Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
[en] The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.
Disciplines :
Microbiology
Author, co-author :
Potluri, Lakshmiprasad; University of Arkansas for Medical Sciences > Department of Microbiology and Immunology
Karczmarek, Aneta; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Verheul, Jolanda; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Piette, André ; Université de Liège - ULiège > Centre d'ingénierie des protéines
Wilkin, Jean-Marc; Université de Liège - ULiège > Centre d'Ingénierie des Protéines
Werth, Nadine; Eberhard Karls Universität Tübingen > Mikrobielle Genetik
Banzhaf, Manuel; Newcastle University > Centre for Bacterial Cell Biology
Vollmer, Waldemar; Newcastle University > Centre for Bacterial Cell Biology
Young, Kevin D; University of Arkansas for Medical Sciences > Department of Microbiology and Immunology
den Blaauwen, Tanneke; University of Amsterdam > Swammerdam Institute for Life Sciences > Molecular Cytology
Language :
English
Title :
Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment
Publication date :
July 2010
Journal title :
Molecular Microbiology
ISSN :
0950-382X
eISSN :
1365-2958
Publisher :
Blackwell Publishing, Oxford, United Kingdom
Volume :
77
Issue :
2
Pages :
300–323
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
UE - Union Européenne NIH - National Institutes of Health ABI - Arkansas Biosciences Institute
Funding text :
We thank Norbert Vischer for writing the Object-J macros.
We thank Rene van der Ploeg for the gift of LMC512 cells, Eefjan Breukink (University of Utrecht) for the gift of 14CGlcNAc-labelled lipid II and Mohammed Terrak (University of Liège) for the gift of 3H-lipid II. We thank Piet de Boer (Case Western Reserve University) for providing plasmid
pMLB1113. We are also very much in debt to Thomas Bernhardt (Harvard Medical School) for communicating unpublished information and for providing the Superfolder GFP dsbA-SS-sfgfp gene construct on plasmid pTB263. This work was supported by the European commission within the
‘EUR-INTAFAR’ LSHM-CT-2004-512138 network, by Grant R01-GM061019 from the US National Institutes of Health, and by the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.
Aarsman, M.E., Piette, A., Fraipont, C., Vinkenvleugel, T.M., Nguyen-Disteche, M. den Blaauwen, T. (2005) Maturation of the Escherichia coli divisome occurs in two steps. Mol Microbiol 55 : 1631 1645.
Addinall, S.G., Cao, C. Lutkenhaus, J. (1997) Temperature shift experiments with an ftsZ84(Ts) strain reveal rapid dynamics of FtsZ localization and indicate that the Z ring is required throughout septation and cannot reoccupy division sites once constriction has initiated. J Bacteriol 179 : 4277 4284.
Begg, K.J., Takasuga, A., Edwards, D.H., Dewar, S.J., Spratt, B.G., Adachi, H., et al. (1990) The balance between different peptidoglycan precursors determines whether Escherichia coli cells will elongate or divide. J Bacteriol 172 : 6697 6703.
Bernhardt, T.G. de Boer, P.A. (2003) The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway. Mol Microbiol 48 : 1171 1182.
Bernhardt, T.G. de Boer, P.A. (2004) Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity. Mol Microbiol 52 : 1255 1269.
Bertsche, U., Breukink, E., Kast, T. Vollmer, W. (2005) In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase- transpeptidase PBP1B from Escherichia coli. J Biol Chem 280 : 38096 38101.
Bertsche, U., Kast, T., Wolf, B., Fraipont, C., Aarsman, M.E., Kannenberg, K., et al. (2006) Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli. Mol Microbiol 61 : 675 690.
de Boer, P.A., Crossley, R.E. Rothfield, L.I. (1989) A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell 56 : 641 649.
Chen, J.C. Beckwith, J. (2001) FtsQ, FtsL, and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division. Mol Microbiol 42 : 395 413.
Dai, K., Xu, Y. Lutkenhaus, J. (1993) Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts). J Bacteriol 175 : 3790 3797.
Davies, C., White, S.W. Nicholas, R.A. (2001) Crystal structure of a deacylation-defective mutant of penicillin-binding protein 5 at 2.3-A resolution. J Biol Chem 276 : 616 623.
Den Blaauwen, T., Buddelmeijer, N., Aarsman, M.E., Hameete, C.M. Nanninga, N. (1999) Timing of FtsZ assembly in Escherichia coli. J Bacteriol 181 : 5167 5175.
Den Blaauwen, T., Aarsman, M.E., Vischer, N.O. Nanninga, N. (2003) Penicillin-binding protein PBP2 of Escherichia coli localizes preferentially in the lateral wall and at mid-cell in comparison with the old cell pole. Mol Microbiol 47 : 539 547.
Den Blaauwen, T., de Pedro, M.A., Nguyen-Disteche, M. Ayala, J.A. (2008) Morphogenesis of rod-shaped sacculi. FEMS Microbiol Rev 32 : 321 344.
Denome, S.A., Elf, P.K., Henderson, T.A., Nelson, D.E. Young, K.D. (1999) Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis. J Bacteriol 181 : 3981 3993.
Dougherty, T.J., Kennedy, K., Kessler, R.E. Pucci, M.J. (1996) Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli. J Bacteriol 178 : 6110 6115.
Draper, G.C., McLennan, N., Begg, K., Masters, M. Donachie, W.D. (1998) Only the N-terminal domain of FtsK functions in cell division. J Bacteriol 180 : 4621 4627.
Edwards, D. Donachie, W.D. (1993) Construction of a triple deletion of Penicillin-binding proteins 4,5, and 6 in Escherichia coli. In Bacterial Growth and Lysis. de Pedro, M., Höltje, J.-V. Löffelhardt, W. (eds).
van den Ent, F., Vinkenvleugel, T.M., Ind, A., West, P., Veprintsev, D., Nanninga, N., et al. (2008) Structural and mutational analysis of the cell division protein FtsQ. Mol Microbiol 68 : 110 123.
Feilmeier, B.J., Iseminger, G., Schroeder, D., Webber, H. Phillips, G.J. (2000) Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182 : 4068 4076.
Ferreira, L.C., Schwarz, U., Keck, W., Charlier, P., Dideberg, O. Ghuysen, J.M. (1988) Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12. Eur J Biochem 171 : 11 16.
Fishov, I., Zaritsky, A. Grover, N.B. (1995) On microbial states of growth. Mol Microbiol 15 : 789 794.
Geissler, B., Elraheb, D. Margolin, W. (2003) A gain-of-function mutation in ftsA bypasses the requirement for the essential cell division gene zipA in Escherichia coli. Proc Natl Acad Sci USA 100 : 4197 4202.
Ghosh, A.S., Chowdhury, C. Nelson, D.E. (2008) Physiological functions of d-alanine carboxypeptidases in Escherichia coli. Trends Microbiol 16 : 309 317.
Glauner, B. (1988) Separation and quantification of muropeptides with high-performance liquid chromatography. Anal Biochem 172 : 451 464.
Gordon, E., Mouz, N., Duee, E. Dideberg, O. (2000) The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance. J Mol Biol 299 : 477 485.
Harris, F., Brandenburg, K., Seydel, U. Phoenix, D. (2002) Investigations into the mechanisms used by the C-terminal anchors of Escherichia coli penicillin-binding proteins 4, 5, 6 and 6b for membrane interaction. Eur J Biochem 269 : 5821 5829.
Ishidate, K., Ursinus, A., Holtje, J.V. Rothfield, L. (1998) Analysis of the length distribution of murein glycan strands in ftsZ and ftsI mutants of E. coli. FEMS Microbiol Lett 168 : 71 75.
Kohlrausch, U. Höltje, J.-V. (1991) One-step purification procedure for UDP-N-acetylmuramyl-peptide murein precursors from Bacillus cereus. FEMS Microbiol Lett 62 : 253 257.
Koppelman, C.M., Aarsman, M.E., Postmus, J., Pas, E., Muijsers, A.O., Scheffers, D.J., et al. (2004) R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division. Mol Microbiol 51 : 645 657.
van der Linden, M.P., Mottl, H. Keck, W. (1992) Cytoplasmic high-level expression of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography. Eur J Biochem 204 : 197 202.
van der Linden, M.P., de Haan, L., Dideberg, O. Keck, W. (1994) Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli. Biochem J 303 : 357 362.
Markiewicz, Z., Broome-Smith, J.K., Schwarz, U. Spratt, B.G. (1982) Spherical E. coli due to elevated levels of d-alanine carboxypeptidase. Nature 297 : 702 704.
Meberg, B.M., Paulson, A.L., Priyadarshini, R. Young, K.D. (2004) Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli. J Bacteriol 186 : 8326 8336.
Morlot, C., Noirclerc-Savoye, M., Zapun, A., Dideberg, O. Vernet, T. (2004) The D,D-carboxypeptidase PBP3 organizes the division process of Streptococcus pneumoniae. Mol Microbiol 51 : 1641 1648.
Nanninga, N. (1991) Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol 5 : 791 795.
Nelson, D.E. Young, K.D. (2000) Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli. J Bacteriol 182 : 1714 1721.
Nelson, D.E. Young, K.D. (2001) Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli. J Bacteriol 183 : 3055 3064.
Nelson, D.E., Ghosh, A.S., Paulson, A.L. Young, K.D. (2002) Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli. J Bacteriol 184 : 3630 3639.
Nicholas, R.A., Krings, S., Tomberg, J., Nicola, G. Davies, C. (2003) Crystal structure of wild-type penicillin-binding protein 5 from Escherichia coli: implications for deacylation of the acyl-enzyme complex. J Biol Chem 278 : 52826 52833.
O'Daniel, P.I., Zajicek, J., Zhang, W., Shi, Q., Fisher, J.F. Mobashery, S. (2010) Elucidation of the Structure of the Membrane Anchor of Penicillin-Binding Protein 5 of Escherichia coli. J Am Chem Soc 132 : 4110 4118.
Park, J.T. Burman, L. (1973) FL-1060: a new penicillin with a unique mode of action. Biochem Biophys Res Commun 51 : 863 868.
de Pedro, M.A., Quintela, J.C., Holtje, J.V. Schwarz, H. (1997) Murein segregation in Escherichia coli. J Bacteriol 179 : 2823 2834.
Peters, P.C., Migocki, M.D., Thoni, C. Harry, E.J. (2007) A new assembly pathway for the cytokinetic Z ring from a dynamic helical structure in vegetatively growing cells of Bacillus subtilis. Mol Microbiol 64 : 487 499.
Phoenix, D.A. Pratt, J.M. (1990) pH-induced insertion of the amphiphilic alpha-helical anchor of Escherichia coli penicillin-binding protein 5. Eur J Biochem 190 : 365 369.
Pichof, S. Lutkenhaus, J. (2002) Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli. EMBO J 21 : 685 693.
Pinho, M.G. Errington, J. (2003) Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery. Mol Microbiol 50 : 871 881.
Pinho, M.G. Errington, J. (2005) Recruitment of penicillin-binding protein PBP2 to the division site of Staphylococcus aureus is dependent on its transpeptidation substrates. Mol Microbiol 55 : 799 807.
Pratt, R.F. (2008) Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins). Cell Mol Life Sci 65 : 2138 2155.
Pratt, J.M., Jackson, M.E. Holland, I.B. (1986) The C terminus of penicillin-binding protein 5 is essential for localisation to the E. coli inner membrane. EMBO J 5 : 2399 2405.
Sauvage, E., Kerff, F., Terrak, M., Ayala, J.A. Charlier, P. (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32 : 234 258.
Stefanova, M.E., Davies, C., Nicholas, R.A. Stoker, W.G. (2002) pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5. Biochim Biophys Acta 1597 : 292 300.
Stoker, N.G., Broome-Smith, J.K., Edelman, A. Spratt, B.G. (1983) Organization and subcloning of the dacA-rodA-pbpA cluster of cell shape genes in Escherichia coli. J Bacteriol 155 : 847 853.
Sung, M.T., Lai, Y.T., Huang, C.Y., Chou, L.Y., Shih, H.W., Cheng, W.C., et al. (2009) Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc Natl Acad Sci USA 106 : 8824 8829.
Sykes, R.B. Bonner, D.P. (1985) Discovery and development of the monobactams. Rev Infect Dis 7 (Suppl. 4 S579 S593.
Tarry, M., Arends, S.J., Roversi, P., Piette, E., Sargent, F., Berks, B.C., et al. (2009) The Escherichia coli cell division protein and model Tat substrate SufI (FtsP) localizes to the septal ring and has a multicopper oxidase-like structure. J Mol Biol 386 : 504 519.
Taschner, P.E.M., Huls, P.G., Pas, E. Woldringh, C.L. (1988) Division behaviour and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments. J Bacteriol 170 : 1533 1540.
Templin, M.F., Ursinus, A. Höltje, J.-V. (1999) A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli. EMBO J 18 : 4108 4117.
Uehara, T., Dinh, T. Bernhardt, T.G. (2009) LytM-domain factors are required for daughter cell separation and rapid ampicillin-induced lysis in Escherichia coli. J Bacteriol 191 : 5094 5107.
Varma, A. Young, K.D. (2004) FtsZ collaborates with penicillin binding proteins to generate bacterial cell shape in Escherichia coli. J Bacteriol 186 : 6768 6774.
Varma, A. Young, K.D. (2009) In Escherichia coli, MreB and FtsZ direct the synthesis of lateral cell wall via independent pathways that require PBP 2. J Bacteriol 191 : 3526 3533.
Varma, A., de Pedro, M.A. Young, K.D. (2007) FtsZ directs a second mode of peptidoglycan synthesis in Escherichia coli. J Bacteriol 189 : 5692 5704.
Vollmer, W. Bertsche, U. (2008) Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta 1778 : 1714 1734.
Wang, L. Lutkenhaus, J. (1998) FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response. Mol Microbiol 29 : 731 740.
Weiss, D.S., Chen, J.C., Ghigo, J.M., Boyd, D. Beckwith, J. (1999) Localization of FtsI (PBP3) to the septal ring requires its membrane anchor, the Z ring, FtsA, FtsQ, and FtsL. J Bacteriol 181 : 508 520.
Wientjes, F.B., Olijhoek, T.J., Schwarz, U. Nanninga, N. (1983) Labeling pattern of major penicillin-binding proteins of Escherichia coli during the division cycle. J Bacteriol 153 : 1287 1293.
Wilkin, J.M., Dubus, A., Joris, B. Frere, J.M. (1994) The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase. Biochem J 301 : 477 483.
Woldringh, C.L., Huls, P., Nanninga, N., Pas, E., Taschner, P.E.M. Wientjes, F.B. (1988) Autoradiographic analysis of peptidoglycan synthesis in shape and cell division mutants of Escherichia coli LMC500, p. 66-78.
