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 hull sensorscommunications propulsion control systemspower 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 trans- membrane proton gradient. Photon capture Respiration Fermentation Trans-membrane proton gradient ATP/ADP ratio Motility Transport processes DNA, RNA and protein synthesis F 1 F 0 ATPase

Motility Swimming motility: rotating flagella powered by proton influx. Helical filaments: 20 nm diameter, 5 to 20  m long. CM PG OM L-ring P-ring MS-ring hook filament motor protein switch protein (Gram negative bacteria only) EM of flagellar base structure

Two-component sensor systems Sense external stimuli. External stimulus causes modification of internal protein. Cell membrane CYTOPLASM PERIPLASM ligand Sensor Kinase P Response Regulator

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. MCP ligand CheW CheA CheY P phosphatase promotes tumbling CheB P increases MCP sensitivity phosphatase Glu-Me methylase demethylase:

Intercellular communication Cells sense population density by ‘quorum sensing’. Detect critical density of an autoinducer, usually a homoserine lactone (LuxI/LuxR-type system) luxCluxDluxAluxBluxEluxI luxG lux promoter LuxR OHHL O O N O O Picture of squid

Life cycle 1: shaken broth cultures Exponential growth phase followed by stationary phase with different genes expressed. lag phase exponential phase stationary phase decline phase Biomass/ Optical density

Life Cycle 2: in nature Cells prefer to grow attached at solid-liquid interfaces (biofilm). swimming cells attached cellsmicrocoloniesmature biofilm detachment surface- associated motility quorum sensing swimming motility, chemotaxis

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.