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

2. Proteins are made of amino acids www.personal.psu.edu G A V LI M W F P ST C Y N Q D E K R H

3. Cystine: two cysteines with a disulfide bond

4. Escherichia coli Protein production factory Lecture 2

5. 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

6. Plasma membrane The plasma membrane is a lipid bilayer

7. Plasma membrane E. coli plasma membrane -phosphatidylethanolamine 65% -phosphatidylglycerol 18% -cardiolipin 12%

8. Lipid bilayers Bilayers -plasma membrane -soap bubbles -vesicles

9. The three layers of defense of E. coli Periplasmic space Outer membrane Cell wall Plasma membrane

10. Cell wall: made of peptidoglycan Strands of carbohydrates cross-linked by short peptides carbohydrate peptide

11. Penicillin, ampicillin Penicillin -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(NH 2 )C 6 H 5 ] 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  -lactam

12. Antibiotic resistance Penicillin 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

13. The three layers of defense of E. coli Gram 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

14. Outer membrane Complex composition, containing -lipopolysaccharides (LPS) -phospholipids -proteins Unusual polysaccharides that help recognize the host cells, but also allow the immune system to detect bacteria as foreign Some of the unusual monosaccharides:

15. Lipid A lipid 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

16. How to get DNA inside… Cells treated with CaCl 2 spontaneously take up DNA -Nobody knows how and why… -Treat cells on ice with CaCl 2 for 45 min, discard supernatant, then treat again on ice with CaCl 2 for 30 min -The phospho-groups of the inner and outer membrane are negatively charged -Ca 2+ is positively charged and has affinity for phosphate -DNA is negatively charged E. coli digests single-stranded and non-circular DNA -Accepts plasmids CaCl 2 treated cells can be frozen in 10% glycerol and stored

17. Electroporation A 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

18. Cytoplasm, periplasm Overexpressed 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

19. Proteins in the periplasm Proteins 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)

20. Life and death of E. coli - replication DNA was lightly labelled with radioactive [ 3 H]thymidine for visualization. A large amount of [ 3 H]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 oriC Replication forks meet at the opposite side of the DNA circle -replication ends at the ter site Each plasmid also has an origin of replication DNA must be duplicated before cell division

21. Replication fork

22. DNA replication DNA was lightly labelled with radioactive [ 3 H]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 min How can the cell doubling time be 20 min? DNA replication can start again even before the current round of DNA replication has been completed

23. 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 gene Additional copies are available during chromosome replication

24. 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

25. 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=play er_detailpage&list=ULKIpcCyuypzg&v=KIpcCyu ypzg

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

27. Bacteriophage life cycle

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

29. Bacterial lawn on culture plate where bacteriophages have formed plaques Bacteriophage infection

30. 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

31. 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 CaCl 2 treated cells (“competent cells”) spontaneously take up DNA Summary

32. 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 K 2 -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

33. Wear gloves, lab coat, covered shoes, safety glasses Protect bacteria by working under sterile conditions Biological safety

34. Conjugation: exchange of genetic material via pili Conjugation

35. 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

36. Which compound is a mutagen? Ames test -10 9 his - tester strains of Salmonella typhimurium in medium with little His -Place mutagen in the centre -Grow for 2 days at 37 o C -Halo of revertant colonies indicates mutagenesis Add rat liver homogenate to test for mutagenesis of metabolites Chemical safety