Microbiology - Bacteriology Exam Notes PDF

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These are notes that were gathered from Helen's lectures on bacteriology and cover most things needed for the exam. ...

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MICROBIOLOGY – Helen P – Bacteriology Bacterial Cell Structure Cell Membranes

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Bacterial can be divided into 2 major groups: o Gram-positive bacteria  Single type of molecule  Thick structure o Gram-negative bacteria  Multi-layer complex

Classical bilayer membrane structure – 8nm thick Vital barrier Non-essential  chromosome Essential  plasmid Composed of phospholipids and proteins In E. coli, 7 phospholipids and about 200 proteins have been identified Most prokaryotes do not have sterols (stabilizers) present unlike eukaryotes, some exceptions such as mycoplasmas Prokaryotes have hopanoids instead of sterols

Bacterial Cell Wall - Bacterial cytoplasm has a high concentration of dissolved salts – a turgor pressure of 2 atm in E. coli - To withstand pressure, bacteria have cell walls to protect barrier against o Osmotic pressure changes o Other environmental stresses - Cell walls also give shape and rigidity - Cell wall + membrane is called cell envelope

Figure 1 - Cell envelope

MICROBIOLOGY – Helen P – Bacteriology Peptidoglycan (murein) in Bacteria - Rigid layer that is primarily responsible for the strength of the cell wall - Layers build into complex meshwork structure - Chemically similar between gram +ve and –ve cells - Each layer making up peptidoglycan is a thin sheet composed of 2 sugar derivatives o N-acetylglucosamine and N-acetylmuramic acid - And a number of amino acids o L-alanine, D-alanine, D-glutamic acid, and either lysine or diaminopimelic acid

Peptidoglycan structure - Basic structure is a sheet of Glycan chains connected by peptide crosslinks formed by the amino acids - The glycan chains are very strong due to the glycosidic bonds but chains alone are not rigid in all directions - Cross-linking by the amino acids gives full strength and rigidity - Extent of cross-linking depends on the bacterium – the more crosslinking the more rigidity - Gram-negative cross-linking is usually direct peptide linkage of the amino group of diaminopimelic acid to the carboxyl group of terminal D-alanine - Gram-positive cross-linkage is usually by a peptide interbridge

MICROBIOLOGY – Helen P – Bacteriology - The kinds and numbers of cross-linking amino acids varies depending upon the species o E.g. Staphylococcus aureus’ each interbridge consists of 5 glycines connected by peptide bonds - In G+ve cells, up to 90% of cell wall is peptidoglycan - In G-ve cells, only about 10% of cell wall is peptidoglycan - G+ve cells have several to 25 layers of peptidoglycan - Shape in both is thought to be due to length of and amount of crosslinkage of peptidoglycan chains Cell Walls of Gram +ve and –ve bacteria

Figure 2 - Overall structure of gram positive wall

Cell Walls – Gram Positive - Have teichoic acids, i.e. acidic polysaccharides embedded in cell wall (not found in gram-ve cells) - Two forms of teichoic acids o Wall teichoic acids – go part way through the wall o Lipoteichoic acids – go completely through the wall and link to the plasma membrane - Both forms protrude above the wall, which gives the bacterial cell a negative charge - Teichoic acids are cells that recognize our immune system

The outer membrane of Gram negative bacteria - Outer membrane is an additional layer of lipopolysaccharide - Lipid bilayer with polysaccharide and lipid - The lipid and polysaccharide are linked in the outer layer of the outer membrane to form lipopolysaccharide structures - Often called lipopolysaccharide layer or LPS - Outer membrane is relatively permeable to small molecules unlike cytoplasmic membrane - Permeability is due to presence of proteins known as porins - Several different porins exist both specific and non-specific - Porins are expressed in different environmental conditions - Is it not permeable to enzymes or large molecules and thus keeps vital enzymes trapped in the periplasm Periplasm - Space between the outer surface of the cytoplasmic membrane and the inner surface of the outer membrane - Has a gel like consistency due to presence of periplasmic proteins - 12-15 nm in E. coli

MICROBIOLOGY – Helen P – Bacteriology Chemistry of LPS - Complex chemistry - The polysaccharide consists of two portions o The core polysaccharide o The O-polysaccharide attached to a lipid portion – Lipid A - Lipid A anchors to the phospholipid bilayer portion of the outer membrane

Lipoprotein - A lipoprotein complex is found on the inner side of the outer membrane in many G-ve bacteria - This is a small protein and acts as an anchor between the outer membrane and peptidoglycan

Figure 3 - Overall gram negative cell envelope Building the Bacterial Cell Wall - The linking together of NAG and NAM subunits is facilitated by enzymes: o Transglycosylase, transpeptidase, polymerase, and hydrolase which combine to construct the backbone

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Cell wall construction mechanisms are targets for antibiotics Cell wall construction is a continuous process – being damaged and then re-built again There are 3 phases of peptidoglycan assembly of a new wall o Cytoplasmic phase  NAG and NAM building-blocks are formed in the cytoplasm of the cell  Cell wall must be constantly refurbished during division  The enzymes coded for by the murA-F genes attach 5 amino acids to each molecule of NAG and NAM  The cytoplasmic phase is an important target for antibiotics  Antibiotics prevent the formation of the peptidoglycan subunits e.g. fosfomycin o Membrane-associated phase  Specific enzymes link the NAG and NAM subunits with the lipid portion of the bacterial cell plasma membrane  First step in the cycle is the formation of a bond between peptidoglycan and the side of the plasma membrane facing the cytoplasm  Somehow the bound subunits cross (flip-flop) the membrane (mechanism still unknown)  Arrive at the exocytoplasmic side  Membrane associated enzymes react with the subunits  The new components are integrated into the growing wall  It is an important target for antibiotics  The last step in the formation of the meshwork is the binding together of the peptidoglycan layers  This is done with polypeptide chains  These connections give the wall many layers and an increased strength o Extra-cytoplasmic phase

MICROBIOLOGY – Helen P – Bacteriology Bacterial Locomotion - Many bacterial cells move about – in liquid or over surfaces - Movement requires a high energy expenditure - Different solutions to movement: o Swimming (directed) o Gliding Flagella & Motility - The flagellum – an organelle of bacterial motility - Long, thin helical structure o (20 nm, 15-20 microns in diameter) - Number and arrangement varies from species to species - Polar flagellation o Attached to one or both ends of cell - Peritrichous o Many flagella attached around the cell o E. coli or salmonella - Monotrichous o One flagellum The Flagellum - Consists of: - The filament o Rigid helix with a constant species dependent wavelength o Composed of subunits of one protein – flagellin o The filament is connected at its base to the motor by the hook which acts as a universal joint - The hook o The motor is anchored in the cytoplasmic membrane o Rotary motor o Driven by proteins or sodium ions o Speeds up to 1000 Hz have been measured - The basal body - The C-ring and the C-rod

Figure 4 - Flagellar Structure Flagellar Synthesis - E. coli and Salmonella tryphimurium need 40 genes for motility - Genes encode different functions - A filament extends from its top (not its base) - Flagellin is synthesized in the cytoplasm and passes through a 3 nm channel in the filament to the terminus of the growing flagellum - At the end of the flagellum a protein cap is present which is involved in the assemblage

Figure 5 - Flagellar Biosynthesis

MICROBIOLOGY – Helen P – Bacteriology Swimming Motility - Flagellum is a semi-rigid structure rotated at its base – a propeller - Cells tethered by the flagellum rotate - Motor is driven by proton motive force (or Na+ ions) through the Mot complex - 1000 protons per revolution - Can swim at speeds of 5-60 body lengths/s (cheetah = 25 bl/s) - Different bacteria show different swimming patterns Gliding Motion - Many bacteria move along surfaces but lack flagella (and no other obvious structures) - It is slow – up to 10 um/s - Many filamentous and rod shaped bacteria glide e.g. filamentous cyanobacteria, Myxococcus xanthus - Gliding requires contact with a surface Behavioral responses – taxes - Motile bacteria respond to physical and chemical gradients in their environment - They respond by swimming up a favorable gradient e.g. nutrient or down a repellent gradient - This movement is known as taxis - Chemotaxis – movement in response to chemicals o In the absence of a gradient bacteria swim in a random 3D pattern o When faced with a gradient they are too small to sense, the gradient along their length – unlike larger organisms (spatial sensing) o Instead whilst moving they compare their current environment with that sense a few seconds ago (temporal sensing) o Bacteria respond to the temporal gradient of signal molecules o When a gradient is sensed bacteria alter their direction changing frequency to bias their random walk up a favorable gradient

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o E. coli is the best understood and has one of the least complex chemotaxis pathway Phototaxis – movement in response to light

How does Chemotaxis work? - Gradient of signal molecules - Membrane receptors – methyl accepting chemotaxis proteins (MCPs) - A signaling cascade – the Che system - The memory – methylation and demethylation of the MCPs controlled by the signal - Control of the flagellar motor – CheY and CheZ Appendages – Fimbriae and Pili - Many bacteria have proteinaceous appendages - Fimbriae and Pili – similar structure o About 1 um in length and 3-5 nm in diameter o Involved in surface attachment and cell/cell interactions Glycocalyx, Slime Layers and Capsules - Many prokaryotes excrete sticky or gummy substances on their surfaces - Usually polysaccharide are a few protein layers - Often called capsule, slime later or glycocalyx - The polysaccharide layers are important in pathogenicity and also may bind water to prevent desiccation Internal Cell Structures - DNA o Circular o Appears as diffuse, dispersed, fibrous structure o Not membrane bound so it’s called a nucleoid - Gas Vesicles and Vacuoles o Found in aquatic species o Protein shelled structure to provide buoyancy o Gas vacuoles comprise vesicles o Appear under phase as bright refractive spots - Intracellular Reverse Materials

MICROBIOLOGY – Helen P – Bacteriology o Bacteria store inorganic and organic materials as polymers o Storage as polymers maintains internal osmotic pressure o Main ones are starch, glycogen, poly-B-hydroxybutyric acid (PHB), cyanophycin Endospores - Some bacteria produce endospores by a process known as sporulation - These are differentiated cells, very resistant to heat and are nongrowing - Remain dormant and viable for decades – perhaps indefinitely – then convert back to a vegetable cell rapidly

Population Growth - Growth is defined as an increase in the number of cells in a population - Growth is also measured as microbial mass - Growth rate is the change in cell number or mass per unit of time - The time taken for the formation of 2 cells from one is the doubling time or generation time - The mass and cell number also doubles during a single generation - Generation times vary depending upon the species and growth conditions Binary Fission - The bacterial cell reproduces by binary fission - It grows until it divides into 2 new cells – e.g. E. coli elongates to 2x the length of the smallest cells - A partition, the septum, forms from the inward growth of the CM - During division, the replicated DNA attaches to the membrane and separates with one copy to each daughter cell - Each daughter cell receives a complete chromosome and enough constituents to function as an individual cell

Figure 6 - Life Cycle of an Endospore Forming Bacterium

Bacterial Cell Division - First the cell elongates and DNA replication starts - A series of proteins are involved in all prokaryotic cell division Microbial Growth

MICROBIOLOGY – Helen P – Bacteriology o Filamentous temperature sensitive (Fts) proteins interact to form a cell division apparatus called the divisome o First FtsZ attaches in a ring around the center of the cell o FtsZ cells polymerise to form a ring. This later becomes the plane of cell division o The ring attracts other divisome proteins:  FtsA – aids in connecting the FtsZ ring to the cytoplasmic membrane and recruits other divisome proteins  ZipA – anchor connects FtsZ ring to the membrane and stabilizes it Cell Wall Synthesis - During division new cell wall material must be synthesized and added in without any loss of structural integrity - Enzymes called AUTOLYSINS make small openings in wall - The autolysins are present in the divisome complex - New cell wall material is added across the openings - The new cell wall precursors (NAG-NAM) are spliced in a coordinated manner to prevent a breach in the peptidoglycan integrity - If a breach occurs at the splice point the cell would lyse spontaneously - In gram positive cells the junction between the old and new material forms a ridge on the cell surface (analogous to a scar) - Gram positive cocci is only localized at one point – the FtsZ defines the cell division plane

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Growth Phases

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Lag Phase o A microbial growth population inoculated into fresh medium growth does not grow immediately, but after a lag phase o The duration of the lag phase depends upon the history of the culture o Period where the cell must gear up to the new conditions e.g. metabolic pathway synthesis o An inoculum from an old culture (stationary phase) gives a long lag as the cells must re-synthesis depleted essential components o Transfers from rich medium to poor have a lag phase as the cell must synthesis essential metabolites Exponential/Log Phase o Cellular reproduction is most active during this phase o Generation time reaches a constant minimum Stationary Phase o In batch culture, the exponential phase must end as growth becomes limited by:  An essential nutrient being used up  A waste product e.g. ethanol building up o The exponential phase ceases and the population has now reached the stationary phase of growth o The number of cells remains constant o However, the cells may continue to metabolize and there may be slow division but this is balanced by cells dying so there is no net increase in cell numbers o Several genes have been identified in E. coli that are involved with survival in the stationary phase o It is thought that in nature, cells will normally be in a nongrowing or slow growing state Death Phase o If incubation continues after a population has reached stationary phase, some cultures may continue to metabolize but others die o If they do become non-viable and die then they are in the death phase and cells may actually lyse

MICROBIOLOGY – Helen P – Bacteriology Environmental Factors Influencing Growth - Temperature o Protein denaturation - pH o Optimum range: 6.5 – 7.5 o Effects stability of membrane, enzymes, and macromolecule stability - Water Activity – biologically available water o 1.0 is max found in pure water o Bacteria prefer to inhabit Aw of >0.9 o Only very xerophilic fungi survive 0.8 o Below 0.8, only spores survive - Gaseous Activity Methods of Measuring Bacterial Populations - Total Microscopy Count o Cannot visualize less than  10,000 yeast cells per ml  30,000 bacterial cells per ml o Cannot distinguish between live and dead cells o Useless for dilute samples - Viable Plate Counts o Method requires the cultures to be diluted so that the plates can be counted over a range of dilutions and eliminate errors due to cells touching etc. o Optimum – 30-300 colonies per plate o Many potential errors therefore care must be taken with design and replicate plates must be made o Choice of media and growth conditions influences range of species that will grow - Turbidity o Fast and reliable o A cell suspension looks cloudy (turbid) to the eye as cells scatter the light passing through o The more cells the more light scattering and the cloudier it looks

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o The turbidity can be measured using a photometer or spectrophotometer which pass light through the sample and measure the un-scattered light passing through giving an optical density reading o The wavelength of light is controlled o For unicellular organisms the OD is proportional to cell number o In practice, a standard curve must be generated for each organism relating dry weight or cell number to the indirect turbidity reading o At high cell concentrations dilution of the suspension is required as the relationship between turbidity and cell numbers breaks down due to problems of light scattering off too many cells Cell Mass Biochemical Activity Automated Systems

MICROBIOLOGY – Helen P – Bacteriology Microbial Systematics Bacterial Phylogenics - Some macromolecules e.g. ribosomal RNA, of bacteria are highly conserved i.e. have changed very slowly during evolution - Analysis of their sequences enables the detection of relatedness between organisms - Ribosomal RNA in particular has been used an evolutionary marker - In prokaryotes the 5S (120 bases) and 16S (1500 bases) have been used extensively - The sequence is determined of the complementary DNA of 16s rRNA that is 16S rDNA - Some parts of the molecule are very highly conserved and these are used for determination of relatedness - From the sequence analysis a phylogenic tree can be created showing relatedness between species Taxonomy or Systematics - Taxonomy – science of classification + major discipline of microbio - Consists of identification and nomenclature - A new organism must be characterized, named, and assigned to a group of similar type - Taxonomy enables identification, understanding of relationships between organisms, predicting characteristics Classical (artificial) Taxonomy - Uses phenotypic analysis as basis of classification e.g. morphology, gram stain energy metabolism, enzymes, specialized structures, GC ratio of DNA - Levels of Classification Domain e.g. Monera Division Graticutes (G-ve) Class Scotobacteria (non-photosynthetic) Order Spirochaetales (helical) Family Spirochaetaceae Genus Borrelia (tiny irregular coiled spirochetes) Species burgdorferi (causative agent of Lyme disease)

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Currently about 1000 genera of bacteria distinguished and 5000 species Many to be discovered including ‘unculturables’ To identify an organism taxonomists go from the general to specific GC Ratio o Can be used as part of classical taxonomy o Determination of the guanine + cytosine content of an organisms genomic DNA o Expressed as (G+C)/(A+T+G+C) x 100% o GC ratios vary between about 20-80% in prokaryotes o It is useful for identification but not definitive as unrelated organisms can have the same GC ratio o Determined by melting temperature, southern blotting, or chromatographic methods o %GC can be determined by measuring the melting temperature of bacterial DNA o When DNA is heated the strands separate and every DNA has a characteristic temperature at which this happens known as the melting temperature o GC bp’s have 3 H atoms, and AT bp’s have only 2 H bonds, so AT-rich DNA ‘melts’ at a lower temp than GC-rich DNA

Molecular (Chemotaxonomy) Taxonomy - Uses the molecular analysis of any of a number of key biomolecules in the cell - Methods mainly used are: o DNA:DNA hybridization  GC ratio can be used as part of a molecular characterization but gives no identification of the sequence of the DNA  If 2 organisms have many identical sequences in their genome they probably contain many similar (if not identical) genes  Two pieces of DNA’s hybridize to one another in proportion to their sequence similarity  Hybridization – if DNA is denatured to SS it can then form hybrid molecules with other SS-DN...