Lecture 1: Metabolism and Assembly Reactions

Reading assignments in Text: Lengeler et al. 1999Text:pages 110-113 Metabolic overviewText:pages 568-569, 573-574 Pili (Fimbriae) and flagellaText:pages 825-829 Surface virulence factors

Candle Flame analogy

Dynamic process that looks static

Birth and death

Self sustaining:

Acquire food and remove wastes

Wastes = CO H O2 2

2Food = -(CH ) -n

O “respiration”2

“Learning should not be the filling of a bucket, but the lighting of a flame.” - roughly W.B. Yeats

Metabolic overview

2 nFood = -(CH ) -

Wastes = CO H O2 2

E. coli

~4,000 genes / reactions

~ 50% are expressed

~2,000 proteins / reactions

AssemblyFuelling Biosyn. Polymer.

Lecture 1

Pili

Flagella

DNARNAProteins

Lecture 2Lectures 3,4

ATPNAD(P)H12 Precursor Metabolites

Assimilation reactions

C N S

P

Assembly reactions / general concepts

Self assembly:

protein foldingribosome assembly

membrane bi-layers

Directed assembly: Scaffolds (leader-peptides, pro-proteins)

Protein chaperones (SecB, PapD, others)

Ordered substrate productionB

C

ACheck-point control

1

23

Cell divisionDNA replication

DNA segregation

Pili assemblyIllustrates: Protein chaperones and “ordered substrate production”

Many types: E. coli F-plasmid “sex-conjugation pili”

Myxobacteria Social motility “gliding/type IV pili”

E. coli Urinary track infection “pathogenic pili”

“Pap” Structure

IMPeriplasmic space

OM

Genetic analysis

Wt

papA

papG

EMRB Cellclumping

yes

yes

no ?

?

PapG = lectin (Gal)1-4(Gal)

?G

papA

AG

Wt

Pap Genetic system

DNA

APilin “tube”

GTip “lectin”

Other genes

= 11 Genes !

RNA

Pr Ter

3 Types of proteins: 6 structural C = anchorA H = tube

E F G = tip2 regulatory On/Off “phase variation”

On/Off

3 assembly J K ?

PapD = chaperone

PapD periplasmic chaperone

Ig-like fold selectively binds Pap proteins

Binds secreted and unfolded Pap on IM

Release at site on OM

Folding at nucleation point

Protein:Protein recognition

Assembled PapA programs released PapA folding

Folding information

released

free

Pap assembly model

IM

OM

General secretion, SecAY

SecB

PapD

PapC G FG

Ordered substrate production

FG

E

EAA

FG

EE

HAAAA

FG

EE

PapH = cap

Model explains:Sub-unit order

Pili length (PapH)

Other issues:Polar vs lateral placementMany pili typesPili retraction (locomotion)Pili sensory (touch) transduction

uropathogenic Escherichia coli

E. coli (gold) tethered to the lumenalsurface of bladder cells (blue).

Flagellum assembly

Illustrate: Check-point control

Salmonella Caulobacter

Flagella Flagellum

Basal body assembled

Cell wall

in out

Make filament

?

The flagellum organelle

~50 genes = ~20 structural ~30 regulatory + assembly ?

Basal body

Mot

(M S P L-rings/rods)

Turbine/rotary engine

H+

H+

Switch

CheY~P

Secretion system

Hook

Flagellar filament

(universal joint)

(propeller)

Leader peptide proteinsuse regular SecAY/SecBsecretory system

Lack leader sequences

Secretion and placement ?“Protein pump”

ATP

Open?

Capped

Nucleation point

Inside view

Open siteAccepts flagellin

Cap = Hap (Hook associated protein)

Hook

Flagellum organelle assembly

“Type IIIsecretionsystems”

Hook

Hap

“Protein pump”

ATP

Hap HapHap Hap Hap

Flagellum growth/Hap movement

Secretion systems (Type III)

Many: Type I

Type IIType IIIType IVSignal Recognition Particle/Receptor system

Selective for Hemolysins/S-layer proteins

General SecA,Y/SecBSelective for key pathogenic proteins “virulence factors”Selective, conjugation DNA/proteins or virulence proteins

(like eukaryotes, e.g. yeast)

in out

Flagellum

“Needle complex” of Type III sec

Homologous, also similar toF0F1 ATPases

ATP

ATP

= direct injection

Toxins

Yersinia pestis cytoplasm

Macrophage cytoplasm

“contact-dependent sec”

Anti-phagocyticAnti-cytokinePro-apoptosis

Shigella flexneri

Flagellum assembly check-point control

Basal Body assembled

Make filament

Salmonella typhimurium

Species specific controls

Last of 4 check-points

Trigger stage 1 gene expression

~50 genes in stages 1, 2, 3, 4

2

3

1DNA

4Hook and flagellin genes

? Passage from stage 3 to 4? Sensor

? Triggering RNA polymerase

? Sensing BB structure/function

BB

Flagellum assembly check-point control MECHANISM

Trigger stage 1 gene expression

2

3

1DNA

4Hook and flagellin genes

1, 2, 3 stages made structure proteins

Plus Regulators

Sigma factor-28FlgM =Anti-Sigma

FlgM looks/pumps like flagellin

Sigma-28 stays inside the cell

Together trigger transcription

RNA

Freed Sigma-28 binds RNA polymerase

FlgM is pumped out

Stage 4 protein synthesis