Bacterial Cell Biology.docx

Bacterial Cell Biology Bacterial Cell Biology 1 Earth formed around 4.8 billion years (byr) ago. Microbial cells appeared 3.8-4.3 byr ago. During first 2 byr the atmosphere was anoxic (N2 + CO2) Evolution of phototrophic microorganisms (anoxygenic) occurred within 1 byr of formation of Earth. Cyanob...

Bacterial Cell Biology Bacterial Cell Biology 1 Earth formed around 4.8 billion years (byr) ago. Microbial cells appeared 3.8-4.3 byr ago. During first 2 byr the atmosphere was anoxic (N2 + CO2) Evolution of phototrophic microorganisms (anoxygenic) occurred within 1 byr of formation of Earth. Cyanobacteria (oxygenic) evolved 2 byr after formation of Earth. Subsequent oxygenation allowed for multicellular life. Domain: one of the three main evolutionary lineages of cells: the Bacteria, the Archaea, and the Eukarya Prokaryote: cell lacking a membrane-enclosed nucleus and other organelles Nucleoid: the aggregated mass of DNA that makes up the chromosome(s) of prokaryotic cells Plasmid: an extrachromosomal genetic element that is replicated independently from the genome Eukaryote: cell which has a membrane-enclosed nucleus and various other membrane-enclosed organelles Nucleus: a membrane-enclosed structure in eukaryotic cells that contains the cell’s DNA genome Organelle: a bilayer-membrane-enclosed structure such as the mitochondrion, found in eukaryotic cells Extremophiles: microorganisms that inhabit environments characterised by extremes of temperature pH, pressure, or levels of salinity and other chemicals. These microorganisms allow polymerase chain reaction (PCR) to be conducted, which is an effective method of replicating genetic material. Prokaryotic cell structure Sizes range from 0.15 um – 700 um long 30 major phylogenetic lineages have been defined. More than 90% of cultural bacteria belong to one of four phyla: Actinobacteria (pronounced ascinobacteria) Firmicutes Protobacteria Bacteroidetes (pronounced bacteroidetees) Can exist as single-cell (swimming, planktonic) or in biofilms. Some species undergo differentiation (sporulation) Essential to life on earth including human health. Gram Stain Gram-stain: a differential staining procedure that stains cells either purple (gram-positive) or pink (gram-negative) Gram-negative: a bacterial cell with a cell wall containing small amounts of peptidoglycan and an outer membrane Eg: Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Bacteroidetes spp. Gram-positive: a bacterial cell whose cell wall consists chiefly of peptidoglycan; it lacks the outer membrane of gram-negative cells Eg: Staphylococcus aureus, Streptococcus mutans, Fermicutes spp. (majority), Some bacterial cells can be neither gram+ve or gram-ve: Synthetic genome of Mycoplasma mycoides transplanted into M. capricolum Phospholipid Bilayer and Cell Membrane Function of cell membrane Permeability barrier: prevents leakage and functions as a gateway of transport. Protein anchor: site of proteins that participate in transport, bioenergetics and chemotaxis. Energy conservation: site of generation and dissipation of the proton motive force. Peptidoglycan Cross-linking of these polysaccharides provides additional strength. Gram positive bacterial cell wall Gram negative bacterial cell wall Lipopolysaccharide layer (LPS) LPS simulates CD14 and triggers immune response. Commensal bacteria can prevent inflammatory endocytosis. Bacterial Cell Biology 2 Cell surface structures Capsules Fimbriae Pili, secretion systems, horizontal gene transfer Cell inclusions Carbon, phosphate, sulfur and carbonite storage Magnetosomes Cell division and growth Binary fission, generation time, growth curves Endospores Formation, properties of endospores Cell surface structures Slime layer and Capsule Polysaccharide or protein excretions on cell surface If layer is easily deformed and loose it is a slime layer If layer is organised into a tight matrix and excludes small particles it is a capsule Function Aid in attachment to solid surfaces Biofilm formation Prevent dehydration and desiccation Virulence factors (and protect from animal immune system) Fimbriae and Pili Thin (2-10nm) filamentous structures made of protein that extend from the cell surface Many different functions Fimbriae Enable cells to stick to surfaces including animal tissues Help in biofilm formation Many fimbriae per cell Pili Longer than fimbriae Typically one one (or a few) per cell All gram negatives and many gram positives have pili Very diverse in function cell movement called twitching motility (a form of gliding motility) secretion systems genetic exchange (horizontal gene transfer) Gram-negative secretion systems Secretion systems: enable movement of molecules from the cell interior to its exterior. Secretion systems transport molecules including proteins enzymes DNA toxins Type IV pili associated with Type IV secretion systems involved in conjugation transformation effector translocation (virulence) Conjugation Transformation (DNA uptake and release) Effector translocation This process releases toxin into host cells. Horizontal gene transfer: The transfer of genes between cells through a process uncoupled from reproduction (transformation, transduction, conjugation) Cell Inclusion Bacterial inclusions can be defined as discrete structures seen within the confines of prokaryotic cell, generally intracytoplasmic, but in some instances in the periplasmic region of the cell Generally they function as energy or metabolite stores Carbon Storage: PHB and PHA Poly-beta-hydroxybutyrate acid (PHB): a common storage material of prokaryotic cells consisting of a polymer of beta-hydroxybutyrate or other beta-alkanoic acids Sometimes the C monomers differ and these cases are referred to as poly-beta-hydroalkanate (PHA) PHB (and PHA) are synthesized by cells when there is an excess of carbon. Broken down again when required for cellular processes. Polyphosphate Many bacteria accumulate and store inorganic phosphate PO43- Used to synthesize nucleic acids and phospolipid bilayers Sulphur Many gram negatives can oxidise reduced sulphur compounds such as hydrogen sulphide H2S. This generates electrons that can be used in metabolism or CO2 fixation. Elemental sulphur can accumulate before being oxidised and removed. Interestingly the sulphur globules are stored in the periplasm Carbonate minerals Some bacteria also store carbonate minerals in a process called biomineralization. In cyanobacteria it may provide ballast to help stay deep in the water column Magnetosomes (Not really a storage mechanism) A particle of magnetite (Fe3O4) enclosed by a nonunit membrane in the cytoplasm of magnetotactic bacteria They can be used in Magnetotaxis Gas vesicles (in buoyant cyanobacteria) Planktonic bacteria need to be able to float. Gas vesicles are gas-filled cytoplasmic structures bounded by protein and conferring buoyancy in cells. Allow cells to position themselves in a column of water. -Gas vesicle architecture: Composed of proteins GvpA and GvpC GvpA is a small hydrophobic protein that forms the ribs of the vesicle and a watertight shell. GvpC strengthens the shell by cross-linking. Bacterial microcompartments (BMCs or MCPs) organelles that are functionally similar to those of eukaryotes; they establish and contain a microenvironment that is distinct from the rest of the cell. shell is composed of proteins similarly to gas vesicles Carboxysome: stores RuBisCo and concentrates carbon dioxide for photosynthesis (anabolic) Metabolosomes: allows for higher concentrations of toxic molecules. EUT: ethanolamine utilisation Ethanolamine is product of the breakdown of plant and animal cell membranes GI tract and other sites of epithelial turnover ethanolamine abundant in inflamed gut -> tetrathionate Salmonella enterica, Enterococcus faecalis, enterohaemorrhagic Escherichia coli (EHEC), Clostridium difficile flourish by utilizing EUT BMCs BMCs are also found in organisms in cancer-associated gut dysbiosis. F. hwasooki and F. nucleatum species contain a EUT2 BMC How salmonella uses BMCs and secretion systems Cell Division – Binary fission Cell division following enlargement of a cell to twice its minimum size Generation time: the time required for a population of microbial cells to double (or equivalently time required for one cell to double) Budding and other processes Cell division process whereby new cell material is produced from a single point instead of along the entire cell Microbial Growth Growth refers to growth in cell numbers. Lag phase Can be short or long depending on the state of the cells and the environment Exponential growth Growth of a microbial population in which cell numbers double at a constant rate Stationary phase The point at which growth slows from exponential. Usually a nutrient is depleted, waste has built up or there is no space Death Very slow process. Cells can remain alive for months. Endosporulation (sporulation) Endospores (spores) are a highly heat-resistant, thick-walled, differentiated structure produced by certain gram-positive bacteria. They are survival structures in case of extreme. temperature drying nutrient depletion Life cycle: vegetative (growing) -> endospore -> vegative Easily dispersed by wind, water, animal gut (faeces) Bacillus spp. and Clostridium spp. are classic examples present in soil. Clostridium difficile is an example of a spore forming pathogen. Life cycle of an endospore-forming bacteria Spore Formation and Generation Spore formation cells only sporulate when they cease growing (eg lack of nutrient) in Bacillus often when carbon or nitrogen becomes limiting Spore activation occurs when spores heated for several minutes. Germination occurs when activated spores are exposed to certain nutrients. ~ several minutes Outgrowth visible swelling due to water uptake (following calcium uptake) and biosynthesis. vegetative cell emerges. Types of Endospores Structure of the bacterial endospore Dipicolinic acid: a substance unique to endospores that confers heat resistance on these structures. Helps dehydrate the spores and projects DNA from denaturing. Small acid-soluble spore proteins (SASPs) bind to DNA. Some spores can withstand heating to 150 degrees C. Endospore formation as differentiation Bacterial Cell Biology 3 Flagella and swimming motility Flagellum: a long, thin cellular appendage that rotates (in bacteria) and is responsible for swimming motility. Peritrichous flagellation: having flagella located in many places around the surface of the cell. Polar (monotrichous) flagellation: having flagella emanating from one pole of the cell. Example: E. coli Example: P.aeruginosa Structure of the flagellum in gram-negative bacteria Stator: fixed, Rotor: free Structure of the flagellum motor The flagellum motor is referred to as the basal body. This is a reversible rotating motor anchored in the cytoplasmic membrane and cell wall, formed of a central rod and a number of rings: L ring in outer membrane (gram-negative only) P ring in peptidoglycan layer (gram-negative only) MS ring in cytoplasmic membrane C ring in cytoplasm Mot proteins surround the inner rings (MS and C rings) and is known as the stator (fixed). The inner rings are known as the rotor proteins. Flagella as proton turbines Proton translocation through Mot proteins exerts electrostatic forces on the helically arranged charges on the rotor proteins. Alternating attractions between positive and negative charges cause the rotor proteins to rotate. Flagella biosynthesis In Escherichia and Salmonella there are upwards of 50 genes involved in biosynthesis The flagella filament grows from the tip Flagellin molecules pass up through a 3nm channel inside the filament. Gliding motility Diverse mechanisms Cyanobacteria secrete a polysaccharide slime Twitching motility via type IV pili Other mechanisms possible eg Flavobacterium johnsoniae Chemotaxis and phototaxis Bacterial cells respond to their environment. Chemotaxis: directed movement of an organism in response to a chemical gradient toward agent (positive chemotaxis) away from agent (negative chemotaxis) Phototaxis: movement of an organism in response to light The number of tumbles is reduced when an attractant is present. In absence of gradient cell randomly switch between: Runs: flagellar rotates counterclockwise (CCW) and forward movement Tumbles: flagellar rotates clockwise (CW); cell stops and jiggles randomly Following a tumble the cell direction is randomized. If a cell senses a chemical attractant is present, the behaviour is biased towards runs. If the cell senses it is moving towards higher concentrations then the runs become longer and the cell tumbles less often. Sensing in time vs space Bacterial cells are too small to sense the change in chemical gradient directly. Cells respond to temporal rather than spatial differences. Essentially sampling the concentration then comparing the current value to what was sensed previously. This “memory” is implemented in a signalling and gene regulatory network. Phototaxis and other ‘taxes’ Phototrophic bacteria can position themselves in order to optimise photosynthesis. Eg Rhodospirillum centenum Aerotaxis: movement towards or away from oxygen Osmotaxis: movement towards or away from ionic strength Hydrotaxis: movement towards or away from water Sensing and regulation Chemotaxis is an example of a sensory response. In general cells need to sense many different environmental fluctuations including temperature, pH, oxygen etc Mechansims exist for cells to receive signals from the environment and transmit these signals to specific targets. Often this means turning on or off a gene (or set of genes) Two component systems are ubiquitous in bacteria. Two component regulatory systems Two-component system (TCS): a regulatory system consisting of two proteins: a sensor kinase and a response regulator. Sensor (histidine) kinase protein: one of the members of a TCS; a protein that phosphorylates itself in response to an external signal and then transfers the phosphoryl group to a response regulator. Response regulator protein: one of the members of a TCS; a protein that is phosphorylated by a sensor kinase and then acts as a regulator, often binding to DNA Regulation of chemotaxis This is an example of a two component system. Quorum sensing Quorum sensing: a regulatory system that monitors the population level and controls gene expression based on cell density. Engineered quorum sensing Vibrio cholerae secrete virulence factors at low population density. Presence of autoinducers AI-2 and CAI-1 increases. Engineer E coli to to produce AI-2 and CAI-1 Biofilms Microbial cells can grow either in suspension or attached to surfaces. Growing in suspension is referred to as the planktonic lifestyle. Bacterial cells growing on surfaces can form complex multispecies biofilms. A biofilm is an attached polysaccharide matrix with embedded bacterial cells. They form in stages and can form complex layered structures containing different species. Biofilms: oral microbiome Ecological interactions Human gut microbiota/microbiome Essential to human health Complex carbohydrates are digested by gut microbiome Inhabitants of the GI tract Stomach: 104 microbial cells per gram (pH 2) Small intestine: 108 microbial cells per gram (pH 5) Colon: 1011 microbial cells per gram (pH 7) Colon contains diverse species that perform: fermentation of complex carbs (fibre) into short chain fatty acids (acetate, butyrate, propionate). Butyrate is an energy source for colonic epithelial cells. Production of vitamins including thiamine, folate, biotin, riboflavin, panthothenic acid and vitamin K Dysbiosis