Pvan-nstADD], SN1145 [WT cckA(D364G) + pMT335-Pvan-
50 nstADD], SN1151 [WT cckA(R356C) + pMT335-Pvan-nstADD] and SN1152 [∆nstA
51 cckA(F496C) + pMT335-Pvan-nstADD] were made by electroporating pSKR126 (pPvan-
52 nstADD) (1) into SN208 [∆nstA cckA (L228P)], LK109 [WT cckA(L228P)], LK122 [WT
53 cckA(A317V)], LK124 [WT cckA(F493C)], LK128 [WT cckA(F392L)], LK135 [WT
54 cckA(D364G)], LK98 [WT cckA(R356C)] and LK100 [∆nstA cckA(F496C)] respectively,
55 The strains SN377 (WT; xylX::Pxyl-gfp-parB), SN379 [∆nstA cckA(L228P);
56 xylX::Pxyl-gfp-parB] and SN559 (∆nstA; xylX::Pxyl-gfp-parB) were made by
57 electroporating pSN190 (pXGFP4C1-Pxyl-gfp-parB) into WT, SN208 and SKR1797
58 respectively.
59 The strains SN461 [∆nstA cckA(L228P) + pJSX-dnaA], SN465 [∆nstA
60 cckA(L228P) + pJSX-dnaA (R357A)] and SN467 [∆nstA cckA(L228P)+pJS14] were
37 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
61 made by electroporating pJSX-dnaA, pJSX-dnaA (R357A) (2) and pJS14 into SN208,
62 respectively.
63 The strains SN505 (WT; xylX:: Pxyl-gfp-parB + pMT335) and SN1153 ( WT;
64 xylX::Pxyl-gfp-parB + pMT335-Pvan-nstADD) were made by electroporating pMT335 (3)
65 and pSKR126 into the strain SN377, respectively.
66 The strains SN740 (WT + pLac290-PpilA-lacZ), SN742 (∆nstA + pLac290-PpilA-
67 lacZ) and SN744 [∆nstA cckA(L228P) + pLac290-PpilA-lacZ) were made by
68 electroporating pJS70 (pLac290-PpilA-lacZ) (4) into WT, SKR1797 and SN208,
69 respectively.
70 The strains SN741 (WT + pLac290-PtacA-lacZ), SN743 (∆nstA + pLac290-PtacA-
71 lacZ) and SN745 [∆nstA cckA(L228P) + pLac290-PtacA-lacZ) were made by
72 electroporating pMV05 (pLac290-PtacA-lacZ) (5) into WT, SKR1797 and SN208,
73 respectively.
74 The strains SN1361 (WT + pLac290-PfliM-lacZ), SN1365 (∆nstA + pLac290-PfliM-
75 lacZ) and SN1369 [∆nstA cckA(L228P) + pLac290-PfliM-lacZ) were made by
76 electroporating pLac290-PfliM-lacZ into WT, SKR1797 and SN208, respectively.
77 The strains SN1406 (WT + pLac290-PflbT-lacZ), SN1407 (∆nstA + pLac290-PflbT-
78 lacZ) and SN1408 [∆nstA cckA(L228P) + pLac290-PflbT-lacZ) were made by
79 electroporating pLac290-PflbT-lacZ into WT, SKR1797 and SN208, respectively.
80 The cckA(L228P) back cross strain SN769 [ΔnstA; cckA(L228P)] was made by
81 backcrossing the cckA(L228P) point mutation in SN208 into SKR1797. pSN155
38 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
82 (pNPTS-cckA backcross construct) was used to transform SN208 and the transformants
83 were plated on PYE supplemented with Kanamycin. Further øCr30 lysates of the
84 transformants were made and was used for transducing into SKR1797 thereby
85 generating SN769. The backcross strain, SN769, was electroporated with pSKR126, to
86 obtain SN771 [ΔnstA; cckA(L228P) + pMT335-Pvan-nstADD].
87 The strain SKR1800 (WT + pMT335-Pvan-nstADD) is previously described (1).
88 Plasmid construction
89 The plasmid pSN155 (pNPTS-cckA-backcross) construct was made by
90 PCR amplifying a region 750bp (approx.) upstream of cckA. The PCR fragment was
91 digested with EcoRI/HindIII. The digested fragment was ligated into pNPTS138 (M.R.K
92 Alley, unpublished) cut with EcoRI/HindIII.
93 Plasmid pSN190 (pXGFP4-C1-Pxyl-gfp-parB) was made by PCR amplifying parB
94 and cleaving it with BgIII/EcoRI, wherein the predicted the start codon ATG was
95 replaced with GTG that carried an overlapping BglII recognition site to allow proper
96 placement of parB facilitating N-terminal GFP fusion. The alleles were ligated into
97 pXGFP4-C1 vector (M.R.K Alley unpublished) cut with BglII/EcoRI.
98 To make pSN205 (PfliM-lacZ, a kind gift from Patrick Viollier) nucleotides
99 2298862-2299971 of NA1000 genome (CP001340) was amplified and ligated as
100 EcoRI/HindIII fragment into a medium-copy plasmid pJGZ290 (6) to drive the
101 transcription of the promoterless lacZ gene.
102
39 bioRxiv preprint doi: https://doi.org/10.1101/300178; this version posted April 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
103 Plasmid pSN206 (PflbT-lacZ, a kind gift from Patrick Viollier) was made by
104 amplifying nucleotides 1633750-1634327 of the NA1000 genome (CP001340) and
105 ligating as EcoRI/HindIII fragment into the medium-copy plasmid pJGZ290 (6) to drive
106 the transcription of the promoterless lacZ gene.
107 References
108 1. Narayanan, S., Janakiraman, B., Kumar, L. and Radhakrishnan, S.K. (2015) A 109 cell cycle-controlled redox switch regulates the topoisomerase IV activity. Genes 110 Dev, 29, 1175-1187. 111 2. Fernandez-Fernandez, C., Gonzalez, D. and Collier, J. (2011) Regulation of the 112 activity of the dual-function DnaA protein in Caulobacter crescentus. PLoS One, 113 6, e26028. 114 3. Thanbichler, M., Iniesta, A.A. and Shapiro, L. (2007) A comprehensive set of 115 plasmids for vanillate- and xylose-inducible gene expression in Caulobacter 116 crescentus. Nucleic Acids Res, 35, e137. 117 4. Skerker, J.M. and Shapiro, L. (2000) Identification and cell cycle control of a 118 novel pilus system in Caulobacter crescentus. Embo J, 19, 3223-3234. 119 5. Marques, M.V., Gomes, S.L. and Gober, J.W. (1997) A gene coding for a 120 putative sigma 54 activator is developmentally regulated in Caulobacter 121 crescentus. J Bacteriol, 179, 5502-5510. 122 6. Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R. (1980) Broad host range 123 DNA cloning system for gram-negative bacteria: construction of a gene bank of 124 Rhizobium meliloti. Proc Natl Acad Sci U S A, 77, 7347-7351.
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