Microbiology and Molecular Biology for Engineers IGEM, 20 June 2006 There are three types of cell • A: Archaea • B: Bacteria (Gram positive, Gram negative) • E: Eukarya (Animals, plants, yeasts, others) Eschericha coli • Kingdom/Division Proteobacteria • Class/Subdivision Gammaproteobacteria • Family Enterobacteriaceae sensors communications hull propulsion control systems power plant Cell envelope • Typical of Gram negative bacteria • Outer membrane: repels hydrophobic molecules. • Peptidoglycan sacculus: resists osmotic pressure. • Cell membrane: main permeability barrier. Energy generation • Energy generated by oxidation or disproportionation of organic molecules. • Stored as non-equilibrium ATP/ADP ratio and transmembrane proton gradient. Photon capture Respiration Motility Trans-membrane proton gradient Transport processes F1F0 ATPase Fermentation ATP/ADP ratio DNA, RNA and protein synthesis Motility • Swimming motility: rotating flagella powered by proton influx. • Helical filaments: 20 nm diameter, 5 to 20 mm long. filament hook MS-ring switch protein (Gram negative bacteria only) L-ring OM P-ring PG CM motor protein EM of flagellar base structure Two-component sensor systems • Sense external stimuli. • External stimulus causes modification of internal protein. ligand PERIPLASM Cell membrane Sensor Kinase Response Regulator CYTOPLASM P Chemotaxis • Swimming towards an attractant or away from a repellant is accomplished by a biased random walk – variable length runs interspersed with random changes of direction (tumbles). Mechanism of chemotaxis • Attractant / repellent chemicals are detected by chemotaxis receptors (MCPs). • Phosphorylation state of CheY alters frequency of tumbles. • Methylation of MCPs decreases sensitivity. methylase Glu-Me CheY phosphatase CheY P promotes tumbling ligand MCP CheA CheW CheB phosphatase CheB P demethylase: increases MCP sensitivity Intercellular communication • Cells sense population density by ‘quorum sensing’. • Detect critical density of an autoinducer, usually a homoserine lactone (LuxI/LuxR-type system) Picture of squid O N O OHHL O O luxE luxG LuxR lux promoter luxI luxC luxD luxA luxB Life cycle 1: shaken broth cultures • Exponential growth phase followed by stationary phase with different genes expressed. Biomass/ Optical density lag exponential phase phase stationary phase decline phase Life Cycle 2: in nature • Cells prefer to grow attached at solid-liquid interfaces (biofilm). swimming cells detachment swimming motility, chemotaxis surfaceassociated motility attached cells quorum sensing microcolonies mature biofilm Stochastic vs. mean field models • Simplistic models often treat biomass as a single compartment. • More realistically, billions of individual cells which may be in quite different states. • Therefore, oscillators etc. must include a cell synchronization mechanism unless individual cells are to be monitored (eg by FACS or fluorescence microscopy). Hosts other than E. coli • Salmonella: related to E. coli but better secretion of proteins into the medium. Problem: pathogen. • Bacillus: good secretion of proteins, forms highly stable resting state (endospores). • Budding yeast (Saccharomyces cerevisiae): eukaryotic cells. • Plant, insect and mammalian cells. endospores yeast Coming up… • What the stuff inside the cell is made of, and how it works. • How to modify it.