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Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB
Molecular Microbiology (2004)

53

(6), 17711783
doi:10.1111/j.1365-2958.2004.04242.x
© 2004 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004

? 2004

53

617711783

Original Article

Complex spatial distribution and dynamics of LamBK. A. Gibbs
et al.

Accepted 1 June, 2004. *For correspondence. E-mail
theriot@stanford.edu; Tel. (

+

1)
650
725
7968; Fax (

+

1) 650 723
6783.

Present address: Miami Valley Laboratories, The Proctor and
Gamble Company, Cincinnati, OH 45252, USA.

Complex spatial distribution and dynamics of an
abundant

Escherichia coli

outer membrane protein,
LamB

Karine A. Gibbs,

1

Daniel D. Isaac,

2

Jun Xu,

3


Roger W. Hendrix,

3

Thomas J. Silhavy

2

and
Julie A. Theriot

1,4

*

1

Department of Microbiology and Immunology, Stanford
University School of Medicine, Stanford, CA 94305, USA.

2

Department of Molecular Biology, Princeton University,
Princeton, NJ 08544, USA.

3

Pittsburgh Bacteriophage Institute and Department of
Biological Sciences, University of Pittsburgh, Pittsburgh,
PA 15260, USA.

4

Department of Biochemistry, Stanford University School
of Medicine, Stanford, CA 94305, USA.

Summary
Advanced techniques for observing protein localiza-
tion in live bacteria show that the distributions are
dynamic. For technical reasons, most such tech-
niques have not been applied to outer membrane pro-
teins in Gram-negative bacteria. We have developed
two novel live-cell imaging techniques to observe the
surface distribution of LamB, an abundant integral
outer membrane protein in

Escherichia coli

res-
ponsible for maltose uptake and for attachment of
bacteriophage lambda. Using uorescently labelled
bacteriophage lambda tails, we quantitatively des-
cribed the spatial distribution and dynamic movement
of LamB in the outer membrane. LamB accumulated
in spiral patterns. The distribution depended on cell
length and changed rapidly. The majority of the pro-
tein diffused along spirals extending across the cell
body. Tracking single particles, we found that there
are two populations of LamB one shows very
restricted diffusion and the other shows greater
mobility. The presence of two populations recalls the
partitioning of eukaryotic membrane proteins
between mobile and immobile populations. In this
study, we have demonstrated that LamB moves along
the bacterial surface and that these movements are
restricted by an underlying dynamic spiral pattern.
Introduction

Over the past decade, advances in techniques for mea-
suring protein localization in bacteria have revealed that
proteins involved in cell division and cell shape (e.g. FtsZ
and MreB), virulence proteins (e.g. IcsA), chemoreceptors
and agella all have specic cellular distributions (Gold-
berg

et al

., 1993; Maddock and Shapiro, 1993; Steinhauer

et al

., 1999; Lybarger and Maddock, 2000; 2001; Fu

et al

.,
2001; Robbins

et al

., 2001; Shapiro

et al

., 2002). Not
restricted to one bacterial phylum, non-uniform subcellular
localization reects the complexity of the bacterial cell
(Lybarger and Maddock, 2001). With an emphasis on
cytoplasmic and inner membrane components, recent
live-cell imaging using green uorescent protein (GFP)
and its derivatives (Feilmeier

et al

., 2000; Southward and
Surette, 2002) has shown that these subcellular protein
patterns can vary from indistinct compact accumulations
to elegant helical structures and that they can change
rapidly. With the advancement of deconvolution micros-
copy, the resolution of subcellular distributions has
increased, revealing that a number of the indistinct accu-
mulations, such as MinCDE in

Escherichia coli

and

Bacil-
lus subtilis

, are in fact helices (Marston and Errington,
1999; Shih

et al

., 2003). Most prominent of these helices
in Gram-negative bacteria are structures containing MreB,
SetB, and the complement of Min proteins, MinCDE
(Espeli

et al

., 2003; Shih

et al

., 2003), all of which are
crucial for cell shape and division (Jones

et al

., 2001; Shih

et al

., 2003). GFP, however, does not fold properly for
uorescence when exported from the cytoplasm via the
general secretory pathway, therefore prohibiting its use for
localizing outer membrane components (Feilmeier

et al

.,
2000).
While the eld of bacterial cell biology is beginning to
reveal the dynamic architecture inside bacteria, we still
have little insight into the dynamics of the Gram-negative
bacterial outer membrane surface in live cells. The overall
architecture of the outer membrane, proteins interspersed
with phospholipids and lipopolysaccharides (LPS), has
been biochemically detailed with respect to composition
ratios, uidity and asymmetry of the inner versus outer
leaet (Jaffe and DAri, 1985; Souzu, 1986; Rodriguez-
Torres

et al

., 1993). However, the spatial distribution of
membrane components, specically proteins, along the
1772

K. A. Gibbs

et al.

© 2004 Blackwell Publishing Ltd,

Molecular Microbiology

,

53

, 17711783

surface has been elusive. A few macromolecular com-
plexes, such as agella and pili, are readily observed
anchored peritrichously along the bacterial surface in

E.
coli

, or exclusively at one pole in other Gram-negative
organisms (e.g.

Pseudomonas aeruginosa

and

Vibrio
cholerae

) (McCarter, 2001; Mattick, 2002; Shapiro

et al

.,
2002). These organelles differ from other outer membrane
structures in that they are anchored through the cell body
to both periplasmic and plasma membrane components,
similar to the type II and type III secretion systems (Tha-
nassi and Hultgren, 2000).
IcsA (VirG) in

Shigella exneri

is one of the few outer
membrane proteins in Gram-negative Enterobacteriaceae
whose spatial localization has been determined. A viru-
lence protein that promotes actin polymerization, IcsA is
secreted at one pole of

S. exneri

and diffuses laterally
across the cell body (Goldberg

et al

., 1993; Goldberg and
Theriot, 1995; Steinhauer

et al

., 1999; Charles

et al

.,
2001; Robbins

et al

., 2001). In predivisional cells, IcsA
localizes asymmetrically to both cell poles, but is not found
in the middle of the cell (Goldberg

et al

., 1993; Robbins

et al

., 2001). Unlike the agella and pili components, IcsA
is believed to have no periplasmic or intracellular binding
partners and is integrated fully into the outer membrane
as a beta-barrel. Localization of IcsA raises the question
of whether other outer membrane proteins on the surface
of Gram-negative bacteria might be localized to the poles.
To address this, we sought to understand the dynamics
and localization of the nutrient uptake protein, LamB.
LamB (maltoporin) is an abundant integral outer mem-
brane porin, responsible for maltose uptake. Found in
many species of Gram-negative bacteria, it is also the
receptor for bacteriophage lambda (Randall-Hazelbauer
and Schwartz, 1973; Boos and Shuman, 1998). It has
been studied for the past 40 years, and its structure, func-
tion and regulation are understood on both the genetic
and molecular levels. The synthesis of LamB is controlled
by carbon metabolism and increases in the presence
of maltose and just before division (Vos-Scheperkeuter

et al

., 1984; Boos and Shuman, 1998). LamB accepts
many insertions without loss of activity, thus allowing for
a wide range of epitope tags (Boulain

et al

., 1986; Newton

et al

., 1996; Andersen

et al

., 1999; Etz

et al

., 2001).
Found mainly as a trimer in the outer membrane, there
are

ª

30 000 copies of LamB monomer present on the
bacterial surface at the height of induction (Neidhardt

et al

., 1987). Early descriptions of LamB located it at the
poles and septum when

E. coli

K12 cells were probed with
live bacteriophage lambda, and localization depended on
bacterial cell length. This led the authors to propose that
LamB insertion in the outer membrane occurs at the sep-
tum in a subset of predivisional cells (Ryter

et al

., 1975).
Other groups, using

lamB

on a high-copy plasmid under
external (

lac

promoter) control, used indirect immunogold
electron microscopy (with a primary antibody to LamB and
a protein A-gold probe) to localize LamB. They demon-
strated that LamB insertion into the

E. coli

K12 outer
membrane occurred randomly across the entire cell body
and that there was no apparent preferential insertion of
LamB either at the poles or at midcell in predivisional cells
(Vos-Scheperkeuter

et al

., 1984; Jaffe and DAri, 1985).
While these studies both indicated the presence of