The ECF sigma factor PvdS regulates the type I-F CRISPR-Cas system in Pseudomonas aeruginosa

During infection, successful colonization of bacteria requires a fine-tuned supply of iron acquired via iron transport systems. However, the transport systems serve as phage attachment sites and entry portals for foreign nucleic acid. Most bacteria possess the CRISPR-Cas system, which targets and destroys foreign nucleic acids and prevents deleterious effects of horizontal gene transfer. To understand the regulation of the CRISPR-Cas system, we performed genome-wide random transposon mutagenesis which led to the identification of the Extracytoplasmic Function (ECF) Sigma factor, PvdS as a regulator of the Type I-F CRISPR-Cas system in P. aeruginosa. We show that under iron-depleted conditions PvdS induces the expression of the type I-F CRISPR-Cas system. This regulatory mechanism involves direct interaction of PvdS with specific binding sites in the promoter region of cas1. Furthermore, activation of the CRISPR-Cas system under iron-depleted conditions increases horizontal gene transfer (HGT) interference and adaptation. The PvdS activation of the CRISPR-Cas system under iron limitation highlights the versatility of the P. aeruginosa in multitasking its regulatory machinery to integrate multiple stress factors. Importance P. aeruginosa infects a wide range of host organisms and adapts to various environmental stress factors such as iron limitation due to its elaborate regulatory system. P aeruginosa possesses the type I-F CRISPR-Cas system as a defense mechanism against phages infection and HGT. This work highlights the ability of P. aeruginosa to multitask its iron regulatory system to control the CRISPR-Cas system under a physiologically relevant stress factor such as iron limitation where the bacteria are vulnerable to phage infection. It also adds to the knowledge of the regulation of the CRISPR-Cas system in bacteria and presents a possible target that could prevent the emergence of phage resistance via the CRISPR-Cas system during the development of phage therapy.

under iron limitation highlights the versatility of the P. aeruginosa in multitasking its regulatory 7 The expression of pvdS is repressed by Fur when iron is abundant in the environment thus, 129 resulting in the downregulation of the PvdS-regulated genes. However, under iron-depleted 130 conditions, Fur relieves PvdS, allowing it to bind to the consensus binding sites in the promoter 131 region of genes and inducing their expression (26). To investigate whether Fur influences the 132 PvdS-CRISPR-Cas regulation, we analyzed the expression of the cas genes in the fur and pvdS 133 mutants under iron-depleted and normal conditions. In the fur mutant expression of the cas 134 genes were upregulated under both normal and iron-depleted conditions relative to the wild 135 type ( Fig. S2A and S2B). This was contrary to the expression of cas genes in the pvdS mutants as 136 they were downregulated under iron-depleted condition ( Fig S2A). This shows that the deletion 137 of fur derepressed the pvdS even under normal conditions and therefore allows PvdS to 138 regulate the cas genes. This is in agreement with the Fur-PvdS regulatory system shown by 139 previous studies (5)(27)(28). From our analysis, exogenous pyoverdine (PVD) did not 140 significantly increase cas gene expression in both wild type and pvdS mutant (Fig. 1C), which 141 signifies the possibility that PvdS regulation of the CRISPR-Cas system may be independent of 142 siderophore production and uptake. 143 binding site are required for interaction and regulation of the cas genes, we created an 151 integrative cas1 promoter-lacZ reporter strain with altered PvdS binding site sequence and 152 examined the effect on cas1 expression under iron-depleted conditions ( Fig 2B). Initially, we 153 investigated the effect of the mutations on the interaction between PvdS and PvdS-cRNAP 154 complex with the cas1 promoter region. The binding assay revealed that the mutation of one 155 binding site did not affect the interaction between PvdS-cRNAP and the cas1 promoter region 156 ( fig 2C). However, PvdS-cRNAP binding was abolished when the sequences of both binding sites 157 were altered ( fig 2C). Also, no interaction was observed between the intact and altered PvdS 158 binding sites in the absence of cRNAP (Fig. S3), which depicts a cRNAP-dependent PvdS 159 interaction. 160 161 Furthermore, alteration of either PvdS binding sites significantly reduced the expression of cas1 162 in the wild type PA14 under iron-depleted conditions (Fig3A). The decrease in cas1 expression 163 was more significant when we altered both binding sites in the wild type PA14 compared to the 164 single binding alteration in the wild type background ( Fig 3A). Additionally, alteration of single 165 PvdS binding sites resulted in similar expression as the intact sequence in the pvdS mutant 166 background (Fig. 3). These results show that PvdS interaction with a consensus binding site in 167 the promoter region of cas1 is required for cas gene expression. Since PvdS is known to require 168 multiple sites for full activity in vivo and in vitro (26), the decrease in cas gene expression as a 169 result of mutations in the binding sites may imply the requirement of both sites for optimal 170 regulation by PvdS. 171

PvdS influences CRISPR-Cas interference of horizontal gene transfer and adaptation 173
Since PvdS regulates the cas genes expression under iron-depleted condition, we further 175 investigated how this regulation influences the function of the CRISPR-Cas system in interfering 176 with HGT and the acquisition of spacers from the foreign nucleic acid. Acquisition of spacers is 177 required for the development of immunological memory against specific nucleic acid sequences 178 (29). To test the impact of PvdS on the interference and adaptation of the CRISPR-Cas system, 179 we engineered CRISPR-targeted constructs containing sequences similar to CRISPR 2 spacer 1 of 180 the type I-F CRISPR-Cas system in PA14. In the three constructs designed, pUCPTsp1 contained 181 matching sequences to CRISPR 2 spacer while pUCPTSp2n and pUCPTSp4n contained 2 and 4 182 nucleotide substitutions respectively after the GG PAM sequence recognized the Type I-F 183 CRISPR-Cas system. These mutations in the protospacer reduce interference but can initiate 184 iron-depleted and normal conditions compared to the wild type (Fig. 5A). Also, a significantly 206 lower expansion (~60% less than wild type) of the CRISPR2 array was detected in the pvdS 207 mutants compared to the wild type under iron-depleted conditions. However, there were no 208 significant differences in spacer acquisition observed under normal conditions between the wild 209 type and the pvdS mutant (Fig. 5A). 210 Next, we examined the influence of PvdS on the CRISPR-Cas system in eliminating incoming 211 nucleic acid sequences to which it has previously encountered. For this assay, we determined 212 the efficiency of conjugal transfer of CRISPR-targeted plasmid (pUCPTSp1) and non-targeted 213 plasmid (pUCPT) from E. coli S17 to P. aeruginosa strains. prior attachment to specific receptors followed by transfer of their nucleic acids into the cell. 248 Iron limitation induces the expression of iron transport channels (30)(31), which can serve as 249 phage attachment sites and entry channels for nucleic acids (10)(11)(12). Hence the evolution 250 of the regulatory system for the CRISPR-Cas system to defend bacteria under such conditions is 251 a vital adaptive mechanism. As an adaptive immune system, expression of the CRISPR-Cas 252 system is known to be metabolically costly to the bacteria (20) (32), Therefore, selective 253 induction of the system under stress condition such as iron depletion, where the bacteria are 254 most vulnerable to phage attack is more beneficial to the bacteria. As such expression of the 255 cas genes is indirectly repressed by fur via the PvdS under normal conditions (Fig S3). This also 256 reduces the risk of autoimmunity imposed by constitutive expression of the system. 257

258
In an era where increased resistance to antibiotics is on the rise, alternative therapies are being 259 experimented for the effective treatment of pseudomonas infections. The use of phage therapy for the treatment of bacterial infections seems like a plausible venture (33). TonB-dependent 261 siderophore uptake systems also serve as targets for antibiotics (34). The development of the 262 trojan horse approach (9) which targets the siderophore transport channels to increase 263 susceptibility to drugs and reduce resistance gene acquisition presents a good platform for a 264 possible combinatory strategy with phage therapy. As seen with antibiotic therapy, bacteria 265 continuously evolve resistance mechanisms against harmful agents. The CRISPR-Cas system 266 remains a potent defense mechanism for bacteria and thus to develop effective phage therapy, 267 targeting a key regulator such as PvdS may present a promising approach to downregulate the 268 phage defense system and prevent resistance during phage therapy. The bacterial strains, plasmids, and oligonucleotides used in this study are listed in Table S1  gentamicin (30g/mL) and sucrose (10% w/v). Bacterial growth media were supplemented with 281 antibiotics at the following concentrations: tetracycline, 10 g/ml for E. coli and 50 g/ml for P. aeruginosa; carbenicillin 200g/ml for E. coli and 300g/ml for P. aeruginosa; kanamycin, 283 50g/ml for E. coli. X-gal was added at a concentration of 50g/ml. 284 The pUCPT, a derivative of pUCP19 containing oriT fragment from pK18, which allows for 285 effective conjugal transfer from E. coli to P. aeruginosa was used to design the CRISPR-Cas 286 targeted constructs. CRISPR-Cas targeted constructs pUCPTSp2n and pUCPTSp4n respectively 287 were created by inserting the CRISPR 2 spacer1 fragments with 2 and 4 nucleotide substitutions 288 adjacent to the GG PAM sequence , into the HindIII/EcoRI digested pUCPT. Sequences were 289 verified by PCR and sequencing using the M13 forward and M13 reverse primers. The Mariner 290 transposon, pBT20 which confers gentamicin resistance was used for random transposon 291 mutagenesis to screening for regulators of the Type I-F CRISPR-Cas system. The plasmid 292 pK18mobsacB with the gentamicin resistance cassette was used to design constructs for in-293 frame deletion and chromosomal lacZ integration. 294

In-frame deletion and integrative Pcas1-lacZ reporter construction 295
To create in-frame deletion mutants in P. aeruginosa strains, Upstream and Downstream DNA 296 fragments flanking the region of interest in the genome were amplified by PCR using primers 297 listed in Table S2 and ligated with the EcoRI/HindIII digested suicide vector pK18mobsacB using 298 the MultiS cloning kit (Vazyme, China). The ligation products were transformed into E. Coli 299 DH5 and positive colonies selected by blue/white screening on gentamicin (5g/mL) and X-gal 300 selection plates and verified by colony PCR and DNA sequencing. Selected constructs were 301 transformed into E. Coli S17-1 for conjugation with P. aeruginosa strains. Transconjugants 302 were selected on Minimal medium containing gentamicin (30g/mL), followed by the selection of in-frame deletion mutants on MM supplemented with sucrose (10% w/v). Mutants were 304 further confirmed by PCR and DNA sequencing. 305

Transposon mutagenesis 306
The Mariner transposon, pBT20 was transferred from E. coli S17 to P. aeruginosa PA14 307 (pMEPcas1-lacZ ) strains by conjugation. Aliquots (10 -3 ) of the mating mix were spread on MM 308 agar (1.5% w/v) supplemented with Tetracycline (50 g/ml) and 5-bromo-4-chloro-3-indoyl-D-309 galactopyranoside(X-gal)(50 g/ml). Single colonies of the transconjugants were picked onto 310 the selection plate composed of MM agar (1.5% w/v) and X-gal (50 g/ml). Plates were 311 incubated for 48 hours and transposon mutants visually inspected for altered blue coloration 312 due to the up/downregulation of the Pcas1-lacZ activity in comparison to the wild type. 313 Colonies with altered coloration were selected tail colony PCR and DNA sequencing using 314 primers in Table S2. We screened over 5000 colonies on the selective media plates. Transposon 315 insertion site mapping was achieved after BLAST search of the sequences against the P. 316 aeruginosa genome (www.pseudomonas.com).

Electromobility Shift Assay (EMSA) 341
The DNA fragments were generated by PCR using the indicated primers in Table S2 and end  342 labeled with biotin using the biotin 3 I end DNA-labeling kit (thermo scientific) as described in 343 the kit protocol. EMSAs were performed using the LightShift chemiluminescent EMSA kit (Thermo scientific) according to the kit protocol. Briefly, 1nm of DNA fragments were incubated 345 with cRNAP-PvdS or PvdS and the binding buffer containing 1μg/μl Poly (dI.dC), 50% Glycerol, 1% 346 NP-40 1M KCl 100mM MgCl 2 and 200mM EDTA supplied with the kit for 25 minutes at 25 o C. 347 The cRNAP was added at a concentration of 1nM. The samples were then resolved on 6% native 348 polyacrylamide gel in 0.5X TBE buffer and transferred to the nylon membrane at 380mA (~100V) 349 for 30 minutes. DNA was crosslinked at 120mJ/cm2 for 45-60 seconds followed by detection of 350 the biotin-labeled DNA by chemiluminescence using the Tanon 5200 imaging system. 351

Plasmid loss and spacer acquisition 352
The plasmids pUCPT (non-targeted naïve control), pUCPTSp2n and pUCPTSp4n (2 and 4 353 nucleotide substitutions in respectively) were transferred into P. aeruginosa strains via 354 conjugation with E. coli S17. Selected colonies were cultured overnight in 5 ml LB with or 355 without 2,2-DP and passaged for 5 days by a subculture of 20l into 5 ml fresh media. Each 356 passage was serially diluted and 10 -6 dilutions plated on LB with or without carbenicillin to 357 count colonies that retain the plasmid. P. aeruginosa strains initially passaged with pUCPTSp2n 358 were further transformed with pUCPTSp4n and passaged for 5 days to assay for primed 359 adaptation. The genomic DNA was then extracted for the identification of expanded CRISPR2 360 arrays using primers stated in Table S2. PCR products were resolved by 3% agarose gel 361 electrophoresis for detection of the CRISPR array expansion.
E. coli S17 was used to transfer CRISPR-targeted pUCPTSp1 (pUCPT containing CRISPR2 364 spacer1) and untargeted pUCPT into P. aeruginosa strains. Overnight culture of E. coli and P. 365 aeruginosa were mixed at a ratio of 1:1, washed twice and pellets resuspended in LB from 366 which 50l was spotted on LB agar gently to prevent splatter. The mating spot was allowed to 367 dry and incubated for 16 hours at 37 0 C. The mating spot was scrapped completely and 368 resuspended in 250 l TSB from which serial dilutions of 10 -5 were platted on LB and LB+2,2-DP 369 agar with carbenicillin. The efficiency was calculated as the percentage transformants with the 370 targeted plasmid compared with the transformants with the non-targeted plasmid.