Prokaryotic Cell Structures Lecture Notes PDF

Prokaryotic cell structures Cell envelope: include cytoplasmic membrane, cell wall & if present, glycocalyx/capsule. Cytoplasmic membrane: barrier between the external & interior environment of the cell. Check Table 3.3 for summary Cel...

Prokaryotic cell structures Cell envelope: include cytoplasmic membrane, cell wall & if present, glycocalyx/capsule. Cytoplasmic membrane: barrier between the external & interior environment of the cell. Check Table 3.3 for summary Cell Wall: A rigid barrier that keeps the cell contents Cytoplasm: Viscous fluid made from bursting out. of variety of substances  Nutrients Glycocalyx/capsule: layer  Ribosomes outside the cell wall.  Enzymes Appendages: important for Nucleoid: gel-like region where motility & adherence the chromosome resides. –Flagellum –Pili Unlike eukaryotic nucleus, the nucleoid is not enclosed within a membrane.  Cytoplasmic membrane Defines the boundary of the cells & consists of a phospholipid bilayer. Contains embedded membrane proteins which constantly move around (Fluid mosaic model). Regulates the movement of proteins & other molecules (ions) into & out of the cell. Selectively permeable: only few types of molecules can pass thoroughly. Movement of molecules across membrane is regulated by  Simple diffusion and osmosis: No energy is expended.  Directed movement of molecules by Active transport: energy is used.  Cell wall Unlike the cytoplasmic membrane, it is a rigid structure and maintains the integrity of the cell. Determines the shape of the organism. Cell wall architecture and differences in its arrangement distinguishes Gram negative and Gram-positive bacteria. Contains certain unique structures & molecules and some of which are recognized by immune system as the sign of invaders. Antimicrobial agents target some of these structures. Peptidoglycan Only found in bacterial cell wall. Polymer of NAM & NAG. Its structure contributes to the differences between Gram +ve & -ve bacteria. Major target for antibiotics.  Gram positive cell wall Thick layer of peptidoglycan (~30 layers of glycan chains). Permeable to many substances (sugars & amino acids). Composed of peptidoglycan & teichoic acid Some also contain lipoteichoic acids which are attached to cytoplasmic membrane. Both teichoic and lipoteichoic acids stick out above the peptidoglyccan layer and give the cell its negative polarity. A gel like structure called periplasm is present in some Gram-positive bacteria. Gram negative cell wall More complex than Gram positive cell wall. Thin layer of peptidoglycan. Periplasm: filled with a gel-like fluid which contains proteins involved in a variety of cellular activities.  Gram negative cell wall Outer membrane: –a unique lipid bilayer with many proteins. –The outside leaflet is made up of lipopolysaccharides (LPS). –Protective barrier to passage of most molecules & thus Gram -ve bacteria are less sensitive to many medications. This layer is joined to peptidoglycan layer through lipoprotein molecules. Also have some porin proteins. -  The lipopolysaccharide (LPS) Extremely important from a medical standpoint. Also called endotoxin to reflect its toxic activity. When injected into an animal, it elicits symptoms characteristic of infections caused by live Gram -ve bacteria.  Structure of LPS Two parts are particularly notable 1. Lipid A: – anchors the LPS molecule in the lipid bilayer. – recognized by immune system as invader & is responsible for symptoms associated with endotoxin. 2. O-specific polysaccharide side chain or O antigen: made up of a chain of sugar molecules. Number & composition of chain varies among different bacterial species & is used to identify certain species or strains. For example, the ‘O157’ in E. coli O157:H7 refers to that particular strain’s characteristic O-side chain or O antigen.  Antibacterial Compounds that target peptidoglycan Differences between prokaryotic and eukaryotic cells allow the development of agents targeting life processes ONLY PRESENT in bacteria. Some compounds interfere with the synthesis or alter the structural integrity of peptidoglycan. This weakens peptidoglycan rigid molecule such that it is not strong enough to prevent the cell from bursting.  Antibacterial compounds that target peptidoglycan Penicillin – interferes with peptidoglycan synthesis. – binds to proteins/enzymes involved in cell wall synthesis & prevents cross-linking of adjacent glycan chains by inhibiting ‘peptide interbridge’ formation. – far more effective against Gram-positive cells than Gram-negative cells as outer membrane prevents it from reaching to peptidoglycan layer.  Antibacterial Compounds that target peptidoglycan Lysozyme: – An enzyme found in bodily secretions including tears and saliva. – breaks the bonds that links the alternating NAM and NAG of peptidoglycans and thus destroy the structural integrity of glycan chain. – Outer membrane of gram-Ve bacteria prevents the enzyme from reaching to peptidoglycan layer; thus, lysozyme is generally more effective against Gram +ve bacteria.  Bacteria that lack a cell wall Example: Mycoplasma Species of Mycoplasma have extremely variable shapes as they lack a rigid cell wall. Some Mycoplasma cause a mild form of pneumonia. Neither penicillin nor lysozyme effects these organisms. Mycoplasma & other bacteria can survive without cell wall as their cell membrane is stronger than that of other bacteria. They have sterols in their membrane which make them stronger. Comparison of Gram-positive and Gram- negative bacteria  Structures outside the bacterial cell wall Capsules and Slime Layers Many bacteria have a gel-like layer outside the cell wall that either protects the cell or allows it to attach. If the layer is distinct & gelatinous, it is a capsule (Fig a). But if the layer is diffuse & irregular, it is a slime layer (Fig b). Colonies of bacteria that form capsule & slime layer often appear Some bacteria cause moist & glistening. diseases only if have capsule e.g. Important in adherence & Streptococcus pneumoniae. pathogenesis & often enable microbes to grow as a biofilm e.g. Unencapsulated cells are dental plaque caused by quickly engulfed & killed by Streptococcus mutans. phagocytes.  Other surface layers/structures Some bacteria have filamentous protein appendages that are anchored in the membrane & protrude out from the surface e.g. Flagellum/Flagella: A long protein structure generally associated with motility. May be involved in pathogenesis e.g. Helicobacter pylori Filament of flagellum Peritrichous flagella is composted of flagellin. Polar flagellum Flagellum in Gram-negative bacterium.  Other surface layers/structures Pili: Shorter and thinner than flagella and are used for motility and adherence. Some pili enable attachment of cells to specific surfaces and are called fimbriae. Fimbriae are important in pathogenesis of enterotoxigenic E. coli. Another type of pilus called ‘sex pilus’ is involved in conjugation (mechanical transfer of DNA from one bacterial cell to another) e.g. F pilus of E. coli.  Internal components of bacterial cell Nuclear Material Chromosome: – Typically, a single, circular, tightly packed, double stranded supercoiled DNA molecule that contains all the genetic information required by a cell. – It resides in nucleoid. Plasmids: – are extra-chromosomal DNA containing additional genetic material. – Most plasmids are circular double-stranded DNA molecules & carry from a few to several hundred genes. – A single cell can carry multiple types of plasmids.  Plasmids contd.. A cell generally does not require the genetic information carried by a plasmid, but encoded information may be advantageous in certain situations. Some plasmids can code enzymes which can degrade antibiotics allowing them to resist effects of antibiotics. Antibiotics resistance can be spread from one bacterium to another through transfer of plasmids. Important in genetic technology & pathogenesis of bacteria (particularly in the area of antibiotic resistance).  Ribosomes Are organelle involved in protein synthesis (this is where translation takes place). Each ribosome is composed of a large and small subunits, which are made up of ribosomal proteins and ribosomal RNAs. – Prokaryotic ribosomes are 70S (svedberg) in size & are composed of 30S & 50S subunits. – Eukaryotic ribosomes are 80S. Differences in the structures of ribosomes serve as targets for certain antibiotics which preferentially bind to 70S ribosomes and prevent protein synthesis in bacteria.  Endospores Unique type of dormant bacterial cells, produced by a process called sporulation, when conditions inhospitable to life develop. May remain dormant for years and are extremely resistant to heat, desiccation, toxic chemicals and UV irradiation etc. Under favorable conditions, endospores germinate to become a typical, actively multiplying cell, called a vegetative cell.  Endospores Cause some medically important diseases. – Tetanus caused by Clostridium tetani – Botulism caused by C. botulinum – Gas gangrene by C. perfringens – Anthrax caused by Bacillus anthracis  Methods to observe bacterial cells It is difficult to observe living microorganisms in the bright- field as mostly transparent and move rapidly. Cells are usually immobilized and stained with dyes. Stains/dyes provide contrast between bacteria & surrounding media (Refractive index differences). If only a single dye is used to stain a specimen, the procedure is called simple staining.  Dyes and Staining techniques Basic dyes: – Carry positive charge & are more commonly used. – Stain many negatively charged component of cells e.g. nucleic acid & many proteins. Examples: methylene blue, crystal violet, safranin & malachite green. – Simple staining uses one of these basic dyes for staining. Acidic dyes: are sometime used to stain backgrounds against which colorless cells can be observed and technique is called ‘negative staining’.  Differential staining Differential staining is used to distinguish one group of bacteria from another. Different bacteria have certain fundamental chemical differences in their cell walls & this is the basis of differential staining. Two most frequently used differential staining methods: 1. Gram staining 2. Acid-Fast staining Gram staining : Most widely used procedure for staining bacteria and developed by Dr. Hans Christian Gram. Based on this staining procedure, bacteria can be separated into two major groups: – Gram positive – Gram negative.  Steps in Gram staining Precautions in Gram staining: 1. Do not decolorize smear for too long otherwise even Gram-positive cell will appear pink after counterstaining. 2. Do not use old cultures as they lose their ability to retain crystal violet-iodine dye complex and even Gram-positive cultures may appear pink. Cell wall type & the Gram stain  Acid-Fast stain A multi step staining procedure used to stain organisms that do not readily take up stains. Cell wall of these organisms contain high amounts of lipids which prevents the uptake of dyes including those used for Gram staining. Primary stain in this procedure is ‘Carbol fuschsin’, a red dye and ‘methylene blue’ is used as a counter-stain. Members from genus Mycobacterium, that cause tuberculosis (M. tuberculosis) & leprosy (M. leprae) are acid-fast organisms. Once stained these cells are very resistant to decolorization by acid- alcohol & thus called acid-fast.  Fluorescent dyes and tags Fluorescence can be used to observe – total cells, – a subset of cells (live or dead) or – cells with certain proteins on their surface. Some dyes bind to compounds found in all cells e.g. – Acridine orange binds DNA & is used to determine the total number of cells. It does not discriminate between live and dead cells.  Fluorescent dyes and tags Certain dyes can be used to detect live cells only e.g. CTC dye is made fluorescent by cellular proteins involved in respiration & only fluoresces when bound to viable cells. Certain dyes when bind to live cells give green fluorescence while give red florescence upon binding to dead cells e.g. Bac light. Green –live cells Red –dead cells Calcofluor white binds to fungi and certain bacterial cell wall components and cause them to fluoresce bright blue. Fig. 3.8  Fluorescent dyes and tags Fluorescence dyes auramine and rhodamine bind to the mycolic acids found in cell walls of members of genus Mycobacterium so can be used analogous to acid-fast stain. Cells of Mycobacterium emit a bright yellow fluorescence upon binding to auramine. Immunofluorescence: used to detect specific microbes/cell components using fluorescence tags antibodies. Immunoflu Auramine orescence  Next lecture Read chapter 4 (Selected topics)