Poles apart: prokaryotic polar organelles and their spatial regulation.

Abstract

While polar organelles hold the key to understanding the fundamentals of cell polarity and cell biological principles in general, they have served in the past merely for taxonomical purposes. Here, we highlight recent efforts in unraveling the molecular basis of polar organelle positioning in bacterial cells. Specifically, we detail the role of members of the Ras-like GTPase superfamily and coiled-coil-rich scaffolding proteins in modulating bacterial cell polarity and in recruiting effector proteins to polar sites. Such roles are well established for eukaryotic cells, but not for bacterial cells that are generally considered diffusion-limited. Studies on spatial regulation of protein positioning in bacterial cells, though still in their infancy, will undoubtedly experience a surge of interest, as comprehensive localization screens have yielded an extensive list of (polarly) localized proteins, potentially reflecting subcellular sites of functional specialization predicted for organelles.

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Poles Apart: Prokaryotic Polar Organelles
and Their Spatial Regulation
Clare L. Kirkpatrick and Patrick H. Viollier
Department of Microbiology and Molecular Medicine, Centre Me ´dicale Universitaire, Faculty of Medicine,
University of Geneva, Rue Michel Servet 1, CH-1211 Geneva 4, Switzerland
Correspondence: patrick.viollier@unige.ch
While polar organelles hold the key to understanding the fundamentals of cell polarity and
cellbiologicalprinciplesingeneral,theyhaveservedinthepastmerelyfortaxonomicalpur-
poses. Here, we highlight recent efforts in unraveling the molecular basis of polar organelle
positioning in bacterial cells. Specifically, we detail the role of members of the Ras-like
GTPase superfamily and coiled-coil-rich scaffolding proteins in modulating bacterial cell
polarity and in recruiting effector proteins to polar sites. Such roles are well established for
eukaryotic cells, but not for bacterial cells that are generally considered diffusion-limited.
Studies on spatial regulation of protein positioning in bacterial cells, though still in their
infancy, will undoubtedly experience a surge of interest, as comprehensive localization
screens have yielded an extensive list of (polarly) localized proteins, potentially reflecting
subcellular sites of functional specialization predicted for organelles.
S
we have known that bacterial cells are polarized
and that they are able to decode the underlying
positional information to confine the assembly
of an extracellular organelle to a polar cellular
site (Fig. 1). Foraging into this unknown terri-
tory has been challenging, but recent efforts
thatexploitthepowerofbacterialgeneticsalong
withmodernimagingmethodstovisualizepro-
teins in the minute bacterial cells has yielded
several enticing entry pointsto dissect polarity-
based mechanisms and explore potentiallycon-
tributing subdiffusive characteristics (Golding
and Cox 2006).
While polar organelles are avisual manifes-
tation of polarity, it is important to point out
ince the first electron micrographs that re-
vealed flagella at the cell poles of bacteria,
that polarity can also be inherent to cells, at
least in molecular terms, even in the absence
of discernible polar structures. In other words,
molecular anatomy can reveal that a bacterial
cell,such as an Escherichiacoli cell, featuresspe-
cialized protein complexes at or near the poles,
despite a perfectly symmetrical morphology
(Maddock and Shapiro 1993; Lindner et al.
2008). Such systemic polarization in bacteria,
likely stemming from the distinctive division
history of each pole, has the potential to be
widespread and to be exploited for positioning
of polar organelles and protein complexes. As
excellent reviews have been published detail-
ing the interplay between cell polarity and pro-
tein localization (Dworkin 2009; Shapiro et al.
2009; Kaiser et al. 2010; Rudner and Losick
Editors: Lucy Shapiro and Richard M. Losick
Additional Perspectives on Cell Biology of Bacteria available at www.cshperspectives.org
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2010), here we focus on recent progress in
understanding the function and localization of
spatial regulators of polarorganelles. Consider-
ing that the ever-growing list of polar protein
complexes emerging from systematic and com-
prehensive localization studies (Kitagawa et al.
2005; Russell and Keiler 2008; Werner et al.
2009; Hughes et al. 2010) is suggestive of multi-
ple polarly confined (organelle-like) functions,
understanding their spatial regulation is also
ofcritical relevanceintherealm ofmedicalbac-
teriology, as many virulence determinants also
underlie polarity (Goldberg et al. 1993; Scott
et al. 2001; Judd et al. 2005; Jainet al. 2006; Jau-
mouille et al. 2008; Carlsson et al. 2009). Below,
we highlight a few prominent examples of
overtly polarorganellesandtheproteinsknown
to date that regulate their polar positioning.
THE POLARITY OF MOTILITY ORGANELLES
Flagellar Motility: FlhFG
The Ras-likeGTPaseFlhF isrequiredfor proper
assembly and placement of the polar flagellum
in several bacterial lineages, including Vibrio,
Pseudomonas, Helicobacter, and Campylobacter
species (Pandza et al. 2000; Niehus et al. 2004;
Murray and Kazmierczak 2006; Kusumoto
et al. 2008; Balaban et al. 2009; Green et al.
2009). Flagellum biogenesis occurs through an
“inside-out” process by which components of
the early structure first assemble a platform on
the cytoplasmic membrane (the MS-ring) that
will facilitate the subsequent polymerization
and growth of the trans-envelope structures
through the periplasm (the flagellar rod), the
peptidoglycan (P-ring), and outer membrane
(L-ring), ultimately to enable assembly of the
extracellular hook and flagellar filament (Mac-
nab 2003) (Fig. 2A). FlhF mutants often exhibit
a reduced ability to assemble flagella and in the
case of Vibrio cholerae this seems to be attrib-
utable to a requirement of FlhF for the polar
recruitment of the flagellar baseplate (MS-ring)
protein FliF (Green et al. 2009). In the absence
of FlhF, FliF is dispersed from the poles and
flagella are formed at a reduced frequency and
at aberrant (nonpolar) locations. Conversely,
functional FlhF-GFP is localized to the flagel-
lated pole in V. cholerae and Campylobacter
Figure 1. Transmission electron micrograph (taken by Jeff Skerker) of a Caulobacter crescentus swarmer cell
showing the polar pili (empty arrowheads), the polar flagellum with the flagellar filament (filled arrowheads),
and the hook (white arrow) (see Fig. 2A).
C.L. Kirkpatrick and P.H. Viollier
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jejuni (Murray and Kazmierczak 2006; Ewing
et al. 2009; Green et al. 2009) independently of
flagellar structural proteins (Ewing et al. 2009;
Green et al. 2009) (Fig. 2B). Thus, FlhF plays a
roleinbothflagellumpositioningandassembly,
and the latter function may in part be mediated
at the level of gene expression (Niehus et al.
2004; Correa et al. 2005). A member of the
(SIMIBI) Ras-like GTPase superfamily, FlhF
associates peripherally with the cytoplasmic
membrane (Green et al. 2009) and exhibits ex-
tensive sequence homology to the signal recog-
nition particle (SRP) component Ffh, a GTPase
that mediates the co-translational insertion of
membrane proteins (Driessen and Nouwen
2008). This event requires a heteromeric inter-
action of Ffhwith its receptor, the FtsYGTPase.
A truncated fragment of Bacillus subitilis FlhF
has been crystallized as a dimer, forming a
composite active site in which the two GTP
molecules are arranged in a head-to-tail config-
uration (Bange et al. 2007). While this same
configuration in the composite active site of
the Ffh-FtsY heterodimer induces GTP hydrol-
ysis, this is not the case for B. subtilis FlhF.
However, recent in vitro assays with recombi-
nant full-length FlhF from C. jejuni harboring
an N-terminal His6-tag exhibited GTPase and
comparatively low ATPase activity (Balaban
et al. 2009). Point mutations that cripple the
GTPase activityof C. jejuni FlhF result in flagel-
lar placement defects that resemble the pheno-
types observed for the flhF deletion mutant of
V. cholerae, Pseudomonas putida, and Pseudo-
monas aeruginosa (Pandza et al. 2000; Murray
and Kazmierczak 2006; Balaban et al. 2009;
Green et al. 2009). Moreover, FlhF with a muta-
tion in the conserved nucleotide-binding
P-loop (K295A) has a reduced ability to bind
GTP and is unable to support motility in
V. cholerae (Green et al. 2009). These findings
indicate a link between flagellar positioning
and the ability to bind and/or hydrolyze GTP.
The FlhF polypetide is sequentially subdivided
into the B-N-G regions. The conserved GTPase
domain of FlhF resides in the C-terminal G-
region. The G-region is preceded by the central
N-regionthatisrichina-helices,whilethebasic
and unstructured (protease-sensitive) B-region
is located at the N-terminus of FlhF (Bange
Filament
Hook
OM
PG
IM
Switch
MS-ring
AB
C
V. cholerae
C. crescentus
FlhF
TipF
PflITipN >
>
Figure 2. Spatial regulation of the polar flagellum (A) by the FlhFGTPase in Vibrio cholerae (B) and by the TipF
c-di-GMPreceptorprotein,theTipNbirthscarprotein,andthePflIpositioningfactorinCaulobactercrescentus
(C).InPanelA,theoutermembrane,peptidoglycanlayer,andinnermembraneareabbreviatedbyOM,PG,and
IM, respectively. In Panels B and C, the wavy and straight lines denote the flagellum and the pili, respectively.
Spatial Regulation of Polar Organelles in Bacteria
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et al. 2007). Unlike the G-region, the B- and N-
regions are quite variable in length among FlhF
orthologs, raising the possibility that these
regions account for the structural and func-
tional differences observed in vitro and in
vivo.Infact,theN-regionincludedinthecrystal
structure of B. subtilis FlhF is significantly
shorter than that from FlhF encoded in the
genomes of polarly flagellated bacteria. Dele-
tion analysis in V. cholerae pointed to a key
role for the N-region in polar localization of
FlhF (Green et al. 2009). FlhF-GFP deleted for
the N-region was not localized to the pole and
unable to confer motility. The N-region could
support polar localization, but not motility,
when fused to either the B region (i.e., as BN-
GFP fusion) or the G-region (i.e., as NG-GFP
fusion). However, the N-region alone (i.e., as
N-GFP construct) was unable to direct GFP to
the pole (Green et al. 2009). It is conceivable
that an association with the membrane is a pre-
requisite for FlhF to recognize localization
determinant(s) that may reside in the envelope,
as isthe case forother polarly localized proteins
(Jacobs et al. 1999; Boyd 2000). If so, the N-
region might be required for this membrane
association, while the B- and G-regions recog-
nize the localization determinant(s). Perhaps
one region is used for polar targeting, while
the other region functions as polar retention
domain. This would imply that the mechanism
and targets for the polar localization for BN-
GFP and NG-GFP is different. In support of
the findings with truncated FlhF derivatives
that are localized but not functional, full length
FlhF-GFP with the GTP-binding K295A muta-
tion did not support motility either, while still
allowing polar recruitment, indicating that
bound GTP is not a prerequisite for polar local-
ization (Green et al. 2009). The identity of the
polar determinant(s) recognized by FlhF is
notknown,but itisnot astructuralcomponent
oftheflagellumasFlhF-GFPisstillatthepolein
Vibrio mutants that do not express any flagellar
subunits (Kusumoto et al. 2008; Green et al.
2009). It would be interesting to examine
whether the BN-GFP and NG-GFP constructs
arestillpolarintheabsenceoftheflagellarcom-
ponents to determine whether each of the FlhF
subdomains relies on flagellar parts for polar
recruitmentand/orretention.Theprincipalde-
terminant for FlhF localization appears to be
widely conserved across bacterial species, since
FlhF-GFP from V. cholerae is also polar in E.
coli (Green et al. 2009). Thus, even a peritri-
chously-flagellatedbacteriumsuchasE.colienc-
odes a positionalsignalthat isrecognized by the
B-and/orG-regionofFlhF.Interestingly,FlhFis
localizedtobothpolesinE.coli,butinthenative
hostFlhF-GFPwasalso seeninapredominantly
unipolar localization pattern (Murray and Kaz-
mierczak 2006; Kusumoto et al. 2008; Green
etal.2009),suggestingthataspecificmechanism
existstoimpartpolarspecificityonFlhFlocaliza-
tion and, thus, the new flagellum assemblysite.
Although FlhF may regulate the correct
positioning of the core flagellum assembly plat-
form in the cytoplasmic membrane through
FliF localization, recent work in C. jejuni has
unearthedflagellarglycosylation
components that are localized to the cell pole
independently of FlhF (Ewing et al. 2009). In
many polarly flagellated bacteria, the flagellar
filament is polymerized from glycosylated fla-
gellins. Glycosylation seems to precede the se-
cretion of the flagellin (Ewing et al. 2009), yet
its general function is not understood. Many
components of the glycosylation pathway are
dispensable for motility. This may be attribut-
able to a certain redundancy in glycosylation
components, as several polarly flagellated bac-
teria of the genera Campylobacter, Helicobacter,
and Caulobacter are unable to polymerize a
flagellarfilamentandunabletosecreteunglyco-
sylated flagellins when other glycosylation pro-
teins are inactivated (Leclerc et al. 1998; Goon
et al. 2003; Schirm et al. 2003). Thus, glycosyla-
tion seems to fulfill an important and mysteri-
ous role in the assembly of polar flagella.
Pertinent to the tentative link between polar
flagellation and glycosylation, the components
of the glycosylation machinery PseC and PseE
are localized to the pole in C. jejuni (Ewing
et al. 2009). Although FlhF is also at the cell
pole in C. jejuni, it is not required for the polar
positioning of PseC and PseE. In support of
this, PseC and PseE are also still at the poles in
the absence of the flagellar export apparatus.
pathway
C.L. Kirkpatrick and P.H. Viollier
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Thus, a FlhF-independent flagellar localization
pathway exists that remains to be uncovered
(Ewing et al. 2009).
FlhF is also found in peritrichously flagel-
lated bacteria and an investigation on FlhF in
Bacillus cereus determined that inactivation of
FlhF reduced flagellation and resulted in a bias
toward polar flagella from a normal peritri-
chous flagellar arrangement (Salvetti et al.
2007). While the function of FlhF in flagel-
lum biogenesis does not appear to be special-
ized for polar flagellation, certain bacteria may
have appropriated FlhF to recruit flagellarcom-
ponents to the pole and, therefore, to confine
the assemblyof the flagellar structure to a polar
site, simply by directing FlhF to the pole. In
other cases, such as B. cereus, FlhF may be lo-
calized to nonpolar sites to obtain a peritrich-
ous flagellar disposition, implying an under-
lying default pathway for polar flagellation in
B. cereus that FlhF acts upon to guide flagellar
assembly factors to lateral sites.
The mechanism(s) of spatial regulation of
FlhF likely holds the key to understanding the
FlhF-dependent polar flagellation in Vibrios,
Pseudomads, and others. Circumstantial evi-
dence has implicated FlhG (also known as FleN
in Pseudomonas spp.), which resembles the
ParA/MinD superfamily of ATPases (cd01983)
thatfeatureadeviantWalkerA-typenucleotide-
binding pocket (the P-loop), in the regulation
of the polar localization of FlhF (Kusumoto
et al. 2008). MinD and ParA adopt key roles as
part of positioning systems that direct the
proper placement of the cytokinetic organelle
or (chromosomal or plasmid) DNA, respec-
tively, within the prokaryoticcellor the eukary-
otic chloroplasts (de Boer et al. 1991; Margolin
2005; Gerdes et al. 2010; Thanbichler 2010). In
the absence of FlhG, the fluorescent cluster of
FlhF-GFP at the cell pole was brighter than
when FlhG was present (Kusumoto et al.
2008). While this result suggested that the polar
localization of FlhF is improved in FlhG-defi-
cient cells, theyalso had higher steady-state lev-
elsofFlhF.Nevertheless,thediffusebackground
fluorescence of FlhF-GFP in the cytoplasm was
also reduced in cells lacking FlhG, pointing
toward a role of FlhG in interference with the
polar sequestration of FlhF (Kusumoto et al.
2008). Interestingly, mutant forms of FlhF
have been identified that show the opposite
phenotype of the FlhG-deficient cells (i.e.,
strong dependency on the presence of FlhG
for polar localization), and GFP-tagged FlhG
is also polar (Kusumoto et al. 2009). These
observations are consistent with the proposed
roleoftheParA/MinDATPasesactingasbinary
regulatory switches that control the localiza-
tion of cellular constituents, a recurring theme
throughout this article. Below we outline the
role of other ParA/MinD-homologs in the
localization of organellar components, includ-
ing that of a chemosensory complex (Thomp-
son et al. 2006), a pilus biogenesis apparatus
(Viollier et al. 2002a), a DNA transfer machine
(Atmakuri et al. 2007), and a polysaccharide
export system (Le Quere and Ghigo 2009).
Flagellar Motility: TipNF-PflI
While FlhG may be a keyelement for the spatial
regulation of FlhF, many polarly flagellated
bacterial lineages, primarily from the a-proteo-
bacteria, do not encode FlhF in their genomes.
Thus, nature has devised another path to polar
flagellation, one that seems to involve a con-
served positioning protein, PflI. PflIwas identi-
fied using a bioinformatic approach and then
characterized in the vibroid-shaped bacterium
Caulobacter crescentus (Obuchowski and Jacobs-
Wagner 2007). In C. crescentus, the flagellum
is always built at the new pole (i.e., the pole
that emerged from the most recent division
event). PflI, a bitopic membrane protein with
a cytoplasmic coiled-coil domain followed by
a proline-rich stretch of 102 residues, is local-
ized to the future flagellated pole well before
the flagellar proteins are expressed. Moreover,
PflI is still localized to the new pole in the
absence of the MS-ring protein FliF, indicating
that, akin to FlhF, PflI does not depend on an
intact flagellar structure for polar localization.
Importantly, inactivation or overexpression of
PflI results in a high proportion of cells with
misplaced flagella, while not affecting the local-
ization of polar marker proteins; for example,
that of the pilus assembly machinery (see
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below) or signaling proteins that normally co-
localize with the flagellum. Thus, PflI seems to
function as a positioning factor specifically for
theflagellum. HowPflIinfluencesthe flagellum
positioning is unknown, but it is likely that
additional localization pathways work in con-
cert with PflI. This conjecture is based on the
finding that in PflI-deficient or -overproducing
cells, the flagellum misplacement is not as per-
vasive, with 30–60% of the flagellated cells still
having a polar flagellum (Obuchowski and
Jacobs-Wagner 2007). Progress has recently
been made in understanding how PflI localiza-
tion is regulated (Davis et al. 2010, in prep). As
hintedbythesimilarlocalizationpatternduring
the cell cycle (Fig. 2C) and in flagellar mutants,
the recruitment of PflI to the future flagellated
pole depends on the EAL-domain protein TipF
(Huitema et al. 2006; Obuchowski and Jacobs-
Wagner 2007). A polytopic membrane protein
previously known to regulate flagellar assembly
and implicated in flagellar positioning, TipF is
also localized to the new pole even in the
absenceofearly flagellarstructuralcomponents
suchastheFliFMS-ring protein(Huitemaetal.
2006) (Davis et al. 2010, in prep). The cyto-
plasmic EAL-domain is responsible for TipF
function in flagellum assembly and the recruit-
mentofPflItothepole(Huitemaetal.2006).In
related proteins, the EAL-domainis responsible
for the binding and subsequent hydrolysis of
cyclic-di-GMP, a signaling molecule that drives
the switch from motility to sessility in eubacte-
ria.(JenalandMalone2006;Hengge2009).The
EAL domain can also act as c-di-GMP binding
domainorprotein–proteininteractiondomain
in the absence of hydrolytic activity (Newell
et al. 2009; Tschowri et al. 2009). The TipF
EAL-domain also lacks c-di-GMP hydrolytic
activity, but has retained the ability to bind c-
di-GMP (Davis et al. 2010, in prep). When
TipF is removed or when c-di-GMP-binding is
preventedeither bypointmutationorbydeple-
tion of cellularc-di-GMP levels, flagella are not
assembled and PflI is not localized (Davis et al.
2010, in prep). TipF is also required for the
localization of the components of the flagellar
switch complex (Fig. 2A), but conversely the
switch complex is not necessary for the polar
positioning of TipF. Intriguingly, in the absence
of an association with c-di-GMP, TipF remains
dispersedinthecellenvelopeanddoesnotclus-
ter at the new cell pole. Moreover, under these
conditions, flagella are also not assembled and
PflI also remains dispersed (Davis et al. 2010,
in prep). Thus, binding of c-di-GMP induces
the polar localization of TipF to enable the
recruitmentofaplacementfactorandstructural
proteins for flagellum assemblyat the new pole.
TipF localization was previously shown to be
determined by the TipN birth scar protein, a
polytopic membrane protein with an extensive
coiled-coil-rich domain facing the cytoplasm,
that is required for proper localization of the
flagellum at the new pole (Huitema et al.
2006; Lam et al. 2006). When TipN was inacti-
vated, flagella and TipF (but not pili) were fre-
quently mispositioned at seemingly identical
aberrant locations (Huitema et al. 2006), hint-
ing that akin to FlhF, TipF regulates flagellar
placement and assembly. In dividing cells,
TipNislocalizedatthedivisionplaneinaman-
ner that requires the FtsZ cytokinetic tubulin
homolog and then retained at the newborn
pole that emerges from division (Huitema
etal.2006;Lametal.2006),thesiteofflagellum
assembly and TipF/PflI localization. Thus,
the protein localization and ensuing flagellum
positioning pathway seems to follow the order:
FtsZ . TipN . TipF . PflI. The key element
of this polarity pathway for flagellar assembly
and positioning is that protein localization
relies on a cytokinetic mark or remnant that is
inherited by the newborn pole of each daughter
cell, thereby preselecting these poles as sites of
flagellum biogenesis that attract the down-
stream assembly factors (Huitema et al. 2006;
Lametal.2006).Intriguingly,TipNwasrecently
shown to serve as the spatial cue that provides
the correct directionality to the new pole in
bacterial chromosome segregation (Ptacin et al.
2010; Schofield et al. 2010), showing that other
polarized cellular functions also rely on the
TipN-dependent polarity axis.
In summary, two mechanisms are known,
FlhFG and TipNF-PflI, that both use a gua-
nine nucleotide derivative to regulate the polar
placement of the flagellum. The physiological
C.L. Kirkpatrick and P.H. Viollier
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significance of the guanine nucleotide-depend-
ent input is unclear, but may be related to the
variation in cellular concentration of the
nucleotides over time. C-di-GMP levels fluctu-
ate during the cell cycle (Paul et al. 2008; Chris-
ten et al. 2010b) and GTP levels change as cells
transition from logarithmic growth into sta-
tionary phase (Freese et al. 1979; Buckstein
et al. 2008; Paul et al. 2008; Christen et al.
2010b). Exploiting such fluctuation(s) would
allow cells to tie the assembly of flagellar struc-
tureswiththecellcycleorwiththegrowthphase
whentheenergeticcostfor theassemblyofsuch
a complex nanomachine can be afforded.
Gliding Motility: MglAB
In a striking parallel to the role of the FlhF
GTPase in determining the polarity of the
flagellum-dependent motility system, recent
research illustrates the role of the Ras-like
GTPase MglA in regulating the dynamic polar-
ity switch of (flagellum-independent) gliding
motility of Myxcoccus xanthus cells on surfaces
(Leonardyet al. 2010; Patryn et al. 2010; Zhang
et al. 2010). M. xanthus cells move by way of
cooperative, but distinct, motility systems,
termed S (conferred by pilus motor proteins)
and A (unidentified motor proteins), whose
activity are modulated by polarized effectors
(see Kaiser et al. 2010). A hallmark of motile
M. xanthus cells is that they exhibit periodic
reversals in the direction of movement and in
a fashion that, at the subcellular level, is exqui-
sitely tuned with the dynamic and often anti-
cyclical polar localization of the A-motility
effectors RomR and AglZ (Leonardy et al.
2007; Mignot et al. 2007) and the S-motility
effectors PilT and FrzS (Mignot et al. 2005;
Bulyha et al. 2009; Mauriello et al. 2010). The
dynamic pole-to-pole oscillation of several of
these effectors shows a strong dependency on
the active, presumably GTP-bound, form of
MglA (Leonardy et al. 2010; Zhang et al.
2010). Mutations in MglA that impair hydroly-
sis but not GTP binding 1) alter the cellular
reversal frequency, 2) block polar oscillations
of RomR, AglZ, and PilTand 3) prevent motil-
ity. Moreover, these MglA point mutant strains
phenocopy those with a deletion in the gene
encoding MglB, which is encoded by the open
reading frame immediately upstream of that
for MglA. MglA is localized to the leading
pole (Mauriello et al. 2010; Patryn et al. 2010)
while MglB clusters at the lagging pole (Leo-
nardy et al. 2010; Zhang et al. 2010). Recent
biochemical studies demonstrated that MglB
essentially functions as a GTPase activating
protein (GAP) to MglA to regulate its polar
localization dynamics and activity (Leonardy
et al. 2010; Zhang et al. 2010). In moving cells,
MglA and MglB oscillate from pole to pole
with a seemingly identical period that is
out-of-phasewithoneanotherandcoordinated
withcellreversals(Fig.3).Theanticyclicalpolar
localization of MglA is ablated when GTP
hydrolysis is crippled by inactivation of MglB
or by GTPase mutations in MglA (Leonardy
et al. 2010; Zhang et al. 2010). Moreover,
MglA also modulates the dynamics of MglB
localization (Leonardy et al. 2010). Based on
the reciprocal regulation of MglAB localization
dynamics and the dependency of downstream
motility effectors on MglAB (Mauriello et al.
2010),itstandstoreasonthattheMglABsystem
is a core constituent of the molecular oscilla-
tor that regulates the dynamic polarity of cell
reversals in M. xanthus. Fine-tuning of the
MglAB localization and activity is accom-
plished through the Frz chemosensory system
and the actin-like MreB cytosleketon, albeit in
an unknown way (Leonardy et al. 2010; Maur-
iello et al. 2010; Zhang et al. 2010). Additional
mechanisms in polarity control remain to be
identified, as the motility effectors are still
recruited to the pole in the absence of MglA,
albeit often to the erroneous one.
POLARITY CONTROL OF PILI AND OTHER
ORGANELLAR PROTEINS BY ACTIN
HOMOLOGS, PARA-LIKE ATPASES,
AND A POLARITY FACTOR
Polar Pili
The pili assembled by P. aeruginosa are very
similar with respect to structure, function,
and localization to the pili that confer gliding
Spatial Regulation of Polar Organelles in Bacteria
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motility in M. xanthus (see the schematic of the
pilus in Fig. 3 of Kaiser et al. 2010). They are
built from homologous components into polar
trans-envelope structures that confer a form of
flagellar-independent motility. In P. aeruginosa,
this second form of motility is called (pilus-
mediated)twitchingmotility.Akintotheafore-
mentioned gliding motility, twitching motility
involves the regular and coordinated exten-
sion/retraction cycles of the pilus filament
from the cell surface(Mattick 2002).Consistent
withtheroleoftheactin-likeMreBcytoskeleton
in maintaining the polar localization of the S-
motility regulator FrzS in M. xanthus (Maur-
iello et al. 2010), MreB seems to play a crucial
role in the localization of pili in P. aeruginosa
(Cowles and Gitai 2010). When P. aeruginosa
cells were pretreated with the small molecule
MreB-inhibitor A22 before being placed on
medium that induces pilus production, cells
accumulated pili at lateral sites instead of the
pole. Also, unlike untreated cells, those pre-
exposed to A22 mislocalized the PilTretraction
ATPasetononpolarsites.Thus,MreBmaintains
the polardisposition of piliand pilus regulators
in an unknown way. A likely model would hold
that pilus structural and regulatory proteins
such as PilT and FrzS travel to the pole along
the helical tracks of MreB filaments lining
the cytoplasmic face of the inner membrane
(Cowles and Gitai 2010; Mauriello et al.
2010). Remarkably, the PilM pilus assembly
protein that is encoded in the pil clusters of
both P. aeruginosa and M. xanthus is a member
of the actin superfamily of proteins (Martin
et al. 1995). As MreB isoforms are thought to
inter-digitate into heteromeric cytoskeletal fila-
ments (Carballido-Lopez et al. 2006), it is con-
ceivable that PilM is also integrated into a
cytoskeletalstructuretoconnectthelocalization
of the pilus structure and its constituents with
the cytoskeleton. It will be interesting to deter-
mine the subcellular distribution of PilM and
whether it is affected by treatment with A22.
MreB has also been shown, directly or indir-
ectly, to influence the polar localization of the
Time
A
B
Figure3. Polarityswitching by the Ras-likeGTPaseMglA (green oval)anditsGTPase-activating protein(GAP)
MglB (red oval) in gliding Myxococcus xanthus cells. (A) A time series of gliding M. xanthus cells expressing
MglA-YFPand MglB-mCherry that were imaged every 20 seconds by fluorescence microscopy (by Yong Zhang
and Ta ˆm Mignot). Theyellowcoloring indicatesthe colocalized MglA and MglB. Thewhite arrow indicatesthe
directionofmovementthatreversesduringthecourseoftheexperiment.(B)Aschematicofthecellsandpolarly
localized proteins from panel A. The straight lines denote the pili that confer S-motility.
C.L. Kirkpatrick and P.H. Viollier
8
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virulence determinant IcsA of Shigella spp., the
E. coli chemoreceptor Tar, several developmen-
tal kinases, and the origin of replication (Cori)
in C. crescentus (Goldberg et al. 1993; Gitai
et al. 2004; Shih et al. 2005). Actin-like proteins
also determine the subcellular distribution of
plasmids in E. coli (Gerdes et al. 2010), organ-
elles like magnetosomes in Magnetospirrillum
magneticum (Komeili et al. 2006; Murat et al.
2010), and cell wall biosynthetic enzymes (Dye
et al. 2005).
Pilus machineries, evolutionarily less-re-
lated to that of P. aeruguinosa and M. xanthus,
are also polarized. The pili of C. crescentus are
assembled from a prototypical pilus/protein
secretion apparatus encoded by a widely-
conserved locus (the cpa or tad locus) at the
new (flagellated) pole (Skerker and Shapiro
2000; Tomich et al. 2007). The CpaE compo-
nent of the C. crescentus pilus system (also
known as TadZ in Aggregatibacter actinomyce-
temcomitans) has extensive sequence homology
to the ParA/MinD-like ATPases and is localized
to the piliated pole, where it inducesthe forma-
tionofthepolarpilussecretionchannel(CpaC)
in the outer membrane (Skerker and Shapiro
2000; Viollier et al. 2002a). How this occurs is
unknown, but it could involve the spatial re-
gulation of the CpaF secretion ATPase or the
CpaD lipopotein that is presumably anchored
to the inner leaflet of the outer membrane.
Both CpaF and CpaD are polarly localized in
C. crescentus (Werner et al. 2009), and a likely
functional analog to CpaD (Tgl) is required
for the formation of the pilus channel at both
M. xanthus poles (Nudleman et al. 2006). In
support of the notion that the ParA/MinD-like
CpaE protein regulates the localization of the
CpaF secretion ATPase, the polarly localized
TadZ protein (the CpaE ortholog) was recently
found to recruit the hexameric TadA secretion
ATPase (the CpaF ortholog) (Bhattacharjee
et al. 2001) to the poles of A. actinomycetemco-
mitans (B. Perez and D. Figurski, pers. comm.;
Perez 2007). Thus, the common theme that
emerges from the CpaEF and FlhGF (or FleN-
FlhF) regulatory pairs is that ParA/MinD-like
regulator influences the correct localization of
the adjacently encoded NTPase.
Polar Polysaccharide Clusters
The central function of ParA/MinD-hom-
ologs in coordinating subcellular organization,
potentially from within a cytoskeletal structure
(Shihetal.2003),isevidentfromseveralstudies
investigatinghowDNAand/orproteinsarepre-
cisely positioned at the cell center or the cell
extremities (reviewed in Gerdes et al. 2010).
RpfA and MipZ determine the medial localiza-
tion of TlpT chemoreceptorcomplex in Rhodo-
bacter sphaeroides and the cytokinetic FtsZ-
ring in C. crescentus, respectively (Thanbichler
and Shapiro 2006; Thompson et al. 2006). The
ParA/MinD-homologs VirC1 and BcsQ are
polarly localized and dictate the recruitment
ofT-DNAnucleoproteincomplexesorcellulose
fibers to the cell pole, respectively (Atmakuri
et al. 2007; Le Quere and Ghigo 2009). The
activity of VirC1 and BcsQ, but not their polar
localization, is impeded by mutations in the
deviant Walker A motif that are thought to be
required for ATP binding and hydrolysis (Leo-
nard et al. 2005; Atmakuri et al. 2007; Le Quere
and Ghigo 2009). Forexample, VirC1 bearing a
mutation(K15Q)oflysineatthecorresponding
position 6 in the Walker-A motif (Fig. 4) is still
Deviant Walker-A motif:

KGGxxKT
S
MinD:
ParA:
MipZ:
PpfA:
FlhG:
VirC1:
CpaE:
TadZ:
BcsQ:
WssJ:




















KGGVGKT
KGGVSKT
KGGAGKS
KGGVGKT
KGGVGKS
KGGAGKT
KGGVGAS
KGGIGAS
RGGVGTT
NGGVGRS
Figure 4. Alignment of conserved residues of deviant
Walker-A boxes found in polarly localized members
of the ParA/MinD family of ATPases. Shown are
Escherichia coli MinD and BcsQ, Caulobacter cres-
centus ParA, MipZ and CpaE, Rhodobacter sphaer-
oides PpfA, Vibrio cholerae FlhG, Agrobacterium
tumefaciens VirC1, Aggregatibacter actinomycetemco-
mitansTadZ,andPseudomonasfluorescensWssJ.Res-
idues that are not identical, but similar, to the
consensus sequence in red are shown in blue. Black
color codes for variable residues.
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localized to the cell poles. Interestingly, the cor-
responding mutation is naturally present in
CpaE and BcsQ, which are also polarly local-
ized.ItisunknownwhetherCpaE,BcsQ,VirC1,
and the K15Q derivative can bind or hydrolyze
ATPon theirown invitro. If this is not the case,
CpaE and BcsQ might rely on ancillary factors
to augment ATP binding/hydrolysis in vivo.
Alternatively, the modified P-loop might ac-
commodate a nucleotide (derivative) other than
ATP, possibly even the signaling molecule c-di-
GMP, which is known to regulate cellulose pro-
ductionatthepost-translationallevel(Jenaland
Malone 2006; Le Quere and Ghigo 2009). Ithas
beenhypothesizedthatBcsQ-orthologsofPseu-
domonas fluorescens, WssA, and WssJ, control
the localization of the cellulose synthase com-
plex (Spiers et al. 2002), raising the intriguing
possibility that this regulation is mediated by
c-di-GMP via BcsQ/WssA/J.
The adhesive holdfast of C. crescentus, the
UPP (unipolar polysaccharide) of its cousins
A. tumefaciens and Rhizobium leguminosarum,
reflect other polar sites where exopolysacchar-
ides are concentrated (Merker and Smit 1988;
Laus et al. 2006; Tomlinson and Fuqua 2009;
Hardy et al. 2010). While studies on the local-
ization of UPPare still in their infancy, a recent
study revealed that the polar localization of
the C. crescentus holdfast attachment proteins
(Hfa `s) requires the PodJ polarity factor that is
localized to the new cell pole (Crymes et al.
1999; Viollier et al. 2002b; Hinz et al. 2003;
Hardy et al. 2010). PodJ is a bitopic membrane
proteinthatfeaturesN-terminalcoiled-coilrich
domain facing the cytoplasm and a C-terminal
putative peptidoglycan-binding domain. The
Hfa proteins are thought to tether the holdfast
polysaccharide to the flagellated cell pole
(Cole et al. 2003; Smith et al. 2003; Levi and
Jenal 2006). PodJ is sequestered to the pole
wellbeforetheHfa `sareexpressed(Janakiraman
and Brun 1999; Viollier et al. 2002b; Hinz et al.
2003). In the absence of PodJ, HfaA/B/D were
no longer polar (Hardy et al. 2010), indicating
that PodJ provides positional information to
enable the polar sequestration of these holdfast
proteins. Interestingly, PodJ is also required for
the polar clustering of the ParA/MinD-paralog
and pilus assembly regulator CpaE (see above)
and the cell fate histidine kinase/phosphatase
PleC (Viollier et al. 2002b; Hinz et al. 2003).
These multifunctional activities are reflected
inthepleiotropicphenotypeofthePodJmutant
that includes a pilus and holdfast assembly
defect and aberrant expression of developmen-
tal genes. It is currently unknown whether PodJ
regulates CpaE, PleC, and/or the Hfa `s directly
or not. However, since the polar localization
of the Hfa `s also depends on the presence of
the holdfast synthesis (hfs) genes, it is possible
that the effect of PodJ on Hfa `s is indirect and
is mediated through hfs-encoded proteins
(Hardy et al. 2010). Recent evidence also sug-
geststhat CpaE contributesto the polar recruit-
ment of PleC (Christen et al. 2010a), arguing
that at least one PodJ-dependent mechanism
indirectly regulates the localization of PleC to
the new (flagellated) pole. How PodJ itself is
attracted to the new cell pole is currently a
AB
C
Figure5.DiscoveryofstalkedproteinX(StpX)ofCaulobactercrescentusfromagenome-widelocalizationscreen
(Hughes etal. 2010).(A) A schematicof the C. crescentus cell in panelB with afluorescentpolarstalk. (B) Fluo-
rescence micrograph from cells expressing StpX-GFP and a red-fluorescent membrane stain (picture taken by
P. Viollier). (C) A mutant with a mispositioned stalk containing StpX-GFP. The left image is a DIC (differential
interference contrast) micrograph, and the image on the right representsthe corresponding (green) fluorescent
image.
C.L. Kirkpatrick and P.H. Viollier
10
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Page 12

mystery. It is possible that PodJ localization is
cued by an analogous mechanism, as that of
TipN (see above). If so, then PodJ must also
recognize a cytokinetic signal left behind at
the newly formed cell poles in the wake of divi-
sion. To be recognized by PodJ, this cytokinetic
signal must remain at the newborn poles until
new PodJ protein is synthesized after the
daughter cells separate. At the time of division,
only a proteolytic fragment of PodJ is present,
while the synthesis of new (full-length)
PodJ occurs during the inter-division period
(Viollier et al. 2002b; Hinz et al. 2003; Chen
et al. 2005; Chen et al. 2006). In support of
the idea that localization of PodJ to the new
pole is cued by a cytokinetic signal, PodJ can
be seen at the division septumwhen it is consti-
tutivelysynthesized(PViollier,unpubl.).More-
over, overexpression of C-terminally truncated
form of PodJ interferes with division and is
lethal (Crymes et al. 1999). These results are
consistent with the view that PodJ, akin to
TipN, interacts with the division apparatus or
an ancillary division protein. PodJ is still polar
in the absence of TipN, indicating that the
signal at the newborn pole is not dependent
on TipN.
WHAT WE KNOW, WE DON’T KNOW
For a large number of polar organelles or pro-
tein complexes, we do not yet have the foot in
thedoortothesecretoftheirpositioningmech-
anism(s).Onesuchcaseisthatofthepolarstalk
of C. crescentus (Fig. 5A,B) that is elaborated by
the cylindrical polaroutgrowth of the cell enve-
lope. Attempts to identify structural genes for
the stalk have been futile, perhaps because of
theessentialnatureofproteinsrequiredforstalk
synthesis(Wagneretal.2005).Alternatively,the
imposed genetic selections or bioinformatic
criteria might have been suboptimal. Compre-
hensive and genome-wide protein localization
efforts may provide an alternative to such recal-
citrant cases. Indeed, they hold the potential to
directly identify the players involved with a cell
biological rather than a genetic bias and, at the
very least, can yield new screening tools that
represent entry points for the dissection of the
organelle positioning and other localization
mechanisms (Werner et al. 2009; Hughes et al.
2010; Ingerson-Mahar et al. 2010) (Fig. 5C).
To put the underlying mechanisms ad-
dressed above into perspective, one must ask
why cells have developed such extensive regula-
tory pathways to ensure the polar placement of
organellesandproteincomplexes.Anappealing
model posits that polar localization serves sim-
ply to ensure the proper partitioning of func-
tions into the corresponding daughter cell that
inherits a particular pole. This is of course
widely accepted in the case of sister chromo-
somesthatmustbesegregatedintobothdaugh-
tercells, but the same principle should apply to
polar organelles or protein complexes that are
present intoolowacopynumber torelyonsto-
chasticdistributionintodaughters(seeFig.2B).
Perhaps not coincidentally, relatives of ParA, a
protein that localizes chromosomal regions
and plasmids to polar regions to ensure their
segregation (Gerdes et al. 2010), have been ap-
propriated by bacteria to fulfill key roles in the
polar positioning of organelles and, by infer-
ence, their partitioning to progeny at division.
ACKNOWLEDGMENTS
We thank Ta ˆm Mignot, Yong Zhang, and Jeff
Skerker for providing micrographs. Funding
support is from the U.S. Department of Energy,
Office of Science (Biological and Environmen-
tal Research, Grant # DE-FG02-05ER64136),
the Swiss National Science Foundation (Grant
# 31003A_127287), and the Human Frontiers
Science Program (Program Grant # RGP0051/
2010).
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