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

Envelope Assembly Mutants Often Lyse at an Elevated Frequency

WT Env-

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

KanR

Trial run with Tn-mutagenized cells

MG1655 (Lac+)

EZTnKanLB +CPRG +IPTG

Screening Plate

ely (elevated frequency of lysis)

High frequency of isolation ca. 0.5-1%

A closer lookLB (1% NaCl)

MG1655 Ely7

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

Screening the Keio collection (LacZ+)

elyC

screening plate (RT LB1) image analysisA B

IRIS: Quantitative Analysis of CPRG

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

ElyC phenotypes (@Room Temp)

LB CPRG

LB 1% NaCl

WT ΔelyC

ΔelyC

WT

3

4

5

6

789

1

181614121086

OD

600

Time (hrs)

LB 1% NaCl

21oC

ElyC phenotypes (continued)

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?

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

AcknowledgmentsBernhardt labAssya YakhninaHongbaek ChoNick PetersRachel YunckMary-Jane Tsang Mui ChingGhee Chuan LaiChris ShamDerek LauTing PangAndrew Fenton

Nassos TypasGeorge Kritikos

Collaborators

CatherineParadis-Bleau

Elizabeth Merrit

Former Lab Members