{'fraction_non_alphanumeric': 0.02923902480246757, 'fraction_numerical': 0.045083872058140025, 'mean_word_length': 4.727976766698935, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

Members of the bacterial T6SS amidase effector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.


98
To understand how sequence diversity within the Tae protein superfamily impacts interbacterial toxicity, 99 we must first probe how each amino acid residue contributes to Tae function. Biochemical assays of 100 Tae protein binding to and degradation of peptidoglycan (PG) use isolated, purified sacculi 10   expressed the variants as fusion proteins with N-terminal periplasm-targeting pelB signal sequences.

146
We transformed E. coli (BW25113) grown in M9 minimal media with our tae1 library and sequenced 147 plasmid barcodes, both before and two hours after library induction ( Figure 1C). Tae1-dependent lytic 148 effects were quantified by using the DMS analysis pipeline Enrich2 17 , which calculates output-to-input 149 ratios for individual variants, yielding fitness scores that are normalized to wild type (Supplemental 150   Table 1). In our assay, the fitness score reflects a difference in the antibacterial capacity of the Tae1 151 variant in vivo when compared to the wild type: relative enrichment of a variant indicates reduced Tae1-152 dependent cell death or loss-of-function (LOF) and yields a positive fitness score, whereas relative 153 depletion of a variant indicates increased cell death or gain-of-function (GOF) and yields a negative 154 fitness score.

156
To validate our approach, we examined the canonical catalytic dyad residues of Tae1 to assess 157 whether our screen accurately identified known functional determinants. Any substitutions of these 158 catalytic residues, Cys30 and His91, should inhibit enzyme activity and lead to LOF. As anticipated, all 159 non-synonymous amino acid changes at these sites resulted in increased fitness scores, indicating 160 their crucial roles in Tae1 toxicity (Figure 1C and D) and confirming that the DMS screen was indeed 161 selecting in vivo functional determinants of Tae PG-degrading toxicity.

165
To obtain a global view of the amino acid residues in Tae1 that affect fitness in our screen, we 166 generated an averaged sequence-function map of variant fitness scores (Figure 2A). The majority of all 167 unique Tae1 protein variants (54%) were LOF variants with positive fitness scores, whereas only 6% 168 were GOF variants. To determine the functional role of each residue of Tae1, we collapsed and 169 averaged scores across all substitutions at each Tae1 site (Figure 2A) and superposed these onto the 170 three-dimensional structure of Tae1 (Supplemental Figure 1). For some Tae1 residues, position 171 averages obscured how the degree or direction of functional change depended greatly on the specific 172 amino acid change introduced. Therefore, we used k-means clustering, a statistical method for 173 analyzing DMS data 18 , to classify Tae1 residues in an unbiased way into one of four categories: 174 residues where mutations generally decreased cell lysis (LOF clus ), those where mutations generally 175 increased cell lysis (GOF clus ), those where mutations had variable effects on lysis (mixed), and those 176 where mutations had little to no effect (tolerant). The vast majority of residues were categorized as 177 Figure 2. DMS identifies functional hotspots extending around the surface of Tae1. (A) Heatmap representation of fitness scores relative to wild-type Tae1 for all mutations in DMS. Each amino acid position is represented on the x-axis, and each possible amino acid substitution is represented on the y-axis, * = stop codon. Each box represents the average fitness score of three replicate experiments. The average fitness score of all substitutions at each residue is depicted across the bottom row. Catalytic residues Cys30 and His91 are indicated with asterisks. Variants in grey were not present in our library. (B) Results of k-means clustering of DMS data. Based on fitness effects, each residue of Tae1 was labelled loss-of-function (LOF CLUS ), gain-of-function (GOF CLUS ), mixed, or tolerant. (C) Ribbon diagram of Tae1 structure. LOF CLUS residues are shown in red, GOF CLUS residues are shown in blue, and all other residues are in gray. The catalytic residues of Tae1 (Cys30 and His91) are indicated. Mutations in the enzyme core lead to loss-of-function phenotypes. (D) Functional residues on the surface of Tae1. The catalytic surface of the enzyme is shown on the left and the opposite surface, after 180-degree rotation, is on the right. Color code as in part C. LOF CLUS mutations are located in large areas of the surface on both sides of the enzyme, and most GOF CLUS mutations are also found on the surface. See also supplemental figure 1.

185
Residues on the enzyme surface, by 186 contrast, were a mix of categories including most of the GOF clus residues.

188
We anticipated that only surface residues within or near to the catalytic cleft would show loss-of-189 function phenotypes. Instead, we found that many surface-exposed residues more distant from the 190 catalytic cleft were also categorized as LOF clus (Figure 2D). Indeed, a zone of LOF clus residues forms a 191 ring around the entire enzyme, suggesting a more distributed set of surfaces may play key roles in cell 192 lysis in vivo than was predicted by in vitro studies 10 . Many of the surface LOF clus residues that our   Nine distal residues on Tae1 that had loss of function phenotypes. The catalytic core is highlighted in yellow. (B) Lysis assays measuring relative lysis of E. coli expressing Tae1 WT , negative controls (vector and C30A) and loss-of-function single amino acid substitutions (G42S, V34S, A52P, S144A, S149A, L21S, G48S, G56S). Data shown are means +/-standard deviation, n = 3, * p < 0.05. Induced with 0.25% arabinose. Lysis experiment was repeated 3 independent times, each time with at least.3 technical replicates. Western blots below show the beta subunit of RNA polymerase (RNAP β) and Tae1 proteins in each assay. All variants and controls showed reduced lysis when compared to wild-type Tae1. (C) Schematic of the multimeric glycan chains used as the substrate in NMR experiments. (D) CSPs calculated from NMR spectra of Tae1 with or without multimeric PG chains mapped onto the Tae1 surface structure. Numerous residues on both sides of the Tae1 display CSPs consistent with direct substrate interactions. (E) 1 H-15 N Best-Trosy NMR spectra from Tae1 alone (black) and Tae1 incubated with multimeric peptidoglycan chains (red) for six residues from A that show CSPs. Residues distal from the Tae1 active site show shifts indicative of interactions with PG. Numerous residues display CSPs consistent with direct substrate interactions. (F) HPLC traces showing degradation of peptidoglycan in the presence of wild type (black, WT) or mutant (red) Tae1 in vitro. Arrows indicate peaks containing the peptide stem that can be cleaved by Tae1. Mutant Tae1 (S144A and G48S) cleaves peptidoglycan less well than wild-type Tae1 (29.1 +/-3.2% and 36.4 +/-4.2% less PG., respectively) See also supplemental figure 2, 3, 4.  Figure 3B). We found that all variants were 239 expressed; five substitutions did not exhibit major 240 visible decreases in protein levels (G42S, S144A, 241 S147A, G48S, G56S). Our results led us to 242 hypothesize that these five surface substitutions may play direct roles in Tae1-PG interactions.

244
We hypothesized that an extended surface of Tae1 may be required for substrate binding and 245 hydrolysis in vivo. Given that Tae1 likely engages with larger and even sometimes insoluble fragments 246 of PG within the intact cell wall, we conducted a series of in vitro biochemical assays of LOF clus variants 247 using either intact PG sacculi or fragments that encompass more than the minimal peptide region. To

257
To map enzyme residues that bind cell wall substrate, we compared NMR spectra of Tae1  interactions 19,20 . Mapping these shifts onto the Tae1 263 structure, we observed CSPs for residues located within 264 the catalytic core as well as for a variety of both buried 265 and exposed residues outside of the core ( Figure 3D).

266
While CSPs for buried residues could be due to  Figure S2. Lysis assay growth curves. Cell growth as measured by OD600 for Tae1 WT , negative controls (Empty, C30A), and the nine distal residues on Tae1 in Figure 3B. Expressing Tae1 proteins containing the nine distal residue substitutions decreases cell lysis, suggesting these are loss-of-function residues. To determine if these functional distal residues that bind PG are also required for hydrolysis, we 279 focused our studies on two highly expressed variants, Tae1 S144A and Tae1 G48S , for which we expressed 280 recombinant forms and purified. Size exclusion chromatography analysis of these variants showed 281 similar elution volumes and apparent hydrodynamic radii compared to Tae1 WT . We then conducted 282 degradative assays with PG sacculi purified from E. coli. Tae enzymes were incubated with PG sacculi, 283 and the resulting fragments were quantitatively assessed using high-performance liquid 284 chromatography (HPLC, Figure 3F). Tae1 S144A and Tae1 G48S cleaved 29.1 +/-3.2% and 36.4 +/-4.2% 285 less PG, respectively, than observed for the wild-type enzyme. Despite being relatively far from the 286 active site, these amino acid substitutions decreased Tae1 activity. Together, these data reveal that 287 numerous sites distributed around the Tae1 surface physically interact with PG, and that these distal

292
The functional landscape of Tae1 is sensitive to PG differences

294
Our results point to a model in which Tae1  The majority of the GOF substitutions in D-Met are condition specific. (F) Lysis assays with wild-type Tae1, empty vector and catalytically inactive Tae1 C30A as negative controls, and 3 gain-of-function Tae1 variants (A116T, D134N, S147I). Data are means +/-standard deviation, n = 3, * p < 0.05. Induced with 0.0025% arabinose. Lysis experiment was repeated 3 independent times, each time with at least.3 technical replicates. Western blots below measure Tae1 protein expression with RNAP as a control. All three gain-offunctions from DMS show increased lysis phenotype in individual lysis assays. (G) Lysis assays with the same Tae1 variants as in F but in M9 media. Western blots below measure Tae1 protein expression with RNAP as a control. The three D-Met GOF substitutions are condition specific. See also supplemental figure 5. would alter Tae1-PG interactions. We hypothesized that such alterations could be captured by 298 comparative DMS analysis across different screening conditions. To address this hypothesis, we 299 conducted a second DMS screen in E. coli grown in the presence of D-methionine (D-Met), a condition 300 known for inducing major featural changes in cell wall composition and organization ( Figure 4A).

301
Specifically, cell walls of E. coli grown in the presence of D-Met (E. coli DMet ) have generally reduced PG 302 density and fewer modified PG crosslinks relative to cell walls of E. coli grown in the absence of D-

303
Met 24,25 . We verified these trends by HPLC analysis, and we confirmed that Tae1 maintained its ability 304 to cleave this distinct cell wall type in vitro (Supplementary Figure 4).

306
We performed the same suite of DMS analyses

328
The E. coli DMet DMS screen also yielded a larger and distinct set of top-performing GOF clus variants with 329 amino acid substitutions that localized to both the catalytic groove and opposing faces of Tae1. When  particularly struck by one variant, Tae1 C110S , which is modified at a partially buried residue sitting 349 directly behind the catalytic Cys30 site (Figure 5A). The Cys110Ser substitution was the top-scoring 350 GOF variant in the D-Met screen and ranked 11 th amongst GOF variants in the M9 screen. Using the E.

351
coli lysis assay, we first confirmed that Tae1 C110S indeed lysed cells at a faster rate than Tae1 WT in cells 352 grown in both D-Met and M9 minimal media (Figure 5B). To rule out indirect effects on toxicity caused 353 by increased protein levels, we assessed Tae1 C110S expression by Western blot analysis and did not 354 observe any increase relative to 355 Tae1 WT (Figure 5B).

357
The observation that Tae1 activity 358 could generally be tuned for greater 359 toxicity led us to ask why tae1 from P.

389
We therefore investigated whether Ser110 naturally occurs in any Tae1 representatives outside of P.

434
aeruginosa may be disfavored to 435 preserve immunity to the toxin. We 436 generated a tree for the Tai1 protein family, similar to our tree for the Tae1 protein family. Consistent 437 with this possibility, the tree for Tai1 has a similar branching pattern that is coincident with the residue 438 variation at this position (Supplemental Figure 6).

440
We tested our prediction through a series of Isothermal titration calorimetry determination of binding constants between wild-type Tae1 and Tai1 (left) and Tae1 C110S and Tai1 (right). Tae1 C110S binds poorly to Tai1. (D) Schematic depicting Tae1 and Tai1 interactions in wild-type P. aeruginosa and Tae1 C110S P. aeruginosa. When wild-type Tae1 is injected into a neighboring cell of the same species, Tai1 binds to it, conferring immunity. Tai1 cannot bind Tae1 C110S , thus the mutant Tae1 can cause cell lysis. (E) Fitness assays of P. aeruginosa expressing wild-type Tae1 or Tae1 C110S from the endogenous locus. P. aeruginosa lacking a Type 6 Secretion System (-T6SS) or expressing catalytically inactive Tae1 (C30A) were used as controls. Data are means +/-standard deviation, n = 3, * p < 0.05. Competition experiment was repeated 3 independent times, each time with at least.3 technical replicates. Cells expressing Tae1 C110S have decreased fitness when compared with wild-type Tae1. See also supplementary figures 6, 7. reduction may preclude the ability of Tai1 to effectively bind its toxin partner in vivo. To test whether 451 Tae1 C110S evades Tai1-dependent immunity in vivo, we constructed a strain of P. aeruginosa harboring 452 this mutation (PA C110S ) with a wild-type strain (PA WT ). We reasoned that if Tae1 C110S escapes Tai1-453 binding and therefore immune protection, we would observe a decrease in PA C110S fitness compared to 454 a wild-type strain (PA WT ) specifically when grown on solid media which induces T6S (Figure 6D).

455
Indeed, for PA C110S we observed substantially 456 reduced growth compared to PA WT on solid media 457 ( Figure 6E) but not in liquid media, where T6S is 458 not induced (Supplemental Figure 7). This

481
Here, we show that sequence-based differences in tae homologs shape Tae proteins and their complex 482 substrate, the PG that constitutes the bacterial cell wall. Our studies revealed that Tae1 engages PG 483 through a circumferential interface that is more extensive than most proteases or other enzymes. This  Tai1s corresponding to Tae1 non-S110 proteins). The tree contains the same species found in the Tae1 tree, except for Mitsuaria (unclassified) and Pseudomonas (multiple). Coloring of the species also matches that in the Tae1 tree. The sequences in the tree were identified using a published heuristic approach that specifically searches for T6S effector toxins. ML tree was inferred using PhyML v20160115 ran with model JTT and

520
Another possibility is that the large functional surface comprises several different PG-binding domains 521 that streamline substrate recognition or underlie enzyme processivity. This has been suggested for a 522 cell wall digesting enzyme, Lysostaphin from Staphylococcus, which exhibits two large PG-binding 523 surfaces on opposite sides of the enzyme 23 . Multi-step binding would support the "smart autolysins" 524 model postulated by Arthur Koch 30 . This model hints at the ability of hydrolase enzymes to recognize 525 "stretched" areas of PG. The resulting advantage for hydrolytic cell wall enzymes is that this type of 526 recognition decreases substrate search dimensionality from random 3-D diffusion to facilitated diffusion 527 by searching along the PG sacculus one plane at a time 31 . This may be particularly important for rapid, 528 effective recognition of substrates within a large cage-like 3D target like the cell wall, which is key for 529 potent, fast-acting toxins. This mechanism is also commonly adopted by DNA-binding proteins, which 530 dock nonspecifically to DNA before searching for specific cut sites 32,33 . Multi-step binding may also lead 531 to processive activity along PG by ensuring that Tae enzymes remain tethered to the cell wall substrate 532 after each hydrolysis event, which would allow for "stepwise chewing." This would be yet another 533 mechanism to increase the odds of localized, lytic activity.

535
In total, our combined use of biophysical and high throughput genetic approaches uncovered novel

575
The authors declare no competing interests.

579
Preparation of long chains of PG 580 PG isolation and HPLC analysis were conducted according to an established protocol 37 with the 581 following modifications. Briefly, E. coli BW25113 cells were grown in 200ml LB media to mid-log phase.

582
The cells were harvested by centrifugation at 5000 x g for 15min, after which they were suspended in 583 300µl of fresh LB media and added to 600µl 6% SDS solution in a 2ml Eppendorf tube. The cells were   The pBAD vector was used to clone Tae1 variants of interest 43 . The Tae1 wild-type was initially 627 amplified from a previously published pET22::Tae1 construct 10 , together with the pelB signal sequence.

628
All variants were constructed based on this pelB_pBAD::Tae1 template. See Table 1 for all the primers 629 used to clone Tae1 and the different variants. SOE-PCR was used to introduce the individual    were harvested by centrifugation at 5000 x g for 15min, after which they were suspended in 300µl fresh 685 LB and added to 600µl of 6% SDS solution in a 2ml Eppendorf tube. The cells were boiled for 3hrs in a 686 heat block and then maintained in the SDS solution at 4°C until needed. Before any treatments, the boiled sacculi were washed 5 times with MQ water to remove the SDS. Each wash consisted of 15min 688 centrifugation at 21000 x g followed by decanting of the supernatant and resuspending in 1ml MQ water 689 by vortexing. After the final wash, the sacculi were suspended in Pronase E digestion buffer (10mM 690 Tris-HCl pH 7.2 and 0.06% NaCl) and were treated for 2hrs at 60°C with Pronase E (100µg/ml final 691 concentration, pre-treated for 2hrs at 60C in the digestion buffer; supplier: VWR). The sacculi were 692 incubated at 100°C for 10min to inactivate the Pronase E. Enzymatic treatments with Tae1 variant 693 enzymes were conducted at this point using 20mM HEPES pH7.0, 100mM NaCl buffer. Tae1 enzyme 694 was added to 1µM final concentration and the reactions were incubated at room temperature for 10min, 695 followed by 10min at 100°C to inactivate the enzyme. The sacculi were washed 3 times as described 696 above. Finally the sacculi were solubilized using 25µg/ml final concentration of mutanolysin (Sigma-697 Aldrich) in 10mM Tris-HCl pH 7.2 and 0.06% NaCl after overnight digestion in a 37°C shaker. To 698 prepare PG fragments for HPLC analysis, the mutanolysin treatment was incubated at 100°C for 10min, 699 followed by centrifugation to remove any debris, alkalinization with 0.5M borate buffer and reduction 700 with sodium borohydride for 1hr at room temperature. The reduced PG fragments were acidified to pH 701 3-4 with 50% o-phosphoric acid and filtered (PVDF syringe filters, 0.22µm, 0.4mm -Tisch Scientific) 702 before the HPLC injection. The HPLC instrument, method, and buffers were the same as described 703 previously 45 .

705
Protein purification 706 All Tae proteins, and Tai1 protein, used in this study were purified according to an established 707 protocol 10 using pET29b+ vector and 3hrs induction in a 37°C shaker with 1mM IPTG. Buffers for metal 708 affinity chromatography were 20mM HEPES pH7, 100mM NaCl, 20mM imidazole and 20mM HEPES 709 pH7, 100mM NaCl, 400mM imidazole. Buffer for size exclusion chromatography (SEC) was 20mM 710 HEPES pH7, 100mM NaCl. The SEC was performed using an isocratic gradient on an AKTA pure 711 instrument with a HiPrep 16/60 Sephacryl S-200 HR column. For isotopically labeled Tae1 C30A , the 712 same plasmid constructs were used, but the expression was conducted in M9 minimal medium 713 prepared with 15 N-ammonium chloride (5g per liter media) (M9 minimal medium prepared according to 714 Cold Spring Harbor Protocols). The cells were grown and induced as described above. The same 715 purification procedure was used as described above.

717
Phylogenetic trees 718 Initial candidates of the Tae1 and Tai1 families as identified in Russell et al. 7 were aligned and used as 719 a seed in a PSI-BLAST search. Significant results were downloaded and aligned using MAFFT.

720
Candidates containing the critical Cys-His dyad were kept, and other candidates discarded. Final

726
P. aeruginosa strain construction 727 P. aeruginosa strains used in this study were derived from the sequenced strain PAO1 46 . E. coli strain 728 SM10 was used for conjugal transfer of pEXg2 plasmids for homologous recombination as described 729 previously 5 . All deletions were in-frame and were generated by exchange with deletion alleles 730 constructed by SOE PCR, followed by restriction enzyme cloning into pEXg2 vector. See Table 1 for 731 the primers used to delete genes pppA and retS, and to introduce the C110S mutation into tae1.

733 734
Contact-dependent fitness assays 735 Fitness assays were performed with overnight P. aeruginosa cultures. The assays were performed on 736 solid media (LB without added NaCl containing 3% agar) using nitrocellulose membranes (Amersham