A type IVB secretion system adapted for bacterial killing, bio lm invasion and biocontrol

14 Many bacteria utilize contact-dependent killing machineries to eliminate rivals in their 15 environmental niches. Here, we show that Pseudomonas putida IsoF is able to outcompete a 16 wide range of bacteria with the aid of a novel type IVB secretion system (T4BSS) that can 17 deliver toxic effectors into bacterial competitors. This extends the host range of T4BSSs, which 18 were so far thought to transfer effectors only into eukaryotic cells, to prokaryotes. Bioinformatic 19 and genetic analyses showed that this killing machine is entirely encoded by a rare genomic 20 island, which has been recently acquired by horizontal gene transfer. IsoF utilizes this secretion 21 system not only as a defensive weapon to antagonize bacterial competitors but also as an 22 offensive weapon to invade existing biofilms, allowing the strain to persist in its natural 23 environment. Furthermore, we show that IsoF can protect tomato plants against the plant 24 pathogen Ralstonia solanacearum in a T4BSS-dependent manner, suggesting that IsoF 25 capabilities can be exploited for pest control and sustainable agriculture. 26


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In this study, we identified a T4BSS that can deliver toxic effectors into bacterial 76 competitors, breaking the paradigm that T4BSSs are only used for effector transfer into 77 eukaryotic cells 43,44 . This novel bacterial killing machine is encoded by a rare genomic island 78 that was probably horizontally acquired by Pseudomonas putida IsoF, which is an effective 79 colonizer of plant roots 45, 46 . We demonstrate that IsoF utilizes this secretion system not only as 80 a defensive weapon to antagonize phylogenetically diverse bacterial competitors but also as an 81 offensive weapon to invade an existing biofilm by contact-dependent killing. We also show that 82 IsoF can protect tomato plants against the pathogen R. solanacearum in a T4BSS-dependent 83 manner, suggesting that this killing machine can be exploited for pest control.

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IsoF exhibits contact-dependent antagonism against a wide range of Gram-negative 86 bacteria 87 We observed that P. putida IsoF (marked with Gfp; IsoF::Gfp) inhibited the growth of P. putida 88 KT2442 (marked with mCherry; KT2442::mCherry) when culture samples were inoculated in a 89 1:1 ratio on a minimal medium plate. After 24 hours in contact-dependent competition (CDC), 90 no red fluorescence could be observed from the macrocolony, indicating that the KT2442 had 91 been outcompeted. We next determined the CFUs of the two strains after 24 h and 48 h and 92 found that after two days IsoF had completely eliminated KT2442 (Fig. 1a). No adverse effect 93 was seen when the two strains were separated by a 0.2 m pore size filter, suggesting that 94 killing depends on cell-to-cell contact (Extended Data Fig. 1). To obtain further insight into the 95 underlying molecular mechanism, we performed competition experiments on plates 96 supplemented with propidium idiode (PI), which allowed us to assess dead cells. After 48 hours 97 of incubation, PI staining (magenta) was observed in the region where the two drops of the 98 inoculated cultures overlapped, whereas dead cells were absent from the pure cultures regions 99 (Fig. 1b). Time-lapse confocal laser scanning microscopy (CLSM) was used to demonstrate 100 that KT2442 cells were killed after they had been in direct contact with IsoF::Gfp (Fig. 1c,

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Extended Data Video 1). We noticed that dead cells did not lyse or change their morphology.

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To determine the host range of the antagonistic activity of IsoF we fluorescently marked several 103 soil-and plant-associated bacteria as well as some phytopathogens, and tested whether they 104 are susceptible to killing by IsoF. All tested strains were outcompeted after 24 h of co-culture 105 with IsoF (Fig. 1d, e). Collectively, our data suggest that IsoF possesses a highly efficient, broad 106 host-range, contact-dependent killing machinery.

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IsoF utilizes a type IVB secretion system for bacterial killing

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To identify the mechanism responsible for contact-dependent killing by IsoF, we constructed a 110 mini-Tn5 transposon insertion library of this strain and tested about 5,000 mutants for their 111 ability to outcompete P. aureofaciens::mCherry when grown as mixed macrocolonies (Fig. 2a).
insertion sites of eight of these mutants were determined by arbitrary PCR 47 and were found to 114 be located in four genes of a large gene cluster, which we designated kib (killing, invasion, 115 biocontrol, see below), that appears to encode several elements of a T4BSS (Fig. 2b). While

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T4BSSs of intracellular pathogens are well known for their capacity to deliver effectors to their 117 eukaryotic hosts 42,48,49 , they have so far not been reported to be involved in interbacterial killing.

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To validate the results of the mutant screen, we constructed defined T4BSS mutants: ΔdotHGF, 119 which lacks the main structural components of the secretion system channel and Δ23-trbN-120 dotD, which lacks Piso_02323 encoding the hypothetical protein TrbN, which has a conserved 121 transglycosylase domain that has been proposed to assist DNA transfer across the PG in  The region comprising the kib locus has a GC content of 58.8 %, whereas the IsoF genome has 132 an average GC content of 62.6 % (Extended Data Table 1), which suggests the kib locus is part 133 of a genomic island that has been recently acquired by horizontal gene transfer. This hypothesis 134 was further strengthened by analyzing the IsoF genome with ICEfinder, allowing the detection 135 of mobile integrative and conjugative elements in bacteria 54 . This algorithm classified the IsoF 136 genomic island containing kib as a putative conjugative element, since it contains T4SS-related 137 genes and an integrase within the region. ICEfinder defined the borders of the gene cluster with 138 genes Piso_02313 and intA_3. Hence, the entire island has a size of 66,917 bp and encodes 139 61 genes, 17 of which share homology with described T4BSS structural genes, 37 were defined as hypothetical proteins, and four encode a Type I Restriction Modification (RM) system and an 141 integrase at the 3'-end of the island (Fig. 2b,c). The basic local alignment search tool (BLAST) 142 was used to interrogate the cluster and revealed that kib genes are also present in 11 other

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Pseudomonas strains, 10 of which are environmental isolates and one is a clinical isolate. Eight 144 of these strains were classified as P. putida (Extended Data Table 1). Interestingly, all 145 orthologous kib gene clusters showed conserved synteny and were located at the same 146 chromosomal position (Extended Data Fig. 4), suggesting a common ancestor. Notably, the 147 orthologous clusters showed deletions at the 3'-end of the island, including the Type I RM

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The kib gene cluster encodes an effector-immunity (E-I) pair 158 Contact-dependent killing systems deliver toxic effector molecules into bacterial competitors.

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To avoid self-killing, the attacking bacterium produces a cognate immunity protein that 160 neutralizes the toxin. The immunity and toxin genes form a so-called effector-immunity (E-I) 161 pair, and are often co-transcribed 22,32,55,56 . The kib gene cluster does not contain any previously 162 described or obvious candidate toxin genes but many hypothetical genes (Fig. 2b), which could 163 potentially encode effector molecules. However, as effector proteins are very heterogeneous 164 both in sequence and function 22,57 , we were not able to identify promising candidates using in 165 silico analysis. To investigate whether an E-I pair was present within the kib region, we deleted 166 49.5 kb of the genomic island containing all kib genes (Piso_02313 to Piso_02360, Extended 167 Data Fig. 5). The resulting mutant, designated ΔT4B, no longer killed P. aureofaciens and formed mixed macrocolonies with this strain (Fig. 2d). Interestingly, the ΔT4B mutant became 169 susceptible to KT2442 in co-culture on nutrient plates, likely because KT2442 possesses a 170 T6SS that was shown to efficiently kill other bacteria 27 (Extended Data Fig. 3). Our original 171 observation that IsoF kills KT2442 (Fig. 1a), suggests that kib-mediated killing is faster or more 172 efficient than T6SS-mediated killing of KT2442. We hypothesized that the absence of an E-I 173 pair would render the ΔT4B mutant sensitive to the wild type strain, while its presence would 174 confer resistance. In competition experiments between IsoF and ΔT4B, the mutant strain was 175 indeed killed, while it was able to co-exist with the ΔdotHGF mutant, which lacks the structural 176 components of the secretion channel required for killing (Fig. 3a). These experiments 177 demonstrate that the genes required for killing and self-protection are present within the kib 178 cluster. Moreover, IsoF was unable to kill mutants ΔdotHGF and Δ23-trbN-dotD, indicating that 179 both deletion mutants are protected against effector toxicity from the wild type strain and that

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Immunity genes are essential since cells lacking an immunity protein would either be killed by 185 neighboring bacteria or die due to self-intoxication 32,58 . We therefore reasoned that it should be 186 possible to identify the genetic elements required for self-protection by transposon sequencing.

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This approach has previously been employed to identify E-I pairs of T6SSs in V. cholerae and 188 P. aeruginosa 58,59 . To this end, we generated a saturated transposon insertion library in the IsoF 189 wild type strain. The pooled library, which consisted of approximately 700,000 mutants, was 190 subjected to three different growth regimes: (i) growth in liquid medium with shaking to prevent 191 cell-to-cell contact, (ii) growth on an agar surface either alone or (iii) in the presence of the 192 competitor P. aureofaciens to promote competition (Fig. 3b). Sequencing of the genomic DNA 193 resulted in more than 7 million reads per sample as summarized in Extended Data Table 2. We 194 used the unique insertion density approach of the Tn-Seq explorer software to identify genes 195 that provide a fitness benefit for growth under the different growth regimes 60 . This analysis identified one gene, Piso_02332, within the kib region, which was virtually devoid of transposon 197 insertions in all three treatments (Fig. 3c, Extended Data Table 3). This gene appears to be co-198 transcribed with Piso_02333, possibly constituting a novel E-I pair.

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To determine the role of this putative E-I pair in bacterial killing, we deleted both genes 200 in IsoF to generate Δ32-33. We were also able to delete the putative effector gene, giving rise 201 to mutant Δ33. Unexpectedly, we noticed that the Δ32-33 grew slower on ABC minimal media   Fig. 10a, b). When a pre-established KT2442 232 biofilm was challenged with the kib mutants ΔdotHGF or Δ23-trbN-dotD, neither of the mutants 233 was able to form microcolonies or to invade the existing biofilm ( Fig. 4a, b). Importantly,

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ΔdotHGF or Δ23-trbN-dotD mutants in isolation formed biofilms similar to the IsoF wild-type 235 strain (Extended Data Fig. 11a, b). We hypothesized that IsoF employed its T4BSS to kill to those that were not in contact with a green cell, demonstrating that kib-mediated killing is 246 strictly dependent on cell-to-cell contact (Fig. 4d). By contrast, when KT2442 was challenged 247 with either the ΔdotHGF or the Δ23-trbN-dotD mutant (green), very few dead cells were 248 observed, similar to monoculture biofilm controls (Fig. 4d, Extended Data Fig. 12). In 249 conclusion, these results suggest that the kib system not only allows IsoF to defend itself against 250 competitors but also to kill bacteria that live within an established biofilm community, which    encoding an integrase as well as a Type I RM system flanking kib, suggest that it was recently 306 acquired by horizontal gene transfer (Fig. 2b, Extended Data Table 1). This is also in line with 307 the finding that orthologs of kib were only present in 11 Pseudomonas strains. Interestingly, all of these homologous islands lacked the Type I RM system and the integrase (Extended Data 309 Fig. 5).

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We speculated that the kib cluster provides IsoF a competitive advantage for survival in the 312 environment, as IsoF was shown to be an excellent colonizer of tomato roots and most of the 313 other strains carrying kib were also isolated from soil. Our results showed that IsoF indeed 314 outcompeted various environmental strains, most notably P. putida KT2442, which was recently 315 demonstrated to use its K1-T6SS as an antibacterial killing device 27 . IsoF does not harbor a 316 homolog of the K1-T6SS gene cluster and thus was expected to be sensitive to killing by 317 KT2442. In fact, the ΔT4B mutant of IsoF was found to be killed by KT2442 in co-culture.

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However, when the two wild type strains were competed against each other, IsoF eliminated 319 KT2442, indicating that T4BSS-mediated killing may occur before KT2442 can fire its T6SS 320 apparatus (Fig. 1a, Extended Data Fig. 3). Previous work has shown that bacteria have different  Fig. 14).

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We performed a Tn-Seq analysis to identify potential E-I pairs within the kib gene cluster. This 343 strategy, which assumes that inactivation of an immunity gene is lethal for the cell, identified 344 Piso_02332 as an essential gene. Importantly, we found this gene to be essential for growth 345 under all conditions tested, namely in liquid medium, in a macrocolony on the surface of an agar 346 plate or in the presence of a competitor strain, suggesting that this gene is constitutively 347 expressed. We provided evidence that Piso_02332 encodes an immunity protein that 348 neutralizes the toxicity of an effector protein encoded by Piso_02333, which appears to be co-349 transcribed with Piso_02332, a genetic architecture that is frequently found with E-I 350 pairs 22,32,55,56 . A defined Piso_02333 mutant killed neither KT2442 nor P. aureofaciens and this 351 defect was at least partially restored by genetic complementation (Extended Data Fig. 9). At 352 present the cellular target of the Piso_02333 effector is unknown. Given that cells killed by IsoF 353 maintained their shape and did not lyse, it is tempting to speculate that the target is located in 354 the cytosol rather than the cell envelope. Expression of Piso_02332 in the Δ32-33 mutant 355 conferred immunity against the IsoF wild type strain, indicating that Piso_02332 neutralizes the 356 toxicity of the Piso_02333 effector. Unexpectedly, complementation of ΔT4B, which lacks the 357 entire kib locus, did not protect the mutant against IsoF, suggesting that an additional E-I pair 358 may be encoded by the kib gene cluster (Fig 3e). We noticed that the transposon insertion 359 density of another gene within the kib locus, Piso_02351, was reduced, albeit to a lesser degree 360 than Piso_02332 (Extended Data Fig. 7, Extended Data Table 3). Whether this gene together 361 with one of its adjacent genes could comprise another E-I pair remains to be elucidated.

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Natural biofilms have dynamic and heterogeneous structures that are shaped by both environmental forces and microbial interactions, which may be cooperative or antagonistic 78-80 .

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The biofilm matrix protects the cells from various external stresses and restricts the entry of 366 invaders into the biofilm 18,78 . Moreover, many bacteria use defense mechanisms that would 367 effectively kill competitors that attempt to enter the established biofilm community 22,23,33 . In this 368 study we demonstrate that P. putida IsoF has the unprecedented ability to invade and replace 369 an established biofilm of P. putida KT2442. However, when the two wild type strains competed 370 against each other, KT2442 was eliminated, presumably because it was killed before it could 371 fire its T6SS. In accordance with a recent report 81 , we observed that on agar plates dead cells

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which eventually led to the replacement of the existing KT2442 biofilm (Fig. 4a, b). It is worth 376 mentioning that IsoF produces the powerful biosurfactant putisolvin, which was previously

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IsoF was originally isolated from the rhizosphere of a tomato plant and was shown to form a 382 biofilm on root surfaces 83 . Here, we demonstrate that IsoF is able to antagonize several 383 economically relevant phytopathogens (Fig. 1d, e). Moreover, we show that IsoF can protect 384 tomato plants from the soil-borne pathogen R. solanacearum, which can infect over 250 385 different plant species, among them important agricultural crops 61 (Fig 5). The presence of 386 microbes secreting bacteriocins, antifungals or antibiotics in the rhizosphere was shown to be 387 an effective strategy to suppress plant pathogens 21,80,84 . In this study we demonstrate that IsoF 388 uses its T4BSS not only to kill phytopathogens but also to invade biofilms. Given that a major 389 limitation in biocontrol applications is that inoculants are unable to establish themselves in the 390 environment, IsoF, which utilizes kib for attack as well as for defense, is a very strong candidate 391 for a novel bioinoculant for plant protection.

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Bacterial strains used in this study have been listed in Table 1 Table   412 1. Mini-Tn7 tagged strains were obtained by tri-parental mating using the donor strain E. coli

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Briefly, the flow cell chambers were inoculated with P. putida cultures at an OD600 of 0.1, biofilm development was followed every 24 h up to 5 days. For the competition experiment, the strain 560 inoculated on top of the pre-established biofilm was adjusted to an OD600 of 0.5. For the two-561 species biofilm, strains were mixed in a 1:1 ratio and cultivated for up to 48 h. Shortly before 562 40 h of cultivation, PI was added. Photomicrographs were taken every 24 h with a confocal

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Prediction ellipses were used to display the 95% confidence intervals. Root parameters were clustered using correlation distance and average linkage, plant samples were clustered using 588 Euclidean distance and average linkage. n = 144. Three independent biological replicates were 589 performed with a total of 28 plants for each treatment. Shoot area and chlorophyll estimations 590 were obtained from calibrated RGB photographs using Fiji 103