An analog to digital converter controls bistable transfer competence development of a widespread bacterial integrative and conjugative element

Conjugative transfer of the integrative and conjugative element ICEclc in Pseudomonas requires development of a transfer competence state in stationary phase, which arises only in 3–5% of individual cells. The mechanisms controlling this bistable switch between non-active and transfer competent cells have long remained enigmatic. Using a variety of genetic tools and epistasis experiments in P. putida, we uncovered an ‘upstream’ cascade of three consecutive transcription factor-nodes, which controls transfer competence initiation. One of the uncovered transcription factors (named BisR) is representative for a new regulator family. Initiation activates a feedback loop, controlled by a second hitherto unrecognized heteromeric transcription factor named BisDC. Stochastic modelling and experimental data demonstrated the feedback loop to act as a scalable converter of unimodal (population-wide or ‘analog’) input to bistable (subpopulation-specific or ‘digital’) output. The feedback loop further enables prolonged production of BisDC, which ensures expression of the ‘downstream’ functions mediating ICE transfer competence in activated cells. Phylogenetic analyses showed that the ICEclc regulatory constellation with BisR and BisDC is widespread among Gamma- and Beta-proteobacteria, including various pathogenic strains, highlighting its evolutionary conservation and prime importance to control the behaviour of this wide family of conjugative elements.


Introduction
domains. Further structural analysis using Phyre2 30 suggested three putative domains with low 136 confidence (between 38% and 53%, Fig. S1). One of these is a predicted DNA-binding domain, 137 which hinted at the possible function of BisR as a transcriptional regulator itself. BlastP analysis 138 showed that BisR homologs are widely distributed and well conserved among Beta-,  and Gammaproteobacteria, with homologies ranging from 43-100% amino acid identity over 140 the (quasi) full sequence length (Fig. S2). 141

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In order to investigate its potential regulatory function, bisR was cloned on a plasmid 143 (pMEbisR) and introduced into P. putida UWC1-ICEclc. Inducing bisR by IPTG addition from 144 P tac triggered high rates of ICEclc transfer on succinate media (Fig. 3A). Deletion of bisR on 145 ICEclc abolished its transfer, even upon overexpression of tciR, but could be restored upon 146 ectopic expression of bisR (Fig. 3A). This showed that the absence of transfer was due to the 147 lack of intact bisR, and not to a polar effect of bisR deletion on a downstream gene (Fig. 1A). 148 In addition, transfer of an ICEclc deleted for tciR 26 could be restored by ectopic bisR expression 149 (Fig. 3A). This indicated that TciR is 'upstream' in the regulatory cascade of BisR, and that 150 TciR does not act anywhere else on the expression of components crucial for ICEclc transfer. 151 152 IPTG induction of bisR in P. putida without ICE again did not yield activation of the single-153 copy P int or P inR transcriptional reporter fusions (Fig. 3B). In contrast, BisR induction in P. 154 putida UWC1 with ICEclc led to a massive activation of the same reporter constructs in 155 virtually all cells (Fig. 3C), compared to a vector-only control (Fig. 2C, pME6032). This 156 suggested that BisR was an(other) intermediate regulator step in the complete cascade of 157 activation of ICEclc transfer competence. Of the tested ICE-promoters within this 7-kb region, 158 directly controlling expression of P inR and/or P int were encoded in this region, which we tried 171 to identify by subcloning different gene configurations. 172

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Removing alpA from the initial construct had no measurable effect on expression of the 174 fluorescent reporters, but replacing P tac by the native P alpA promoter abolished all P int reporter 175 activation (Fig. 4B). This suggested that P alpA is silent without activation by BisR (see below) 176 and no spontaneous production of regulatory factors occurred. Removing three genes at the 3' 177 extremity (i.e., orf96323, orf95213 and inrR) reduced P int -echerry reporter expression, but a 178 fragment with a further deletion into the bisC gene was unable to activate P int (Fig. 4B). 179 Induction of inrR alone did not result in P int activation (Fig. 4B). Deletion of parA and shi at 180 the 5' end of the fragment still enabled reporter expression from P int , narrowing the activator 181 factor regions down to two genes, previously named parB and orf97571, but renamed here to 182 bisD and bisC (Fig. 4B). Neither bisC or bisD alone, but only the combination of bisDC resulted 183 in reporter expression from P int in P. putida UWC1 without ICEclc (Fig. 4B), and similarly, of 184 P inR (Fig. S3). In the presence of ICEclc, inducing either bisC or bisD from a plasmid yielded 185 a small proportion of cells expressing the P int reporters (Fig. 4C), which was absent in a P. 186 putida carrying an ICEclc with a deletion of bisD (Fig. S4). In contrast, induction of bisDC in 187 combination caused a majority of cells to express fluorescence from P int in P. putida containing 188 ICEclc (Fig. 4C) or ICEclc-∆bisD (Fig. S4). These results indicated that BisDC acts as an 189 ensemble to activate transcription, and this pointed to bisDC as the last step in the regulatory 190 cascade, since it was the minimum unit sufficient for activation of the P int -promoter, which is 191 exclusively expressed in the subpopulation of tc cells of wild-type P. putida with ICEclc 11 . 192 induced transfer of ICEclc-variants deleted for tciR or for bisR (Fig. 4D). This confirmed that 196 both tciR and bisR relay activation steps to P bisR and P alpA , respectively, but not to further 197 downstream ICE promoters (Fig. 1B). Moreover, an ICEclc deleted for bisD could not be 198 restored for transfer by overexpression of tciR or bisR, but only by complementation with bisDC 199 (Fig. 4D). Interestingly, the frequency of transfer of an ICEclc lacking bisD complemented by 200 expression of bisDC in trans was two orders of magnitude lower than that of similarly 201 complemented wild-type ICEclc, ICEclc with tciR-or bisR-deletion (Fig. 4D). This was similar 202 as the reduction in reporter expression observed in P. putida ICEclc-∆bisD complemented with 203 pMEbisDC compared to wild-type ICEclc (Fig. S4), and suggested the necessity of some 204 'reinforcement' occurring in the wild-type configuration that was lacking in the bisD deletion 205 and could not be restored by in trans induction of plasmid-cloned bisDC. To investigate this potential 'reinforcement' in wild-type configuration, we revisited the 210 potential for activation of the alpA promoter. Induction by IPTG of the plasmid-cloned 211 fragment encompassing the gene region parA-shi-bisDC caused strong activation of reporter 212 gene expression from P alpA in P. putida without ICEclc (Fig. 5A). The minimal region that still 213 maintained P alpA induction encompassed bisDC, although much lower than with a cloned parA-214 shi-bisDC fragment (Fig. 5A). Interestingly, when the parA-shi-bisDC fragment was extended 215 by alpA itself, reporter expression from P alpA was abolished, whereas also a fragment containing 216 only alpA caused significant repression of the alpA promoter (Fig. 5A). These results would 217 imply feedback control on activation of P alpA , since its previously mapped transcript covers the 218 complete region from alpA to orf96323 on ICEclc, including bisDC (Fig. 1A) 25 . Although 219 induction of BisDC was sufficient for activation of transcription from P alpA , this effectively only 220 yielded a small subpopulation of cells with high reporter fluorescence values (Fig. 5B, C), in 221 contrast to induction of the larger cloned gene region encompassing parA-shi-bisDC that 222 activated all cells (Fig. 5B, C). The feedback loop, therefore, seemed to consist of a positive 223 forward part that includes BisDC (reinforced by an as yet unknown other mechanism) and a 224 modulatory repressive branch including AlpA. forms to and unbinding from their respective nodes (i.e., the linked promoters P bisR , P alpA and 241 P int ). Binding is assumed to lead to protein synthesis and finally, protein degradation (Fig. 6A). BisDC value (magenta) (Fig. 6B). The output zero results when BisDC levels stochastically fall 252 to 0 (as for the light blue line in the panel STOCHASTIC of Fig. 6B), since in that case there is 253 no BisDC to stimulate its own production. Parameter variation showed that the proportion of 254 output zero from the loop is dependent on the binding and unbinding constants for the alpA 255 promoter, and the BisDC degradation rate (Fig. 6B,  Since the feedback loop cannot start without BisDC, it is imperative to kickstart the alpA 260 promoter by BisR (Fig. 6C). Simulations of a configuration that includes activation by BisR, 261 showed how upon a single pulse of BisR, the feedback loop again leads to a bimodal population 262 with zero and positive BisDC levels (Fig. 6C). Increasing the input level of BisR resulted in 263 increasing the proportion of cells with positive BisDC state, but did not influence their mean 264 value (Fig. 6C). Bimodally distributed BisR input also gave rise to bimodal BisCD output, but 265 with a higher proportion of zero BisDC state (Fig. 6C, bimodal). In contrast to the BisDC loop Simulations thus predicted that the ICE regulatory network faithfully transmits and stabilizes 286 analog input (e.g., a single regulatory factor expressed in all cells) to bistable output (e.g., a 287 subset of cells with transfer competence and the remainder silent). To demonstrate this 288 experimentally, we engineered a P. putida without ICEclc, but with a single copy 289 chromosomally inserted IPTG-inducible bisR, a plasmid with parA-shi-bisDC under control of 290 P alpA , and a single-copy dual P int -echerry and P inR -egfp reporter (Fig. 7A). Induction from P tac 291 by IPTG addition yields unimodal (analog) production of BisR, the mean level of which can be 292 controlled by the IPTG concentration (Fig. S5). In the presence of all components of the system, 293 induction was converted by the feedback loop into an increased proportion of fluorescent cells 295 (Fig. 7C). This effectively created a scalable bimodal (digital) output from unimodal input, 296 dependent on the used IPTG concentration (Fig. 7C, Fig. S5). The proportion of fluorescent 297 cells was in line with predictions from stochastic simulations as a function of the relative 298 strength of P tac activation (Fig. 7D). Furthermore, in agreement with model predictions (  The gene synteny from bisR to inrR of ICEclc was maintained in several genomes (Fig. S2), 323 suggesting them being part of related integrated ICEs. Notable exceptions included a region in 324 P. aeruginosa Carb01_63, which carried an integrase gene upstream of bisR but that was still 325 downstream of a tRNA gly gene (Fig. S2). This region may encompass an ICE that has retained 326 the same integration specificity as ICEclc but carrying a different modular architecture where 327 upstream of bisR with 53% and 51% amino acid identity to TciR (overlap lengths 95%), 331 respectively. Regulation of these two elements might thus involve a cis-acting LTTR, rather 332 than the trans-acting TciR. In the genomes of Xanthomonas campestris strain AW13 and 333 Cupriavidus nantongensis X1 (Fig. S2) BisR and BisDC, acting sequentially on singular (TciR, BisR) or multiple nodes (BisDC). The 360 network has an 'upstream' branch controlling the initiation of transfer competence, a 'bistability 361 generator' that confines the input signal, and maintains the 'downstream' path of transfer 362 competence to a dedicated subpopulation of cells (Fig. 1B). 363

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The previously characterized mfsR-marR-tciR operon 26 , whose transcription is controlled 365 through autorepression by MfsR, is probably the main break on activation of the upstream 366 branch. This was concluded from effects of deleting mfsR, which resulted in overexpression of 367 TciR, and massively increased and deregulated ICE transfer even in exponentially growing 368 cells 26 . We showed here that TciR activates the transcription of a hitherto unrecognized 369 transcription factor gene named bisR, but not of any further critical ICEclc promoters. suggested that BisR triggers and transmits the response in a scalable manner to the bistability 379 generator, encoded by the genes downstream of P alpA . Triggering of P alpA stimulated expression 380 of (among others) two consecutive genes bisD and bisC, which code for subunits of an activator 381 complex that weakly resembles the known regulator of flagellar synthesis FlhDC 31,32 . BisDC 382 production was sufficient to activate the previously characterized bistable ICEclc promoters P int 383 and P inR , making it the key regulator for the 'downstream' branch (Fig. 1B). Importantly, BisDC 384 was also part of a feedback mechanism activating transcription from P alpA , and therefore, 385 regulates its own production. Simulations and experimental data indicated that the feedback 386 loop acts as a scalable analog-to-digital converter, transforming any positive input received 387 from BisR into a dedicated cell that can regenerate sufficiently high BisDC levels to activate 388 the complete downstream transfer competence pathway. Plasmid DNA was purified using the Nucleospin Plasmid kit (Macherey-Nagel) according to 480 manufacturer's instructions. All enzymes used in this study were purchased from New England 481 Biolabs. PCR reactions were carried out with primers described in Table S2. PCR products 482 were purified using Nucleospin Gel and PCR Clean-up kits (Macherey-Nagel) according to 483 manufacturer's instructions. E. coli and P. putida were transformed by electroporation as  Table S2, with genomic DNA of P. putida UWC1-ICEclc as template. Amplicons were 495 digested by EcoRI and cloned into EcoRI-digested pME6032 using T4 DNA ligase, producing encompassing parA-inrR was recovered from pTCB177 29 and cloned into pME6032 (producing 498 pMEreg∆alpA, Table S1). An alpA-parA-shi-bisD' fragment was amplified by PCR (Table S2) 499 and cloned into pME6032 using EcoRI restriction sites (Table S1). The resulting plasmid was 500 digested with SalI and the 4.8-kb fragment containing the P tac promoter, alpA-parA-shi-bisD' 501 was recovered and used to replace the parA-shi-parB part of pMEreg∆alpA. This generated a 502 cloned fragment encompassing alpA all the way to inrR (pMEreg, Table S1). Further 3' 503 deletions removing orf96323-inrR or bisC-inrR were generated by PstI and AfeI digestion and 504 religation (Table S1). A DNA fragment containing P alpA , alpA, parA, shi and the 5' part of bisD 505 was synthesized (ThermoFisher Scientific), and ligated by Quick-Fusion cloning (Bimake) into 506 pMEreg∆alpA digested with PmlI and BamHI to remove the part containing lacI q , P tac , parA, 507 shi and bisD. This plasmid was then digested by PstI to remove orf96323-inrR and religated 508 (Table S1). 509 The promoter regions upstream of bisR or alpA were amplified in the PCR (Table S2) and 510 cloned into the promoterless egfp reporter miniTn5 delivery plasmid pBAM1 54 or into a 511 pUC18-derived miniTn7 delivery plasmid 55 . The P inR -egfp insert was recovered from the 512 miniTn5-based reporter system 10 using HindIII and KpnI, and subsequently cloned into 513 pUC18miniTn7 digested by the same enzymes. The dual miniTn7 P inR -egfp; P int -echerry 514 reporter has been described previously 10 . All reporter constructs were integrated in single copy 515 into the chromosomal attB Tn7 site of P. putida by using pUX-BF13 for miniTn7, or randomly 516 for miniTn5-based constructs 54,56 , in which case three independent clones were recovered, 517 stored and analyzed. 518 Deletions of bisR or bisD in ICEclc were constructed using the two-step seamless chromosomal 519 gene inactivation technique as described elsewhere 57 . images. Up to ten images at different positions were acquired using Visiview software (Visitron 557 systems GMbH), with exposures set to 40 ms (phase contrast, PhC) and 500 ms (eGFP and 558 previously 11 , from which their fluorescence (eGFP or eCherry) was quantified. Subpopulations 560 of tc cells were quantified using quantile-quantile-plotting as described previously 27 . 561 Fluorescent images for display were scaled to the same brightness in ImageJ 60 as indicated, 562 saved as 8-bit gray tiff-files and cropped to the display area in Adobe Photoshop (Adobe, 2020). 563

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Fluorescent reporter intensities were compared among biological triplicates. In case of mini-566 Tn5 insertions, this involved three clones with potentially different insertion sites, each 567 measured individually. For mini-Tn7 inserted reporter constructs, we measured three biological 568 replicates of a unique clone. Expression differences between mutants and a strain with the same 569 genetic background but carrying the empty pME6032 plasmid were tested on triplicate means 570 of individual median values in a one-sided t-test (the hypothesis being that the mutant 571 expression is higher than the control). Coherent simultaneous data series were tested for 572 significance of reporter expression or transfer frequency differences in ANOVA, followed by 573

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For strain numbers, see Table S1.