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Thomas BernhardtDepartment of Microbiology & Immunobiology
Harvard Medical School, Boston, MA
A high throughput genetic screen for the identification of cell envelope assembly
factors in gram-negative bacteria
Outer Membrane (OM)
Inner Membrane (IM)
Peptidoglycan (PG)
Periplasm
Cytoplasm
The Gram-negative Cell EnvelopeLPS
Envelope Assembly Systems
Lol System
Sec
Bam Complex
PBPs-Lpos Lpt System
LP
LP
LP
β-barrel
IMprotein
Most of the essential factors identified
Hypothesis: Additional factors important for envelope assembly remain to be identified
Over 1/3 of ca.1000 genes encoding envelope proteins in E. coli remain unannotated
Hu et al. PLoS Bio 2009
CPRGLacZZ
LacZ
CZZZZZZZZZZ LacZacccccccccZZZZZZZZZZZZZZZZZZZZZZZZZ
LacZLLLLLLLLLLLLLLacZ
LLLLLLLLLLLLLaaaaaaaacZLLLLLLLLLLLLLLaaaaaaaaaaaaaaaaaaacccZ
LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLaaaaaaLacZ
CPRG
CPRCPRG
LacZ
LacZ
LacZLacZLaaccZaacccZZZaaccZZZ
ZZLLLaaacLLLLLaa
LaccccccccccZZZZZZZZZLLLLLLLLLaaaaaaaaaaaaaaccccccccccccLLLLLLacZ
LacZLacZ
LacZ CPRG CPR
WT Env-
Identify Mutants Using a Membrane Impermeable LacZ Substrate
Screen E. coli knockout library
E. coli Keio collection
in collaboration with Carol Gross & Nassos Typas
Screen E. coli knockout library
E. coli Keio collection
in collaboration with Carol Gross & Nassos Typas
Problem: parent strain LacZ-
Converting the Keio collection to Lac+Keio RecipientΔgeneX::kanR
lacZ
MOB
DonorMobile lacZ plasmid; CamR
pin ontolawn of Donor
Converting the Keio collection to Lac+Keio RecipientΔgeneX::kanR
lacZ
MOB
DonorMobile lacZ plasmid; CamR
pin ontolawn of Donor
lacZ
MOB
ExconjugantΔgeneX::kanR/lacZ plasmid
pin ontoLB Kan + Cam
ResultsScreened: LB no salt & LB 1% salt @ RT and 30oC
Numerous CPRG+many known envelope assembly genes:
bamB, imp4213, lolA*, lpp, mrcB, surA, smpA, tatB, tolA....
102 genes of unknown function
Chose to characterize ElyC (YcbC)
N CDUF218periplasm
cytoplasm
- Strong Ely phenotype at RT- Highly conserved domain of unknown function
IM
PG
Does loss of ElyC function affect the PG biogenesis pathway?
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
IM
PG
Does loss of ElyC function affect the PG biogenesis pathway?
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
DAP
Cell wall biosynthesis is blocked in ΔelyC cells
MG1655
Time (hrs)
WT
SDS-
inso
lubl
e C
PM x
103
ΔelyC
LB room temperature25
20
15
10
5
0
x103
1086420
IM
PG
The PG biogenesis pathway
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
Can overexpression of PG biogenesis factors suppress the lysis phenotype of ElyC- cells?
overexpressed gene
ΔelyC
IM
PG
Result support a role for ElyC in PG biogenesis
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
IM
PG
Result support a role for ElyC in PG biogenesis
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
IM
PG
Result support a role for ElyC in PG biogenesis
MraY
lipid I
pp Mp
undecaprenol-P
polyisoprenoid biosynthesis
UppS
lipid IIGUDP-
pp M G
MurG
pp M G
GUDP-
MurAGUDP- -EP
MurBUDP- M
MurC-FUDP- M
PBPs
Many important surface polysaccharides
SUMMARY
- developed a screen for envelope assembly mutants in E. coli suitable for a high throughput format
- identified over 100 y-genes with a potential role in envelope assembly
-characterized one of these factors, ElyC, and found that it likely plays an important role in Und-P metabolism
- Screen should be adaptable to other organisms