Introduction to Microbiology Study Materials

1. what is microbiology: study of life that can't be seen with the naked eye 2. who discovered the existence of microorganisms: Antonie Van Leeuwenhoek 3. what is three domain system: - organisms are grouped using differences in the sequence of nucleotides in the cells' ribosomal RNA, the cells' membrane lipid structure, their sensitivity to antibiotics - Archaea - Bacteria - Eukaryota 4. three domain system: Archaea: - unicellular prokaryotes without peptidoglycan in cell walls - have 70s ribosomes - RNA polymerase of different organisms contains between 8 and 10 protiens. is very similar to eukaryotic ribosome 5. three domain system: bacteria: - unicellular prokaryotes with cell wall containing peptidoglycan - have 70s ribosomes - RNA polymerase contains 5 proteins 6. three domain system: eukaryota: - the eukaryotes: protista, fungi, plantae, animalia - have 80s ribosomes - RNA polymerase (responsible for most mRNA transcription) contains 12 proteins 7. what are prokaryotic cells: - are extremophiles (organisms that live in extreme conditions under high pressure and temp) - found in hydrothermal vents and salt lakes, soil, human digestive system - unicellular - DNA not contained within nucleus - few organelles - organelles not membrane bound 8. prokaryotic DNA: - extensively folded & condensed into an nucleoid with no nuclear membrane - single (haploid) circular chromosome with double stranded DNA which encodes all genes required - genes on chromosomes grouped into operons: can be switched on and off - plasmids: small circular, double stranded DNA molecule distinct from the cell's chromosomal DNA - genes on plasmids provide bacteria with genetic advantages e.g. antibiotic resistance 9. prokaryotic ribosomes: - 70s - aren't present in nucleoid area, they float freely in cytoplasm 1/7 - made from protein and RNA and consist of two subunits - 30s subunit: 16s rRNA and 21 proteins - 50s subunit: 5s and 23s rRNA and 34 proteins - site of translation during protein synthesis 10. prokarytoic cells wall: - rigid, surrounds all bacterial cells except mycoplasmas - gives cell its shape - protects cell from mechanical disruption and from being burst by osmotic pressure, maintaining a high conc of solutes inside the cell 11. why is the cell wall useful for identification: - it can be gram stained - it provides ligands for adherence to host cell receptors and receptor sites for drugs or viruses - provides immunological distinction between bacteria 12. what is gram staining: - technique used to separate bacteria into two groups: gram positive and gram negative - how: crystal violet applied to specimen, then iodine, then washed with alcohol -iodine/crystal violet complexes are retained in peptidoglycan scaffold in gram positive cell wall there is is stained purple - complexes are readily extracted from gram negative wall therefore it is stained pink with counterstain safranin 13. exception of gram staining: - mycobacteria - fails to stain due to unique cell wall structure there an acid fast technique is used instead for the diagnosis of mycobacterium tuberculosis 14. structure of the peptidoglycan cell wall: - polysaccharide made of 2 glucose derivatives: N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) alternated in long chains (linked by 1,4-glycosidic bonds) - the glycosidic bonds aren't strong enough to give rigidity in all direction so the chains are cross-linked to each other by tetrapeptide that extends off the NAM units,a llowing a lattice-like structure to form 15. what is tetrapeptide: - peptide consisting of four amino acids joined by peptide bonds - the four amino acids alternate between L- and D- amino acids: - L-alanine - D-glutamic acid - L-lysine (some gram positives) or meso-diaminopimelic acid (DAP) (all gram negatives and some gram positives) - D-alanine 16. tetrapeptides more: - can be directly linked: the D-alanine on one tetrapeptide is bound to the L-lysine on another tetrapeptide - can be indirectly linked: cross bridge of 5 amino acids such as glycine that connects 2/7 one tetrapeptide to another - cross-links between other tetrapeptides connect the peptidoglycan sheets to form a 3D rigid matric which forms a scaffold like molecule referred toa s the peptidoglycan sac. - the shape of the cell is determined by length of peptidoglycan chains and the manner and extent of cross linking 17. what are the differences between gram negative and gram positive bacterial cell walls: - gram positive: thicker, 20 layers of peptidoglycan (90% of the wall) and teichoic acid - gram negative: multilayered and complex, thin single layer of peptidoglycan (10% of wall) and a periplasm and outer membrane 18. explain teichoic acid in gram positive cell walls: - teichoic acid: acidic polysaccharides. Are either polymers of glycerol phosphate or ribitol phosphate - up to 50% of the wall may be teichoic acid - can either be covalently linked to peptidoglycan or connected to the cell membrane via a lipid anchor -> lipoteichoic acid - generates an overall negative charge, essential for development of a proton motive force (fore that promoted movement of protons across membranes down electrochemical gradient) and helps the bacterium evade phagocytosis 19. what is periplasmic gel: - when some of the peptidoglycan forms a single layered sheet around the cell - the rest, with minimal cross linking, forms a gel like substance -> periplasmic gelo 20. can all substances pass through the cell wall: - peptidoglycan is porous so most substances can pass - some nutrients are too big, so need exoenzymes - exoenzymes: extracellular enzymes made within the cell's cytoplasm and then secreted past the cell membrane, through cell wall, where they function outside of the cell to breakdown large macromolecules into smaller components 21. How much of the gram positive cell wall is composed of peptidoglycan?: peptidoglycan represents 5-20% of total cell wall 22. what is the outer membrane is a gram positive peptidoglycan cell wall: the plasma membrane located outside the peptidoglycan layers - acts as a permeability barrier against most lysozyme, bile salts, digestive enzymes and many antibiotics - made of lipid bilayer - different from cell membrane due to presence of lipopolysaccharide (LPS) anchored into outer membrane and projecting from the cell into the environment 23. what is lipopolysaccharide (LPS) structure and functions: - lipid A: phospholipid containing glucosamine (toxic) rather than glycerol. it anchors the LPS into 3/7 outer membrane. acts as toxin, endotoxin, causing general symptoms of illness e.g. fever, diarrhoea. if a large amount of lipid A is released into the bloodstream it can trigger endotoxic shock - core polysaccharide: contains unusual carb residues - O-antigen polysaccharides: major surface antigen of gram positive galactose, glucose, rhamnose, mannose and unusual dideoxy sugars 24. what is the periplasm and where is it (gram positive): - between outer surface of membrane and inner surface of outer membrane - contains gram negative peptidoglycan - contains proteins: enzymes with hydrolytic functions, antibiotic-inactivating enzymes, various binding proteins with roles in chemiotaxis and active transport of solutes into the cell 25. what are porins: - transmembrane proteins which form a pore across the membrane - some are non-specific and transport any molecules that fits, while some are specific and only transport substance that they recognize 26. how do substances pass through the cell wall structure (gram positive): large molecules broken down by periplasmic enzymes stored in periplasm to get past LPS - nutrients now need to pass outer membrane - porins are used which allows the hydrophilic molecules to diffuse into the periplasm - now molecules go through porous peptidoglycan layers before being transported by integral proteins across the cell membrane 27. how are peptidoglycan layers inked to the outer membrane: - by a lipoprotein named Braun's lipoprotein - at one end the lipoprotein is covalently bound to the peptidoglycan while the other end is embedded into the outer membrane via its polar head - this linkage provides additional structural integrity and strength 28. prokaryotic inclusion bodies: - distinct granules that may occupy a substantial part of the cytoplasm - serve as storage vessels of carbon, phosphorus ... - highly variable - are carbs, lipids, proteins, inorganic substances - not essential for cell survival - are dense, spherical 29. prokaryotic flagellum structure: - whip like extensions that protrude from cells - provide swimming movement for motile bacteria - thinner than eukaryotes 4/7 - energy to rotate supplied from chemiosmosis - attached to cell membrane by a basal body and rotated by a molecular motor 30. prokaryotic flagellum are distributed around the cell differently. how?: monotrichous (one hair) - lophotrichous (tuft of hair) - amphitrichous (hair on both sides) - peritrichous (around the cell) 31. what are the tactic responses of prokaryotic flagellum: - flagella propel the cell by rotating at the point of insertion in the envelope and lalow tactic behaviour: - chemotaxis: motile response to certain chemicals, bacteria are attracted to useful substances or repelled form toxic substances - phototaxis: motile response to light - aerotaxis: motile response to magnetic fields, useful for sourcing ferrous metals 32. what is flagellin: - polymerised molecule of a single protein - makes up flagellar filaments - protein forms the H-antigen of the bacterium often used for serological identification of bacterial strains 33. prokaryotic pili: - molecular hair like projections on surface of cells - composed of molecules of protein called pilin - pilin: arranged to form tube with a small, hollow core - common pili (fimbriae): cover surface of cell and act as adhesions allowing the bacteria to colonise surfaces and cells 34. medically important examples of pili: - neisseria gonorrhoeae: adhered strongly to human cervical or urethral epithelium using lots of pili - streptococcus pyogenes: have pili all around the cell wall that protect it against phagotrophic engulfment - sex pili: involved in exchange of genetic material between mating bacteria 35. prokaryotic endospores: - small, metabolically inert cells with thick cell walls - oval or round in vegetative cells - form when cells are under stress: UV radiation, high temps, strong acids, disinfectant - enclose DNA to help it survive - have no signs of life, are dormant 36. prokaryotic capsules and slime layers: - bacterial cells surround themselves with hydrophilic gel, if the material is defined in shape it is a (true) capsule. if it is amorphous (without a clearly defined shape) it is a slime layer (biofilm) - sticky outermost layer made of polysaccharides - some made of polypeptides - helps cells cling to each other and various surfaces 5/7 - helps prevent cell from drying out - helps trap nutrients - do not contribute to growth and multiplication but can protect bacteria - assist in colonisation: primarily by assisting the cell to attach to surfaces and allow bacteria to form biofilms 37. cell membrane: - made up of phospholipid bilayer with integrated membrane proteins - eukaryotes: membranes contain sterols (cholesterol) - bacterial: membranes contain hopanoids instead 38. where are the integrated proteins on a cell membrane: - confined to one surface - buried within the membrane - spread across the membrane (transmembrane proteins) 39. cell membrane properties: - act as selective permeability layer - regulates movement of substances in and out of cell - for example hydrophilic and charged molecules can't pass through the hydrophobic bilayer - regulates nutrients uptake by active transport mechanisms - extracellular enzymes are secreted from the cell membrane - cell wall components are synthesised in and exported from the cell membrane - energy generating functions take place here 40. what are the 4 stages of peptidoglycan synthesis: 1. autolysins (bacterial enzymes) break the glycosidic bonds between the peptidoglycan monomers and break the peptide cross-bridges - peptidoglycan monomers are synthesized in the cytosol and carried across the cytoplasmic membrane by bactoprenols - transglycosidase enzymes insert and link new peptidoglycan monomers into the breaks in the growing peptidoglycan chains - transpeptidase enzymes reform the peptide cross-links between the rows and layers or peptidoglycan to make the walls strong 41. what is the lag phase in microbial growth in a liquid growth medium: - after inoculation, cells do not multiply during lag phase - the cells are increasing in metabolic activity due to synthesis of enzymes proteins, DNA - may be increase in cell size 42. what is the log phase in microbial growth in a liquid growth medium: - cell population double at regular intervals or grow exponentially depending upon the growth conditions 6/7 43. what is the stationary phase in microbial growth in a liquid growth medium: - when the log phase ceases, there is no net increase or decrease in cell number 44. what is the death phase in microbial growth in a liquid growth medium: the number of viable cells decreases exponentially 45. what is the generation time: - time taken by bacteria to double - varies between 12 mins to 24 hrs - growth calculated by calculating mean generation time 7/7