Direct interaction between MreB and the RodA-PBP2 complex organizes lateral cell wall synthesis in Escherichia coli

Abstract

The elongasome, or the Rod complex, orchestrates lateral peptidoglycan (PG) synthesis in many rod-shaped bacteria. It consists of the actin-like protein MreB, the PG synthase RodA-PBP2 complex, as well as MreCD and RodZ. Although the loss or disruption of any elongasome component results in a loss of rod shape, previous studies found that a constitutively active RodA-PBP2 complex can partially bypass the requirement of MreCD and RodZ for lateral PG synthesis and restore rod shape. However, how MreB is connected to RodA-PBP2 under this situation and whether this linkage is important for elongasome activity in wild-type cells remain unknown. Here, we report the isolation of additional RodA and PBP2 variants that can partially compensate for the absence of MreCD and RodZ in lateral PG synthesis. Taking advantage of these mutants and guided by an AlphaFold 3 structural model of the elongasome complex, we discover that both the cytoplasmic region of PBP2 and the C-terminal tail of RodA interact with MreB. Moreover, disruption of these interactions results in a loss of rod shape, indicating that the interaction between MreB and RodA-PBP2 is critical for elongasome function. Taken together, our results uncover how the MreB cytoskeleton is coupled to RodA-PBP2 to organize lateral PG synthesis. These findings provide mechanistic insights into cell wall biogenesis in bacteria and offer strategies for the development of new antibiotics targeting the elongasome.

References

[1] Rohs PDA, Bernhardt TG. Growth and division of the peptidoglycan matrix. Annu Rev Microbiol. 2021; 75: 315–336.

[2] Egan AJF, Errington J, Vollmer W. Regulation of peptidoglycan synthesis and remodelling. Nat Rev Microbiol. 2020; 18: 446–460.

[3] Höltje JV. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev. 1998; 62: 181–203.

[4] Vollmer W, Bertsche U. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta. 2008; 1778: 1714–1734.

[5] Bouhss A, Trunkfield AE, Bugg TDH, Mengin-Lecreulx D. The biosynthesis of peptidoglycan lipid-linked intermediates. FEMS Microbiol Rev. 2008; 32: 208–233.

[6] Meeske AJ, Riley EP, Robins WP, Uehara T, Mekalanos JJ, Kahne D, et al. SEDS proteins are a widespread family of bacterial cell wall polymerases. Nature. 2016; 537: 634–638.

[7] Emami K, Guyet A, Kawai Y, Devi J, Wu LJ, Allenby N, et al. RodA as the missing glycosyltransferase in Bacillus subtilis and antibiotic discovery for the peptidoglycan polymerase pathway. Nat Microbiol. 2017; 2:16253.

[8] Sjodt M, Brock K, Dobihal G, Rohs PDA, Green AG, Hopf TA, et al. Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis. Nature. 2018; 556: 118–121.

[9] Sjodt M, Rohs PDA, Gilman MSA, Erlandson SC, Zheng S, Green AG, et al. Structural coordination of polymerization and crosslinking by a SEDS-bPBP peptidoglycan synthase complex. Nat Microbiol. 2020; 5: 813–820.

[10] Taguchi A, Welsh MA, Marmont LS, Lee W, Sjodt M, Kruse AC, et al. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat Microbiol. 2019; 4: 587–594.

[11] Bendezú FO, de Boer PAJ. Conditional lethality, division defects, membrane involution, and endocytosis in mre and mrd shape mutants of Escherichia coli. J Bacteriol. 2008; 190: 1792–1811.

[12] Jones LJF, Carballido-López R, Errington J. Control of cell shape in bacteria. Cell. 2001; 104: 913–922.

[13] Figge RM, Divakaruni AV, Gober JW. MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus. Mol Microbiol. 2004; 51: 1321–1332.

[14] Leaver M, Errington J. Roles for MreC and MreD proteins in helical growth of the cylindrical cell wall in Bacillus subtilis. Mol Microbiol. 2005; 57: 1196–1209.

[15] Bendezú FO, Hale CA, Bernhardt TG, de Boer PAJ. RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. EMBO J. 2009; 28: 193–204.

[16] Shiomi D, Sakai M, Niki H. Determination of bacterial rod shape by a novel cytoskeletal membrane protein. EMBO J. 2008; 27: 3081–3091.

[17] Spratt BG. The mechanism of action of mecillinam. J Antimicrob Chemother. 1977; 3(Suppl B): 13–19.

[18] van den Ent F, Johnson CM, Persons L, de Boer P, Löwe J. Bacterial actin MreB assembles in complex with cell shape protein RodZ. EMBO J. 2010; 29: 1081–1090.

[19] Salje J, van den Ent F, de Boer P, Löwe J. Direct membrane binding by bacterial actin MreB. Mol Cell. 2011; 43: 478–487.

[20] Gitai Z, Dye NA, Reisenauer A, Wachi M, Shapiro L. MreB actin-mediated segregation of a specific region of a bacterial chromosome. Cell. 2005; 120: 329–341.

[21] Bean GJ, Flickinger ST, Westler WM, McCully ME, Sept D, Weibel DB, et al. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry. 2009; 48: 4852–4857.

[22] Kruse T, Bork-Jensen J, Gerdes K. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Mol Microbiol. 2005; 55: 78–89.

[23] Dersch S, Mehl J, Stuckenschneider L, Mayer B, Roth J, Rohrbach A, et al. Super-resolution microscopy and single-molecule tracking reveal distinct adaptive dynamics of MreB and of cell wall-synthesis enzymes. Front Microbiol. 2020; 11: 1946.

[24] Domínguez-Escobar J, Chastanet A, Crevenna AH, Fromion V, Wedlich-Söldner R, Carballido-López R. Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria. Science. 2011; 333: 225–228.

[25] Garner EC, Bernard R, Wang W, Zhuang X, Rudner DZ, Mitchison T. Coupled, circumferential motions of the cell wall synthesis machinery and MreB filaments in B. subtilis. Science. 2011; 333: 222–225.

[26] van Teeffelen S, Wang S, Furchtgott L, Huang KC, Wingreen NS, Shaevitz JW, et al. The bacterial actin MreB rotates, and rotation depends on cell-wall assembly. Proc Natl Acad Sci USA. 2011; 108: 15822–15827.

[27] Liu X, Biboy J, Consoli E, Vollmer W, den Blaauwen T. MreC and MreD balance the interaction between the elongasome proteins PBP2 and RodA. PLoS Genet. 2020; 16:e1009276.

[28] Morgenstein RM, Bratton BP, Nguyen JP, Ouzounov N, Shaevitz JW, Gitai Z. RodZ links MreB to cell wall synthesis to mediate MreB rotation and robust morphogenesis. Proc Natl Acad Sci USA. 2015; 112: 12510–12515.

[29] Bratton BP, Shaevitz JW, Gitai Z, Morgenstein RM. MreB polymers and curvature localization are enhanced by RodZ and predict E. coli's cylindrical uniformity. Nat Commun. 2018; 9: 2797.

[30] Colavin A, Shi H, Huang KC. RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape. Nat Commun. 2018; 9: 1280.

[31] Hussain S, Wivagg CN, Szwedziak P, Wong F, Schaefer K, Izore T, et al. MreB filaments align along greatest principal membrane curvature to orient cell wall synthesis. eLife. 2018; 7:e32471.

[32] Tamaki S, Matsuzawa H, Matsuhashi M. Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli. J Bacteriol. 1980; 141: 52–57.

[33] Nygaard R, Graham CLB, Belcher Dufrisne M, Colburn JD, Pepe J, Hydorn MA, et al. Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex. Nat Commun. 2023; 14: 5151.

[34] Li Y, Gong H, Zhan R, Ouyang S, Park KT, Lutkenhaus J, et al. Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes. PLoS Genet. 2021; 17:e1009366.

[35] Rohs PDA, Buss J, Sim SI, Squyres GR, Srisuknimit V, Smith M, et al. A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery. PLoS Genet. 2018; 14:e1007726.

[36] Martins A, Contreras-Martel C, Janet-Maitre M, Miyachiro MM, Estrozi LF, Trindade DM, et al. Self-association of MreC as a regulatory signal in bacterial cell wall elongation. Nat Commun. 2021; 12: 2987.

[37] Shlosman I, Fivenson EM, Gilman MSA, Sisley TA, Walker S, Bernhardt TG, et al. Allosteric activation of cell wall synthesis during bacterial growth. Nat Commun. 2023; 14: 3439.

[38] Rohs PDA, Qiu JM, Torres G, Smith MD, Fivenson EM, Bernhardt TG. Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery. J Bacteriol. 2021; 203:e00493–20.

[39] Contreras-Martel C, Martins A, Ecobichon C, Trindade DM, Matteï PJ, Hicham S, et al. Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex. Nat Commun. 2017; 8: 776.

[40] Shlosman I, Fivenson EM, Gilman MSA, Sisley TA, Walker S, Bernhardt TG, et al. Allosteric activation of cell wall synthesis during bacterial growth. Nat Commun. 2023; 14: 3439.

[41] Shiomi D, Toyoda A, Aizu T, Ejima F, Fujiyama A, Shini T, et al. Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ-deficient cells. Mol Microbiol. 2013; 87: 1029–1044.

[42] Yang X, McQuillen R, Lyu Z, Phillips-Mason P, De La Cruz A, McCausland JW, et al. A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW. Nat Microbiol. 2021; 6: 584–593.

[43] Du S, Pichoff S, Lutkenhaus J. FtsEX acts on FtsA to regulate divisome assembly and activity. Proc Natl Acad Sci USA. 2016; 113: E5052–E5061.

[44] Park KT, Du S, Lutkenhaus J. Essential role for FtsL in activation of septal peptidoglycan synthesis. mBio. 2020; 11:e03012-20.

[45] Park KT, Pichoff S, Du S, Lutkenhaus J. FtsA acts through FtsW to promote cell wall synthesis during cell division in Escherichia coli. Proc Natl Acad Sci USA. 2021; 118:e2107210118.

[46] Ago R, Tahara YO, Yamaguchi H, Saito M, Ito W, Yamasaki K, et al. Relationship between the Rod complex and peptidoglycan structure in Escherichia coli. MicrobiologyOpen. 2023; 12:e1385.

[47] Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989; 171: 4617–4622.

Rights & permissions

2026 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.