[BIC-announce] Seminar in Biomedical Engineering - Wednesday Nov 6th - 1 pm - Room 333

[Christophe Grova]

Dear all,

We will have our next Biomedical Engineering seminar today

Wednesday – November 6th,  at 1 pm

Location: Room  333 Lyman Duff Building (Biomedical Engineering Dpt, 3775  University Street).

Speaker: Dr. Catherine Paradis-Bleau PhD, Département de microbiologie, infectiologie et immunologie, Université de Montréal

Title: Novel genetic screens revealed key players in Gram-negative envelope assembly

Abstract:


Gram-negative bacteria are surrounded by a complex cell envelope composed of two membranes

sandwiching the cell wall. Most of the essential envelope biogenesis systems have now been

identified in Escherichia coli and related proteobacterial pathogens including: (i) the Sec system

that transports proteins across the inner membrane or inserts them into it, (ii) the Lol system for

lipoprotein transport to the outer membrane, (iii) the Bam system for outer membrane beta-barrel

protein assembly, (iv) the Lpt system for lipopolysaccharide transport to and assembly in the

outer membrane, and (v) the penicillin-binding proteins (PBPs) that construct the cell wall layer.

What remains unclear is how these different processes are controlled and coordinated with one

another so that the envelope grows uniformly and maintains its integrity as it is remodeled. Given

that genes coding for envelope proteins constitute roughly one quarter of the E. coli genome, and

that about half of these have an unknown or poorly understood function, it is likely that many

factors important for modulating envelope assembly remain to be identified.


I will first describe the design of synthetic lethal screens to directly identify factors essential for

the in vivo activity of the bifunctional enzymes PBP1a and PBP1b that construct the cell wall

layer in the model gram-negative rod Escherichia coli. These screens allowed the discovery of

two outer membrane lipoproteins of previously unknown function as new cell wall assembly

factors. We have designated them LpoA and LpoB for lipoprotein modulators of PBP1 function

from the outer membrane. LpoA was found to be specifically required for PBP1a function while

LpoB was found to be specifically required for PBP1b function. These factors directly modulate

the activity of their cognate PBPs through the formation of specific trans-envelope lipoprotein-

PBP complexes. The conserved LpoA and LpoB factors probably direct and guide cell wall

biogenesis to assure coordination with the outer membrane in Gram-negative envelope.


I will then present the development of a large-scale genetic method to discover new factors

involved in Gram-negative envelope biogenesis. We decided to use an old reporter, ?-

galactosidase (LacZ), in a new way. The LacZ substrate chlorophenyl red-?-D-galactopyranoside

(CPRG) fails to penetrate the E. coli envelope and cannot be processed by cytoplasmic LacZ.

Because mutants impaired for envelope assembly typically either lyse at an elevated frequency to

release LacZ into the medium and/or are more permeable to small molecules like CPRG, they are

readily identifiable based on CPRG hydrolysis and the formation of red colonies on CPRGcontaining

agar. We proceeded to systematically screen an ordered E. coli deletion library for

mutants with a CPRG+ phenotype. We identified 463 mutants with a CPRG+ phenotype and

implicating 102 genes of unknown function in proper envelope assembly. As a proof of principle,

we further analyzed a mutant inactivated for ycbC, which encodes a factor with a highly

conserved domain of unknown function (DUF218). Loss of YcbC function was found to cause a

severe growth defect at low temperature accompanied by an elevated frequency of cell lysis

resulting from impaired metabolism of the essential lipid precursor required for cell wall

biogenesis. We have therefore renamed this factor ElyC (elevated frequency of lysis) to reflect

this phenotype. Further analysis of the function of ElyC and other CPRG+ mutants is likely to

uncover a wealth of new information about the biogenesis of the Gram-negative envelope and its

control under different environmental conditions. Importantly, the CPRG screen should also be

transferable to other organisms to study the biogenesis of different envelope architectures.


A list of upcoming seminars can be found at : http://www.mcgill.ca/bme/news/seminars

See you there


Christophe Grova

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Christophe Grova, PhD
Assistant Professor
Biomedical Engineering Dpt
Neurology and Neurosurgery Dpt

Multimodal Functional Imaging Lab (Multi FunkIm)
Montreal Neurological Institute
Centre de Recherches en Mathématiques

Biomedical Engineering Department - Room 304
McGill University
3775 University Street, Montreal, Quebec, Canada,
H3A 2B4
email : christophe.grova at mcgill.ca<mailto:christophe.grova at mcgill.ca>
tel : (514) 398 2516
fax : (514) 398 7461

Web:
http://www.bic.mni.mcgill.ca/ResearchLabsMFIL/PeopleChristophe
http://www.bmed.mcgill.ca/
MultiFunkIm Lab:
http://www.bic.mni.mcgill.ca/ResearchLabsMFIL/HomePage

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