Class representatives:Keiran Walters u5010982@anu.edu.auEllen Rykers u5024183@anu.edu.au

Proteins are made of amino acids

www.personal.psu.edu

G A V L I M W F P

S T C Y N Q

D E K R H

Cystine: two cysteines with a disulfide bond

Escherichia coli

Protein production factory

Lecture 2

Lecture overview

More on what E. coli look like

And the stuff they are made of

Cell division

DNA, RNA, protein synthesis

Bacteriophage infection

Biological safety

Plasma membraneThe plasma membrane is a lipid bilayer

Plasma membrane

E. coli plasma membrane

-phosphatidylethanolamine 65%

-phosphatidylglycerol 18%

-cardiolipin 12%

Lipid bilayersBilayers-plasma membrane-soap bubbles-vesicles

The three layers of defense of E. coli

Periplasmic space

Outer membrane

Cell wall

Plasma membrane

Cell wall: made of peptidoglycanStrands of carbohydrates cross-linked by short peptides

carbohydrate

peptide

Penicillin, ampicillinPenicillin -antibiotic secreted by the mold penicillium notatum-variable R group bonded to the -lactam ring via a peptide linkage

Ampicillin-R group is aminobenzyl [-CH(NH2)C6H5]

Penicillins inactivate enzymes that cross-link the peptidoglycan strands -Bacterial enzymes that degrade cell walls (essential for cell-division) lead to cell lysis when the bacteria grow in the presence of penicillins

-lac

tam

Antibiotic resistancePenicillin disrupts the normal balance between cell wall biosynthesis and degradation-Penicillin-treated bacteria that are kept in a hypertonic medium remain intact, even though they have no cell wall. They become spherical.Penicillin inhibits no human enzyme-lactamases hydrolyse the amide bond of the -lactam ring -Confer resistance to penicillins

The three layers of defense of E. coliGram negative bacteria like E. coli have an outer membrane that protects (to some degree) against penicillin and lysozyme

Periplasmic space

Outer membrane

Cell wall

Plasma membrane

Outer membraneComplex composition, containing-lipopolysaccharides (LPS)-phospholipids-proteinsUnusual polysaccharides that help recognize the host cells, but also allow the immune system to detect bacteria as foreign

Some of the unusual monosaccharides:

Lipid Alipid component of a lipopolysaccharide in the outer membrane -very potent stimulant of the immune system-may cause shock by an “out of control” immune reaction -therapeutic proteins produced in E. coli must be made free of “endotoxin”-alkaline phosphatase in the gut detoxifies lipid A

Structure of a lipopolysaccharide: Lipid A with six alkyl chains is most toxic:

Poly-saccharide

Lipid A

How to get DNA inside…Cells treated with CaCl2 spontaneously take up DNA-Nobody knows how and why…-Treat cells on ice with CaCl2 for 45 min, discard supernatant, then treat again on ice with CaCl2 for 30 min

-The phospho-groups of the inner and outer membrane are negatively charged -Ca2+ is positively charged and has affinity for phosphate-DNA is negatively charged

E. coli digests single-stranded and non-circular DNA-Accepts plasmids

CaCl2 treated cells can be frozen in 10% glycerol and stored

ElectroporationA large electric field punches holes into the cell wall-Holes close again in the absence of the electric field-Alternating fields are used in pulses to prevent DNA from migrating to the anode

http://www.nepagene.jp

Cytoplasm, periplasmOverexpressed proteins accumulate in the cytoplasm-Redox potential in cytoplasm is reducing, i.e. no disulfide bonds can form

Accumulation in the periplasm is possible-Needs an N-terminal signal peptide (15-30 residues) for transport across the plasma membrane- The transporter proteins that recognize the signal peptide also cleave it from the protein-Redox potential in periplasm is oxidizing and presence of protein disulfide isomerases (PDI) aids in the formation of correct disulfide bonds

Inclusion body of a protein that has been directed to the periplasm

From http://mbel.kaist.ac.kr/lab/research/protein_en2.html

Proteins in the periplasmProteins are easier to purify from the periplasm

-There are fewer proteins in the periplasm than in the cytoplasm

-There is no DNA or RNA in the periplasm

-To release the proteins, only the outer membrane needs to be disrupted, e.g. by osmotic shock (the cell wall is porous)

Life and death of E. coli - replication

DNA was lightly labelled with radioactive [3H]thymidine for visualization. A large amount of [3H]thymidine was added for a few seconds before isolating the DNA. The picture shows that the DNA is replicated in both directions (“bidirectional replication”).

Replication of the chromosome starts at the origin of replication -a specific 250 bp sequence named oriCReplication forks meet at the opposite side of the DNA circle-replication ends at the ter siteEach plasmid also has an origin of replication DNA must be duplicated before cell division

Replication fork

DNA replication

DNA was lightly labelled with radioactive [3H]thymidine for visualization of a replication eye. In the interpretive drawing: black: non-radioactive DNA template; brown: newly synthesized DNA. About a tenth of the DNA has been replicated in the replication eye.

The DNA replication rate in E. coli is ~1000 nucleotides/s. -Two replication forks, 5 mio base pairs → 2500 s = 42 minHow can the cell doubling time be 20 min?DNA replication can start again even before the current round of DNA replication has been completed

RNA synthesis

RNA polymerase (RNAP) transcribes DNA to RNA at 20-70 nucleotides/s-ribosomal RNA is needed in large quantities-Initiation of transcription is sterically possible only about once per second

Electron micrograph of three contiguous ribosomal genes undergoing transcription.

What holds back cell division?

Not limiting: E. coli has seven copies of each rRNA geneAdditional copies are available during chromosome replication

Electron micrograph, showing simultaneous transcription and translation of an E. coli gene.

Coupled RNA and protein synthesis

Ribosomes get to work quickly-commence translation near 5’ end of a nascent mRNA as soon after it is extruded from RNAP

mRNA is a transient molecule-Most mRNA is enzymatic-ally degraded within 1 – 3 min of their synthesis-5’ ends can degrade before 3’ ends have been made

E. coli can divide no faster than once per 20 min.

Time limit set by the time it takes to form the septum between the old and new cell

http://www.youtube.com/watch?feature=player_detailpage&list=ULKIpcCyuypzg&v=KIpcCyuypzg

Bacteriophages - when E. coli catches a cold…E. coli with bacteriophages T5 adsorbed to its surface

Bacteriophage life cycle

Electron micrograph of bacteriophage T2 with its DNA spilled by osmolytic lysis in distilled water

Bacterial lawn on culture plate where bacteriophages have formed plaques

Bacteriophage infection

With great difficulties!

-Bleach, UV irradiation

Phages exist for weeks to months as an aerosol

In the lab, careful sterile techniques are important

Phages can replace antibiotics in phage therapy, but they are specific rather than broadband…

Bacteriophages – how to get rid of them

E. coli

Single circular chromosome

Additional DNA in plasmids/vectors

Plasma membrane, cell wall, outer membrane-Lipid bilayer, peptidoglycan, glycolipid bilayer

Oxidising conditions in the periplasm

At best, 20 minutes cell doubling time

Bacteriophages infect bacteria

CaCl2 treated cells (“competent cells”) spontaneously

take up DNA

Summary

500 – 1000 different species of bacteria live on every human (skin and gut)

Bacterial cells outnumber human cells 10 to 1!

About 10% of human body weight is bacteria

E. coli:

About 30% of the contents of the human colon is E. coli

-producing vitamin K2

-preventing growth of pathogenic bacteria

laboratory strains BL21(DE3) and K12 are closely related-99% sequence identity for >90% of the genome-completely unrelated to toxic strains-BL21 does not survive in blood, does not persist after 6 days

Biological safety

Wear gloves, lab coat, covered shoes, safety glasses

Protect bacteria by working under sterile conditions

Biological safety

Conjugation: exchange of genetic material via pili

Conjugation

Can happen between bacteria of different species

-Spread of antibiotic resistance is problem in hospitals

-Typically, the DNA transferred via the pili is plasmid DNA that contains the genes required to make the pili and transfer of DNA

We work only with non-conjugative plasmids

E. coli BL21 and K12 strains do not naturally contain F-plasmids (“F” meaning fertility)

Horizontal gene transfer

Which compound is a mutagen?

Ames test-109 his- tester strains of Salmonella typhimurium in medium with little His

-Place mutagen in the centre

-Grow for 2 days at 37 oC

-Halo of revertant colonies

indicates mutagenesis

Add rat liver homogenate to test for

mutagenesis of metabolites

Chemical safety