Pseudomonas can survive bacteriocin-mediated killing via a persistence-like mechanism

Phage tail-like bacteriocins (tailocins) are bacterially-produced protein toxins that can mediate competitive interactions between co-colonizing bacteria. Both theoretical and empirical research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive, sensitive outcompete resistant, and resistant outcompete producers. These so-called ‘rock-paper-scissor’ dynamics explain how all three populations can be maintained in the same environment, without one genotype driving the others extinct. Using Pseudomonas syringae as a model system, we demonstrate that otherwise sensitive bacterial cells have the ability to survive bacteriocin exposure through a physiological mechanism. This mechanism is similar to the persister phenotype that allows cells to survive antibiotic exposure, without acquiring antibiotic resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival in subsequent exposure (i.e. they survived through a persistence-like mechanism). Tailocin persister cells were more prevelant in stationary rather than log phase cultures. Of the fraction of cells that gained detectable tailocin resistance, there was a range of resistance from complete (insensitive) to incomplete (partially sensitive). By genomic sequencing and genetic engineering we showed that a mutation in a hypothetical gene containing 8-10 transmembrane domains causes tailocin high-persistence and genes of various glycosyl transferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result, combined with previous research, indicates that bacteria likely utilize persistence as a means to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance, and expands our understanding of how sensistive bacterial populations can persist in the presence of lethal competitors.

sensitive outcompete resistant, and resistant outcompete producers. These so-called 'rock-paper-23 scissor' dynamics explain how all three populations can be maintained in the same environment, 24 without one genotype driving the others extinct. Using Pseudomonas syringae as a model 25 system, we demonstrate that otherwise sensitive bacterial cells have the ability to survive 26 bacteriocin exposure through a physiological mechanism. This mechanism is similar to the 27 persister phenotype that allows cells to survive antibiotic exposure, without acquiring antibiotic 28 resistance. We show that a significant fraction of the target cells that survive a lethal dose of 29 tailocin did not exhibit any detectable increase in survival in subsequent exposure (i.e. they 30 survived through a persistence-like mechanism). Tailocin persister cells were more prevelant in 31 stationary rather than log phase cultures. Of the fraction of cells that gained detectable tailocin 32 resistance, there was a range of resistance from complete (insensitive) to incomplete (partially 33 sensitive). By genomic sequencing and genetic engineering we showed that a mutation in a 34 hypothetical gene containing 8-10 transmembrane domains causes tailocin high-persistence and 35 genes of various glycosyl transferases cause incomplete and complete tailocin resistance. 36 Importantly, of the several classes of mutations, only those causing complete tailocin resistance 37 compromised host fitness. This result, combined with previous research, indicates that bacteria 38 likely utilize persistence as a means to survive bacteriocin-mediated killing without suffering the 39 costs associated with resistance. This research provides important insight into how bacteria can 40 can employ multiple strategies to survive toxin exposure without acquiring an otherwise fitness-109 reducing mutation. One strategy was physiological persistence, a mechanism that enables a sub-110 population of sensitive cells to transiently survive lethal doses of the bacteriocin without 111 undergoing genetic changes. The second strategy relies on acquiring subtle genetic changes 112 (incomplete resistance) that do not impose a detectable fitness cost, while still allowing the 113 mutants to better survive bacteriocin exposure. We found pronounced differences between the 114 frequencies of persistent cells depending on growth phase of the target cells. Moreover, we 115 identified ten unique mutant alleles with likely roles in the lipopolysaccharide (LPS) O-antigen 116 biosynthesis leading to various degrees of tailocin resistance. We also recovered a mutation in an 117 open reading frame, located in an LPS biosynthesis operon, that results in increased bacteriocin 118 persistence. In addition to increasing the basal persistence frequency, this mutation results in a 119 loss of growth phase-dependent difference in tailocin peristence. Finally, we demonstrated that 120 the complete mutants suffered a fitness cost within a susceptible host plant, whereas both high 121 persisteter, as well as incomplete resistant mutants were equally fit compared to the wild-type. 122 This work suggests that bacterial cells can employ mechanisms to survive antagonistic toxins but 123 still preserve their host colonization potential. This work has important implications for how 124 bacteria can potentially avoid rock-paper-scissor dynamics widely understood to be important in 125 mediating interactions between toxin producing, sensitive, and resistant genotypes and in using 126 bacteriocins as potential therapeutic agents. 127 128 129 130

Results 131
A sub-population of Pph cells survived tailocin by persistence that increased in the 132 stationary state 133 The relative activity of the purified tailocin was determined to be 103-104 activity units (AU) and 134 1.25×107-4.25×109 lethal killing units/ml. The minimum inhibitory concentration was estimated 135 to be 100 AU when exposed to ~106 viable target cells at their logarithmic growth. No loss of 136 tailocin activity was observed for a period of over six months in the buffer (10 mM Tris PH 7.0, 137 and 10 mM MgSO4) at 4•C. 138 Purified tailocin was used to test its killing effects on stationary and log phase cultures of the 139 Pph target cells in a broth environment. After an hour of 100 AU tailocin treatment, a consistent 140 reduction (3.59 ± 0.12 log) in the viable population occurred for logarithmic cultures, while a 141 significantly lower reduction (1.38 ± 0.14 log) occurred for the stationary cultures. Further 142 analysis showed that, upon treatment of equivalent number of viable cells, stationary cells 143 consistently survived 10 to 100-fold more than the logarithmic cells (Fig 1 and Fig S1). 144 Surviving colonies, especially those from the stationary phase, were predominantly sensitive 145 upon tailocin re-exposure suggesting survival by persistence mechanism (see below). 146

147
The persistent sub-population was maintained under prolonged exposure time and 148 increased concentration of tailocin 149 Tailocin treatments were applied to both stationary and log cultures of Pph for up to 24 hours 150 with enumeration of surviving population before and after 1, 4, 8, and 24 hours of tailocin 151 treatment to generate a tailocin death curve. After a steep reduction in the population within the 152 first hour of treatment, further killing of the cells that survived the first hour treatment, did not 153 occur in either culture (Fig 2A). Twenty four hours post-treatment, although the overall 154 population increased (Fig 2A), individual treatments showed different results: for some replicate 155 treatments, the population remained constant suggesting maintenance of the persistent state, 156 while for some other replicates, population increased due to replication of cells that acquired 157 tailocin resistance (see Fig S2). 158 Upon tailocin re-treatment, >90% of stationary and >60% of log cells that survived the first hour 159 treatment, were as sensitive as the wild type (i.e. persistent) as in (Fig 2B). The proportion of 160 persistent survivors was higher in the stationary cultures than in the log cultures at all time points 161 ( Fig 2B). Tailocin persistent cells were recovered from both cultures even after 24 hours of 162 tailocin treatment, although the proportion decreased over time (Fig 2B). Tailocin activity was 163 detected in the supernatants recovered from the treated samples that contained persistent cells 164 (Fig S3), confirming saturation of tailocin in the treatment. Although a slight reduction of 165 activity was observed when the tailocin preparation was mixed with undiluted stationary 166 supernatant compared to log supertanant, no difference was detected upon diluting the supertants 167 up to 1,000-20,000-fold before mixing with tailocin (similar to how the cultures were diluted for 168 tailocin treatment) (Fig S4). This suggested that the increased tailocin persistence in the 169 stationary phase is not related to inhibition of tailocin activity by an extracellular component. 170 Upon treating the cells with a concentrated tailocin (900 AU) the surviving population decreased 171 such that no difference in survival between the stationary and log cultures was detected (Fig 3A). 172 However, even with this higher level of tailocin applied, the proportion of tailocin persistent cells 173 remained higher for stationary phase survivors than that for the log phase survivors (Fig 3B). 174

Tailocin exposure selected for heritable mutants showing increased persistence and 175
heterogenous resistance 176 In addition to the recovery of tailocin persistent sub-population, we recovered an unique mutant, 177 refered here as high persistent-like (HPL), which survived significantly greater than the wild type 178 under liquid-broth treatment (Fig. 4A). However, under a long-term exposure of overlay 179 condition, it showed similar sensitivity to the wild type (Fig. 4B). Furthermore, the HPL 180 phenotype did not differ in survival between the stationary and log phases even at a higher 181 concentration of tailocin applied (Fig 5). Next category of mutants recovered were conditionally-182 sensitive and are referred here as incomplete resistant (IR) mutants (see Fig 2B and 3B for 183 proportion). These mutants lost sensitivity in the broth even at high tailocin concentration ( Fig.  184 4A), but displayed some sensitivity in the overlay (Fig. 4B). Lastly, complete tailocin resistant 185 (R) mutants that were insensitive to tailocin under both treatment conditions were also recovered. 186 Of the four complete resistant mutants we selected, two (R1 and R4) showed an unique rough 187 colony morphotype. 188 189

Mutations involved various genes likely associated to LPS biogenesis and modification 190
Genome sequencing and variant identification of the high-persistent like (n=1), incomplete 191 resistant (n=9), and resistant mutants (n=4) was performed by mapping the Illumina reads with 192 the parental reference sequence. Mutants isolated at different experiments showed mutation in a 193 different locus. A specific region (Fig 6) was identified in the Pph genome that showed the most 194 prominent role in tailocin activity. The HPL mutant contained a 16 bp deletion in an ORF that 195 caused frameshift near the C-terminal of a hypothetical protein that is co-transcribed with the 196 LPS genes. No functional evidence could be found for the hypothetical protein by in silico 197 analysis except that it was predicted to contain 8-10 transmembrane domains. Majority of genes 198 identified for complete and incomplete resistance were glycosyl transferases and related proteins 199 that are likely involved in LPS biogenesis ( Table 1  In this study, we addressed these questions using a phage-tail like bacteriocin (i.e. tailocin) 242 produced by P. syringae pv. syringae strain B728a in killing target cells of P. syringae pv. 243 phaseolicola 1448A. We showed that, upon exposure of a lethal dose of tailocin, a sub-244 population of sensitive cells survives without undergoing genetic changes. The fraction of this 245 sub-population, termed here as tailocin persistent sub-population, increased significantly in the 246 stationary phase than in the logarithmic phase of growth. By repeated exposures of this sub-247 population to same or higher doses of tailocin, we showed that they have not gained any 248 heritable resistance, and physiological persistence is the only mechanism for their survival. 249 Moreover, a prolonged tailocin exposure generated a killing pattern similar to that reported for 250 persistent sub-population upon antibiotic treatment (41, 42). Persistence was maintained for at 251 least 24 hours with tailocin exposure, a phenomenon that was more evident in some treatment 252 replicates in which resistant evolution did not occur (see Fig. S2). Although increasing the 253 tailocin concentration killed some of the persistent survivors, and the difference in survival 254 between the two growth phases was no longer seen, stationary phase-derived cells still exhibited 255 higher persistence than the log phase cells upon re-exposure. This indicated that the stationary 256 cells may require multiple hits by tailocin particles as opposed to the one-hit-one-kill mechanism 257 of killing described for tailocins (32, 33), or that the probabilty of a successful hit in stationary 258 phase is lower than in log phase. Since tailocins are thought to be target cell specific, and are not 259 known to have off-target effects, higher concentration of tailocin could be used to achieve a more 260 effective pathogen control. However, although at a low level, persistence was still maintained 261 even with high-dose tailocin treatment and inherent emergence of either complete or incomplete 262 resistance was frequently observed. As such, although a significant reduction in pathogen 263 population and disease pressure can be obtained with tailocins, a stand-alone tailocin treatment 264 might not be enough to achieve a sustainable pathogen control. 265 The use of the term 'persistence' in relation to antimicrobial survival is disputed to some extent 266 and is sometimes used interchangably with 'tolerence' and 'viable but not culturable state'. In 267 this paper, we used 'persistence' as this phenotype was only seen in a sub-population, resulted in 268 a bi-phasic death curve, cells resuscitated almost immediately upon tailocin removal, and were 269 equally sensitive to the wild type cells upon re-exposure. This definition of persistence has been 270 suggested previously (42). Persistence to antimicrobials is being increasingly recognized for its However, It was also shown that activation of TA system does not always induce persister 278 formation (50). Additionally, recent findings have indicated a mechanism mediated by the 279 guanosine penta-or -tetraphosphate (ppGpp) for persister formation that is not dependent on a 280 TA system (51). A strong stationary state effect, that likely involved starvation response, was 281 shown to increase persistence by 100-1,000 fold in Staphylococcus aureus with ciprofloxacin 282 treatment (50). Whether similar mechanisms of TA and independent ppGpp systems regulate 283 tailocin persistence or a specific mechanism for tailocin and/or related bacteriophage exists, 284 remains to be determined. Nevertheless, our data of the difference in tailocin persistence between 285 the stationary and log cultures suggests that metabolic inactivity and starvation-induced stress 286 could be a strong factor in tailocin persistence. 287 Few previous studies have demonstrated growth-phase dependent differences in LPS O-antigen 288 chain length and composition or their regulatory pathways (52, 53). In a previous study with P. 289 fluorescens, exponentially growing cells had a significantly higher rate of cell lysis than 290 stationary or decline phase cells with bacteriophage PhiS1 (54). Moreover, a recent study 291 showed that sensitivity to colicin, a bacteriocin from Eschericia coli, could be altered by growth 292 condition dependent LPS O-antigen changes (55). Growth phase dependent LPS modification, 293 although has not been reported so far in P. syringae, could be contributing to the difference in 294 tailocin persistence. We also observed that undiluted stationary phase supernatant inhibited 295 tailocin activity to some extent compared to the log phase supernatant. However, whether this is 296 linked to differences in the secreted LPS between the two supernatants or other cellular factors 297 needs further assessment. Moreover, we also demonstrated that mutation in one of the 298 hypothetical proteins containing a signal peptide and several trans-membrane domains caused 299 increased tailocin persistence. Since the hypothetical protein occurs in the same operon as other 300 LPS biogenesis genes, it is likely that it plays a role in O-antigen biogenesis and/or modification, 301 thereby reducing tailocin interaction with the cells. 302 Phase variation is another mechanism that is known to cause increased survival to surface active 303 antimicrobials (eg. phages and host immune defenses) (56). Phase variation is a gene regulation 304 system that induces heterogenous expression of specific genes in a clonal population (56-58). 305 Although phase variation is heritable, the 'ON' 'OFF' switch from variant to wild type 306 phenotype occurs randomly amounting to 10-4 to 10-1 per generation, significantly greater than 307 what is expected by mutational events (59). Phase variation has been shown to modify LPS 308 and persister) that can be detected following bacteriocin-mediated selection. In particular, we 352 view the persister sub-population as one that can survive bacteriocin exposure, without paying 353 any long term fitness cost. In the short term, however, we believe that persister cells have a 354 fitness of zero because they are likely not replicating (see Figure S2). Thus, bacteriocin persisters 355 may best be thought of as a means to switch at high frequency between being phenotypically 356 resistant and phenotypically sensitive. 357 358

Materials and Method 359
Bacterial strains, media, and culture conditions 360 All bacterial strains, plasmids, and mutants used in this study are listed in Table 1 and 2. P. 361 syringae pv. syringae (Psy) wild type (WT) strain B728a and its tailocin defective mutant ∆Rrbp 362 were used to prepare the treatment supernatants. P. syringae pv. phaseolicola (Pph) 1448A was 363 used as the target strain. Tailocin high-persistent like, resistant, and selected complementated 364 strains of Pph generated in this study are described in Table 1 Table 3. The PCR fragment contained gateway cloning sites added through the primer 482 extension. Purified PCR fragment was cloned into pDONR207 and further recombined into 483 pMTN1907 using BP and LR clonase enzymes, respectively. pMTN1907 containing a desired 484 clone was transformed to S17-1 and conjugated to the Pph wild type or mutant background by 485 bi-parental mating. TetR merodiploids of Pph selected on Tet, Rif, and NFT plates were counter-486 selected in KB supplemented with 10% Sucrose, followed by PCR confirmation of the desired 487 allele-swapped strains. Allele swap of the high persistent-like (HPL) mutant was performed 488 similarly, but using a one-step gateway vector pDONR1K18ms and the merodiploids were 489 selected in Km, Rif, NFT plates.    and optical density was measured at 600nm every two hours until 24 hourrs. Growth curve 839 experiment was performed twice. One representive experiment is shown. 840