Transcript How do bacteria respond to their environment?

How do bacteria respond
to their environment?
• Myxococcus
Starvation
Fruiting bodies
• Bacillus
Starvation
Spores
• Anabaena
Starvation
Heterocysts
How is starvation sensed and interpreted?
Response of E. coli
to lactose deprivation
Economize
Don’t make enzymes for lactose utilization
How does E. coli sense
starvation for lactose?
+ Lactose
- Lactose
lactose
lac expression repressed
(use other carbon source)
Response of E. coli
to nitrogen deprivation
+ Nitrogen
Protein synthesis
Fast growth
- Nitrogen
Economy
Slow growth
Response of E. coli
to nitrogen deprivation
Stringent response
• Make less ribosomal protein
• Make less ribosomal RNA
• Make less transfer RNA
Problem: Multiple outputs
• Control scores of genes
Problem: Multiple inputs
• Deficiency in ANY amino acid = starvation
How does E. coli sense
starvation for nitrogen?
Is there a protein that binds ALL amino acids?
The
Ribosome
How does E. coli sense
starvation for nitrogen?
Plenty of
amino acids
How does E. coli sense
starvation for nitrogen?
Plenty of
amino acids
How does E. coli sense
starvation for nitrogen?
Plenty of
amino acids
How does E. coli sense
starvation for nitrogen?
Plenty of
amino acids
How does E. coli sense
starvation for nitrogen?
Plenty of
amino acids
How does E. coli sense
starvation for nitrogen?
Starvation for
amino acid
Stuck!
Ribosomes can sense absence of any amino acid
How does E. coli sense
starvation for nitrogen?
???
Low alanine
Low cysteine
…
Low valine
Stringent
response
One small
molecular signal
How does E. coli sense
starvation for nitrogen?
What’s the signal?
Translocation
(requires energy)
GTP
How does E. coli sense
starvation for nitrogen?
What’s the signal?
Translocation
Pi
GDP
How does E. coli sense
starvation for nitrogen?
What’s the signal?
pppGpp
GTP
RelA
RelArelaxed
(no stringent response)
ATP
AMP
Is response to N-starvation
mediated through pppGpp?
Critical Questions
1. Does pppGpp affect genes of stringent response?
2. Does uncharged tRNA
pppGpp
???
Ogawa & Sy (1977) J Biochem 82:947. Synthesis of guanosine polyphosphates
(pppGpp and ppGpp) and its regulation by aminoacyl t-RNA
Does uncharged tRNA
pppGpp?
How to tell?
Experiment
• Add uncharged tRNA to ribosomes
• See if pppGpp increases
Need:
• In vitro system where charging of tRNA can be controlled
• Assay for level of pppGpp
Does uncharged tRNA
pppGpp?
In vitro system (~Nirenberg & Matthaei)
Ribosomes:
From differential centrifugation
tRNAs: store bought
tRNA charging enzymes:
From soluble fraction
Amino acids: store bought
RNAs: homopolymers, e.g.
AAAAAAAAA...
GTP and ATP: store bought
or
or
Does uncharged tRNA
pppGpp?
Assay for level of pppGpp
GTP*
ATP
+
pppGpp
Thin layer chromatography
+
GTP
+
(ATP)
+
GDP
+
ppGpp
(magic spot)
+ amino acids
- amino acids
Does uncharged tRNA
pppGpp?
What’s required to get pppGpp?
Condition
AAAAA…
% GTP*
(p)ppGpp
+ Poly(A)
- Poly(A)
No aa’s
30.6
0.4
All aa’s
2.8
0.3
Only lysine
8.9
0.4
30.1
0.2
(AAA=lys)
All but lysine
Maximal pppGpp depends only on absence of lysine
Does uncharged tRNA
pppGpp?
Is [pppGpp] correlated with [lys-tRNA]?
Dose-response curve
Yes
Does uncharged tRNA
pppGpp?
• In vitro, Lys induces pppGpp
• In vitro, Lys induces Lys-tRNA
• In vitro, Lys-tRNA correlated with pppGpp
What happens in vivo?
What does ppGpp do?
Stuck!