Lesson #2.1: Eukaryotes and Prokaryotes

[Virtual Presenter] Lesson 3.4 Structure, Function and Synthesis of Bacterial Surface Components, Appendages, and Internal Organs Lotis M. Balala College Of Veterinary Medicine, Visayas State University.

[Audio] Learning Outcomes 1. Introduce the structures of bacteria including surface components, appendages and internal organs. 2. Discuss the synthesis and functions of these bacterial structures. 3. Illustrate the location in the bacteria expressing the structures..

[Audio] Prokaryotes pro = before, karyon = nucleus Eukaryotes eu = true, karyon = nucleus Madigan et al., 2003.

[Audio] Prokaryotic Cell Madigan et al., 2003. Prokaryotic Cell Madigan et al., 2003.

[Audio] Cell Wall  Layer that is usually fairly rigid that lies outside the plasma membrane  One of the most important structures because  confers shape  protects the cell from osmotic lysis  anchors the flagellum  adds to pathogenicity of the cell  protects the cell from toxic substances and pathogens  used in identification  Bacteria can be divided into two big groups based on cell wall structure Gram positive Gram negative.

[Audio] Gram Positive Cell Wall Characteristics A. Thick layer of peptidoglycan  peptidoglycan (PG); a.k.a. murein; mucopeptide  a polymer of disaccharide linked by polypeptide  insoluble, porous, big polymer  > 50% of the cell wall's dry weight  isolatable as murein sacculus.

Table 2- Amino acid variations in peptide stem (Vollmer et al-, 2008)- Position 1 2 3 Residæ encountered Gly I-Ser D-lsoglutarnate D-lsoglutamine• thno.3.Hy&axygIutamate• meso-Apprn I-Lys I-Orn t-LyML-Orn t -Lyfi-Lys LL.A2prn meso-La n thionine 1-2.4- Dtarnin±uty rate I-HornaEtine I-Glu Amidated meso-Aa prn• 2.6Dianno.3hydroxyphIate' L-5-Hydraxybinet N'4cetyl+2,a-d nabu$ate• D-Ser Examples Most spcies inperiaje 8Gtyribcteäum rettgeri Most Grarn-mgatöæ »eciB Most Grarn-positive species. %obacteria Micro&ctMum Iacöctrn r.•lost Grarn-rpgatræ speciz. Baalli, Myc±acteria Most Grarn-p<tive merrnLß therrno@'i.rus Edd±acteäum maritim Streptomycs albus. Propima»cterium n LRkaturn awafcurn pomseajae Eryopekgl-tå HI Lßig'a±sae J. 39 Bacillt$ wbüfis AmpuraFeNa regdarb Jrzk*osurn All Most bactena Enteræoccus LactÖacNus casei. Enterococö with acqüred resistance to vancorrycin *These residæs result from reactims occurring posterior the action of Mur ligases. 'The of fTrruban af tyese residuæs (diret b,' MurÉ or lydroxylaton of the nonhydroxylated rztdue) undear (Mui•oz 1966; Perkins, 1969). aln this a I O : I ratio of Vie to tydroxyqysire et al. (1969-.

[Audio] B.PresenceofTeichoicAcids polymersofrepeatingunitsofglycerolorribitoljoinedbyphosphates aminoacids(D-ala)orsugars(glu)areattachedtoglycerol/ribitol covalentlylinkedtomureinthroughmuramicacid connected/embeddedinPGlayerortomembranelipids.

[Audio] Properties/functions of teichoic acids 1. highly antigenic 2. anchor the wall to the cell membrane 3. provide high density of regularly oriented charges 4. storage of phosphorus 5. facilitate attachment of bacteriophage 6. inhibit activity of autolytic enzymes which hydrolyze the murein lipoteichoic acid -linear polymers of 16-40 phosphodiester-linked glycerophosphate residues covalently linked to the cell membrane.

[Audio] Other substances which may be found in the cell wall:  Teichuronic acids – acidic polysaccharides containing uronic acids  Neutral polysaccharides – important in classification of some gram positive bacteria  Mycolic acids – waxy lipids found in Mycobacterium.

[Audio] Substances active against peptidoglycan synthesis: 1. Phosphonomycin (Fosfonomycin) - prevents synthesis of UDP-NAM from UDP-NAG 2. Cycloserine - inhibits the formation of the pentapeptide 3. Bacitracin - inhibits incorporation of lysine into the peptidoglycan - prevents dephosphorylation of the carrier lipid 4. Vancomycin, Tunicamycin, Ristocetin - inhibits translocation step of peptidoglycan 5. Penicillin - prevents cross-linking.

[Audio] Localization of cell wall synthesis Incorporation of new cell wall in differently shaped bacteria. Rod-shaped bacteria such as B. subtilis or E. coli have two modes of cell wall synthesis: new peptidoglycan is inserted along a helical path (A), leading to elongation of the lateral wall, and is inserted in a closing ring around the future division site, leading to the formation of the division septum (B). S. pneumoniae cells have the shape of a rugby ball and elongate by inserting new cell wall material at the so called equatorial rings (A), which correspond to an outgrowth of the cell wall that encircles the cell. An initial ring is duplicated, and the two resultant rings are progressively separated, marking the future division sites of the daughter cells. The division septum is then synthesized in the middle of the cell (B). Round cells such as S. aureus do not seem to have an elongation mode of cell wall synthesis. Instead, new peptidoglycan is inserted only at the division septum (B). Elongation-associated growth is indicated in red, and division-associated growth is indicated in green. doi: 10.1128/MMBR.69.4.585-607.2005.

[Audio] Gram Negative Cell Wall 1.Peptidoglycan - thin: 1-2 layers in E. coli - constitute not more than 5 – 10% of wall's dry weight - may be more of a gel than a compact layer 2. Outer membrane - located above/external to peptidoglycan layer - like the cytoplasmic membrane - other main components - a. lipopolysaccharides (LPS) - b. lipoproteins - c. outer membrane proteins (porins).

[Audio] Gram Negative cell wall. Gram Negative cell wall.

[Audio] A. Lipopolysaccharides/endotoxin - lipids and carbohydrates - outer layer of the outer membrane Endotoxin consists of three parts: Lipid A - embedded in the membrane as part of the lipid bilayer - hydrophobic - composed of 2 glucosamine residues linked β-1,6 (backbone) with saturated fatty acids.

[Audio] Core Region - consists of an outer and inner core Outer core - shows high to moderate variability - consists of hexoses Inner core - shows low structural variability - consists of 2-keto-3-deoxyoctonate (KDO), heptose, ethanolamine and phosphate.

[Audio] O-antigen - short polysaccharide extending outward from the core - consists of peculiar sugars which varies between bacterial strains - not essential for viability.

[Audio] Importance of LPS: avoidance of host defenses (O-antigen) contributes to the negative charge on the cell's surface stabilizes membrane structure acts as endotoxin B. Lipoproteins (Braun's Lipoprotein) - mediate interconnection between the OM and murein - synthesized within the cell and contains a leader sequence of ~ 20 amino acids at its amino terminal end - facilitates integration into the OM.

[Audio] C. Porins - form small hydrophilic channels through the outer envelope allowing the diffusion of neutral and charged solutes of MW <600 daltons.

[Audio] Importance of the OM: proteins in OM are used as attachment sites by bacteriophages permeability barrier to heavy metals, lipid-disrupting agents and larger molecules outer surface with strong negative charge is important in evading phagocytosis highly variable O-antigen reduces complement binding O-antigen provides host with multiple antigenic structures in various strains LPS complex is a bacterial endotoxin causing a variety of pathophysiological reactions ranging from fever to death may be involved in adherence.

[Audio] Periplasm - A separate compartment between the cell membrane and outer membrane in Gram (-) bacteria seen in electron micrographs as space but should be considered an aqueous compartment activities: redox reactions osmotic regulation solute transport protein secretion hydrolysis.

[Audio] Synthesis of LPS Lipid A - synthesized from UDP-NAG (uridine diphosphate-Nacetylglucosamine) made in the CM by peripheral membrane proteins Roles of Lipid A: 1. primer 2. vehicle for transport of the core-lipid A to the periplasm where the LPS is completed by attachment of O-antigen Core - synthesized on Lipid A inner core: synthesis is not fully understood outer core: each sugar is added one at a time to Lipid A O-antigen synthesized on a separate lipid carrier (undecaprenyl PO4) completed oligosaccharide chain is transferred to Lipid A-core.

[Audio] Archaeal cell wall  lacks peptidoglycan  typically lack an outer membrane  exhibit different cell wall profiles  most common type possesses a surface layer or "S" layer S layer: paracrystalline surface layer consists of interlocking molecules of protein or glycoprotein Konig et al., 2007 S layer subunits Madigan et al., 2015.

Cell wall profiles Jcthar:othermuf, PM. GG Methanosphaera. Konig etal.„ 2007 S layer Pesqxionvrcio Abets and Meyer, 2011.

[Audio] Cellwall-lessbacteriamanyareparasiticandpathogeniccontainunusuallytoughcytoplasmicmembraneduetothepresenceofsterolssomecontainlypoglycanswhicharecovalentlylinkedtomembranelipidsembeddedintheplasmamembrane Mycoplasmaspp..

[Audio] Other cell surface structures Glycocalyx general term for the thick cover or layer of polymers deposited outside the cell Capsule - well organized glycocalyx - attached firmly to the cell wall - compact - excludes particles like India ink Slime layer - zone of diffused, unorganized material - loose association - does not exclude particles.

[Audio] Importance: exclude viruses and most hydrophobic toxic substances protection from physical injury aid attachment to surfaces provide resistance to phagocytes reservoir of stored food prevent desiccation confers pathogenicity "cellular garbage dump" antigenicity.

[Audio] Capsule synthesis: Exopolysaccharide capsules a. from sugar nucleotide precursors - synthesis of nucleoside diphosphate-sugar precursors - formation of polysaccharide chain involving successive transfers of the glycosyl residues via a lipid carrier in the cell membrane - translocation across the membrane b. from external sugar source: Sucrose - involves successive addition of glycosyl units to an acceptor molecule of sucrose - no expenditure of energy - cell elongation occurs by transglycosylation Sugars are synthesized by the cell through the normal processes of intermediary metabolism..

(a) FIGURE 6.32 (b) The formation of extracellular polysaccharides by bacteria. Two plates of Leuconostoc mesenteroides, streaked on glucose medium (a) and sucrose medium (b) The large size and mucoid appearance of the colonies on sucrose are caused by the massive synthesis and deposition around the cells of dextran..

[Audio] Pili -hair-likestructuresonthesurfacesofprokaryoticcell -composedofproteinsub-unitscalled"pilins" -sometimesreferredtoas"fimbriae" Synthesisofpili 1.Chaperone-usherpathway 2.Sortase-mediatedassembly 3.ATP-mediatedassembly.

[Audio] Inclusions - distinct bodies that may occupy a substantial part of the cytoplasm - may be organic or inorganic - some inclusion bodies lie free in the cytoplasm or enclosed by a shell consisting of proteins or a membranous structure composed of proteins and phospholipids - usually used for storage 1. Granules and globules a. glycogen – a polymer of glucose units composed of long chains formed by α(1→ 4) glycosidic bonds and branching chains connected to them by α(1→ 6) glycosidic bonds b. Poly-β-hydroxybutyric acid (PHB) – contains β-hydroxybutyrate molecules joined by ester bonds.

[Audio] Polyphosphate granules (volutin or metachromatic granules) - linear polymer of orthophosphates joined by ester bonds Functions: a.energy source and ATP substitute b.source of phosphate c. chelators of various metal ions d.serve as internal buffers against cations in alkaline environments e.form channels for DNA entry f. as regulators in response to environmental stresses g.function in developmental changes in microorganisms.

[Audio] Cyanophycin granules - composed of large polypeptides containing approximately equal amounts of amino acids arginine and aspartic acid 2. Carboxysomes - polyhedral bodies about 120 nm in diameter - contain the enzyme ribulose-1,5-bisphosphate carboxylase - may serve as site for CO2 fixation.

[Audio] 3. Sulfur globules - used to store sulfur temporarily - accumulate in the periplasmic space of in special cytoplasmic globules 4. Gas vesicles - small, hollow, cylindrical structures composed entirely of a single small protein - impermeable to water but freely permeable to gases.

[Audio] 5. Magnetosomes intracellular chains of magnetite (Fe3O4), gregrite (Fe3S4) and pyrite (FeS2) particles bounded by a membrane aids in orienting bacteria in the earth's magnetic field.

Some inclusions in bacterial cells Cytoplasmic mcluslons glycogen polybetahydroxybutytic acid (PHB) polyphosphate (volutin gtanules) sulfur globules gas vesicles paraswal crystals magnetosomes carboxysomes phycobilisomes chlorosomes Where found many bacteia e.g. E coir many bactetia e.g. Pseudomonas many bactetia e.g. phototrophic purple and green sulfur bacteäa and lithotrophic colodess sulfur bactefia aquatic bactena especially cyanobactelia endospore-forming bacilli (genus Bacillus) certain aquatic bacteria many autotrophic bacteria cyanObacte1ia Green bacteria Composition polyglucose polymerized hydroxy linear or qclical polymers of P04 elemental sulfur protein hulls or shells inflated with gases protein magnetite (iron oxide) Fe304 enzymes for autotrophic C02 fixation phycobifiptoteins lipid and protein and bactetiochlorophyll Function resetve catbon and energy source resenre catbon and energy source resetve phosphate; possibly a resewe of high energy phosphate resetve of electrons (reducing source) in phototrophs; teserve energy source in Vithotrophs buoyancy (floatation) in the vertical water column unknown but toxic to cerein insects orienting and migtating along geo- magnetic field fines site of C02 fixation fight-harvesting pigments tight-harvesting pigments and antennae.

[Audio] BacterialEndospore atypeofrestingstructureformedbysomegroupsofbacteria cryptobiotic highlyresistanttoenvironmentalstresses formedbyvegetativecellsinresponsetoenvironmentalsignals amechanismofsurvival usedinclassificationandidentificationofbacteria considershape,locationandabilitytoswellthesporangium.

[Audio] Endospore staining Madigan et al., 2012. Endospore staining Madigan et al., 2012.

[Audio] Parts: exosporium - thin, delicate covering surrounding the spore spore coat - composed of several spore-specific protein layers cortex - made of peptidoglycan core wall - surrounds the protoplast spore core - contains normal cell structures.

[Audio] Sporogenesis - commences when growth ceases - involves a complex series of events in cellular differentiation Stage of spore formation Willey et al., 2008.

[Audio] Cytoplasmic membrane - absolute requirement of all living organisms chief point of contact with the environment Functions: 1. Transport of nutrients 2. Location of a variety of crucial metabolic processes synthesis of membrane lipids wall murein synthesis assembly and secretion of extracytoplasmic proteins respiratory electron transport 3. Chromosome segregation 4. Establishment of electrochemical gradient 5. Motility 6. ATP synthesis 7. Intercellular signaling 8. Respond to environmental signals.

[Audio] Functions: Madigan et al., 2012. Functions: Madigan et al., 2012.

[Audio] Composition: 1. Phospholipids - most membrane associated lipids are structurally asymmetric 2. Proteins - two types of membrane proteins have been identified a. Integral proteins - embedded in the CM - amphipathic - bound to the fatty acids of the phospholipids via hydrophobic bonding b. Peripheral proteins - attached to membrane surfaces by ionic interactions 3. Hopanoids - rigid, planar molecules found associated with bacterial CM molecules similar to sterol.

[Audio] Fluid Mosaic Model - most widely accepted model of the CM - shows that the CM is a lipid bilayer with which proteins and lipids "float" freely.

[Audio] Intracytoplasmic membrane/Cytomembranes - connected to the plasma membrane - found in methanotrophs, nitrosifiers, nitrifiers, phototrophs, and nitrogen fixers Felter et al., 1970.

[Audio] The Cytoplasm consists of aqueous solution of three groups of molecules macromolecules small organic molecules various inorganic ions and ribosomes structural components: nucleoid ribosomes inclusion bodies Nitrosomonas eutropha DOI 10.1007/s00203-005-0074-4.

[Audio] Inorganic ions present in the cytoplasm of a growing bacterial cell.

[Audio] Small molecules present in the cytoplasm of a growing bacterial cell in glucose minimal medium.

[Audio] Prokaryotic Cytoskeleton - cytoskeletal filaments are structurally similar to their eukaryotic counterparts MreB – cell shape Cresentin – cell shape FtsZ – cell division Madigan et al., 2012.

[Audio] Ribosomes - complex structures made of both protein and ribonucleic acid - present in the cytoplasmic matrix or loosely attached to the plasma membrane - site of protein synthesis - composition: 52 different proteins → 3 RNA (23S, 16S and 5S).