Escherichia coliSYSTEMS BIOLOGY

Suh-Chin WuInstitute of Biotechnology

Department of Life ScienceNational Tsing Hua University

The Escherichia Coli Paradigm

• Part I: Bacterial Cell Structure

• Part II: Metabolic Networks

Part I: Bacterial Cell Structure

Reference Books• Microbial Physiology, fourth edition, by Moat, Foster,

Spector, Wiley-Liss (2002)– Chapter 1 Introduction to microbial physiology– Chapter 2 Macromolecular synthesis and processing:

DNA, RNA, and protein synthesis– Chapter 7 Cell structure and function

• Microbiology, third edition, by Prescott, Harley, Klein, Wm C Brown Publishers (1996)

• Growth of the Bacterial Cell, by Ingraham, Maaloe, Neidhardt, Sinauer Associates (1983)

Molecular Structure of Bacterial Cell Parts

• Peptidoglycan cell wall• Outer membrane of the Gram-negative cells• Capsule• Flagella• Pili• Cytoplasm• Ribosome• Nucleoid

Peptidoglycan (or murein)• A peptidoglycan monomer consists of two joined

amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a tetrapeptidecoming off of the NAM.

• Glycosidic bonds link the monomers together to form chains of peptidoglycan subunits.

• These individual chains are then joined to one another by means of peptide cross-links between the tetrapeptides coming off of the NAMs.

The peptidoglycan monomer in E. coli, most gram-negative bacteria, and many gram-positive bacteria. These monomers join together to form chains and the chains are then joined by cross-links between the tetrapeptides to provide strength.

E. Coli: Gram-negative Bacteria

• Both gram-positive and gram-negative bacteria have a cell wall made up of peptidoglycan and a phospholipid bilayer with membrane-spanning proteins.

• Gram-negative bacteria have an unique outer membrane, a thinner layer of peptidoglycan, and a periplasmic space between the cell wall and the membrane.

• The outer membrane of gram-negative bacteria has lipopolysaccharides (LPS), porin channels, and murein lipoprotein all of which gram-positive bacteria lack

Electron Micrograph of a Gram-Negative Cell Wall

Electron Micrograph of a Gram-Positive Cell Wall

Gram-negative

Gram-positive

Appendix:Gram Staining Procedures

• First, stained with the basic dye crystal violet– appear purple for all bacteria

• Second, treated with gram's iodine solution. – retained better by forming an insoluble crystal violet-iodine complex– remain purple for all bacteria

• Third, stained with Gram's decolorizer, a mixture of ethyl alcohol and acetone (the differential step)– Gram-positive bacteria retain the crystal violet-iodine complex– Gram-negative bacteria are decolorized

• Finally, treated with the counterstain safranin (also a basic dye)– Gram-positive bacteria are already stained purple, they are not affected

by the counterstain (appear purple). – Gram-negative bacteria, that are now colorless, become directly stained

by the safranin (appear pink).

Gram stain of Escherichia coli Gram stain of Staphylococcus aureus

gram-positive (purple) cocci in clusters gram-negative (pink) bacilli

Outer Membranes of E. coli

• Lipopolysaccharides– O-antigen– Outer/Inner core– Lipid A

• OMP (porin)– OmpA, OmpB– OmpC, OmpF– LamB

Outer membrane• composition: a lipid bilayer composed of

phospholipids, lipoproteins, lipopolysaccharides (LPS), and proteins.

• Phospholipids are located mainly in the innerlayer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan.

• The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A embedded in the membrane and a polysaccharide portion extending outward from the bacterial surface.

Capsules of E. coli

• E coli produces some sorts of glycocalyx, an outer viscous covering of fibers

• Capsule– If the glycocalyx appears as an extensive,

tightly bound accumulation of gelatinous material adhering to the cell wall

• Slime layer– if the glycocalyx appears unorganized and

more loosely attached

Negative staining of group A streptococci viewed by TEM. The "halo" around the chain of cells (approximately equal in thickness to the cell diameter) is the remnants of the capsule that may be found surrounding the exterior of certain strains of group A streptococci. The septa between pairs of dividing cells may also be seen.

(28,000X)

Functions of Glycocalyx• Resisting phagocytosis

– capsules enabling bacteria to resist phagocytosis by evading the complement and antibody body defense responses

• Adhering to and colonizing environmental surfaces – capsules enabling bacteria to adhere to

environmental surfaces (rocks, root hairs, teeth, etc.), colonize, and resist flushing

– “biofilm” formation: consists layers of bacterial populations adhering to host cells and embedded in a common capsular mass

The S-layer

• Many gram-negative and gram-positive bacteria, as well a many Archaea possess a regularly structured layer called an S-layer attached to the outermost portion of their cell wall.

• It is composed of protein or glycoproteinand in electron micrographs, has a pattern resembling floor tiles.

Capsular K antigens (103)

• Capsular group I (heat-stable)– K30

• Capsular group II (temp regulated)– K5

Flagella of E. coli• Filament

– the longest and most obvious portion– A hollow, rigid cylinder composed of the protein flagellin

arranged in helical chains so as to form a hollow core• Hook

– is a flexible coupling between the filament and the basal body

• Basal body– consists of a rod and a series of rings that anchor the flagellum

to the cell wall and the cytoplasmic membrane– acts as a molecular motor, enabling the flagellum to rotate

and propell the bacterium through the surrounding fluid– L, P, S, M rings in E. coli (most gram-negative bacteria)

Insertion Structure of Bacterial Flagellum

Flagella and Motility

• Flagella are the organelles of locomotion for most of the bacteria that are capable of motility.

• Two proteins in the flagellar motor, called MotAand MotB, form a proton channel through the cytoplasmic membrane and rotation of the flagellum is driven by a proton gradient.

• The flagellar motor rotates very rapidly (The motor of E. coli rotates 270 revolutions per second!).

Flagellar H antigens (56)

• H typing– E coli associated with diarrheal disease

• Non-motile (NM) or H-: those strains not developing motility

Transmission Electron Micrograph of Escherichia coli O157:H7

Flagellar Synthesis

• Flagellin gene(s)• 10 or more genes coding for hook and

basal body proteins• An excellent example of self-assembly

E coli K12 MG1655

Chemotaxis

• Chemotaxis is the phenomenon in which bacteria to direct their movements according to certain chemicals in their environment.

• This is important for bacteria to find food (for example, glucose) by swimming towards the highest concentration of food molecules, or to flee from poisons (for example, phenol).

E coli K12 MG1655

Pili of E. coli• Pili are thin, protein tubes originating from the

cytoplasmic membrane and are found in virtually all gram-negative bacteria but not in many gram-positive bacteria.

• Short attachment pili, also known as fimbriae, usually quite numerous – Allowing bacteria to colonize environmental surfaces or cells and

resist flushing (Adhesion)– constantly losing and reforming pili as they grow in the body and

the same bacterium may switch the adhesive tips of the pili in order to adhere to different types of cells and evade immune defenses

• Long conjugation pili, also called "F" or sex pili, very few in number

Long Conjugation (F, Sex) PiliShort Attachment Pili

Cytoplasm of E. coli

• Refers to everything enclosed by the cytoplasmic membrane

• Cytosol- the liquid component• the site of most bacterial metabolism

– catabolic reactions in which molecules are broken down in order to obtain building block molecules for more complex molecules and macromolecules

– anabolic reactions used to synthesize other molecules and macromolecules

Nuclear body (Nucleoid) of E. coli

• The genome is composed of chromosomal deoxyribonucleic acid or DNA and represents the bacterium's nucleoid.

• Bacterial nucleoid has no nuclear membrane or nucleoli, does not divide by mitosis, has only one chromosome, and only reproduce asexually.

Prokaryotic Cell (Bacillus megaterium)

Chemical Structure of DNA

Nucleoid DNA Structure

• A one long, single molecule of double stranded, helical, “supercoiled” DNA

• Two ends of the double-stranded DNA covalently bond together to form both a physical and genetic circle.

• E. coli has a chromosome approximately 1400 µm long.

Topoisomerase

• Enzyme to alter the topological form (supercoiling) of a circular DNA molecule

• Type I– Type IA: E coli TopA– Type IB: E coli TopIII

• Type II– DNA gyrase (GyrA, GyrB)

• introduce negative supercoils• ~ 100 supercoils per min

– toposiomerase IV

Type I topoisomerase working on single strand

Type II topoisomerase handling double stands

Nucleoid domains

• Bacterial chromosome contains 30-200 negative supercoiled loops or domain– Repetitive extragenic palindrome (REP)

sequence• Gathering these DNA loops compact the

chromosome, centrally located within the cells

Electron Micrograph of Nucleoid DNA

1: U00096 Escherichia coli K-12 MG1655 complete genomegi|48994873|gb|U00096.2|[48994873]

DNA Res. 8:11-22February, 2001

[PubMed]

88%

1026 bp/gene

5361Genome Atlas

5,498,450

49Escherichia coliStrain: O157:H7 (substrain RIMD 0509952) DDBJ NCBI tax NCBI entrez

Nature 409:529-533

January, 2001[PubMed]

86%

1047 bp/gene

5283Genome Atlas

5,529,376

49Escherichia coliStrain: O157:H7 (substrain EDL93)U. Wisconsin NCBI tax NCBI entrez

Science 277:1453-1474

September, 1997[PubMed]

87%

1055 bp/gene

4397Genome Atlas

4,639,221

49Escherichia coliStrain: K-12, isolate MG1655U. Wisconsin TIGR cmr NCBI taxNCBI entrez

-79%

1135 bp/gene

4085Genome Atlas

4,636,552

49Escherichia coliStrain: K-12, isolate W3110DDBJ NCBI tax

ReferenceCodingdensity

Numberof genes

Atlas Size (bp)%A

TOrganism

E. coli K-12 sequence and annotations NomenclatureRNA GenesAnnotation Updates

Update released June 10, 2004: The Escherichia coli K-12 strain MG1655 sequence and annotations have been updated; see this announcement for further information.

Plasmid of E. coli

• small molecules of double stranded, helical, nonchromosomal DNA

• Not essential for normal bacterial growth (containing 5~ 100 genes)

• code for synthesis of a few proteins not coded for by the nucleoid– R-plasmids (gram negative bacteria) coding

for both production of a conjugation pilus and multiple antibiotic resistance

Ribosome of E. coli

• Contains 65% ribosomal RNA (rRNA) and 35% protein

• Two subunits with densities of 50S and 30S to form a complete 70S ribosome for protein synthesis

• Polyribosomes- Ribosomes hold on a single mRNA

• A typical bacterium may have as many as 15,000 ribosomes.

Mechanisms of Action of Antimicrobial Drugs

• Inhibition of cell wall synthesis• Inhibition of cell membrane function• Inhibition of protein synthesis

– translation– transcription

• Inhibition of nucleic acid synthesis

Antibiotics Action through Inhibition of Cell Wall Synthesis

• Binding to “penicillin-binding proteins”(PBPs) to block peptidoglycan synthesis

• Penicillin, Cepholosporins, Bacitracin, Cycloserine, Vancomycin

• Resistant mechanisms– Production of penicillin-destroying enzymes

(β-lactamase)– Absence of some PBPs in bacterial cells

Peptidoglycan (Murein) biosynthesis of E. coli

• Three stages– cytoplasm, membrane,

wall• cluster genes

– murC, murD, murE, ddl, murF, mraY, murG (2 min region)

– Others (murZ in 69.3 min, murI in 90 min, ..)

PBPs of E. coli• Covalently interact with penicillin and other β-

lactam antibiotics• PBP1A, PBP1B, PBP1C, PBP2, PBP3

– Peptidoglycan biosynthesis• PBP1A, PBP1B

– Association with cell elongation • PBP2

– Cell shape• PBP3

– Septum formation

β-lactamases are enzymes which catalyse the hydrolysis of β-lactams. There are about fifty different known types. The production of β-lactamases by bacterial cells is the most important contributing factor to the development of penicillin-resistant strains of bacteria.

By hydrolysing the labile β-lactam ring bond, the penicillin can no longer react with the transpeptidase enzyme. Thus the antibacterial properties of the penicillin have been deactivated

Role of Penicillins in Blocking Transpeptidase Enzymesfrom Assembling the Peptide Cross-Links in Peptidoglycan

Role of Vancomycin in Blocking Transpeptidase Enzymesfrom Assembling the Peptide Cross-Links in Peptidoglycan

Antibiotics Action through Inhibition of Cell Membrane Function

• Amphotericin B• Colistin• Imidazoles• Polyenes

Antibiotics Action through Inhibition of Protein Synthesis

• Chloramphenicol– Inhibiting peptidyl transferase to interfere with new amino acids to the nascent peptide chain– binding to 50S subunit ribosome (S6, L3, L6, L14, L16, L25, L26, L27)

• Tetracycline– blocking the binding of animoacyl-tRNA to A site on the 30S subunit ribosome (S7, strongest

binding site)– Resistance is plasmid-mediated in many bacteria (E. coli, S. aureus)

• Macrolides– angolamycin, carbomycin, chalcomycin, erythromycin, kujimycin, leucomycin, macrocin,

megamycin, oleandomycin, spiramycin (all contain a large alctone ring)– erythomycin binding to L15 (50S subunit ribosome) and to 23 rRNA to dissociate of peptidyl-

tRNA from ribosome– erythomycin resistance in E. coli by alternation in L4 or L12

• Aminoglycosides– streptomycin, kanamycin, neomycin, gentamicin, tobramycin,

amikacin,sisomycin,paromocycin, tobramycin, spectinomycin– binding to a specific receptor protein (P12 for streptomycin) on the 30S subunit ribosome– Resistance through alternation of S12

• Lincomycins– lincomycin, clindamycin– Inhibition of the peptidyl transfer by binding to 23 rRNA in 50S subunit ribosome

Role of Tetracyclines in Blocking Translation during Bacterial Protein Synthesis

Mode of Action of Macrolides in Blocking Translation during Bacterial Protein Synthesis (A)

Mode of Action of Macrolides in Blocking Translation during Bacterial Protein Synthesis (B)

Mode of Action of Aminoglycosides in BlockingTranslation during Bacterial Protein Synthesis

Antibiotics Action through Inhibition of Nucleic Acid Synthesis

• Rifampin– binding to DNA-dep RNA polymerase

• Quinolones, fluoroquinolones– blocking DNA gyrase

• Nalidixic acid to GyrA• Novobiocin to GyrB

• Sulfonamides– analogues of PABA to inhibit dihydropteroate synthetase

• Trimethoprim– inhibiting dihydrofolic acid reductase

• Pyrimethamine– inhibiting dihydrofolic acid reductase