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Cell Structure and Function Part-2 Microbial Physiology 3 BIO 336-2 Dr. Shahira Hassoubah 1  Objectives Identify the structure and Properties of cell wall. Define peptidoglycan cell wall synthesis, functio...

Cell Structure and Function Part-2 Microbial Physiology 3 BIO 336-2 Dr. Shahira Hassoubah 1  Objectives Identify the structure and Properties of cell wall. Define peptidoglycan cell wall synthesis, function and structure. The bacterial cell wall is often a target for antibiotic treatment. 2  Cell Wall A cell wall is a layer located outside the cell membrane found in plants, fungi, bacteria, algae, and archaea. 3  Eukaryotic Cell Surfaces The cell walls of vascular plants are largely composed of cellulose, a β-1,4-linked polymer of glucose (Fig. 3- 1). Many algae, as well as higher green plants, contain cellulose as a major cell wall constituent. The cell walls of some protozoa and many fungi contain chitin, a linear polymer of N-acetyl glucosamine (Fig. 3- 1). Most fungi, algae, and higher plants contain microfibrils of either cellulose or chitin as a prominent skeletal component of their cell walls. 4 Fig.3-1 5  The yeast, S. cerevisiae, contains a cell wall that appears as a layered structure when viewed under the electron microscope. The chemical composition of the yeast cell wall is rather uniform over most of the cell surface. Chemical analysis of yeast walls reveals the presence of 29% β-glycans (both 1,6-β-glycan and 1,3-β-glycan are found), 31% mannan, and 13% protein (Fig. 3-1) 6  Yeast cell walls also contain small percentages of lipids and other materials. Chitin (poly-N-acetyl glucosamine) is present in small amounts in vegetative cells of yeast. 7 Prokaryotic Cell Surfaces 8 This Photo by Unknown Author is licensed under CC BY-NC-ND  Prokaryotic Cell Surfaces Rigid structure called peptidoglycan formed the major backbone of the murein sacculus of the cell wall of both gram-positive and gram-negative bacteria. Archaea produce a pseudomurein and an associated surface layer (S-layer) composed of protein or glycoprotein. In many archaebacteria the S-layer may represent the only surface component outside the plasma membrane. 9  Prokaryotic Cell Surfaces In B. subtilis the S-layer is associated with the peptidoglycan-containing sacculus. Gram- negative bacteria such as E. coli produce an outer membrane. The S-layer, if produced, is attached to this outer membrane. See page p 291 fig 7-10. 10  Bacterial Structure Bacterial structure. (Reproduced with permission from Ryan K et al. Sherris Medical Microbiology. 4th ed. Copyright 2004, McGraw-Hill.) The important features of each component are presented in Table 2-1 Pg.6 11  The cell wall of Prokaryotes Structure that lies outside the cell membrane in almost all bacteria. functions: 1. Maintains characteristic shape of cell. 2. Prevents the cell from bursting when fluids flow into the cell by osmosis. 3- Contributes to bacterial ability to cause disease (pathogenicity) 4- Site of action of some antibiotics. 5- Very porous and does not regulate passage of materials into the cell. 12 This Photo by Unknown Author is licensed The cell wall of Prokaryotes: Peptidoglycan and Related Molecules Peptidoglycan is one rigid layer that is primarily responsible for the strength of the wall. The rigid layer of both Gram-negative and Gram-positive bacteria is very similar in chemical composition. 13 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules peptidoglycan (or murein) composed of two sugar derivatives, - N-acetylglucosamine (NAG) - N-acetylmuramic acid (NAM) A small group of amino acids consisting of L-alanine, D-glutamic acid, D-alanine, and either lysine or diaminopimelic acid (DAP). These constituents are connected to form a repeating structure, the glycan tetrapeptide. 14 Peptidoglycan Fig 3-2 15 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules The glycosidic bonds connecting the sugars in the glycan chains are very strong, but these chain alone cannot provide rigidity in all directions. The full strength of the peptidoglycan structure is realized only when these chains are cross-linked by amino acids. 16  This cross-linking occurs to characteristically different extents in different bacteria with greater rigidity coming from more complete crosslinking. In Gram-negative bacteria, cross-linkage usually occurs by direct peptide Iinkage of the amino group of diaminopimelic acid to the carboxyl group of the terminal D-alanine. The Gram-positive bacteria cross-linkage is usually by a peptide interbridge, the kinds and numbers of cross-linking amino acids varying from organism to organism. 17  In Staphylococcus aureus, a gram-positive organism, each interbridge peptide consists of five molecules of the amino acid glycine connected by peptide bonds. 18 19 Gram positive Fig 3-3 20 Fig 3-4 21 Fig 3-5 22 Fig 3-6 23 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules In gram-positive Bacteria, as much as 90% of the cell wall consists of peptidoglycan, although another kind of constituent, teichoic acid is usually present in small amounts. In gram-negative Bacteria about 10% of the wall is peptidoglycan, the majority of the wall consisting of a complex layer. 24  The cell wall of Prokaryotes: Peptidoglycan and Related Molecules However, the shape of both gram-positive and gram- negative cells is thought to be determined by the lengths of the peptidoglycan chains and by the manner and extent of cross- linking of the chains. Peptidoglycan is present only in bacteria; the sugar N-acetylmuramic acid and the amino acid diaminopimelic acid, (DAP) are never found in the cell walls of Archaea or Eukaryotes. However, not all bacteria have DAP in their peptidoglycan. 25 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules Several generalizations regarding peptidoglycan structure can be made. The glycan portion is uniform, with only the sugars N-acetylglucosamine and N- acetylmuramic acid being present, and these sugars are always connected in -1,4 linkage. 26 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules The tetrapeptide of the repeating unit shows major variation only in one amino acid, the lysine-diaminopimelic acid alternation. However, the D-glutamic acid at position 2 can be hydroxylated in some organisms, whereas substitutions occur in amino acids at positions 1 and 3 in a few others. 27 The cell wall of Prokaryotes: Peptidoglycan and Related Molecules More than 100 different peptidogIycan types are known and the greatest variation among them occurs in the interbridge. Any of the amino acids present in the tetrapeptide can also occur in the interbridge. But, in addition, a number of other amino acids can be found there, such as glycine, threonine, serine, and aspartic acid. However, certain amino acids are never found in the interbridge: branched-chain amino acids, aromatic amino acids, sulfur-containing amino acids, histidine, arginine, and proline. 28  Note: The shape of both gram-positive and gram-negative cells is thought to be determined by the lengths of the peptidoglycan chains and by the manner and extent of cross- linking of the chains. Peptidoglycan is present only in bacteria; the sugar N-acetylmuramic acid and the amino acid diaminopimelic acid, (DAP) are never found in the cell walls of Archaea or Eukaryotes. However, not all bacteria have DAP in their peptidoglycan. Because peptidoglycan is present in bacteria but not in human cells, it is a good target for antibacterial drugs. Several of these drugs, such as penicillin , cephalosporins, and vancomycin, inhibit the synthesis of peptidoglycan by inhibiting the transpeptidase that makes the cross-links between the two adjacent tetrapeptides  Peptidoglycan (Murein) Hydrolases Almost all bacteria produce peptidoglycan (murein) hydrolases — enzymes that hydrolyze bonds in the peptidoglycan structure. These enzymes are of three basic types: Glycan-strand hydrolyzing a. Endo-N-acetylmuramidases b. Endo-N-acetylglucosaminidases Endopeptidase hydrolyzing a. Peptide bonds in the interior of the peptide bridges b. Bonds involving the C-terminal D-alanine residue N-acetylmuramoyl-L-alanineamidase Acting a the junction between the glycan strands and the peptide units 30 31 Peptidoglycan (Murein) Hydrolases These enzymes appear to play an important role in a number of cellular activities including septum and wall extension during cell growth, cell separation, turnover of wall components, sporulation, competency for transformation, and the excretion of toxins and exoenzymes. 32  Peptidoglycan (Murein) Hydrolases Activation of certain of these hydrolases, particularly under conditions of external stress, may result in cellular autolysis. In fact, it is now considered that triggering of murein hydrolases or other autolysins by environmental stress is responsible for programmed cell death (sometimes referred to as apoptosis). 33  Peptidoglycan (Murein) Synthesis Three stages: Cytoplasmic stage Synthesis of precursors (NAG and NAM) Membrane stage Transfer of precursors from cytosol to membrane and incorporation into the growing peptidoglycan Extracellular stage (crosslinking) Crosslinking of linear chain of peptidoglycan by membrane bound transpeptidases https://www.youtube.com/watch?v=2T3- https://www.youtube.com/watch?v=fJ3pVJoJD Umt62E8 v8 34  Peptidoglycan (Murein) Synthesis Seven genes in E.coli (murC, murD, murE, ddl, murF, mraY, murG) participate in the pathway of peptidoglycan synthesis from UDP-GlcNAc to the formation of the C55-prenol intermediate undecaprenyl-PP- MurNAc- pentapeptide. 35  Other genes also participate in murein synthesis. For example, the E. coli enzyme UDP- N- acetylglucosamine enolpyruvate transferase (reaction 4 in Fig. 3.7) catalyzes the first committed step in peptidoglycan formation. This enzyme (encoded by murZ ) is inhibited by the bactericidal antibiotic phosphomycin (L-cis-1,2- epoxypropylphosphonic acid; phosphonomycin), a structural analog of phosphoenolpyruvate: 36 Fig 3-7 37  What are the general steps of peptidoglycan biosyntehsis? 1) Inside the cell the precursor is synthesized 2) transport across the plasma membrane 3) final assembly and cross linkage 38  What are the two antibiotics that work on step one of the peptidoglycan synthesis cascade? Phosphonomycin Cycloserine 39  Where does Phosphonomycin work in the peptidoglycan synthesis cascade? Inhibiting UDP muramic acid biosyntehsis 40 41