A Synthetic 5,3-Cross-Link in the Cell Wall of Rod-Shaped Gram-Positive Bacteria

A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria

David A. Dika, Nan Zhanga, Emily J. Sturgellb, Brittany B. Sanchezb, Jason S. Chenb, Bill Webbc, Kimberly G. Vanderpoold, and Peter G. Schultza,1

aDepartment of Chemistry, Scripps Research, La Jolla, CA 92037; bAutomated Synthesis Facility, Scripps Research, La Jolla, CA 92037; cCenter for Metabolomics and Mass Spectrometry, Scripps Research, La Jolla, CA 92037; and dCore Microscopy Facility, Scripps Research, La Jolla, CA 92037

Contributed by Peter G. Schultz, January 25, 2021 (sent for review January 5, 2021; reviewed by Hung-wen Liu and Christopher T. Walsh) Gram-positive bacteria assemble a multilayered cell wall that pro- The arrangement of the peptide cross-links dictates the di- vides tensile strength to the cell. The cell wall is composed of gly- mensionality, proportions, and porosity of the cell wall as well as can strands cross-linked by nonribosomally synthesized peptide the shape of the bacterium. To explore the structural constraints stems. Herein, we modify the peptide stems of the Gram- of the cell-wall architecture that allow it to meticulously or- positive bacterium Bacillus subtilis with noncanonical electrophilic chestrate essential biological processes and maintain cellular D-amino acids, which when in proximity to adjacent stem peptides morphology, we have begun to replace the naturally existing form novel covalent 5,3-cross-links. Approximately 20% of canon- canonical cell-wall cross-links (Fig. 1B) with unnatural synthetic ical cell-wall cross-links can be replaced with synthetic cross-links. cell-wall cross-links. Previously, we used electrophilic non- While a low level of synthetic cross-link formation does not affect canonical D-amino acids (D-AAs) to form unnatural synthetic B. subtilis growth and phenotype, at higher levels cell growth is cell-wall 4,3-cross-links in the Gram-negative bacterium E. coli perturbed and bacteria elongate. A comparison of the accumula- (9). Herein, we extend this approach to the Gram-positive bac- tion of synthetic cross-links over time in Gram-negative and Gram- terium B. subtilis, revealing differences in synthetic cross-link positive bacteria highlights key differences between them. The arrangements, synthetic cross-link–induced effects on cell ability to perturb cell-wall architecture with synthetic building growth and phenotype, as well as differences in the mode of blocks provides a novel approach to studying the adaptability, accumulation of synthetic cell wall within the bacteria. elasticity, and porosity of bacterial cell walls. Results and Discussion BIOCHEMISTRY bacteria | cell wall | transpeptidases | synthetic cross-links Experimental Approach. Our previous studies on the formation of unnatural cell-wall cross-links in Gram-negative bacteria ram-positive bacteria are monoderms and their single depended on the ability of bacterial transpeptidases to incor- – Gmembrane is encased by a multilayered (15 30 nm) cell porate environmental D-AAs into the cell wall, a strategy used by wall. In contrast, Gram-negative bacteria are diderms and the bacteria to reduce the extent of peptide cross-linking (Fig. 2A) cell wall is mono- or bilayered (3–6 nm) and resides between the (9–12). In seminal studies, others have used this approach to inner and outer membranes (1). Notwithstanding this disparity in incorporate fluorophores and biorthogonal photo–cross-linkers general architecture, the chemical structures of the peptidogly- into the cell wall of diverse bacteria for live-cell cell-wall imaging can of each organism are remarkably similar (2). The cell-wall (13, 14). By using noncanonical electrophilic D-AAs as substrates peptidoglycan, or murein, is composed of glycan strands assem- bled from the saccharide constituents N-acetyl muramic acid Significance (NAM) and N-acetyl glucosamine (NAG), and in each class of bacteria a pentapeptide extends from the lactyl group of the A The cell wall of bacteria is a biopolymer formed of glycan NAM saccharide (Fig. 1 ) (3). While variability in the sequence chains cross-linked by interconnecting peptide stems. Eluci- of the peptide stem exists across species, the archetypal structure γ dating the structure, biosynthesis, and recycling mechanisms of of the monoderm stem is L-Ala- -D-Glu-L-Lys-D-Ala-D-Ala, and the cell wall is important to understanding the mechanism of in the diderm L-Lys is typically substituted with the carboxy de- β meso m B action of -lactam antibiotics and the development of new rivative -2,6-diaminopimelate ( -DAP) (Fig. 1 ) (3, 4). antibiotics. Here we use a synthetic biology approach to probe The peptide stems of the cell wall are cross-linked by penicillin- the structural and biosynthetic constraints of cell-wall archi- binding protein (PBP) D,D-transpeptidases, and L,D-trans- tecture by introducing noncanonical building blocks into the peptidases. In Gram-negative bacteria, the epsilon amino group cell walls of living bacteria. We show that a Gram-positive m of -DAP serves as a nucleophile displacing a terminal D-Ala of bacterium remains viable under circumstances where approxi- an adjacent peptide stem in the formation of D,D-transpeptidase- mately 20% of the cell wall is interconnected by synthetic catalyzed 4,3- and L,D-transpeptidase-catalyzed 3,3-peptide 5,3-cross-links, a cross-linking arrangement absent in Nature. cross-links (5). Both cross-linking arrangements stabilize the cell Characterization of these synthetic cell-wall cross-links high- wall and the L,D-transpeptidase cross-link additionally anchors lights key differences in cell-wall recycling between Gram- ’ the cell wall to the outer membrane via Braun s lipoprotein (6). negative and -positive bacteria. In Gram-positive bacteria, the epsilon amino group of lysine acts as the corresponding nucleophile in cross-link formation. Due to Author contributions: D.A.D. and P.G.S. designed research; D.A.D., N.Z., E.J.S., B.B.S., the absence of an outer membrane in Gram-positive bacteria, J.S.C., B.W., and K.G.V. performed research; N.Z. contributed new reagents/analytic tools; L,D-transpeptidase cross-linking is a rare event that may aid in D.A.D., N.Z., J.S.C., and P.G.S. analyzed data; and D.A.D. and P.G.S. wrote the paper. providing mechanical stability. Functional redundancy is evident Reviewers: H.L., The University of Texas at Austin; and C.T.W., Stanford University. in cell-wall synthesis as Escherichia coli encodes at least five D,D- The authors declare no competing interest. transpeptidases and six L,D-transpeptidases, while Bacillus subtilis Published under the PNAS license. encodes at least 10 D,D-transpeptidases and two L,D-trans- 1To whom correspondence may be addressed. Email: [email protected]. peptidases (5, 7, 8). This redundancy implies that bacteria have This article contains supporting information online at https://www.pnas.org/lookup/suppl/ evolved a careful balance in cell-wall assembly that forms an doi:10.1073/pnas.2100137118/-/DCSupplemental. ideal biopolymer for bacterial life. Published March 8, 2021.

PNAS 2021 Vol. 118 No. 11 e2100137118 https://doi.org/10.1073/pnas.2100137118 | 1of7 Downloaded by guest on September 24, 2021 unnatural synthetic 5,3-cross-links (Fig. 2A). To this end, we synthesized five D-AAs as potential structures for synthetic cross- link formation (9). Amino acids 1a, 1b, and 1c are fluorosulfates capable of reacting with amino and hydroxy groups of amino acids in close proximity, and 2a and 2b are somewhat more re- active vinyl sulfonamides (Fig. 2B) (19). Variants with different ring substitution patterns and sidechain lengths were assessed for efficiency of incorporation into cell walls. Previously we had shown that the sidechain length and the nature of the electro- phile significantly affected the incorporation of the D-AA and the efficiency of synthetic cross-link formation. Additionally, the L-enantiomers of 1c (1d) and 1a (1e) were prepared as controls.

Synthetic Cross-Link Formation by Noncanonical D-AAs. We first determined whether the noncanonical D-AAs are toxic to B. subtilis ΔdacA at elevated concentrations. The L-enantiomers of fluorosulfate and vinyl sulfonamide substituted phenylalanine have previously been incorporated into proteins in live bacteria with minimal effect on bacterial growth at 1 mM concentrations (19, 20). In E. coli, addition of the D-AAs and the cognate L-enantiomer to growth media resulted in identical growth curves with significant effects on growth rates beginning at concentrations above 8 mM (9). However, in B. subtilis ΔdacA, we observed increased detrimental effects on cell growth for the noncanonical D-AAs compared to the L-AAs starting at 4 mM Fig. 1. (A) The cell wall of Gram-positive bacteria is anchored to the outer (Fig. 2C and SI Appendix, Fig. S1 A–H). Compounds 1a, 1b, 1c, leaflet of the membrane by lipoteichoic acids (LTAs; shown in yellow). 1d, 1e, 2a, and 2b completely inhibit growth at 8 mM, 8 mM, 8 Walled teichoic acids (WTAs; shown in orange) provide additional support > > > > between branches (36). (A, B) The peptidoglycan is composed of glycan that mM, 8 mM, 8 mM, 8 mM, 8 mM concentrations, assembles from the disaccharide pair NAG (shown and labeled in dark respectively. green)-NAM (light green). Peptide stems (blue) connect the glycan via To determine if the noncanonical D-AAs are incorporated into enzyme-catalyzed cross-linking reactions. The glycan of the cell wall is de- the cell wall, we initially treated cultures of B. subtilis ΔdacA with graded by muramyl hydrolases, and to a lesser extent by lytic trans- 1 mM of each D-AA. Briefly, an overnight culture of B. subtilis glycosylases, the latter producing anhydroNAM (shown in red) as the ΔdacA was inoculated into Luria Bertani (LB) media supple- reaction product (37). mented with chloramphenicol and grown to optical density600nm (OD600) = 0.05. Subsequently, cultures were independently treated with 1 mM of each D-AA and grown to OD600 = 1.10. for the transpeptidases, we were able to replace ∼30% of the Cells were then placed on ice, pelleted, and the cell wall of each natural cell-wall cross-links in E. coli with unnatural synthetic culture was isolated following a modified version of the estab- cell-wall cross-links with no accompanying observable effect on lished methodology (9, 21, 22). We analyzed each sample by bacterial phenotype (9). To determine if a similar strategy can be liquid-chromatography mass spectrometry (LC-MS). The con- used to form unnatural cell-wall cross-links in Gram-positive centration of each sample was normalized to the most abundant bacteria, we selected B. subtilis. B. subtilis is a model bacterium native non–cross-linked muropeptide, NAG-NAM-pentapeptide for the study of the structure of the rod-shaped, spore-forming at a mass spectra signal of ∼106 (SI Appendix, Fig. S2 A and B). capable Gram-positive bacterium (15). A key distinction between Masses corresponding to the incorporation of noncanonical the cell wall of B. subtilis and other Gram-positive bacteria is the D-AAs into the fifth position of the peptide stems were detected presence of the archetypal Gram-negative m-DAP at the third for each compound and the corresponding mass spectra signal is position of the peptide stem, which in B. subtilis commonly exists provided: 1a (106), 1b (106), 1c (106), 2a (105), and 2b (106) as an enzyme-catalyzed amidated amino acid (16). (Fig. 2E,2G, and 2I, and SI Appendix, Fig. S2 C and D). No In Gram-negative bacteria, noncanonical D-AAs are incorpo- incorporation was detected for samples treated with the L-AA rated into the peptide stem at the fourth position by L,D-trans- controls 1d and 1e. peptidases and at the fifth position by D,D-transpeptidases (17). Next, we analyzed the MS data for a mass corresponding to In our previous efforts to generate unnatural synthetic cell-wall the synthetic cross-link. The expected structure for a synthetic cross-links, we primarily observed amino acid replacement at the 5,3-cross-link formed by 1c is shown in Fig. 2D. Mass spectral fourth position, likely a result of D,D-carboxypeptidases removing analysis confirmed the expected synthetic cell-wall 5,3-cross-links any noncanonical D-AAs incorporated at the fifth position of the for all three fluorosulfates 1a, 1b, and 1c (a complete list of stem peptide (9, 17). However, in Gram-positive bacteria, L,D- structures is provided in SI Appendix, Figs. S3–S8). The structure transpeptidases seldom form cross-links, which limits the ability of the synthetic 5,3-cross-link formed by 1c was confirmed by LC- to incorporate noncanonical D-AAs at the fourth position MS/MS (SI Appendix, Fig. S9). The synthetic cross-linked mur- (17, 18). opeptides formed by 1a, 1b, and 1c gave comparable mass As an alternative approach, it was shown previously that spectra signals of ∼106 and were ∼4-fold lower than the signal knocking out the dacA gene, which encodes the primary D,D- observed for the synthetic non–cross-linked muropeptides carboxypeptidase PBP5, permits accumulation of fluorescent (Fig. 2 E–J). A synthetic cross-linked muropeptide was not de- D-AAs into the fifth position of the peptide stems of B. subtilis tected for vinyl sulfonamides 2a or 2b, which previously formed (13). We reasoned that this same strategy might be used to in- synthetic cross-links in E. coli (9). Furthermore, we do not detect corporate noncanonical electrophilic D-AAs into the fifth posi- the reaction product of m-DAP and the free D-AA. tion of the peptide stems of B. subtilis ΔdacA. These D-AAs Notably, in our previous studies with E. coli, 1c was poorly might be appropriately positioned to covalently cross-link with incorporated and much less efficient at forming cross-links the amidated m-DAP of an adjacent peptide stem resulting in compared to 1a or 1b (9). This disparity in cross-linking efficiency

2of7 | PNAS Dik et al. https://doi.org/10.1073/pnas.2100137118 A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria Downloaded by guest on September 24, 2021 BIOCHEMISTRY

Fig. 2. (A) Comparative mechanisms of D,D-transpeptidase-mediated canonical cell-wall cross-linking and noncanonical cell-wall cross-linking by exogenous D-AAs in B. subtilis.(B) Structures of electrophilic noncanonical D-AAs and cognate L-AA controls. (C) Bacterial growth curves of B. subtilis untreated or treated with D-AA 1c or L-AA 1d.(D) Structure of the noncanonical 5,3-cross-linked NAG-NAM-(pentapeptide)-NAG-anhydroNAM-(tetrapeptide) formed by 1c. The gray boxes show a comparison of the synthetic cross-link and the native cross-link. For each noncanonical D-AA the primary synthetic cross-linked muropeptide formed comprises the canonical NAG-NAM-(tetrapeptide) (R′) and NAG-anhydroNAM-(tetrapeptide) (R′′), where the noncanonical D-AA is installed adjacent to the fourth-position D-Ala of the R′ stem, replacing the fifth-position D-Ala. The mass spectra corresponding to the synthetic non–cross-linked and cross-linked cell-wall species formed by (E, F) 1a,(G, H) 1b, and (I, J) 1c are shown. Compounds 2a and 2b form synthetic non–cross-linked muropeptides, but not synthetic cross-linked muropeptides. The structures and masses of each unnatural synthetic cell-wall muropeptide is provided in SI Appendix (SI Appendix, Figs. S3–S8).

is likely due to the spatial arrangement of the peptide stems, such m-DAP of the adjacent peptide stem. An unexpected feature in that the extended 1c sidechain places the fluorosulfate in close our analysis is that the m-DAP donor strand of the primary D-AA proximity to the epsilon amino group of m-DAP in B. subtilis. cross-linked muropeptide is a tetrapeptide (Fig. 2D), while in the Because noncanonical D-AAs are incorporated at the fourth po- primary native cross-linked muropeptide this same strand is a sition in E. coli, this extension likely worsens interactions with pentapeptide (SI Appendix, Fig. S3). While the cell encodes other

Dik et al. PNAS | 3of7 A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria https://doi.org/10.1073/pnas.2100137118 Downloaded by guest on September 24, 2021 low-activity D,D-carboxypeptidases (23), this structural disparity untreated wild-type and ΔdacA cells were used as controls. Cells was unexpected in the ΔdacA strain, and may point to key dif- were prepared for SEM analysis as described previously (9). B. ferences in cell-wall processing for the two muropeptides. We subtilis wild-type cells show a characteristic linear rod shape with next focused on further characterizing the cross-links formed by division at the midcell (Fig. 4A). In comparison, B. subtilis ΔdacA D-AA 1c. is slightly shorter, but otherwise shares a similar morphology (Fig. 4B). The observation that B. subtilis ΔdacA shows a Quantification of Synthetic Cell-Wall Composition. We evaluated the shortened morphology was previously reported and literature effect of varying concentrations of 1c on the density of synthetic suggests that the shortening of the cells becomes more profound cell-wall cross-links. Cultures were prepared as previously de- as cells enter into stationary phase (24). In contrast, B. subtilis scribed and independently treated with 1, 2, and 4 mM of 1c. ΔdacA treated with 4 mM 1c undergo significant cell lysis, as Additionally we prepared an untreated culture and a culture shown in part, by the white arrows (Fig. 4 C and E). Cells that treated with compound 1d as controls. After cell-wall extraction survive treatment appear elongated and spiraled (Fig. 4 C–E). and digestion, the samples were analyzed by high-performance For comparison, cells treated with the L-enantiomer 1d divide liquid chromatography (HPLC). At the highest concentration successfully and exhibit a cell length comparable to untreated tested (4 mM of 1c), the percentage of the most abundant cells (Fig. 4 F–H), however, compared to untreated cells they noncanonical D-AA containing synthetic non–cross-linked spe- appear curled which may be due to an off-target interaction of cies (SnC), an amidated NAG-NAM-tetrapeptide-1c, accounted the fluorosulfate with cellular components. This curl effect, for ∼19% of the total non–cross-linked peptidoglycan; the most when compounded by the elongation of cells caused by treat- abundant native non–cross-linked species (nC) was an amidated ment with 1c, may contribute to the observed spiraling (Fig. 4 D NAG-NAM-pentapeptide (Fig. 3 A and B). The primary D-AA and E). B. subtilis ΔdacA treated with 1 mM 1c or 1d show no containing synthetic cross-linked species (SC), the amidated significant change in morphology, while cells treated with 2 mM NAG-NAM-tetrapeptide-1c-tetrapeptide-NAG-anhydroNAM 1c or 1d show a slight curl, that is more pronounced in the 1c (structure in Fig. 2D), accounted for 19% of the total cross-linked treatment. Additional SEM images at each compound treat- peptidoglycan; the most abundant native cross-linked species (C) ment concentration are provided in SI Appendix, Fig. S13 A–F. was an amidated NAG-NAM-tetrapeptide-pentapeptide-NAG- Collectively, these data show that cells containing synthetic cell- NAM (Fig. 3 A and B). HPLC analysis showed a concentration- wall cross-links elongate, and likely have an impaired ability to dependent increase in D-AA-containing SnC and SC cell-wall divide. species (Fig. 3B and SI Appendix, Figs. S10–S12). These results It has been documented that peptidoglycan at the septum indicate that B. subtilis survives in conditions (4 mM of 1c)where during cell division is often denuded of peptide stems, which are 19% of the cell wall has been replaced with noncanonical building clipped by periplasmic amidases at the lactyl group (25). Sac- blocks. At 2 mM of compound 1c, B. subtilis grows unperturbed charides at the septum are directed and remodeled by the cell- and the cell wall contains 12% synthetic cross-links. division proteins of the divisome as well as by certain cell-wall remodeling enzymes that contain sporulation-related repeat Bacterial Phenotype Imparted by D-AA Cross-Link. We analyzed B. (SPOR) domains, which recognize and bind to denuded glycan subtilis by scanning electron microscopy (SEM) for an observable (25–27). Additionally, gene deletion of periplasmic cell-wall alteration of cellular morphology as a result of the unnatural amidases produces an elongated cell phenotype (28). In our synthetic crosslinks. B. subtilis ΔdacA was grown to OD600= 0.05 LC-MS analysis, we observe the reaction product of the primary and treated with 1 mM, 2 mM, or 4 mM of 1c or 1d for 2 h; native cross-link with cell-wall amidases, but not the reaction product of the primary synthetic cross-link with cell-wall amidases at either end (i.e., NAG-NAM or NAG-anhydroNAM). We hypothesize that the presence of synthetically modified cell wall interferes with the activity of periplasmic amidases and therefore cell-division–related SPOR-domain–containing enzymes, leading to impaired cell division and the observed phenotype at elevated concentrations of 1c. We do not know at this time why this observation is unique to our model Gram-positive bacterium B. subtilis and not found in our model Gram-negative bacterium E. coli, but hypothesize that it may be due to the substrate specificity and scope of the requisite enzymes involved in division. A phenotypic comparison of the spiraling in our B. subtilis cells with that of Campylobacter is intriguing. Campylobacter helical shape is controlled by cell-shape–determining (Csd) proteins which often contain lysostaphinlike metalloprotease domains that act on peptidoglycan (29, 30). The exact function of these proteins is a current topic of study (31). Structural and in vitro analysis suggests that at least some of these enzymes are D,D- Fig. 3. (A) HPLC trace (205 nm) of isolated peptidoglycan from B. subtilis carboxypeptidases (cleave D-Ala from the fifth position of the treated with 1c (4 mM). The peaks corresponding to the most abundant peptide stem) and D,D-endopeptidases (cleave cross-links). These muropeptide species for the native non–cross-linked (nC), native cross-linked enzymatic activities would likely be impaired/modified in a bac- (C), synthetic non–cross-linked (SnC), and synthetic cross-linked (SC) are la- terium with a synthetic cell wall. However, due to the difference beled. SnC and SC correspond to the D-AA-modified structures. The black in enzyme profile and general cell-wall architecture (Campylo- circles denote the amidated species, the gray circles denote the non- bacter amidated non–cross-linked species, and gold circles denote the partially is Gram-negative) between the organisms, we cannot at amidated and nonamidated cross-linked species. (B) The percentage of this time draw conclusions from the comparison. synthetic non–cross-linked and synthetic cross-linked muropeptides for cell – wall isolated from B. subtilis after treatment with 1c. Note, percentages are Activity of Cell-Wall Degrading Enzymes Affects Synthetic Cell-Wall based solely on the most abundant amidated native and synthetic species. A Content. Previously, we observed the formation of an anhy- detailed quantification is provided in SI Appendix. droNAM saccharide on the primary D-AA cross-linked muropeptide

4of7 | PNAS Dik et al. https://doi.org/10.1073/pnas.2100137118 A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria Downloaded by guest on September 24, 2021 Fig. 4. B. subtilis was cultured to OD600= 0.05 and either not treated or treated with 4 mM 1c or 1d for 2 h. The bacteria were imaged by SEM and the results are shown. SEM of untreated bacteria (A) B. subtilis wild type and (B) B. subtilis ΔdacA display a linear rod shape. The ΔdacA mutation gives slightly shortened cells. Images of B. subtilis ΔdacA treated with 1c were captured at (C) 1,500× and (D, E) 15,000× magnification. Significant cell lysis is observed as indicated by white arrows. Bacteria that survive treatment by 1c are able to successfully elongate, but cell division appears impaired. Elongated cells display a spiraled phenotype. Images of B. subtilis ΔdacA treated with 1d were captured at (F) 1,500× and (G, H) 15,000× magnification. No cell lysis was observed for bacteria treated with 1d, although cells display a subtle curl. A 1-μM white scale bar is shown in A, B, D, E, G,andH (Top Right). A 5-μM white scale bar is shown in C and F (Top Right).

in E. coli (9). AnhydroNAM is a reaction product of lytic that the LT activity of E. coli is impaired by both D-AA– transglycosylases (LTs), enzymes that degrade the cell wall by containing non–cross-linked and cross-linked muropeptides, we nonhydrolytically cleaving the glycosidic bond between NAM treated E. coli cells with 1 mM of noncanonical control com- and NAG to form an anhydroNAM reaction product (shown in pound O-methyl-D-tyrosine (structure in SI Appendix, Fig. S14A), Fig. 2D). LT activity is dominant in Gram-negative bacteria and which structurally resembles 1a, but is nonreactive. We again BIOCHEMISTRY in isolated cell-wall samples 3.7% of muropeptides contain observed an accumulation pattern that resembles that of culture anhydroNAM, which caps the termini of glycan strands after treated with 1a (SI Appendix, Fig. S14 B–G), demonstrating that scission (32). Using LC-MS/MS, we mapped this modifica- LT activity is impaired by the D-AA–modified peptide stems, tion to the saccharide affixed to the peptide stem that is not even if a synthetic cross-link is not formed. modified by the D-AA. We did not observe the formation of In B. subtilis the LTs likely produce similar reaction products anhydroNAM on the primary D-AA–containing non–cross- to those formed in E. coli (Fig. 5 E, I, II). However, hydrolytic linked muropeptide, nor the peptide stem of the cross-linked muramidases in B. subtilis cleave and excise both native and muropeptide that is modified by the D-AA. This observation synthetic cell wall, which should result in lower accumulation of suggests that LTs cannot form anhydroNAM caps on NAM synthetic cell wall than in E. coli where degradation is more saccharides adjoined to our D-AA-modified peptide stems. In- impaired (Fig. 5 E, III, IV). HPLC analysis of cell-wall samples terestingly, in the current study we observed an anhydroNAM isolated from B. subtilis as a function of time shows a similar cell- modification at this same position (i.e., the saccharide affixed wall content to E. coli at 2-h posttreatment. However, in B. to the peptide stem that is not modified by the D-AA of the subtilis, significant accumulation of synthetic cell wall past 2 h cross-linked muropeptide) in B. subtilis (Fig. 2D). Gram-positive was not observed (Fig. 5 G and H). Hence, a homeostasis is bacteria predominantly rely on hydrolytic muramidases to quickly reached between incorporation and subsequent synthetic cleave the glycosidic bond between NAM and NAG (33), and cross-link formation of noncanonical D-AAs, and excision of the corresponding reaction products of hydrolysis serve as the synthetic cell wall by hydrolytic muramidases. “caps” on the termini of their glycan strands (34). LT activity in Gram-positive bacteria is less abundant and in isolated na- Conclusion. Herein, we showed that the cell wall of a Gram- tive cell-wall samples only 0.4% of muropeptides contain an positive rod-shaped bacterium can be modified with electro- anhydroNAM (18). philic noncanonical D-AAs that form nonenzyme catalyzed syn- In E. coli we had hypothesized that the noncleavable nature of thetic cell-wall cross-links to the extent of 19% of the total the synthetic cross-link interferes with the exolytic processivity cell-wall cross-links in the live bacterium. In B. subtilis,ahigh (disaccharide cleavage from a glycan terminus) of the LTs such level of synthetic cell-wall cross-links appears to interfere with that the glycosidic bond positioned at C4 of GlcNAc cannot be septal cell-wall synthesis, but not side-wall cell-wall synthesis, cleaved (Fig. 5 A, I-III). As LTs fail to degrade the cell wall of affording the bacterium an elongated morphology. We have also synthetic cross-linked muropeptides, an anhydroNAM imprint is begun to elucidate the enzymatic activities that are impaired by imparted on the synthetic cross-links and the synthetic cell-wall synthetic cell-wall cross-links and the resulting effects on cell- content begins to accumulate as the rate of synthetic cell-wall wall synthesis and degradation. The ability to alter the arrange- formation outcompetes the rate of degradation (Fig. 5 A, IV). To ment of synthetic cross-links in live bacteria by gene ablation of assess whether a disruption in LT activity by synthetic cell wall cell-wall biosynthetic enzymes allows for the study of highly di- leads to accumulation of synthetic muropeptides, we treated verse cell-wall structures. We have shown that nonnative syn- E. coli with 1 mM of 1a for 8 h. After each 2-h interval, we thetic 4,3-cross-links and 5,3-cross-links, the latter of which is an collected 50 mL of culture and performed LC-MS on the isolated arrangement entirely absent in Nature, are accommodated muropeptide to determine the synthetic cell-wall content within the cell wall of live bacteria at high levels. Additionally, it (Fig. 5 B and C). Synthetic cell-wall content increased signifi- was recently shown that synthetic 5,5-cross-links can be formed cantly from 0 to 4-h posttreatment; after 6 h, the accumulation in the cell walls of live bacteria by an azide-alkyne cycloaddition, slows, which may be due to a homeostasis that is reached at late- although this reaction requires exogenous copper and a corre- stationary phase (Fig. 5 C and D). To confirm experimentally sponding copper ligand (35). It is likely that more extensive

Dik et al. PNAS | 5of7 A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria https://doi.org/10.1073/pnas.2100137118 Downloaded by guest on September 24, 2021 Fig. 5. (A) Simplified depiction of the proposed glycan degradation route of synthetically modified cell wall in the Gram-negative bacteria E. coli by glycan- cleaving enzymes. The noncanonical D-AAs are shown as yellow circles. The red lines indicate potential cut sites of the enzymes in each respective panel. (B) Bacteria growth curve of E. coli treated with compound 1a (1 mM). Gray circles indicate growth collection points, at which time growth was halted by pelleting and freezing of the bacterial culture. (C) HPLC trace (Abs. 205 nm) of E. coli bacterial cell wall at each collection point. (D) Quantification of SnC and SC muropeptides relative to the total muropeptide concentration of nC and SnC, and C and SC, respectively. (E) Simplified depiction of the proposed glycan degradation route of synthetically modified cell wall in the Gram-positive bacteria B. subtilis by glycan-cleaving enzymes. TAs not shown. (F) Bacteria growth curve of B. subtilis treated with compound 1a (1 mM) (G) HPLC trace (Abs. 205 nm) of B. subtilis bacterial cell wall. (H) Quantification of SnC and SC muropeptides of B. subtilis. A figure key for cell-wall structures is provided in Fig. 1.

modifications can be made to the cell-wall architecture using Data Availability. All study data are included in the article and/or SI Appendix. similar approaches. ACKNOWLEDGMENTS. We thank Prof. K. B. Sharpless for supplying SO2F2 Materials and Methods gas. We thank Dr. C. S. Diercks and Prof. J. F. Fisher for helpful discussions. The following strain was obtained through the Bacillus Genetic Stock Center: Detailed experimental methods for the biological assays, bacterial growth 1A742, original code: JT175, description: dacA::cat+ trpC2strain. The follow- curves, mass spectrometry analyses, synthetic cell-wall structures, HPLC ing strain was obtained through the E. coli Genetic Stock Center: E. coli analyses, SEM, and compound synthesis procedures and characterization ΔlysA763::kan. We acknowledge Kristen Williams for her assistance in data appear in SI Appendix.SeeSI Appendix, Figs. S1–S14. manuscript preparation.

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