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

sensors communications

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 trans-

membrane proton gradient.

Photon capture

Respiration

Fermentation

Trans-membrane proton gradient

ATP/ADP ratio

Motility

Transport processes

DNA, RNA andprotein synthesis

F1F0 ATPase

Motility• Swimming motility: rotating flagella powered by proton influx.• Helical filaments: 20 nm diameter, 5 to 20 m long.

CM

PG

OML-ring

P-ring

MS-ring

hook

filament

motor proteinswitch 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

SensorKinase

PResponseRegulator

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.

MCPligand

CheW

CheA

CheY

CheY P

phosphatase

promotes tumbling

CheB

CheB Pincreases 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)

luxC luxD luxA luxB luxEluxI luxGlux promoter

LuxR

OHHL

O

O

NO

O

Picture of squid

Life cycle 1: shaken broth cultures

• Exponential growth phase followed by stationary phase with different genes expressed.

lag phase

exponentialphase

stationaryphase

declinephase

Biomass/Optical density

Life Cycle 2: in nature

• Cells prefer to grow attached at solid-liquid interfaces (biofilm).

swimming cells

attached cells microcolonies mature 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.