A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole.
ABSTRACT The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.
- SourceAvailable from: François-Xavier Barre[Show abstract] [Hide abstract]
ABSTRACT: The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (oriI) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the oriI region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and oriI polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.PLoS Genetics 07/2014; 10(7):e1004448. · 8.52 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The replication terminus region (Ter) of the unique chromosome of most bacteria locates at mid-cell at the time of cell division. In several species, this localization participates in the necessary coordination between chromosome segregation and cell division, notably for the selection of the division site, the licensing of the division machinery assembly and the correct alignment of chromosome dimer resolution sites. The genome of Vibrio cholerae, the agent of the deadly human disease cholera, is divided into two chromosomes, chrI and chrII. Previous fluorescent microscopy observations suggested that although the Ter regions of chrI and chrII replicate at the same time, chrII sister termini separated before cell division whereas chrI sister termini were maintained together at mid-cell, which raised questions on the management of the two chromosomes during cell division. Here, we simultaneously visualized the location of the dimer resolution locus of each of the two chromosomes. Our results confirm the late and early separation of chrI and chrII Ter sisters, respectively. They further suggest that the MatP/matS macrodomain organization system specifically delays chrI Ter sister separation. However, TerI loci remain in the vicinity of the cell centre in the absence of MatP and a genetic assay specifically designed to monitor the relative frequency of sister chromatid contacts during constriction suggest that they keep colliding together until the very end of cell division. In contrast, we found that even though it is not able to impede the separation of chrII Ter sisters before septation, the MatP/matS macrodomain organization system restricts their movement within the cell and permits their frequent interaction during septum constriction.PLoS Genetics 09/2014; 10(9):e1004557. · 8.17 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: P. aeruginosa ParA belongs to a large subfamily of Walker-type ATPases acting as partitioning proteins in bacteria. ParA has the ability to both self-associate and interact with its partner ParB. Analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with ParB. Here, a set of the ParA alanine substitution mutants in the region between E67 and L85 was created and analyzed in vivo and in vitro. All mutants impaired in dimerization (substitutions at positions M74, H79, Y82, L84) were also defective in interactions with ParB suggesting that ParA-ParB interactions depend on the ability of ParA to dimerize. Mutants with alanine substitutions at positions E67, C68, L70, E72, F76, Q83 and L85 were not impaired in dimerization but defective in interactions with ParB. The dimerization interface partly overlaps the pseudo-hairpin, involved in interactions with ParB. ParA mutant derivatives tested in vitro showed no defects in ATPase activity. Two parA alleles, parA84, whose product can neither self-interact nor interact with ParB, and parA67, whose product is impaired in interactions with ParB but not in dimerization, were introduced into P. aeruginosa chromosome by homologous gene exchange. Both mutants showed defective separation of ParB foci but to different extents. Only PAO1161 parA84 was visibly impaired in chromosome segregation, growth rate and motilities similarly to a parAnull mutant.Microbiology 08/2014; · 2.84 Impact Factor
A multidomain hub anchors
the chromosome segregation
and chemotactic machinery
to the bacterial pole
Yoshiharu Yamaichi,1,2,5Raphael Bruckner,1,2,5,6Simon Ringgaard,1,2Andrea Mo ¨ll,1,2
D. Ewen Cameron,2,7Ariane Briegel,3Grant J. Jensen,3,4Brigid M. Davis,1,2
and Matthew K. Waldor1,2,4,8
1Division of Infectious Diseases, Brigham and Women’s Hospital,2Department of Microbiology and Immunobiology, Harvard
Medical School, Boston, Massachusetts 02115, USA;
California 91125, USA;4Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA
3Division of Biology, California Institute of Technology, Pasadena,
The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome
segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation,
recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified
HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like
ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the
chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles,
chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella.
Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while
a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the
mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus,
a single polar hub is instrumental for establishing polar identity and organization.
[Keywords: Vibrio cholerae; cell polarity; chemotaxis; chromosome segregation; motility]
Supplemental material is available for this article.
Received June 29, 2012; revised version accepted August 27, 2012.
The formation and recognition of subcellular domains
is critical for numerous cellular processes, even in relatively
simple unicellular organisms such as bacteria (Shapiro
et al. 2009; Rudner and Losick 2010; Thanbichler 2011).
Without an ability to establish intracellular landmarks,
key events in the bacterial cell cycle, such as chromosome
segregation and formation of the cell division plane, are
instead subject to stochastic variation that can lead to
detrimental results. Bacterial morphology and motility
localizationof piliand flagella. A few determinants ofsuch
subcellular patterns have been well studied, such as
MinCDE, which enable a number of bacterial species to
form a division plane at the mid-cell (Raskin and de Boer
1997; Lutkenhaus 2007). In Gram-positive bacteria and
actinomycetes, DivIVA recruits other proteins to the cell
poles and septum and thereby regulates several cell pro-
cesses (Marston et al. 1998; Ben-Yehuda et al. 2003; Fla ¨rdh
Caulobacter crescentus, TipN and PopZ are markers and
determinants of pole formation (Huitema et al. 2006; Lam
et al. 2006; Bowman et al. 2008; Ebersbach et al. 2008).
domains are generated or recognized remain unknown.
In the Gram-negative rod Vibrio cholerae, as in other
rod-shaped bacteria, the cell poles are key subcellular
domains, and the bacterium has the ability to distinguish
poles from sides and the old pole from the new (most
recently generated) pole. For example, following cell di-
vision, the origin region of the larger of V. cholerae’s two
chromosomes (oriC of chrI, oriCI) is always found at the
old pole, as is an array of chemoreceptors with associated
5These authors contributed equally to this work.
Present addresses:6Department of Cell Biology, Harvard Medical School,
240 Longwood Avenue, Boston, MA 02115, USA;7Department of Bio-
medical Engineering, 44 Cummington Street, Boston University, Boston,
MA 02215, USA.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.199869.112.
2348 GENES & DEVELOPMENT 26:2348–2360 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
chemosignaling proteins, and the bacterial flagellum also
develops at this site. We found that targeting of oriCI to
the pole is dependent on ParA1 (Fogel and Waldor 2006),
a homolog of bacterial plasmid partitioning proteins, and
that the localization of the chemotactic apparati is also
regulated by a ParA homolog, ParC (Ringgaard et al. 2011).
FlhG (Correa et al. 2005; Kusumoto et al. 2008), raising the
possibility that these proteins rely on related processes to
govern spatial patterning. Notably, each protein forms
a focus at the old pole in newborn cells, although their
distribution is not always limited to this site (Fogel and
Waldor 2006; Kusumoto et al. 2008; Ringgaard et al. 2011).
As the cell cycle progresses and the cell prepares for
division, which will yield a ‘‘new’’ old pole in one of the
daughter cells, ParA1, ParC, and FlhG all adopt a bipolar
pattern.However, the meansbywhichtheirdistributionis
initiallydetermined andsubsequently shiftedhas not been
established. V. cholerae (a g-proteobacterium) lacks appar-
ent homologs of TipN and PopZ; in fact, these polar
determinants are restricted to a subset of a-proteobacteria
(Lam et al. 2006; Bowman et al. 2008; Ebersbach et al.
ParA family proteins are typically relatively small,
cytoplasmic proteins that contain a deviant Walker
AAA ATPase motif. They often have the ability to self-
associate; e.g., into polymers that may push or pull DNA
and thereby promote its equal distribution between
daughter cells. ParA family proteins have also been found
and MipZ) (Thanbichler and Shapiro 2006; Lutkenhaus
2007), type IV pili (e.g., via TadZ/CpaE) (Viollier et al.
2002; Perez-Cheeks et al. 2012; Xu et al. 2012), and
conjugative transfer machinery (via VirC1) (Atmakuri
et al. 2007). They often have a dynamic distribution
within the cell, and this distribution is key for proper
function. In most cases, the proteins’ ability to bind and/
or hydrolyze ATP is critical for their subcellular localiza-
tion and function (Szardenings et al. 2011; Lutkenhaus
2012). It should be noted, however, that not all ParA-like
proteins, even in V. cholerae, form foci at the cell poles.
The small chromosome (chrII)-associated protein ParA2
and MinD are oscillatory and more diffusely distributed
throughout much of the cell (Raskin and de Boer 1999;
Fogel and Waldor 2006). ParA proteins associated with
plasmid segregation have also been observed to oscillate.
The distinct localization patterns of these proteins are
likely determined by the specific protein partners with
which each interacts.
We performed a genetic screen to identify factors that
contribute to the establishment of polar identity in
V. cholerae and found that a single protein, VC0998
(henceforth called HubP, for hub of the pole), is required
for the proper polar localization of ParA1, ParC, and FlhG.
In the absence of HubP, oriCI is not targeted to the cell
poles, chemotaxis is impaired due to the absence or mis-
localization of polar chemotactic receptor arrays and their
associated signaling proteins, and cells have an increased
propensity to produce multiple, rather than single, flagella.
HubP, like the three ParA family ATPases, is localized to
the cell poles; however, it routinely marks both poles—
rather than the single (old) pole typically observed for the
other proteins in young cells—and can migrate between
the two poles. Despite the homology between ParA1,
ParC, and FlhG, their polar targeting by HubP appears to
rely on different mechanisms, and a distinct region of
HubP is needed to localize each client protein. Thus,
HubP is a multifaceted scaffold necessary for the estab-
point around which multiple polar processes are oriented.
Identification of HubP, an organizer of polar features
Our screen for pole-organizing factors, which was similar
to a screen in C. crescentus (Huitema et al. 2006), was
premised on the idea that V. cholerae lacking a polar
landmark protein is likely to have impaired motility/
chemotaxis, since its flagella and/or chemotaxis proteins
may be mislocalized. Therefore, we focused our analysis
on genes that had previously been linked to a motility
defect (Cameron et al. 2008) yet did not have an obvious
connection to chemotaxis or flagellar assembly. A set of
91 mutants was transformed with an expression construct
YFP, which, in contrast to wild-type ParA1-YFP, forms
discrete bipolar foci throughout the cell cycle (Ringgaard
et al. 2011). Transformants were individually screened
using fluorescence microscopy to identify strains in which
this protein is mislocalized.
We found that disruption of a previously undescribed
gene, vc0998 (hubP), caused ParA1[K11E] to be diffusely
distributed throughout cells, rather than displaying its
typical bipolar distribution (Fig. 1A,E). This pattern was
observed in both a transposon insertion mutant (hubPTTn)
and a strain with an in-frame deletion of hubP. Further-
more, wild-type localization of ParA1[K11E] was re-
stored in the DhubP strain by expression of the protein
in trans, providing strong evidence that polar targeting
of ParA1[K11E] is dependent on the presence of HubP.
HubP is also required for the proper cellular positioning
of several additional polar factors. In wild-type cells, at
least a portion of ParA1, ParC, and FlhG is found within
polar foci at either one or both poles, depending on the
age of the cell. However, in a strain lacking hubP, the
distribution of these three proteins was markedly differ-
ent than in wild-type cells (Fig. 1B–E). ParA1 was diffusely
distributed throughout the cell (Fig. 1B), and FlhG was
either diffusely distributed or found in a nonpolar focus
(Fig. 1C); neither protein formed polar foci. ParC formed
polar foci in a subset of hubP cells (Fig. 1D); however,
;50% of cells contained nonpolar ParC foci either in-
stead of or in addition to a polar focus. An increase in
diffusely distributed ParC was also evident in the hubP
cells. Thus, HubP appears to be important for the proper
subcellular distribution of three ParA-type ATPases in
V. cholerae. However, it is not required for the correct
cellular targeting of all such paralogous proteins. The
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2349
distribution of ParA2, which mediates chrII partitioning,
and of MinD, which likely specifies the V. cholerae
division site, was not altered in the absence of HubP
(Supplemental Fig. S1). Additionally, the absence of HubP
did not cause detectable changes in cell shape or cell size
and did not impair cell growth.
Sequence and genomic analyses revealed several nota-
ble attributes of the HubP protein. HubP is quite large
(1621 amino acids, or ;178 kDa) and extremely acidic
(pI = 3.22). Its N terminus appears to contain a signal
sequence, a potential LysM peptidoglycan (PG)-binding
domain, and a single transmembrane domain. Its C
terminus, which is predicted to reside within the cyto-
plasm, contains 10 copies of an imperfect 46-amino-acid
repeat, in which 19 of the consensus amino acids are
acidic (Fig. 2A; Supplemental Fig. S2). The protein is fairly
well conserved among vibrio and photobacteria species
(Vibrionaceae/Photobacteriaceae); for example, homo-
logs with >40% identity are present in Vibrio vulnificus
and Vibrio parahaemolyticus, and Vibrio fischeri encodes
a protein with 27.5% identity. Homologs appear to have
similar overall structures, although the sizes and copy
numbers of their acidic repeat sequences vary signifi-
cantly (Supplemental Table S1; Supplemental Fig. S2).
Outside of the Vibrionaceae, homologs are found princi-
pally in the g-proteobacteria, where they are significantly
less conserved, but many appear to be encoded within
a similar chromosomal neighborhood. Some of these
proteins (e.g., FimV of Pseudomonas aeruginosa) have
been found to be associated with the assembly of type IV
pili and pilus-associated twitching motility (Semmler
et al. 2000; Wehbi et al. 2011).
localization. (A–D) Subcellular localization of po-
lar proteins fused to YFP (pseudocolored in green
with phase contrast in the top panels) in indicated
V. cholerae strains. (A) ParA1[K11E]. (B) ParA1. (C)
FlhG. (D) ParC. Pictures shown are representative
fields. Bar, 2 mm. (E) Percentage of cells with
mislocalized polar proteins. For ParA1[K11E],
ParA1, and FlhG, mislocalization refers to cells
lacking a polar focus, and for ParC, it refers to cells
that contain a nonpolar focus. Means, standard
deviations, and total number of cells counted (n)
are shown. See also Supplemental Fig. S1.
HubP is a determinant of polar protein
Yamaichi et al.
2350 GENES & DEVELOPMENT
HubP is directed to the V. cholerae pole and mid-cell
by its periplasmic domain
Given the effect of HubP deficiency on subcellular
targeting of polar proteins, we assessed whether HubP
itself is present at the V. cholerae cell poles. For these
experiments, we used fluorescence microscopy and visu-
alized either ectopically or chromosomally expressed
HubP-CFP (or YFP/GFP). The fusion proteins, which are
tion in the hubP mutant, displayed a bipolar distribution,
even in small (young) cells (Fig. 2B–D). Thus, HubP
appears to mark the new pole prior to the arrival of the
ParA family proteins, which are typically not bipolar in
young cells (Fogel and Waldor 2006; Ringgaard et al.
of cells, where it always colocalized with FtsZ, suggesting
that it arrives at this site as cells prepare to divide (Fig.
2E). Time-lapse analyses confirmed that HubP is not
present at the mid-cell in young cells, but instead arrives
there as cells progress through the cell cycle, which
presumably enables it to be present at newly formed
in this study. (Scissors) Signal sequence; (LysM) LysM domain; (TM) transmembrane domain; (103 repeat) repeat sequence (see the
text). (B) Subcellular localization of HubP-CFP in DhubP V. cholerae. (C, top) Time-lapse images of plasmid-borne HubP-YFP in DhubP
V. cholerae. A schematic representation is drawn below. (D,E) Subcellular localization of HubP-CFP and ParA1[K11E]-YFP (D) and FtsZ-
YFP (E) in DhubP V. cholerae. In E, HubP and FtsZ at mid-cell are indicated with arrowheads and arrows, respectively. (F) FRAP
experiment of HubP-GFP. Pictures from a representative experiment (left) are shown, along with a graph of average signal intensities
over time, based on analysis of 30 cells (right). The bleached focus is indicated by an arrow in the pictures and by the green line in the
graph. The magenta line shows the average intensity of the focus at the pole opposite the bleached pole. Pale blue and yellow lines show
the intensities of polar foci from unbleached (control) cells. (G) Fluorescence of -CFP and -mCherry fusions to truncated HubP in V.
cholerae. Representative fields are shown. Bar, 2 mm.
HubP localization and its determinants. (A) Schematic of the HubP polypeptide (top) and truncation mutants (bottom) used
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2351
poles (Fig. 2C). HubP localization was similar using
plasmid- and chromosome-encoded protein, although the
chromosome-encoded protein was less abundant (Supple-
mental Movies S1, S2).
Fluorescence microscopy was also used to explore the
dynamics and determinants of HubP’s subcellular local-
ization. Notably, photobleaching of polar HubP-GFP was
followed by recovery of the polar signal, revealing that
HubP can be directly targeted to the pole; it does not
need to arrive there via the cell division site. Recovery
of fluorescence intensity at the bleached pole was accom-
(unbleached) pole, suggesting that there is an exchange of
HubP between the two poles (Fig. 2F). Additionally, we
found that polar targeting of HubP is independent of its
putative cytoplasmic and membrane-spanning domains.
A fusion protein consisting of the first 324 amino acids of
HubP (the putative periplasmic and transmembrane se-
quences) fused to CFP had a cellular distribution equal to
that of the full-length fusion protein (Fig. 2G). A smaller
with the predicted localization of the HubP N-terminal
domain in the periplasm (Fig. 2A), where CFP does not
fluoresce. However, when smaller N-terminal fragments
were fused to mCherry (which is fluorescent even in the
periplasm), polar targeting was also observed (Fig. 2G,
1–284). The first 161 amino acids of HubP were sufficient
to enable polar targeting of the fusion protein, although
the first 150 amino acids were not (Fig. 2G, 1–161 and
1–150). We also found that polar targeting of HubP re-
quired the putative LysM domain (amino acids 90–134)
(Fig. 2G, DLysM). In the absence of targeting sequences,
HubP appeared to be diffusely distributed in the periplasm
(Fig. 2G, 1–150 and DLysM). The requirementfor the LysM
domain raises the possibility that an interaction between
HubP and PG contributes to its targeting and/or retention
at the V. cholerae pole. However, to date, we have been
unabletodetect adirectinteractionbetweenHubP andPG
using in vitro binding assays.
HubP modulates the localization of oriCI
We explored whether the failure of ParA1 to localize to
the cell poles in cells lacking HubP altered chrI segrega-
tion dynamics. Although ParA1 is not essential for chrI
partitioning, in its absence, oriCI and the origin-associ-
ated centromere-binding protein ParB1 do not localize to
the old pole in young cells, nor are they drawn to the new
pole following chromosome replication (Fogeland Waldor
2006). Instead, in a parA1 deletion mutant, they are
generally found at the mid-cell of young cells and near
the 1/4 and 3/4 positions following replication (Fogel and
Waldor 2006). We found that deletion of hubP has a
similar effect on the localization of ParB1: No polar
ParB1-CFP foci were detected in the hubP mutant, and
cells typically contained two well-separated cytoplasmic
foci (Fig. 3A–C), as observed in parA1 mutants. Thus,
marking of the V. cholerae cell pole by HubP is critical for
the establishment of the normal cellular distribution of
the organism’s major chromosome.
HubP binds directly to ParA1 to control its subcellular
Several lines of evidence suggest that HubP controls
ParA1’s distribution via a direct interaction between the
two proteins. First, when ParA1[K11E]-YFP and HubP-
CFP were coexpressed in V. cholerae, the foci they formed
colocalized (Fig. 2D; see below). Second, these proteins
were found to interact in a bacterial two-hybrid assay,
which also showed that ParA1 can self-associate (Table 1;
(A–C) Subcellular localization of ParA1-YFP and ParB1-CFP in
wild type (A) and DhubP (B) V. cholerae cells. (C) The percentage
of wild-type and DhubP cells lacking polar foci of ParA1 (green)
and ParB1 (magenta) is shown, based on analysis of the number of
cells (n) indicated above. Asterisk indicates 0%. (D) Fluorescence
of HubP-CFP expressed in E. coli. (E,F) ParA1[K11E]-YFP (E) or
ParA1-YFP (F) was expressed in E. coli in the absence (left) or
presence (middle) of HubP-CFP (right). ParA1-YFP localized to
the nucleoid in the cell lacking HubP-CFP expression (marked
with an asterisk). Representative fields are shown. Bar, 2 mm.
HubP governs the subcellular distribution of ParA1.
Yamaichi et al.
2352GENES & DEVELOPMENT
Supplemental Fig. S3), similar to observations for other
ParA-like proteins (Leonard et al. 2005; Ebersbach et al.
2006). Finally, HubP recruited ParA1 to the cell periphery
when the two proteins were coexpressed in Escherichia coli
(Fig. 3D–F). When expressed alone in E. coli, ParA1[K11E]-
YFP and ParA1-YFP were widely distributed within the
3E,F). However, when expressed along with HubP-CFP,
which appears to be membrane-associated but not polar in
E. coli (Fig. 3D), the distribution of both wild-type ParA1
and ParA1[K11E] was shifted toward the cell membrane
A variety of mutations within parA1 was generated in
order to explore which features of the protein are needed
for interaction with HubP. Based on previous analyses of
ParA family members and identification of key conserved
amino acids, mutations were generated to disrupt dimer-
ization (ParA1[G12V]), DNA binding (ParA1[R189E]), ATP
and the subcellular distribution of the mutant proteins
was analyzed in E. coli and V. cholerae. Mutants predicted
to be unable to dimerize or bind DNA still appeared
capable of interacting with HubP in both systems; they
formed HubP-dependent polar foci in V. cholerae and
showed HubP-dependent membrane association in E. coli
(Supplemental Fig. S4). Mutants lacking the ability to bind
or hydrolyze ATP also were recruited by HubP to the cell
periphery in E. coli and formed HubP-dependent polar foci
in V. cholerae (Supplemental Fig. S4). However, nonpolar
ParA1[K16A] and ParA1[D40A] foci were also detected in
both wild-type and hubP cells regardless of the presence or
absence of a chromosomal parA1+copy, suggesting that
ParA1 binding to and/or hydrolysis of ATP is not essential
for, but may influence, ParA1–HubP interactions.
HubP negatively regulates polar accumulation
Our observation of FlhG mislocalization in the hubP
mutant, coupled with the previously noted reduced mo-
tility of this mutant on soft agar plates, led us to further
explore the connection between HubP and V. cholerae
flagellation. The hubPmutantexhibited a >50%reduction
in swarming on soft agar, comparable with the defect
displayed by an flhG mutant and significantly larger than
that of a parC mutant (Fig. 4A). However, deletion of hubP
had a fairly modest effect on flagellation. Electron micros-
copy analyses revealed that a slightly increased fraction
(6%) of V. cholerae lacking HubP produces more than
a single polar flagellum, a feature observed in only 1% of
wild-type cells (Fig. 4B). This frequency contrasts mark-
edly withthat of anflhGmutant, where;80% of cells had
multiple flagella (Fig. 4B). In Vibrio alginolyticus, FlhG is
thought to repress flagellar formation via two mecha-
nisms: (1) by binding to FlhF (which is thought to guide
polar placement of flagella) and preventing its polar re-
cruitment, and (2) by repressing expression of flagellar
genes (Kusumoto et al. 2008). In V. cholerae, deletion of
hubP does not have a significant effect on the distribution
of FlhF, despite its effect on FlhG localization, suggesting
that polar FlhG is not a critical determinant of FlhF
accumulation at this site (Fig. 4C). Furthermore, the
relatively minor effect of FlhG mislocalization on flagella
production in the hubP mutant suggests that mislocaliza-
tion does not substantially impair its efficacy in negatively
regulating flagellar production. Thus, the biological ratio-
nale for polar targeting of FlhG by HubP remains to be
HubP interacts directly with FlhG and FlhF
Like ParA1, FlhG appears to be retained at the V. cholerae
pole through direct interaction with HubP. Bacterial two-
hybrid analyses suggest that the two proteins interact
(Table 1; Supplemental Fig. S3), and coexpression of HubP
along with FlhG in E. coli shifts FlhG from a diffuse
cytoplasmic distribution to a pole-associated distribution
that corresponds to the location of HubP (Fig. 4D). It is not
clear why coexpression of these two proteins also alters the
subcellular distribution of HubP (cf. Figs. 3D–F and 4D).
Unexpectedly, two-hybrid analyses suggest that FlhF
also interacts with HubP (Table1), although neither pro-
tein is required for the polar targeting of the other (Fig.
4C,E). Analyses of FlhF and HubP localization in E. coli
also suggest that they interact; expression of HubP shifts
FlhF from a predominantly polar distribution to a pattern
that matches HubP (Fig. 4F). Notably, FlhF, like HubP, is
required for recruitment of FlhG to the V. cholerae pole
(Fig. 4E). Thus, although the precise reason for an interac-
tion between FlhF and HubP is unknown, it is possible that
they act together to modulate the subcellular distribution
Summary of HubP’s interactions
FPcin E. colid
FP in V. choleraee
FP in E. colid
FP in V. choleraee
FP in E. colid
FP in V. choleraee
FP in E. colid
FP in V. choleraee
FP in E. colid
FP in V. choleraee,h
a(BACTH) Bacterial two-hybrid.
bSee also Supplemental Figure S3.
c(FP) Colocalization of fluorescent proteins.
dSee also Supplemental Figure S5.
eSee also Figure 6.
hNote that FlhF forms polar foci even in the DhubP background.
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2353
Deletion of hubP impairs polar localization
of chemotactic signaling proteins and polar
assembly of chemoreceptor arrays
In contrast to its relatively minor effect on flagellation,
deletion of hubP has a far more dramatic effect on V.
cholerae chemotaxis. Video tracking of swimming bacte-
ria revealed that the hubP strain displays a significant
bias toward straight swimming: Direction reversals were
detected 0.16 6 0.07 sec?1for the mutant versus 0.57 6
0.06 sec?1for the wild-type strain (Fig. 5A). The effect of
hubP deletion on swimming is more marked than that
of a parC deletion (0.28 sec?1), consistent with its more
pronounced effect on swarming on soft agar plates (Fig.
4A). Chemotaxis appears to be impaired due to mislocal-
ization of components of chemotactic apparati in the
hubP mutant. As previously shown (Briegel et al. 2009),
electron cryotomography revealed that 39 of 100 wild-
type cell poles examined had detectable chemoreceptor
arrays (Fig. 5B). In marked contrast, only seven of 61
(11%) hubP cell poles had detectable arrays. Consistent
with the reduced number of polar arrays, chemotaxis
signaling protein CheY3 formed aberrant nonpolar foci in
;60% of hubP cells (Fig. 5C). Furthermore, most of the
nonpolar CheY3 foci colocalized with ParC (Fig. 5D),
which we showed is also mislocalized in the hubP
mutant (Fig. 1C). Together, these observations suggest
that HubP (via ParC) promotes the polar localization of
the entire chemoreceptor/signaling complex. Thus, at
least in V. cholerae, the distribution of receptors and
downstream signaling proteins is not simply the result of
mutants on a representative soft agar plate is pictured (left), and average swarm diameter relative to wild type, with standard deviation
based on three or more experiments, is graphed (right). (B, left) Images of wild-type, hubP, and flhG V. cholerae, generated using electron
microscopy, are shown. The number of flagella observed per cell for each mutant is graphed at the right. (C,E) Subcellular localization of
FlhF-YFP (C), HubP-YFP (E), and FlhG-YFP (E) in the indicated V. cholerae strains. (D,F) FlhG-YFP (D) or FlhF-YFP (F) was expressed in
E. coli in the absence (left) or presence (middle) of HubP-CFP (right). Representative fields are shown. Bar, 2 mm.
HubP is important for V. cholerae chemotaxis and regulation of flagellar production. (A) Swarming of various V. cholerae
Yamaichi et al.
2354GENES & DEVELOPMENT
In contrast to ParA1 and FlhG, ParC does not appear to
be anchored at the pole by directly interacting with HubP.
The two proteins did not interact in a two-hybrid assay
(Table 1); furthermore, there was at most a subtle change
in the distribution of YFP-ParC in E. coli induced by
coexpression of HubP-CFP, and the two proteins do not
appear to be colocalized (Supplemental Fig. S5). Finally,
although HubP-CFP and YFP-ParC are both polar proteins
in V. cholerae, they do not precisely colocalize. Mapping
of ParC’s position relative to HubP (and ParA1[K11E]’s,
for comparison) revealed that ParC was, on average,
approximately two times farther from HubP than was
swimming wild-type and DhubP V. cholerae. (B) Electron cryotomographs of wild-type V. cholerae. The region in the square is
magnified in the inset. The arrow indicates chemoreceptor array. Arrowheads indicate outer membrane (OM), inner membrane (IM),
and base plate (BP) of chemoreceptor array. (C) Foci of CheY3-CFP in wild-type and DhubP V. cholerae (left) and percentage of cells with
mislocalized CheY3-CFP in indicated background (right). (D) CheY3-CFP and YFP-ParC foci in DhubP V. cholerae. Arrowheads indicate
colocalization of nonpolar CheY3-CFP and YFP-ParC foci. (E) HubP-CFP (magenta) colocalizes with ParA1[K11E]-YFP (right; green) but
not with YFP-ParC (left; green). Localization of YFP-ParC and ParA1[K11E]-YFP foci relative to neighboring HubP-CFP focus are
plotted. Inner and outer circles correspond to diameters of 2 and 4 pixels, respectively. (F) Swarming of wild-type, DhubP, and flagella
mutant (lacks both polar and lateral flagella) V. parahaemolyticus on a representative soft agar plate is pictured (left), and average
swarm diameter relative to wild type, with standard deviation based on three or more experiments, is graphed (right). (G) Subcellular
localization of YFP-ParCvpin the indicated V. parahaemolyticus strain. Representative fields are shown. Bar, 2 mm.
Deletion of hubP indirectly alters the distribution and efficacy of vibrio chemotaxis proteins. (A) Positional tracking of
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2355
ParA1[K11E]: 68.4 6 40.4 nm versus 30.9 6 16.4 nm
(Student’s t-test: 1.2 3 10?54) (Fig. 5E). Collectively, these
observations suggest the possibility that HubP modulates
the distribution of ParC indirectly, perhaps via the medi-
ation of an as-yet-unidentified vibrio-specific factor. How-
this function, as previous analyses have shown that ParC
localization is independent of ParA1 and FlhG as well as of
FlhF (Ringgaard et al. 2011).
HubP promotes the polar localization of ParC
and ParA in V. parahaemolyticus
Since the aberrant chemotaxis of the V. cholerae hubP
mutant (Figs. 4A, 5A) was a particularly striking pheno-
type, we wondered whether hubP also modulates chemo-
taxis in other vibrios (e.g., V. parahaemolyticus). Like
all vibrios and photobacteria, V. parahaemolyticus en-
codes homologs of V. cholerae HubP and ParC (69% and
86% similarity, respectively). We found that V. parahae-
molyticus swarming motility, which is dependent on the
organism’s polar flagella, was reduced by ;40% in a V.
parahaemolyticus hubP mutant (Fig. 5F). Furthermore, as
in V. cholerae, YFP-ParC routinely formed polar foci in
wild-type V. parahaemolyticus. In the hubP mutant,
however, YFP-ParC was often observed within nonpolar
HubP’s role as an organizer of chemotactic machinery is
conserved, at least within the Vibrionaceae. Interestingly,
there is a striking correlation in the coincidence of hubP
and parC homologs among g-proteobacteria (Supplemen-
tal Table S1), suggesting that HubP’s influence over
chemotaxis may extend through a broader swath of the
bacteria kingdom. There is much less overlap between
the distribution of HubP and ParA homologs (which are
far more abundant). Nonetheless, we observed that HubP
also appears to mediate the polar localization of ParA1 in
V. parahaemolyticus (data not shown), indicating that
HubP’s role in chromosome partitioning is likewise not
limited to V. cholerae.
Different HubP domains are required to mediate polar
localization of ParA1, FlhG, and ParC
Our observation that HubP interacts directly with ParA1
and FlhG, but not with ParC, suggested that distinct
regions of HubP might be important for determining the
subcellular distribution of client proteins. We explored
this possibility using the bacterial two-hybrid system and
the fluorescence-based assays described above, except
that hubP was altered by the presence of a series of dele-
tion mutations (summarized in Table 1; data presented in
Fig. 6; Supplemental Figs. S3, S5). Results from all of these
systems suggest that the interaction between ParA1 and
HubP requires amino acids within the repeat region of
HubP. Truncation of the protein to remove the final 181
amino acids did not impair HubP’s ability to interact with
or guide the localization of ParA1 (hubP[1–1440] (Fig. 6A,B);
however, deletion of the repeats (hubP[0-repeat]) or of the
HubP’s activity with respect to ParA1 (and ParA1[K11E]). In
contrast, the extreme C terminus of HubP (amino acids
1441–1621) appeared to be essential for interaction between
HubP and FlhG, at least based on FlhG-YFP localization
in V. cholerae expressing HubP from the chromosome
(Fig. 6A,B). Plasmid-based assays in E. coli suggest that
additional regions of HubP may have a weak capacity to
interact with FlhG (Supplemental Fig. S3); nonetheless, it is
clear that amino acids 1441–1621 are important using these
other assays as well. The C-terminal 181 amino acids were
also critical for the interaction between HubP and FlhF
localization appears to depend on yet another domain
within HubP. The first 900 amino acids of HubP were
sufficient to localize ParC normally in V. cholerae (Fig. 6B),
and chemotaxis (soft agar motility) by V. cholerae express-
ing HubP[1–900] was significantly greater than by a strain
lacking the entire protein (Fig. 6C). Chemotaxis by the
hubP[1–900] mutant was not equivalent to that by the
wild-type strain; however, this may reflect the inadequacy
of the truncated HubP for modulation of flagellar proteins.
Consistent with this hypothesis, HubP[0-repeat], which
interacts with both ParC and FlhG, enabled chemotaxis
equivalent to that of the wild-type strain. Collectively,
these results suggest that the cytoplasmic portion of HubP
is likely tohave multiple distinct interactiondomainsthat
mediate its spatial regulation of ParA family proteins.
In rod-shaped bacteria, the cell poles are critical sub-
cellular domains, but knowledge of the mechanisms that
underlie pole formation, recognition, organization, and
perpetuation is restricted to very few model organisms. In
V. cholerae, polarly localized ParA-related proteins are
essential for mediating the polar localization of DNA/
protein (ParA1 and parS1/ParB1) and protein/protein
complexes (ParC and CheY3) that are required for proper
segregation of oriCI and the chemotactic machinery,
respectively. FlhG, a third polar ParA-like protein, mod-
ulatesformation of V. cholerae’s polar flagellum. Here, we
found that the polar localization of ParA1, ParC, and FlhG
all depend on HubP, a large transmembrane protein that
is conserved in all Vibrionaceae/Photobacteriacea as
well as in several other g-proteobacteria. In the absence
of this polar anchor, origin segregation and chemotaxis
are impaired, and cells produce extra flagella. Polar target-
ing of ParA1, ParC, and FlhG requires different regions
within HubP, and the ParA family proteins clearly use
distinct mechanisms (including both direct and indirect
organization of the vibrio cell pole is dependent on a
complex and multifaceted protein hub that anchors chro-
mosome segregation and chemotactic machinery to this
site (Fig. 6D).
Strikingly, although HubP is required for the polar
placementof itsParA-relatedclients, thereis notaperfect
correspondence between their subcellular localizations.
HubP is always detectable at both the old and new pole in
V. cholerae and often at the future division plane at mid-
cell as well. In contrast, ParC does not form detectable
Yamaichi et al.
2356 GENES & DEVELOPMENT
foci at the new pole until late in the cell cycle (Ringgaard
et al. 2011), and FlhG is only bipolar in a small subset of
(typically long) cells (Fig. 1). Distinct ParA1 foci are also
difficult to discern at the new pole until late in the cell
cycle, which corresponds to the migration of replicated
oriCI to this site. However, it is presumed that some
ParA1 must be anchored at the new pole at the start of
chromosome segregation in order for cycles of ParA1
visualized in V. cholerae in which full-length hubP was replaced by a truncated gene. Representative fields are shown. Bar, 2 mm. (B) For
each protein indicated, the frequency with which the aberrantly localized fusion protein was observed is plotted as a function of hubP
genotype. Protein localization was scored in the indicated number (n) of cells. For ParA1 and FlhG, aberrant localization refers to cells
lacking a polar focus; for ParC, it reflects the number of cells with a nonpolar focus. Mean and standard deviations and total number of
cells counted (n) are shown. Wild-type and DhubP data are reiterated from Figure 1E for comparison. (C) Swarming of V. cholerae hubP
mutants on soft agar plates was quantitated, relative to wild type, based on swarm diameter. Truncated HubP (as indicated) was
expressed from an allele inserted within the chromosome in place of the wild-type gene. Plotted values reflect the means and standard
deviations from three or more experiments. (*) P < 0.05; (**) P < 0.01 by Student’s t-test. (D) Schematic of HubP’s role in organizing the
V. cholerae old cell pole. In wild-type cells, different regions of HubP anchor three ParA-related ATPases (ParA1, ParC, and FlhG) to the
old cell pole. These ATPases mediate the polar localization of the centromeric ParB1/parS1 complex and chemotaxis receptor arrays
and signaling proteins and modulate flagellar production, respectively. HubP anchoring of ParC to the pole appears to be mediated
through an intermediate factor. In the absence of HubP (DhubP), ParA1 and FlhG are diffuse, the ParB1/parS1 complex is no longer
polar, ParC and chemotaxis signaling proteins form nonpolar foci (although polar foci are observed as well), and there is a small increase
in cells that contain two flagella; FlhF, which also interacts with HubP, remains at the pole.
HubP appears to have multiple domains for interaction with partners. (A) Fluorescent fusion proteins (as indicated) were
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2357
polymerization/depolymerization to yield a net force
pulling ParB1/oriCI across the cell (Fogel and Waldor
2006). Nonetheless, there appear to be restrictions on
interactions between HubP and its clients at the new pole
(and at the mid-cell prior to division). What limits HubP
interactions with its clients at the new pole? It is possible
that additional factors must be present at this site along
with HubP in order to anchor HubP’s partners (as appears
to be the case for ParC). Alternatively, interactions at the
new pole may be limited by the presence of an additional
factor that is released or inactivated later in the cell cycle.
Client-specific processes, such as interactions with addi-
tional protein partners, ATP, or the nucleoid, may also
regulate protein localization and may account for the
apparently distinct arrival times of ParA1, FlhG, and ParC
at the new pole. It remains to be seen whether there is any
temporal coupling among these processes (e.g., between
oriCI segregation and movement of ParC to the new pole)
or whether they are fully independent.
Similarly, it is noteworthy that some HubP-dependent
proteins are not exclusively targeted to either pole for at
least a portion of the cell cycle. Much of ParA1 is present
within the cytoplasm, although its distribution there is
dynamic, and much of FlhG is diffusely localized. Polar
versus cytoplasmic localization is likely modulated at
least in part by ATP binding and hydrolysis. Binding of
ParA-like proteins to ATP typically enables them to
dimerize, and dimerization is often required for their
interactions with both other proteins (e.g., ParB and
homologs) and DNA (Lutkenhaus 2012). Interestingly,
our analyses indicate that ParA1 dimerization is not re-
quired for its interaction with HubP. In fact, ParA1[G12V],
new poles (Supplemental Fig. S4), suggesting that dimer-
ization may inhibit interaction with HubP. Our analyses
also suggest that ATP binding and hydrolysis are not
essential for the interaction between ParA1 and HubP,
since ParA1[K16A] and ParA1[D40A], which fail to bind
and hydrolyze ATP, respectively, still form some polar foci,
although they do have a reduced presence at the poles
relative to wild-type ParA1. The abilityof client proteins to
bind ATP could also indirectly modulate interactions with
HubP; e.g., by altering their affinity for and positioning by
other interaction partners. Ultimately, the distribution of
HubP’s clients between the cytoplasm and the pole likely
reflects the relative affinities of the client proteins for their
cytoplasmic partners versus HubP.
The means by which nascent HubP is initially recruited
to the mid-cell or poles warrants further investigation.
Polar targeting of HubP could be due to recognition of
membrane curvature, as has been proposed for DivIVA of
Bacillus subtilis (Lenarcic et al. 2009; Ramamurthi and
distribution in the spherical or oval cells formed due to
mutation of rodZ (Shiomi et al. 2008; Alyahya et al. 2009;
Bendezu ´ et al. 2009) and in MreB inhibitor MP265-treated
recognize the membrane curvature associated with polar
regions, as HubP retains a focal distribution even in these
cases. One possibility is that HubP recognizes features of
PG that are stable and specific to the poles. Polar PG is
thought to be relatively inert, and pole-specific PG struc-
tures formed during cell division might serve as a ‘‘birth
scar’’ that might be used in proper cellular targeting of
HubP. There is a precedent for PG fastening other pro-
teins to poles; e.g., C. crescentus SpmX (Radhakrishnan
et al. 2008). Alternatively, HubP association with polar PG
(presumably through its LysM domain) may only stabilize
its association with the poles after it is recruited there by an
unknown factor. Determination of the relative timing of
could aid in distinguishing between these possibilities.
Although HubP routinely localizes at the poles and may
interactwithpolarPG,HubPisnotstably retained atthese
sites; instead, it can migrate from one pole to the other,
apparently bidirectionally. The significance of and mech-
anism underlying this movement, which has not been
reported for other polar markers, remain to be determined.
Additionally, it has not been investigated whether partner
proteins remain associated and move with HubP or
whether they are instead released and recaptured. How-
ever, the fact that HubP is detected at the mid-cell or new
pole of young cells apparently without associated partners
may indicate that partners are released prior to or during
movement. It is also possible that HubP acquires interact-
ing partners during its transits across the cell. If so, bipolar
movement of HubP might serve as a tool for recruiting its
interaction partners to polar sites.
The most profound consequences of hubP deletion that
we observed relate to its regulation of chemotaxis. In the
absence of HubP, polar chemotactic receptor arrays are
present at a markedly reduced frequency, and chemotac-
tic signaling complexes (marked by ParC and CheY3) are
often mislocalized, although they are assumed to be struc-
turally intact. The altered distribution of these proteins
presumably is the major factor underlying the mutant’s
impaired chemotaxis, detectable as reduced swarming on
soft agar plates and a reduced reversal frequency in liquid
culture. Notably, a HubP deficiency has a more profound
phenotype than does a ParC deficiency, probably both
because ParC does not modulate flagellar assembly and
because the frequency at which receptors and associated
signaling proteins accumulate at the pole remains rela-
tively high in the absence of ParC, so signaling is not
perturbed. In contrast, in the absence of HubP, ParC is
frequently mislocalized, causing mislocalization of down-
stream signalingproteins.However,downstream signaling
proteinsare also frequently mislocalized ina strainlacking
hubP and parC (data not shown). Thus, although mislocal-
ization of ParC may be more detrimental to V. cholerae
is not the only factor through which HubP controls the
localization of chemotaxis proteins. Contrasting paradigms
characterize HubP’s impact on FlhG and ParA1. Mislocal-
ization of FlhG has a relatively minor effect on flagellar
assembly, far less than that of FlhG’s absence, perhaps
because some of FlhG’s regulatory roles are not dependent
on its subcellular distribution. Finally, the mislocalization
and absence of ParA1 have similar consequences; both
result in aberrant subcellular placement of oriCI.
Yamaichi et al.
2358GENES & DEVELOPMENT
Although the precise means by which HubP recruits
and/or anchors ParC to the pole and thereby situates the
remainder of the chemotactic machinery remains to be
determined, bioinformatic analyses suggest that it may
be a relatively conserved process. There is a strong corre-
lation between genomes containing hubP and genomes
containing parC, particularly among the g-proteobacteria
(Supplemental Table S1). Notably, since our data suggest
that ParC and HubP do not interact directly, it is likely
that an additional component that serves as a bridge
between these two proteins will also be found to have
HubP bears some functional similarity to TipN and
PopZ, polar determinants and markers in C. crescentus,
the organism in which the molecular bases for pole
establishment and organization are best understood.
Through their presence at the poles, all three of these
proteins enable polar placement/anchoring of several
additional proteins and protein complexes, such as the
chromosome segregation machinery, whose proper activ-
ity depends on their polar localization. TipN, like HubP,
is an early marker of new/nascent poles; both proteins are
present at the mid-cell prior to cell division, thereby
providing cells with cues regarding polar orientation at
the start of their development. However, there are also
notable differences in how these three pole-specifying
proteins localize and function. In particular, HubP differs
from TipN and PopZ in that it is always present at both
poles, rather than marking the new pole (TipN) or
primarily the old pole (PopZ). It seems possible that
additional factors/processes in V. cholerae are therefore
needed for the cell to distinguish between its polar
regions. Additionally, there is no evidence that HubP is
a primary determinant, rather than a marker, of pole
formation, unlike TipN, whose overexpression can in-
duce ectopic pole formation (Lam et al. 2006). Further-
more, HubP’s clients identified to date are limited to
ParA-like ATPases, whereas TipN and PopZ modulate
the localization of structurally diverse proteins, although
these include ParA family members (Schofield et al.
2010). The C. crescentus and V. cholerae pole-organizing
proteins lack amino acid sequence similarity and clearly
evolved through independent processes in a-proteobacteria
and g-proteobacteria. Despite their differences, it is
striking that their core characteristics—polar proteins
that anchor multiple clients to generate and perpetuate
cell polarity—have so much in common. It seems likely
that additional diverse hub proteins will control the
activity of the growing family of ParA-related proteins
that govern the polar localization of numerous subcellu-
Materials and methods
Plasmids and strains
Plasmids used in this study and their construction are described
in the Supplemental Material (Supplemental Table S2; Supple-
mental Material). Oligonucleotides used for plasmid construc-
tion are listed in Supplemental Table S3. Nucleotide sequences
of plasmids were confirmed whenever the construction involved
PCR. Strains used in this study are listed in Supplemental Table
S4. Briefly, we used El Tor O1 strains N16961 (Heidelberg et al.
2000) and C6706 (parental strain of the mapped Tn insertion
library) (Cameron et al. 2008) as wild-type V. cholerae. Chro-
mosomal deletions and insertions were introduced by allelic
exchange (Donnenberg and Kaper 1991). For growth conditions
and supplements including antibiotics, see the Supplemental
The mapped Tn insertion library of V. cholerae contains 147
mutants showing a motility deficiency (<50% compared with
wild type) (Cameron et al. 2008). Of these 147 mutants, the 56
that have Tn insertions in genes obviously linked to motility
(e.g., flagellar biosynthesis genes) were discarded from the pool of
candidates. A reporter plasmid encoding fluorescent fusion pro-
teins, pBAD33 parA1[K11E]-yfp cheY3-cfp, was introduced into
the remaining 91 mutants, and the subcellular localization of the
fusion proteins in log-phase cells was manually examined using
For microscopy, see the Supplemental Material.
Cells were grown to O.D. ;0.5 in LB, immobilized on grids, fixed
with glutaraldehyde, and then repeatedly washed in water.
Imaging was performed on a Tecnai G2Spirit BioTWIN.
For cryomicroscopy, see the Supplemental Material.
We are grateful to Janet Iwasa for making Figure 6D, Paula
Montero Llopis for an expert tutorial in Microbe Tracker, and
John Mekalanos for providing the mapped transposon library. We
also thank Richard Losick, Hubert Lam, and Waldor laboratory
members for helpful discussions and comments on the manu-
script. S.R. was funded with a post-doctoral fellowship from
the Villum Kann Rasmussen Foundation. This work was sup-
ported by the NIAID R37 AI-042347 (to M.K.W.), NIGMS R01
GM094800B (to G.J.J), and HHMI (to M.K.W. and G.J.J.).
Alyahya SA, Alexander R, Costa T, Henriques AO, Emonet T,
Jacobs-Wagner C. 2009. RodZ, a component of the bacterial
core morphogenic apparatus. Proc Natl Acad Sci 106: 1239–
Atmakuri K, Cascales E, Burton OT, Banta LM, Christie PJ.
2007. Agrobacterium ParA/MinD-like VirC1 spatially co-
ordinates early conjugative DNA transfer reactions. EMBO
J 26: 2540–2551.
Bendezu ´ FO, Hale CA, Bernhardt TG, de Boer PA. 2009. RodZ
(YfgA) is required for proper assembly of the MreB actin
cytoskeleton and cell shape in E. coli. EMBO J 28: 193–204.
Ben-Yehuda S, Rudner DZ, Losick R. 2003. RacA, a bacterial
protein that anchors chromosomes to the cell poles. Science
Bowman GR, Comolli LR, Zhu J, Eckart M, Koenig M, Downing
KH, Moerner WE, Earnest T, Shapiro L. 2008. A polymeric
A multidomain hub organizes the cell pole
GENES & DEVELOPMENT2359
protein anchors the chromosomal origin/ParB complex at
a bacterial cell pole. Cell 134: 945–955.
Briegel A, Ortega DR, Tocheva EI, Wuichet K, Li Z, Chen S,
Mu ¨ller A, Iancu CV, Murphy GE, Dobro MJ, et al. 2009.
Universal architecture of bacterial chemoreceptor arrays.
Proc Natl Acad Sci 106: 17181–17186.
Cameron DE, Urbach JM, Mekalanos JJ. 2008. A defined trans-
poson mutant library and its use in identifying motility
genes in Vibrio cholerae. Proc Natl Acad Sci 105: 8736–8741.
Correa NE, Peng F, Klose KE. 2005. Roles of the regulatory
proteins FlhF and FlhG in the Vibrio cholerae flagellar
transcription hierarchy. J Bacteriol 187: 6324–6332.
Donnenberg MS, Kaper JB. 1991. Construction of an eae dele-
tion mutant of enteropathogenic Escherichia coli by using a
positive-selection suicide vector. Infect Immun 59: 4310–4317.
Ebersbach G, Ringgaard S, Møller-Jensen J, Wang Q, Sherratt DJ,
Gerdes K. 2006. Regular cellular distribution of plasmids by
oscillating and filament-forming ParA ATPase of plasmid
pB171. Mol Microbiol 61: 1428–1442.
Ebersbach G, Briegel A, Jensen GJ, Jacobs-Wagner C. 2008. A
self-associating protein critical for chromosome attach-
ment, division, and polar organization in caulobacter. Cell
Fla ¨rdh K. 2010. Cell polarity and the control of apical growth in
Streptomyces. Curr Opin Microbiol 13: 758–765.
Fogel MA, Waldor MK. 2006. A dynamic, mitotic-like mecha-
nism for bacterial chromosome segregation. Genes Dev 20:
Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML,
Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L,
et al. 2000. DNA sequence of both chromosomes of the
cholera pathogen Vibrio cholerae. Nature 406: 477–483.
Huitema E, Pritchard S, Matteson D, Radhakrishnan SK, Viollier
PH. 2006. Bacterial birth scar proteins mark future flagellum
assembly site. Cell 124: 1025–1037.
Kusumoto A, Shinohara A, Terashima H, Kojima S, Yakushi T,
Homma M. 2008. Collaboration of FlhF and FlhG to regulate
polar-flagella number and localization in Vibrio alginolyti-
cus. Microbiology 154: 1390–1399.
Lam H, Schofield WB, Jacobs-Wagner C. 2006. A landmark
protein essential for establishing and perpetuating the polar-
ity of a bacterial cell. Cell 124: 1011–1023.
Lenarcic R, Halbedel S, Visser L, Shaw M, Wu LJ, Errington J,
Marenduzzo D, Hamoen LW. 2009. Localisation of DivIVA
by targeting to negatively curved membranes. EMBO J 28:
Leonard TA, Butler PJ, Lo ¨we J. 2005. Bacterial chromosome
segregation: Structure and DNA binding of the Soj dimer—
a conserved biological switch. EMBO J 24: 270–282.
Lutkenhaus J. 2007. Assembly dynamics of the bacterial MinCDE
system and spatial regulation of the Z ring. Annu Rev
Biochem 76: 539–562.
Lutkenhaus J. 2012. The ParA/MinD family puts things in their
place. Trends Microbiol 20: 411–418.
Marston AL, Thomaides HB, Edwards DH, Sharpe ME, Errington
J. 1998. Polar localization of the MinD protein of Bacillus
subtilis and its role in selection of the mid-cell division site.
Genes Dev 12: 3419–3430.
Perez-Cheeks BA, Planet PJ, Sarkar IN, Clock SA, Xu Q, Figurski
DH. 2012. The product of tadZ, a new member of the parA/
minD superfamily, localizes to a pole in Aggregatibacter
actinomycetemcomitans. Mol Microbiol 83: 694–711.
Radhakrishnan SK, Thanbichler M, Viollier PH. 2008. The
dynamic interplay between a cell fate determinant and a
lysozyme homolog drives the asymmetric division cycle of
Caulobacter crescentus. Genes Dev 22: 212–225.
Ramamurthi KS, Losick R. 2009. Negative membrane curvature
as a cue for subcellular localization of a bacterial protein.
Proc Natl Acad Sci 106: 13541–13545.
Raskin DM, de Boer PA. 1997. The MinE ring: An FtsZ-independent
cell structure required for selection of the correct division
site in E. coli. Cell 91: 685–694.
Raskin DM, de Boer PA. 1999. Rapid pole-to-pole oscillation of
a protein required for directing division to the middle of
Escherichia coli. Proc Natl Acad Sci 96: 4971–4976.
Ringgaard S, Schirner K, Davis BM, Waldor MK. 2011. A family
of ParA-like ATPases promotes cell pole maturation by
facilitating polar localization of chemotaxis proteins. Genes
Dev 25: 1544–1555.
Rudner DZ, Losick R. 2010. Protein subcellular localization in
bacteria. Cold Spring Harb Perspect Biol 2: a000307. doi:
Schofield WB, Lim HC, Jacobs-Wagner C. 2010. Cell cycle
coordination and regulation of bacterial chromosome segre-
gation dynamics by polarly localized proteins. EMBO J 29:
Semmler AB, Whitchurch CB, Leech AJ, Mattick JS. 2000.
Identification of a novel gene, fimV, involved in twitching
motility in Pseudomonas aeruginosa. Microbiology 146:
Shapiro L, McAdams HH, Losick R. 2009. Why and how bacteria
localize proteins. Science 326: 1225–1228.
Shiomi D, Sakai M, Niki H. 2008. Determination of bacterial
rod shape by a novel cytoskeletal membrane protein. EMBO
J 27: 3081–3091.
Szardenings F, Guymer D, Gerdes K. 2011. ParA ATPases can
move and position DNA and subcellular structures. Curr
Opin Microbiol 14: 712–718.
Takacs CN, Poggio S, Charbon G, Pucheault M, Vollmer W,
Jacobs-Wagner C. 2010. MreB drives de novo rod morpho-
genesis in Caulobacter crescentus via remodeling of the cell
wall. J Bacteriol 192: 1671–1684.
Thanbichler M. 2011. Good things come in small packages:
Subcellular organization and development in bacteria. Curr
Opin Microbiol 14: 687–690.
Thanbichler M, Shapiro L. 2006. MipZ, a spatial regulator
coordinating chromosome segregation with cell division in
Caulobacter. Cell 126: 147–162.
Viollier PH, Sternheim N, Shapiro L. 2002. A dynamically
localized histidine kinase controls the asymmetric distribu-
tion of polar pili proteins. EMBO J 21: 4420–4428.
Wehbi H, Portillo E, Harvey H, Shimkoff AE, Scheurwater EM,
Howell PL, Burrows LL. 2011. The peptidoglycan-binding
protein FimV promotes assembly of the Pseudomonas aeru-
ginosa type IV pilus secretin. J Bacteriol 193: 540–550.
Xu Q, Christen B, Chiu HJ, Jaroszewski L, Klock HE, Knuth MW,
Miller MD, Elsliger MA, Deacon AM, Godzik A, et al. 2012.
Structure of the pilus assembly protein TadZ from Eubacte-
rium rectale: Implications for polar localization. Mol Microbiol
Yamaichi et al.
2360 GENES & DEVELOPMENT