WhyD tailors surface polymers to prevent bacteriolysis and direct cell elongation in 1 Streptococcus pneumoniae 2

called penicillin-induced bacteriolysis. We recently that penicillin treatment dramatic shift in biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content 57 of the wall to areas adjacent to active PG synthesis. Our results support a model in which the tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA. pJFK004. The plasmid was confirmed by sequencing.

misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this 48 phenomenon, little is known about the factors that control autolysins and how penicillins 49 subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the 50 major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that 51 penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell 52 wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked 53 lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to 54 its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD 55 (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA 56 misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content 57 of the wall to areas adjacent to active PG synthesis. Our results support a model in which the 58 WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell 59 elongation in addition to preventing autolysis by LytA. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint INTRODUCTION 7 exhibited by wild-type cells whereas its addition to a DlytA DwhyD double mutant resulted in 166 lysis almost immediately after exposure ( Figure 1D). Thus, whyD has the properties expected 167 for a gene encoding a factor that restrains LytA activity at the cell surface. samples by alcian blue-silver staining of polymers released from purified cell wall sacculi. As a 176 control, we analyzed LTA and WTA levels in mutants inactivated for the LTA synthase TacL. 177 As expected, LTAs were undetectable in these cells and WTA levels dramatically increased 178 (Figure 2). In mutants defective for WhyD, a similarly dramatic increase in WTAs was 179 observed. However, in this case, LTA levels were unaffected (Figure 2). Expression of whyD 180 from an ectopic locus restored wild-type levels of WTAs, indicating that the phenotype was due 181 to the absence of the WhyD protein rather than an effect of the deletion on the expression of a 182 nearby gene (Figure 2). 183 184 We previously showed that in cells treated with penicillin or those grown for an extended 185 period in stationary phase, TacL is degraded, leading to a decrease in LTAs and an increase in  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint Importantly, rLytA-Alexa triggered growth-phase dependent autolysis at rates indistinguishable 286 from unlabeled rLytA (Figure 7 -figure supplement 1B) indicating that labeling did not affect 287 LytA activity. As expected, rLytA * -Alexa did not induce lysis and was used for all imaging 288 experiments to avoid complications of PG cleavage (Figure 7 -figure supplement 1B). Since 289 WTA and LTA are identical polymers with the same PCho moieties, we next investigated 290 whether rLytA*-Alexa labels both polymers or exclusively labels WTAs. To do so, we used the 291 Pzn-whyD strain that over-expresses WhyD and reduces WTA levels (Figure 7 -figure  292 supplement 2). Surface labeling by rLytA*-Alexa was readily detectable on wild-type Sp and 293 cells harboring Pzn-whyD without exogenous Zn 2+ . However, rLytA*-Alexa was undetectable 294 when WhyD was over-expressed (+Zn) (Figure 7 -figure supplement 2). Furthermore, we 295 confirmed that rLytA*-Alexa exclusively labels WTAs from Sp (Figure 7 -figure supplement 296 1C) and purified Sp sacculi, provided that WTAs had not been removed (Figure 7 -figure  297 supplement 3). Altogether, these results indicate that rLytA*-Alexa specifically binds WTAs 298 when added to intact cells. 299 300 Having established that rLytA*-Alexa labeling can be used as a proxy for the in vivo 301 localization of WTAs, we monitored the subcellular positions of WTAs relative to newly 302 synthesized PG in exponentially growing cells. To follow nascent PG and recently synthesized 303 wall material that had moved away from midcell during cell elongation, we first pulse-labeled 304 cells with HADA and 5 minutes later added the compatibly labeled FDAA sBADA. We then 305 washed the cells with medium containing 1% choline to remove native choline binding proteins 306 from the WTAs to ensure that the choline moieties were fully accessible to rLytA*-Alexa. Cells 307 were then incubated with rLytA*-Alexa for 30 seconds, washed to remove unbound probe and 308 imaged ( Figure 7A). Elongating cells displayed a weak rLytA*-Alexa signal at midcell that co-309 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint 13 localized with sBADA. The sBADA signal at midcell was flanked by two prominent fluorescent 310 bands of rLytA*-Alexa that co-localized with HADA-labeled peripheral PG (Figure 7B and 7C). 311

Consistent with WhyD hydrolyzing WTAs at midcell, co-localization analysis of GFP-WhyD and 312
WTAs showed an anti-correlation between GFP-WhyD enrichment at midcell and WTA 313 localization (Figure 7 -figure supplement 4). In   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022.

WTA turnover and localization in Sp cells 347
In our previous study, we found that inactivation of the LTA synthase TacL resulted in the that LTAs predominate in the envelope in exponentially growing cells due to TacL 352 outcompeting the WTA ligases (LCP proteins) for their common substrate. However, the 353 discovery that WhyD inactivation also causes a dramatic increase in WTA accumulation in 354 exponentially growing cells without affecting LTA accumulation (Figure 2) indicates that 355 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint instead of substrate competition, it is likely that the continuous degradation of WTAs maintains 356 their low levels in the cell wall of actively growing cells. 357

358
In addition to reducing the total WTA content attached to the PG matrix (Figures 2 and 3), the 359 WTA cleavage activity of WhyD also results in the localized accumulation of these polymers at 360 sites adjacent to areas of active wall growth (Figure 7). Determining how this localization is 361 achieved will require further investigation, but this phenomenon is likely to arise from the 362 enrichment of WhyD at midcell where most (Figure 6) Schaefer et al., 2017). Therefore, the balance between WTA addition and 367 cleavage at midcell could explain the observed pattern of WTA localization. In this scenario, 368 the enrichment of WhyD in the septal region is likely to result in the removal of most but not all 369 WTAs added to nascent PG. Zonal PG synthesis would then be expected to push the WTA-370 decorated PG material away from the cell center (Figure 8). If processing of WTAs from this 371 older material were less efficient due to the lower concentration of WhyD outside midcell 372 and/or the reduced accessibility of WTAs attached to more mature PG, the expected result 373 would be a gradient of WTA accumulation centered at positions adjacent to midcell, as 374 observed (Figure 8). Re-localization of WhyD to the future daughter cell septa to prepare for 375 the next cell cycle could then be responsible for the midcell accumulation of WTAs displayed 376 by cells in the final stages of division (Figure 8). 377 378

Possible role of WTAs in directing the activity of space-making PG hydrolases 379
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022.

WTA cleavage activity of WhyD 401
WhyD has seven predicted N-terminal transmembrane segments in addition to a C-terminal 402 GlpQ-like domain (WhyD CT ; Figure 3A). GlpQ-containing proteins from other gram-positive 403 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022.  (Figures 2 and 3). However, because WTAs and LTAs in Sp cells are built 413 from a common undecaprenyl-linked precursor and have an identical polymeric structure 414 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint A mechanism for controlling the WTA content of the wall and its localization by cleaving a 427 significant portion of the polymers that are made seems wasteful and inefficient. However, 428 such a scenario is not that different from the synthesis of the cell wall itself, which involves the 429 turnover of up to 50% of the PG layer per generation (Borisova et   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint that inhibition of WhyD during exponential phase has the potential to trigger cell lysis (Figure 2

460
Cells were grown in Todd Hewitt (Beckton Dickinson) medium supplemented with 0.5% yeast 461 extract (THY) at 37 °C in an atmosphere containing 5% CO2 or on pre-poured tryptic soy agar 462 5% sheep blood plates (TSAII 5% sheep blood, Beckton Dickinson) with a 5 ml overlay of 1% 463 nutrient broth (NB) agar containing the required additives. When required, TSA agar plates 464 containing 5% defribrinated sheep blood (Northeast laboratory) were used. E. coli strains were 465 grown on Luria-Bertani (LB) broth or LB agar. Wild-type Bacillus subtilis strain PY79 466 (Youngman et al., 1983) was grown in LB broth or LB agar as described previously (Fenton et  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. Sp. Approximately 302,000 (wt) and 305,000 (ΔlytA) transformants were recovered for each 493 library. Genomic DNA was then isolated and digested with MmeI, followed by adapter ligation. 494 Transposon-chromosome junctions were amplified and sequenced on the Illumina HiSEq 495 2500 platform using TruSeq Small RNA reagents (Tufts University Core Facility Genomics). 496 Reads were de-multiplexed, trimmed, and transposon insertion sites mapped onto the D39 497 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint genome. After normalization, a Mann Whitney U test was used to identify genomic regions with 498 significant differences in transposon insertions. Transposon insertion profiles were visualized 499 using the Artemis genome browser (v10.2). 500 501

Isolation and analysis of pneumococcal LTAs. 502
Sp strains were grown in THY medium with required additives at 37 °C in 5% CO2 to the 503 indicated growth phase and normalized to an OD600 of 0.5. 20 ml of the normalized culture 504 were collected by centrifugation at 5000 xg for 5 min and the cell pellet was washed twice with 505 2 ml SMM (0.5 M sucrose, 20 mM maleic acid pH 6.5, 20 MgCl2) and then re-suspended in 2 506 ml SMM. Protoplasts were generated by addition of lysozyme (1mg/ml final concentration) and 507 100 units mutanolysin (Sigma) and incubation at 37 °C for 30 minutes. Complete protoplasting 508 was monitored by light microscopy. Protoplasts were pelleted by centrifugation at 5000 xg for 5 509 min and resuspended in 2 ml cold hypotonic buffer (20 mM HEPES (Na + ) pH 8.0, 100 mM 510 NaCl, 1 mM dithiothreitol (DTT), 1 mM MgCl2, 1 mM CaCl2, 2X complete protease inhibitors 511 (Roche), 6 µg/ml RNAse A, 6 µg/ml DNAse. Unbroken protoplasts were removed by 512 centrifugation at 20,000 xg for 10 min, and the lysate was then subjected to ultracentrifugation 513 at 100,000 xg for 1 hr at 4 °C. Membrane pellets were resuspended in 1ml Tris-tricine sample 514 buffer (200 mM Tris-HCl pH 6.8, 40% glycerol, 2% SDS, 0.04% Coomassie Blue G-250), 515 boiled for 10min, and analyzed by Tris-tricine PAGE followed by immunoblotting. The results 516 in figures analyzing LTA levels are representative of experiments that were performed on at 517 least two independently collected samples. 518 519 520 521 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint

Isolation and analysis of pneumococcal WTAs. 522
Sp strains were grown and harvested as above. The pellets were resuspended in 2 ml of buffer 523 1 (50 mM 2-(N-morpholino ethanesulfonic acid (MES)) pH 6.5) and centrifuged at 7000 xg for 5 524 min. The resulting pellets were resuspended in 2 ml buffer 2 (50 mM MES pH 6.5, 4% (w/v) 525 SDS) and incubated in boiling water for 1 hr. The sample was then centrifuged at 7,000 xg for 526 5 min and the pellet was washed with 2 ml buffer 2. The sample was transferred into a clean 527 microfuge tube and centrifuged at 16,000 xg for 5 min. The pellet was then washed with 2 ml 528 buffer 2, followed by successive washes with 2 ml buffer 3 (50 mM MES pH 6.5, 2% (w/v) 529 NaCl) and 2 ml buffer 1. The samples were then centrifuged at 16,000 x g for 5 min, 530 resuspended in 2 ml of buffer 4 (20 mM Tris-HCl pH 8.0, 0.5% (w/v) SDS) supplemented with 531 2 µl proteinase K (20 mg/ml), and incubated at 50 °C for 4 hr with shaking (1000 rpm). The 532 pellet was then collected by centrifugation and washed with 2 ml buffer 3 followed by 3 washes 533 with distilled water. The pellet was collected by centrifugation and subjected to alkaline 534 hydrolysis in 0.5 ml of 0.1 N NaOH and incubation at 25 °C for 16 h with shaking (1000 rpm). 535 The samples were then pelleted by centrifugation and the supernatants containing the 536 extractable WTA were collected and resuspended in 0.5 ml native sample buffer (62.5 mM 537 Tris-HCl pH 6.8, 40% glycerol, 0.01% bromophenol blue). Samples were analyzed by native 538 containing the pET21amp-lytA or pET21amp-lytA* expression vectors. Cells were grown in LB 545 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint supplemented with 100 µg/mL ampicillin at 37 °C and expression was induced at an OD600 of 546 0.5 with 1 mM IPTG for 2 h at 37 °C. Cells were collected by centrifugation and stored 547 overnight at -20 °C. The cell pellets were resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 548 500 mM NaCl, 200 μg/ml DNase, and 2X complete protease inhibitors (Roche)) and lysed by 549 two passages through a cell disruptor (Constant systems Ltd.) at 25,000 psi. Unbroken cells 550 were discarded by centrifugation. The supernatant was then passed over a DEAE cellulose 551 column (Sigma). After washing with 20 column volumes of wash buffer (20mM NaPO4 pH 7, The C-terminal domain of WhyD (WhyD CT ) was expressed in E. coli BL21(DE3) ΔfhuA using 560 the PT7-His6-SUMO-whyd CT expression vector (pTD68-whyD). Cells were grown in LB 561 supplemented with 100 µg/mL ampicillin at 37 °C to an OD600 of 0.5. Cultures were allowed to 562 equilibrate at room temperature for 30 min and then transferred to 30 °C. his6-sumo-whyD CT 563 expression was induced with 0.5 mM IPTG for 3 hr. Cells were collected by centrifugation, 564 resuspended in 50 ml Buffer A (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM Imidazole, and 565 2X complete protease inhibitor tablets (Roche)), and stored at −80 °C. The cell suspension 566 was thawed on ice and lysed by two passes through a cell disruptor at 25,000 psi. The lysate 567 was clarified by ultracentrifugation at 35 Krpm for 30 min at 4 °C. The supernatant was added 568 to 1 mL Ni 2+ -NTA resin (Qiagen) and incubated for 1 hr at 4 °C. The suspension was loaded 569 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint 24 into a 10 ml column (BioRad), washed twice with 4 ml Buffer A, and eluted with 2.5 ml Buffer B 570 (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 300 mM Imidazole). 10 µL of purified His6-Ulp1 (1.25 571 mg/ml) was added to the eluate, and the mixture was dialyzed into 100mM Tris-HCl pH 8, 100 572 mM NaCl, 10% glycerol) overnight at 4 °C. The next morning 10 µL more His6-Ulp1 was added 573 to the dialysate and incubated for 1 hr at 30 °C. The dialysate was mixed with 1 mL of Ni 2+ -574 NTA resin for 1 hr at 4 °C and then loaded onto a column and the WhyD CT -containing flow-575 through was collected, dialyzed into 100mM Tris-HCl pH 8, 100 mM NaCl, 1mM CaCl2, 10% 576 glycerol overnight at 4 °C and stored at −80°C. The purified protein was used for in vitro 577 assays and to generate rabbit polyclonal antibodies (Covance). 578

579
In vitro WTA and LTA release assays using WhyD CT . 580 For the WTA release assays, the activity of WhyD CT was assayed using purified sacculi (from 581 ΔlytAΔwhyD cells to obtain larger quantities of WTAs attached to sacculi) prepared as 582 described above without the alkaline hydrolysis step to retain WTA. The release assays were 583 conducted with 0.1 mg sacculi and 10 µg/ml WhyD CT , 10 µg/ml WhyD CT + 1 mM EDTA, or no 584 WhyD CT in 1 ml reaction buffer (0.1 M Tris-HCl pH 8, 1 mm CaCl2) incubated at room 585 temperature with gentle shaking. Released WTAs were collected by centrifugation. To recover 586 WTAs that were not released, the sacculi pellets were then treated with 0.1 M NaOH overnight 587 at room temperature with gentle shaking. Akaline-released WTAs were collected by 588 centrifugation and analyzed alongside the WhyD-released WTAs by SDS-PAGE followed by 589 alcian blue-silver staining. 590 591 LTA assays were performed in reaction buffer with 0.1 mg homogenized membrane extracts 592 (from ΔlytAΔwhyD cells) prepared as described above. 0.1 mg of the homogenized 593 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint membranes were incubated with 10 µg/ml WhyD CT , 10 µg/ml WhyD CT + 1 mM EDTA, or no 594 WhyD CT in 1 ml reaction buffer (0.1 M Tris-HCl pH 8, 1 mm CaCl2), and incubated at room 595 temperature with gentle shaking. After incubation, the reactions were quenched with 1mM 596 EDTA. Released and membrane-associated LTAs were then analyzed by 16% Tris-tricine 597 SDS-PAGE and probed with a monoclonal antibody specific for phosphocholine. These 598 assays are representative of experiments that were performed on at least two independently 599 collected samples. Cells were grown to mid exponential phase, labeled with sBADA for 5 min, and sacculi with or 611 without WTAs were prepared as described above. Sacculi from the equivalent of 1ml of cells at 612 OD600 of 0.5 were labeled with 1 µg/ml rLytA*-Alexa as described above the samples were 613 imaged by fluorescence microscopy. These assays are representative of experiments that 614 were performed on at least three independently collected samples. 615 616 Fluorescence microscopy. 617 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint Cells were harvested and concentrated by centrifugation at 6800 x g for 1.5 min, re-suspended 618 in 1/10th volume growth medium, and then immobilized on 2% (wt/vol) agarose pads 619 containing 1XPBS. Fluorescence microscopy was performed on a Nikon Ti inverted 620 microscope equipped with a Plan Apo 100x/1.4 Oil Ph3 DM phase contrast objective, an Andor 621 Zyla 4.2 Plus sCMOS camera, and Lumencore SpectraX LED Illumination. Images were 622 acquired using Nikon Elements 4.3 acquisition software. HADA was visualized using a Chroma 623 ET filter cube for DAPI (49000); sBADA and GFP were visualized using a Chroma ET filter 624 cube for GFP (49002); LytA*-AlexaFluor594 was visualized using a Chroma ET filter cube for 625 mCherry (49008). Image processing was performed using Metamorph software (version (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint (https://www.zeiss.com/microscopy/us/products/super-resolution/elyra-7.html). Laser powers 642 were set up to achieve ~3000 gray values in the 16-bit raw image per channel.

S. pneumoniae deletion strains 662
All Sp deletion strains were generated using PCR fragments as described previously and are 663 listed in Table S1. Briefly, two products representing the regions (~1 kb each) flanking the 664 target gene were amplified, and an antibiotic resistance cassette ligated between them using 665 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint Gibson assembly. Assembled PCR products were transformed directly into Sp as described 666 above. In all cases, deletion primers were given the name: "gene name"_5FLANK_F/R for 5′ 667 regions and "gene name"_3FLANK_F/R for 3′ regions. Antibiotic markers were amplified from 668 ΔbgaA::antibiotic cassette (bgaA gene disrupted with an antibiotic cassette) strains using the 669 AB_Marker_F/R primers. A full list of primer sequences can be found in the Table S3. 670 Extracted gDNA from deletion strains was confirmed by PCR using the AntibioticMarker_R 671 primer in conjunction with a primer binding ∼200 bp 5′ of the disrupted gene; these primers 672 were given the name: "gene name"_Seq_F. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint

29
The whyD ORF, with its native RBS, was amplified using primers whyD_F_optRBS_XhoI and 689 whyD_R_BamHI. The primers introduced XhoI and BamHI sites used for cloning into pLEM023 690 cut with the same enzymes, resulting in plasmid pJFK003. The plasmid was sequenced and 691 used to transform strain D39 Dcps Dbga::kan. Integration into the bga locus was confirmed by preparations treated with WhyD CT to assess its ability to release LTAs. Membranes from ΔlytA 833 ΔwhyD cells were treated with 10 µg/ml WhyD CT , 10 µg/ml WhyD CT + 1 mM EDTA, or no 834 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. WTAs. Cells were then washed twice with 1X PBS and analyzed by fluorescence microscopy. 976 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint 41 Cells were imaged using an Elyra 7 system with SIM 2 as described in Materials and Methods. 977 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint midcell where they recruit PG hydrolases that promote cell separation (yellow). At this stage, 1001 WhyD might not be localized at midcell or its activity could be inhibited. Upon entry into 1002 stationary phase or exposure to cell wall targeting antibiotics (autolysis), WhyD is unable to 1003 keep pace with the increase in WTA synthesis and/or is actively inhibited, leading to an . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 9, 2022. ; https://doi.org/10.1101/2022.01.07.475315 doi: bioRxiv preprint  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m

55
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m      Alcian blue-silver stained gel of WTAs released from purified sacculi (top) and those that remain associated with the PG (bottom) after incubation with 10 µg/ml WhyD CT , 10 µg/ml WhyD CT + 1 mM EDTA, or no WhyD CT . The reactions were incubated overnight at room temperature and then quenched with 1 mM EDTA. To release WTAs that remained associated with the sacculi, the reactions were further treated with 0.1 M NaOH overnight at room temperature. The alkaline-hydrolyzed WTAs were then collected from the supernatant. (C) Immunoblot analysis of membrane preparations treated with WhyD CT to assess its ability to release LTAs. Membranes from ΔlytA ΔwhyD cells were treated with 10 µg/ml WhyD CT , 10 µg/ml WhyD CT + 1 mM EDTA, or no WhyD CT . The reactions were incubated overnight at room temperature. The samples were then resolved by 16% Tris-tricine SDS-PAGE, transferred to nitrocellulose and probed with an anti-phophsocholine monoclonal antibody.    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m

62
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m

63
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  Plates were incubated at 37°C in 5% CO 2 and imaged. (B) Immunoblot analysis of WhyD and GFP-WhyD. Samples from Figure 3A were collected and normalized to an OD 600 of 0.5 and resolved by SDS-PAGE followed by anti-WhyD or anti-GFP immunoblotting. A region of the nitrocellulose membrane used for immunoblot analysis was stained with Ponceau S to control for loading (LC). A degradation product of the GFP-WhyD fusion protein detected with the anti-WhyD antisera is indicated with an asterisk. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m   Figure 1D. M, Molecular weight markers. (B) rLytA-Alexa is functional. Growth curves of the indicated strains before and after the addition of 1 mg/ml rLytA, rLytA-Alexa, or rLytA*-Alexa at an OD 600 of~0.2. The ΔlytA strain incubated with rLytA or rLytA-Alexa lysed in stationary phase in a manner similar to LytA + cells. (C) LytA*-Alexa specifically labels S. pneumoniae cells. Wild-type Bacillus subtilis and S. pneumoniae cells were incubated with rLytA*-Alexa as described in the Materials and Methods. Cells were labeled with sBADA for 5 min prior to imaging. Scale bar, 3 μm.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  LytA that were labeled with rLytA*-Alexa. The ∆lytA mutant was grown in THY to mid-exponential phase and labeled with sBADA for 5m prior to harvest. Sacculi were purified with their WTAs intact (+WTAs) or with their WTAs removed (-WTAs) as described in the Materials and Methods. Sacculi from the equivalent of 1 ml of cells at OD 600 of 0.5 were labeled with rLytA*-Alexa as described in the Materials and Methods and imaged on 2% agarose pads.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  Representative phase-contrast and fluorescent images of cells expressing GFP-WhyD and labeled with LytA*-Alexa (WTA) and HADA (PG). The ∆lytA mutant was grown in THY medium to mid-exponential phase and labeled with sBADA for 5 min. A sample was then collected normalized to an OD600 of 0.5, washed with fresh medium containing 1% choline and then incubated with 1 µg/ml rLytA*-Alexa for 30 sec with gentle shaking to label WTAs. Cells were then washed twice with 1X PBS and analyzed by fluorescence microscopy. Yellow carets, mid-cell localization of nascent PG and GFP-WhyD; pink carets, LytA*-Alexa (WTA) enrichment. sBADA (nascent PG). The ∆lytA mutant was grown in THY medium to mid-exponential phase, labeled with sBADA for 5 min. The sample was collected and normalized to an OD 600 of 0.5 before incubation with rLytA*-Alexa to label WTAs as described in the Material and Methods. The cells were then imaged on 2% agarose pads. (B) Representative deconvolved image of the ∆lytA mutant labeled with rLytA*-Alexa and sBADA as described in (A). Z-stack images were taken every 100 nm from 1.5 µm above and below the focused image plane. Deconvolution was performed using Hyugens Widefield Deconvolution Software. (C) Representative Structured illumination microscopy image of a ∆lytA mutant grown in THY medium to mid-exponential phase, labeled with HADA for 5 min, washed with fresh THY, and then labeled with sBADA for 5 min. The sample was then collected, normalized to an OD600 of 0.5, washed with fresh medium containing 1% choline and incubated with 1 µg/ml recombinant LytA(H26A) coupled to Alexa Fluor 594 (rLytA*-Alexa) for 30 sec with gentle shaking to label WTAs. Cells were then washed twice with 1X PBS and analyzed by fluorescence microscopy. Cells were imaged using an Elyra 7 system with SIM 2 as described in Materials and Methods. Carets indicate nascent PG (green), recently synthesized PG (white), and the final stage of cell separation (pink)

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  Representative phase-contrast and fluorescence images of wild-type cells labeled with LytA*-Alexa (WTAs) and HADA (nascent PG) and sBADA (recently synthesized PG). The ∆lytA mutant was grown in THY medium to mid-exponential phase, labeled with HADA for 5 min, washed with fresh THY medium, and then labeled with sBADA for 5 min. A sample was collected and normalized to an OD 600 of 0.5 prior to incubation with LytA*-Alexa to label WTAs as described in the Materials and Methods. The images are not normalized and were adjusted to best highlight the LytA*-Alexa distribution along the cell envelope. Each image is representative of a distinct stage in the pneumococcal cell elongation cycle: (i) early division/elongation, (ii) mid-division/elongation, (iii) early constriction, (iv) late constriction/separation.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m  Schematic model of WhyD function. WhyD releases the majority WTAs attached to the cell wall during nascent PG synthesis at midcell. A subset of the WTAs remain intact and as the cell elongates these polymers recruit PG hydrolases with choline binding domains (yellow, orange, red Pac-Men) to the zone of peripheral PG synthesis, promoting expansion of the cell wall meshwork and cell elongation. At a late stage of cell constriction, WTAs accumulate at midcell where they recruit PG hydrolases that promote cell separation (yellow). At this stage, WhyD might not be localized at midcell or its activity could be inhibited. Upon entry into stationary phase or exposure to cell wall targeting antibiotics (autolysis), WhyD is unable to keep pace with the increase in WTA synthesis and/or is actively inhibited, leading to an increase in WTAs throughout the sacculus. Recruitment of LytA and other PG hydrolases leads to cell wall cleavage and lysis.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is m