ORIGINAL RESEARCH

Asymmetric chromosome segregation in Xanthomonas citri
ssp. citri
Amanda P. Ucci1, Paula M. M. Martins2, Ivy F. Lau1, Maurı́cio Bacci Jr2, Jos�e Belasque Jr3 &
Henrique Ferreira1,2

1Depto. de Ciências Biol�ogicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Rodovia Araraquara/Jaú Km 1, CP 502,

Araraquara, São Paulo 14801-902, Brazil
2Depto. de Bioquı́mica e Microbiologia, Instituto de Biociências, Universidade Estadual Paulista, Av. 24A, 1515, Rio Claro, São Paulo 13506-900, Brazil
3Fundo de Defesa da Citricultura, Av. Dr. Adhemar P. de Barros, 201, Araraquara, São Paulo 14807-040, Brazil

Keywords

Cell division arrest, chromosome segregation,

citrus canker.

Correspondence

Henrique Ferreira, Depto. de Ciências

Biológicas, Faculdade de Ciências

Farmacêuticas, Universidade Estadual

Paulista, Rodovia Araraquara/Jaú Km 1, CP

502, Araraquara, São Paulo 14801-902

Brazil. Tel: +55 19 3526 4187;

Fax: +55 16 3301 6940;

E-mail: henrique.ferreira@linacre.oxon.org

Funding Information

A. P. U. and P. M. M. M. received

scholarships from FAPESP (2010/02041-8)

and CNPq (142293/2009-1), respectively. This

work was funded by FAPESP (grants 2004/

09173-6; 2010/05099-7). Beneficiário de

auxı́lio financeiro da CAPES, Brasil.

Received: 21 August 2013; Revised: 24

October 2013; Accepted: 4 November 2013

MicrobiologyOpen 2014; 3(1): 29–41

doi: 10.1002/mbo3.145

Abstract

This study was intended to characterize the chromosome segregation process of

Xanthomonas citri ssp. citri (Xac) by investigating the functionality of the ParB

factor encoded on its chromosome, and its requirement for cell viability and

virulence. Using TAP tagging we show that ParB is expressed in Xac. Disrup-

tion of parB increased the cell doubling time and precluded the ability of Xac

to colonize the host citrus. Moreover, Xac mutant cells expressing only trun-

cated forms of ParB exhibited the classical phenotype of aberrant chromosome

organization, and seemed affected in cell division judged by their reduced

growth rate and the propensity to form filaments. The ParB-GFP localization

pattern in Xac was suggestive of an asymmetric mode of replicon partitioning,

which together with the filamentation phenotype support the idea that Xac may

control septum placement using mechanisms probably analogous to Caulobacter

crescentus, and perhaps Vibrio cholerae, and Corynebacterium glutamicum. Xac

exhibits asymmetric chromosome segregation, and the perturbation of this pro-

cess leads to an inability to colonize the host plant.

Introduction

Citrus canker is a serious disease present in the major cit-

rus-producing areas around the world. Currently, there

are no effective curative measures to safeguard the orch-

ards, where the eradication of affected trees is the only

reliable method to prevent the spread of the causal agent

Xac to regions where it has not been detected (Gottwald

et al. 2002). The eradication of plants is a highly contro-

versial, costly, and difficult method to control citrus can-

ker. In addition, its effectiveness is only possible in areas

with very low incidence of the disease (Belasque et al.

2010). Other control measures have been adopted in areas

where citrus canker is endemic or the eradication of

affected trees is not mandatory. These methods, collec-

tively known as the disease management approach,

include distinct cultivation strategies, the use of chemical

formulations to contain the spread of Xac, and the plan-

tation of citrus genotypes displaying relative resistance to

citrus canker. The disease management approach is there-

fore intended to minimize and/or prevent damages and

losses promoted by citrus canker, such as blemish on

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of

the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,

provided the original work is properly cited.

29



fruits, symptomatic fruit drop, defoliation, and stem die-

back (Behlau et al. 2010).

Citrus canker has an endemic status in some of the

main citrus-producing areas in the world, the state of

Florida, USA, and in the southern portion of South

America. In Florida, the eradication program was aban-

doned in January 2006, and as a consequence, citrus can-

ker is spreading throughout the region. In both areas,

growers adopt the disease management system. On the

other hand, the state of São Paulo, Brazil, still keeps an

eradication program with mandatory elimination of foci

of the disease; however, due to a diminished effort to

detect and eradicate infected trees, citrus canker is becom-

ing an epidemic (see references within Ferreira and Belas-

que [2011]). As a result, in some years growers may have

to manage citrus canker in the state of São Paulo as it is

already done in the southern states of Brazil. For these

reasons in depth knowledge of the biology of Xac may

help in the development of strategies to control and mini-

mize the losses caused by this plant pathogen.

Recently, we characterized some esters of gallic acid

that interfere with the subcellular localization of ParB-

green fluorescent protein (GFP) and also GFP-ZapA in

Xac (Silva et al. 2013). As ParB and ZapA are compo-

nents of the bacterial chromosome segregation (segro-

some) and cell division (divisome) machineries

(Gueiros-Filho and Losick 2002; Mierzejewska and Jagu-

ra-Burdzy 2012), respectively, alkyl gallates are believed

to target components of the segrosome and/or the divi-

some (Silva et al. 2013). ParB-like factors are DNA-

binding proteins encoded on plasmids or on the chro-

mosomes of many bacteria (Leonard et al. 2005b; Gerdes

et al. 2010; Mierzejewska and Jagura-Burdzy 2012). They

act together with cognate ATPases, ParA-like factors, to

operate efficient replicon segregation in prokaryotes. Seg-

regation of low copy number plasmids and chromo-

somes requires the specific interaction of ParB with

short cis-acting elements designated parS, which on bac-

terial chromosomes are normally located around the ori-

gins of replication (Livny et al. 2007). Homology

searches showed that Xac possesses at least one of these

cis elements (TGTTCCACGTGGAACG; genomic coordi-

nates c2753..2768), which is located right at the 3′-end
of the dnaN gene on the bacterial chromosome (Livny

et al. 2007). Active segregation of ParB–parS complexes

(also known as the bacterial centromeres), and conse-

quently the origins of replication, occurs with the help

of ParA filaments that help to orient newly replicated

chromosomes to opposite positions within the cells, a

mechanism elegantly demonstrated for the models

Vibrio cholerae (V. cholerae) and Caulobacter crescentus

(C. crescentus) (Fogel and Waldor 2006; Ptacin et al.

2010). In the case of ZapA, this protein is well con-

served among Bacteria, and shows septal localization

dependent on FtsZ (Gueiros-Filho and Losick 2002).

ZapA stimulates FtsZ polymer bundling, as well as the

cross-linking of FtsZ filaments, which is believed to sta-

bilize the Z-ring during cytokinesis (Gueiros-Filho and

Losick 2002; Low et al. 2004; Mohammadi et al. 2009;

Dajkovic et al. 2010).

The fact that alkyl gallates are able to target at once chro-

mosome segregation and cell division in Xac is supported

by demonstrations that these processes are interlinked in

other bacteria, for example, C. crescentus, Streptomyces

coelicolor (S. coelicolor), Mycobacterium smegmatis (M.

smegmatis), and Corynebacterium glutamicum (C. glutami-

cum) (Easter and Gober 2002; Thanbichler and Shapiro

2006a; Jakimowicz et al. 2007; Donovan et al. 2010; Ginda

et al. 2013). In S. coelicolor, for instance, ParAB is required

for proper distribution of chromosomal copies within the

aerial hyphae during sporulation (Jakimowicz et al. 2005,

2007). ParB bound to the oriC region organizes segregation

complexes, while ParA coordinates their spatial distribution

for a concomitant assembly of multiple septa. Therefore,

lack of ParA interferes not only with chromosome segrega-

tion but also with septum placement in S. coelicolor

(Jakimowicz et al. 2007). In C. crescentus, MipZ, an inhibi-

tor of FtsZ polymerization, follows ParB/parS precluding

the formation of the Z-ring at sites other than in the middle

of the cells (Thanbichler and Shapiro 2006a; Kiekebusch

et al. 2012). In a recent model, ParB/DNA was proposed to

catalyze the conversion of MipZ monomers into the active/

dimeric inhibitor of FtsZ, and given the polar localization

of ParB/DNA in C. crescentus, a cloud of active inhibitor

is formed and surrounds the polar regions of the cells (Kie-

kebusch et al. 2012). In the case of M. smegmatis, deletion

of parA produced aberrant septum placement (Ginda et al.

2013); this phenotype was attributed to overall chromo-

some disorganization, and it was suggested that another

factor, maybe analogous to MipZ, could link both

processes. Finally, C. glutamicum possesses an orphan

parA-like gene that codes for a protein designated PldP

(ParA-like division protein) (Donovan et al. 2010). Muta-

tion of pldP had just a mild effect on segregation producing

a slight increase in the number of anucleate cells, and over-

expression led to cell elongation; however, a fluorescent

form of PldP localized close to the septum, and protein

interaction analysis showed that PldP associated with both

ParB and ParA, making it a possible candidate for a septum

inhibitor.

Here, we report on the role of ParB in the chromo-

some segregation process of Xac. Using a tandem affinity

purification (TAP)-TAG version of ParB (ParB-TAP), we

demonstrate that this protein is stably expressed in Xac.

Disruption of ParB from Xac severely compromised cell

division and the ability to induce disease symptoms in

30 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Chromosome Segregation in Xanthomonas citri ssp. citri A. P. Ucci et al.



planta. Subsequent analyses of the ParB-GFP subcellular

localization pattern coupled to the observation that chro-

mosome segregation and cell division may be interlinked

in this plant pathogen raised the hypothesis that the strat-

egies used by Xac to deal with these tasks resembles

those that have been described for the model organism

C. crescentus (Thanbichler and Shapiro 2006b).

Materials and Methods

Bacterial strains and plasmids

The strains and plasmids used are listed in Table 1; oligo-

nucleotides are listed in Table S1. E. coli was cultivated at

37°C in luria broth (Sambrook et al. 1989); Xac was

cultivated at 30°C in nutrient yeast glycerol (NYG) (Daniels

et al. 1984). The TAP-tag expression vector pHF5Ca

(FJ562210) is a variant of pPM2a (Martins et al. 2010). To

construct pHF5Ca, we substituted the gfpmut1 cassette of

pPM2a by the tap1479, which was extracted from pBS1479

(Puig et al. 2001) using PCR with primers PBS1479F/

PBS1479R; gfpmut1 was released from pPM2a by a BamHI/

XbaI digest, whereas tap1479 was treated with the same

enzymes prior to ligation. The ParB-TAP expression vector,

pAPU1, was constructed by the ligation of parB (PCR

amplified from the chromosome of Xac with primers Par-

BF20070822/ParBR20080530) into the BamHI site of

pHF5Ca. pAPU2 was generated by the ligation of parB124-

769 (PCR amplified using primers parBintF/parBintR), into

the cloning vector pCR-2.1-TOPO (Invitrogen, Grand

Island, NY). The ParB-GFP expression plasmid, pAPU3, is

a derivative of pPM7g, which carries the parB gene (PCR

amplified with primers ParBF20070822/ParB20100309)

ligated between BamHI/XhoI restriction sites.

General methods

Electrotransformation of Xac was followed (Ferreira et al.

1995). Southern and Western blot analyses were carried

out following the instructions contained into the DIG

(Roche, Indianapolis, IN) and the Amersham (GE, Fair-

field, CT) ECL Western blotting kits, respectively. To

detect ParB-TAP we used a horseradish peroxidase-

conjugated anti-horse (IgG) raised in rabbits as an unique

antibody (Sigma-A6917, Sigma-Aldrich, St. Louis, MO).

Pathogenicity tests

The host plant used was Rangpur lime (C. limonia

Osbeck). Citrus plants were cultivated under greenhouse

conditions at 25–35°C. For the infiltration tests, Xac cells

were cultivated in NYG medium until the OD600 nm of

~1. Cells were subsequently diluted to 105 colony forming

unit (CFU)/mL in PBS 1X, and infiltrated on the abaxial

surface of leaves using hypodermic syringes without nee-

dles. Symptoms were observed during the course of

60 days. All the tests were performed in triplicates.

Microscopy

Wild-type and mutant strains of Xac were immobilized

onto agarose-covered slides for microscope observations

as previously described (Martins et al. 2010). Cells were

visualized using an Olympus BX-61 microscope and doc-

Table 1. Strains and plasmids.

Relevant characteristics1 References

Strain

Xanthomonas citri ssp. citri IBSBF 1594; formerly known as Xanthomonas axonopodis

pv. citri strain 306 (Xac); ApR
da Silva et al. (2002),

Schaad et al. (2005, 2006)

E. coli DH10B Cloning strain Invitrogen

E. coli BL21(DE3) Protein expression strain (pET system) Novagen

Xac amy::pAPU1 pHF5Ca-parB integrated into the amy locus of Xac; ApR KmR This work

Xac parB::pAPU1 pHF5Ca-parB integrated in parB; ApR KmR

Xac parB::pAPU2 pCR-2.1-TOPO-parB124-769 integrated in parB; ApR KmR

Xac parB::pAPU3 pPM7g-parB integrated in parB; ApR KmR

Plasmids

pPM2a and pPM7g GFP expression vectors; xylR pxyl gfpmut1 bla neo Martins et al. (2010)

pHF5Ca TAP-tag expression vector; xylR pxyl tap1479 bla neo This work

pAPU1 pHF5Ca-parB: xylR pxyl parB-tap1479 bla neo

pAPU2 Derivative of pCR-2.1-TOPO (Invitrogen) carrying parB124-769; bla neo

pAPU3 pPM7g-parB: xylR pxyl parB-gfpmut1 bla neo

1ApR and KmR, ampicillin and kanamycin resistance, respectively; bla and neo, genes for b-lactamase and Neomycin phosphotransferase, respec-

tively.

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 31

A. P. Ucci et al. Chromosome Segregation in Xanthomonas citri ssp. citri



umented with a monochromatic XM-10 camera. Image

processing and analyses were conducted using the soft-

ware Cell^F (Olympus, Center Valley, PA).

Statistics

Statistical analysis was performed using one-way analysis

of variance (ANOVA) followed by Tukey posttest

(P < 0.05).

Results

ParB is expressed in Xac

The annotation of the genome sequence of Xac showed

the presence of at least five parA-like open reading frames

(ORFs) with the following designations: XAC0192,

XAC1907, XAC2205, XAC2433, and XAC3905. The last

one, XAC3905, was found in a small operon (parAB) with

XAC3906, which was identified as a chromosomal parB

homologue (da Silva et al. 2002). In order to verify if

parB was expressed in Xac, we monitored the activity of

the parAB promoter (pparAB) by the ability of a Xac

mutant strain to express a TAP-tagged version of ParB

(ParB-TAP). This mutant was prepared by the integration

of the suicide plasmid pAPU1 (carrying the fusion parB-

tap1479) by a single crossover event into the parB locus

of Xac (Xac parB::pPAU1) using an established protocol

(Martins et al. 2010). Following pAPU1 integration, parB

was duplicated, where the native copy of the gene was

adjacent to the xylose promoter (pxyl) carried by the plas-

mid, whereas the parB-tap1479 fusion had its expression

governed by the native promoter of the parAB operon

(Fig. 1B). The genomic structure of two selected Xac

parB::pPAU1 mutants was evaluated by Southern blot

using the parB gene as a probe (1C), and the detection of

the hybridization bands of 1.031 bp and 5.615 bp con-

firmed the integration of pAPU1 into the parB locus

(compare lanes 1–3 and 2–3). To detect the production

of ParB-TAP by Xac parB::pPAU1, cells were cultivated in

NYG medium until the mid-log phase, and subsequently

processed for the immune detection of the TAP-tagged

protein by Western blot (Fig. 1D). A signal of ~54 kDa

was observed for the two mutant strains analyzed, which

is consistent with the size expected for ParB-TAP (lanes

5–6). No bands could be detected for the wild-type strain

(lane 7), whereas Xac transformed with the empty vector

(Xac amy::pHF5Ca) produced a signal relative to the TAP

tag of ~21 kDa (lane 8). We also included in these analy-

ses two mutants in which pAPU1 had integrated into the

amy locus through recombination between the amy106-

912 fragment carried by pAPU1 and the a-amylase gene

of Xac (Xac amy::pPAU1), hence, they express ParB-TAP

(A)

(B)

(C) (D)

Figure 1. Activity of the Xac parAB operon. The activity of the parAB

operon was demonstrated by the ability of Xac parB::pPAU1 mutants

to express ParB-TAP under the control of the native parAB promoter.

The ParB-TAP expression vector was integrated into the parB locus of

Xac by a single crossover event (B) generating the genomic structure

depicted at the bottom of (B). (A) Genomic coordinates of the parAB

operon. (B) Schematics of the integration of pPAU1 (carrying the

parB-tap fusion) into the parB locus. Numbers above the map and

around the circle indicate the sizes in base-pairs of the DNA

fragments delimited; ERV, EcoRV restriction sites. (C) Southern blot

analysis of Xac parB::pPAU1 mutants: genomic DNA was digested

with EcoRV and subsequently probed with a DIG-labeled parB. The

sizes of the DNA fragments detected correspond to those illustrated

in (B). Lanes 1 and 2, Xac parB::pPAU1 mutants 1 and 2, respectively;

lane 3, wild-type Xac. (D) Western blot was used to detect ParB-TAP

(54 kDa) in the protein extracts of Xac parB::pPAU1 and Xac amy::

pPAU1 mutants. Lanes: 1–2, Xac amy::pPAU1 mutant 1; 3–4, Xac

amy::pPAU1 mutant 2; 5, Xac parB::pPAU1 mutant 1; 6, Xac parB::

pPAU1 mutant 2; 7, Xac wild type (negative control); 8, Xac amy::

pHF5Ca (expresses only the TAP tag of 21 kDa). The inductor xylose

was added as indicated.

32 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Chromosome Segregation in Xanthomonas citri ssp. citri A. P. Ucci et al.



ectopically to serve as a control. Note that both Xac amy::

pPAU1 mutants also express ParB-TAP, but at higher lev-

els (compare lanes 1 and 3 with 2 and 4 for noninduced

and xylose-induced cultures, respectively, with lanes 5–6).
The lower expression of ParB-TAP by the Xac parB::

pPAU1 mutants is probably due to the fact that these

cells have parB-tap1479 under the control of pparAB, and

therefore, they express ParB-TAP at physiological/natural

levels. In summary, the stable production of ParB-TAP

from the native parAB promoter confirmed the activity of

the parAB operon in Xac.

Xac parB mutant is unable to induce citrus
canker symptoms

Several unsuccessful attempts were made to delete the

parB gene of Xac. To circumvent this problem and to be

able to extend our functional characterization of the

parAB operon of Xac, we decided to construct a mutant

strain that can only express truncated forms of ParB. This

was achieved by transforming Xac with the suicide vector

pAPU2 that carried a DNA fragment corresponding to

parB124-769. Upon integration into the parB locus

(Fig. 2B) two new forms of parB should arise: one under

the control of pparAB, which lacks the coding sequence

for the 52 C-terminal residues of ParB (codes for an

equivalent of ParBΔC52, and that has a DNA addition

from the vector coding for extra 18 residues not related

to LacZ [Fig. 2B]), and another that can only be

expressed by the activity of the plac promoter of pPAU2,

and that codes for a LacZa-ParB chimera (LacZa1-26-
ParB42-308, which corresponds to a ParB deleted for the

first 41 N-terminal aminoacids, ParBΔN41). Although

ParBΔC52 carries a predicted HTH motif (Fig. 2B; resi-

dues 158–179 identified using GYM2.0; Narasimhan et al.

2002), this protein is not expected to form dimers and

(A)

(B)

(C)

Figure 2. The strategy used to disrupt parB in Xac. The suicide vector pPAU2, which carries the parB124–769 fragment, was integrated into the

parB locus of Xac by a single crossover event; integration generated the genomic structure depicted underneath the vector in which ParBΔ52 can

be expressed under the control of the native promoter of ParB (pparAB). (A) Genomic coordinates of parAB. (B) Schematics of integration: upon

plasmid insertion, two new forms of parB will be generated: one coding for ParBΔN41, a fusion between the first 26 amino acids of LacZa (coded

by the expression vector) plus residues 42–308 of ParB, and another that codes for ParBΔC52, which lacks the 52 C-terminal residues of ParB and

has an addition of the 18 new amino acids shown. Both truncated ParB proteins still carry the helix-turn-helix (HTH) motif shown. The numbers

above the map and around the circle indicate the sizes of the DNA fragments delimited; the predicted helix-turn-helix motif of ParB is depicted

below the drawings; ERV, EcoRV restriction sites. (C) Southern blot analysis: total DNA of the three Xac parB::pAPU2 mutants was digested with

EcoRV, and probed with parB. The sizes of the hybridization fragments detected correspond to those estimated based on the genomic

coordinates shown in B.

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 33

A. P. Ucci et al. Chromosome Segregation in Xanthomonas citri ssp. citri



should be impaired in DNA binding (by analogy to data

from the biochemical characterization of Spo0JΔ20, and
also of a C-terminal deletion form of ParB from Thermus

thermophilus (Leonard et al. 2004; Murray et al. 2006);

and H. Ferreira and J. Errington, unpublished data). On

the other hand, ParBΔN41, if correctly expressed and

folded, would be compromised in its ability to interact

and/or stimulate the ParA partner as demonstrated for

the ParAB system of T. thermophilus (Leonard et al.

2005a).

Upon transformation of Xac with pAPU2, and the

expected integration of this plasmid into the bacterium

chromosome, the genomic context surrounding the parB

locus of three independently selected Xac parB::pAPU2

mutants, which were derived from the same disruption

event, was checked by Southern blot (Fig. 2C), where the

observation of the diagnostic hybridization band of

4.560 bp confirmed the alterations. The three mutant

strains exhibited a slower growth rate when compared

with the wild-type strain. Growth curves of these mutants

displayed a slower doubling time on the first 24 h,

being only able to reach the usual culture limit for Xac

(OD600 nm ~2) after 30 h of growth. This contrasts with

the 18 h normally taken by the wild-type strain to

approach the growth peak (Figure S1). Note that it took

twice as much for the mutant to get to the same culture

limit of OD600 nm ~2. Furthermore, the recovery of kana-

mycin-resistant mutants after transformation took almost

5 days (120 h), whereas wild-type colonies become visible

in up to 48 h.

As a plant pathogen, the ability to colonize its host is a

vital process for Xac. Since the disruption of parB altered

the growth pattern of the Xac parB::pAPU2 mutants on

rich medium, we tested whether these cells would still be

able to colonize citrus plants. The three Xac parB::pAPU2

mutants were infiltrated in leaves of Rangpur lime along-

side the wild-type strain. While the region inoculated

with the wild-type strain evolved erumpent–corky and

brownish lesions surrounding the infiltration point, none

of the mutant strains was capable of inducing the typical

symptoms of citrus canker (Fig. 3), even after the long

incubation periods of 60 days. We conclude that disrup-

tion of parB in Xac retarded the cell doubling time and

promoted a loss of the ability to colonize the host citrus.

Alteration of the normal ParB function
disrupts cell division in Xac

Disruption, deletion, and/or the overexpression of ParB-

like proteins in several microorganisms, including C. cres-

centus, C. glutamicum, and Pseudomonas aeruginosa (P.

aeruginosa), may lead to pleiotropic effects such as cell

filamentation, loss of motility, and perturbations in the

basic processes of chromosome segregation and cell divi-

sion (Mohl and Gober 1997; Bartosik et al. 2009; Dono-

van et al. 2010). In this work, the inability to colonize

citrus observed for Xac parB::pAPU2 could be a conse-

quence of at least two other deficiencies beyond the per-

turbation of the segrosome: (1) metabolic and/or

physiological alterations, and (2) the perturbation of

pathogenicity systems/factors indirectly triggered by the

disruption of parB. To eliminate such possibilities, we

began by examining the mutants under the microscope in

order to detect any morphological changes in the cells

that could support a segregation defect.

In general, cultures of the parB mutant Xac parB::

pAPU2 had a mix of filaments and rods (Figs. 4, 5). Per-

centages of the entire cell types documented are shown in

Table 2. Filaments and/or irregular chains were approxi-

mately 5% of the cell types in a culture (n = 400)

(Fig. 5). Close inspection of these filamented cells showed

the presence of septal constrictions (arrows in Fig. 5), in

which the division pattern displayed seems as if a particu-

lar rod started to grow and divide normally, losing the

ability to form septa in successive cellular cycles; thus, fil-

amentation seems to happen with elder rods attached to

their tips. Despite the propensity to grow in long fila-

ments, the division machinery seems operative in these

mutants.

Considering the nonfilamented cells of Xac parB::

pAPU2, they looked apparently longer than the wild-type

strain (compare Fig. 4A and B; Table 2). In order to

quantitatively evaluate this, we measured 200 individuals

Figure 3. Xac mutants disrupted for parB are unable to colonize the

host citrus. Three Xac parB::pPAU2 mutants were inoculated in leaves

of Rangpur lime alongside the wild-type Xac and the diluent PBS 1X.

Only Xac wild type was capable of inducing the typical symptoms of

citrus canker as eruptive lesions within chlorotic spaces. Infected

plants were kept in green house for a period of 60 days in order to

score for the appearance of citrus canker symptoms. Here, we show a

representative experiment; tests were performed in triplicates.

34 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Chromosome Segregation in Xanthomonas citri ssp. citri A. P. Ucci et al.



of each culture (wild-type and Xac parB::pAPU2), and

calculated the average cell length. We observed significant

differences between them: the average wild-type cell

length was 1.44 � 0.31 lm, whereas the Xac parB::

pAPU2 mutants measured 2.56 � 0.42 lm.

To subsequently estimate the effects of parB disruption

on the chromosome organization of Xac parB::pAPU2,

cells were labeled with 40,6-diamidino-2-phenylindole

(DAPI) and visualized by fluorescence microscopy

(Figs. 4, 5). Cultures of Xac parB::pAPU2 exhibited an

increase in the number of anucleate rods (~30% of the

cell types, Fig. 4B and C), which contrasts with an

absence of DNA-free cells in cultures of wild-type Xac

(Table 2). When we looked at filamented cells of the Xac

parB mutant, we observed a considerable accumulation of

chromosomal mass, sometimes adopting the form of a

continuum in which the nucleoid fills up the whole of a

cell compartment extending through and preventing the

closure of a septum (Fig. 5; arrow on the right). Minicells

Figure 4. Morphological analysis of Xac parB::

pPAU2 mutant cells. The Xac parB::pPAU2

mutant, which expresses only truncated forms

of ParB, was compared with Xac wild type

using various combinations of phase contrast

(PhC), differential interference contrast (DIC),

and fluorescence microscopy; the membrane

and nucleoid stains FM 4–64 and DAPI,

respectively, were used as indicated. Wild-type

and mutant cells were cultivated in NYG

medium at 30°C, and inspected around the

OD600 nm ~0.3; cells were immobilized onto

1% agarose-covered slides. (A) Xac wild type.

(B–C) Xac parB::pPAU2. Scale bar corresponds

to 4 lm.

Figure 5. The filamentation phenotype of Xac parB mutant. The Xac

parB mutant (Xac parB::pPAU2) was investigated under phase

contrast (PhC) and fluorescence microscopy of DAPI-stained cells

(DAPI) in order to evaluate their chromosome organization. Here, we

show the picture of a representative filament from cells cultivated in

NYG medium at 30°C, and visualized around the OD600 nm ~0.3;

arrows show the closure of septa; on the right (DAPI), septum is

closing without chromosome clearance. Scale bar corresponds to

4 lm.

Table 2. Morphological analysis of Xac parB::pAPU2.

Cell length

(n = 200), lm

Filaments

(n = 400) Anucleate Minicells

Xac wild type 1.44 � 0.31 0 0 0

Xac parB::pAPU2 2.56 � 0.42* 5% 30% 0

Data correspond to the average cell length � standard deviation.

*P < 0.05; One-way ANOVA with Tukey posttest.

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 35

A. P. Ucci et al. Chromosome Segregation in Xanthomonas citri ssp. citri



were not detected neither in cultures of Xac wild-type nor

in the parB mutant (Table 2). Altogether, data show that

the disruption of parB in Xac leads to a severe impair-

ment of the chromosome segregation process, which also

culminated in an unexpected alteration of its ability to

stimulate citrus canker symptoms.

Subcellular localization of ParB-GFP

The localization patterns of several chromosomally

encoded ParB-like proteins have been documented in vivo

and it constitutes an excellent indicative of function in

chromosome segregation (Glaser et al. 1997; Mohl and

Gober 1997; Jakimowicz et al. 2005; Fogel and Waldor

2006; Bartosik et al. 2009; Maloney et al. 2009; Donovan

et al. 2010). To further extend our characterization of

Xac ParB, we prepared a mutant strain, Xac parB::pAPU3,

which expresses ParB-GFP under the control of pparAB at

physiological levels (Fig. 6) (Xac parB::pAPU3 was con-

structed using the same strategy depicted above for the

expression of ParB-TAP). In Figure 6A (I and II), we

show the localization of ParB-GFP in cells without a clear

sign of septal constriction. Note the presence of two foci

per compartment, each occupying one of the cellular

poles. This localization pattern was very similar to the

one documented for the ParB homologue of C. crescentus

(Mohl and Gober 1997). Inspection of several fields

revealed another localization pattern in which the ParB-

GFP focus is splitting in two in just one of the cellular

compartments (Fig. 6A-III, arrow), whereas in the other

it apparently remains as a single entity. The pattern seen

in Figure 6A-III is probably a resultant of either the initi-

ation of a new replication event in only one of the cell

compartments, at a late stage of septation (which implies

an asynchrony in the process of initiation of chromosome

replication), or the brighter individual focus would in fact

be two ParB-GFP foci superimposed. We favor the latter

possibility as we also observed localization patterns in

which the cells seemed to be in synchrony with regard to

the initiation of replication (Fig. 6B). In Figure 6B-I, we

see dividing cells and two closely positioned ParB-GFP

foci occupying in each compartment just one of the polar

regions. Here, new replication events that would separate

the origin regions of the chromosomes are taking place at

the poles; hence, the replisomes should be nearby. The

cellular type shown in Figure 6B-II (a nondividing rod)

seems to support the idea that the origins in Xac initiate

replication toward one of the cellular poles, which is simi-

lar to the replication patterns exhibited by C. crescentus,

V. cholerae, and C. glutamicum (Mohl and Gober 1997;

Fogel and Waldor 2006; Donovan et al. 2010). Finally, we

performed DAPI staining of cells and captured individuals

in late stages of septum closure judged by the placement

of the ParB-GFP foci within the cells (Fig. 6C). We

observed four ParB-GFP foci evenly distributed inside the

two linked cell compartments, where ParB-GFP colocaliz-

es with the edges of the nucleoids. If we consider that

these edges comprise the origins of replication, similar to

B. subtilis and many other systems (Leonard et al. 2005b;

Mierzejewska and Jagura-Burdzy 2012), parS in Xac is

located in this region (at ~3 kb from the origin in the

chromosome of Xac (Livny et al. 2007)) and ParB-GFP is

expected to be bound to it.

In Figure 6B-II, we showed evidence that Xac initiates

chromosome replication close to a cellular pole. This sug-

gests that chromosome segregation is asymmetric in this

plant pathogen as it was also proposed for C. crescentus,

V. cholerae, and C. glutamicum (Fogel and Waldor 2006;

(A) (B)

(C)

Figure 6. Localization of ParB-GFP. The

localization pattern of ParB-GFP was analyzed

in different cell types of a Xac parB::pAPU3

expressing ParB-GFP from the native pparAB

promoter. (A, B, C) represent cells

photographed during different moments of the

cell cycle; arrows mark recently divided origins

of replication (see text). Cells were cultivated

in NYG medium at 30°C until the OD600nm of

~0.3, immobilized onto 1% agarose-covered

slides, and visualized using fluorescence

microscopy. Scale bar corresponds to 4 lm.

PhC, phase contrast; DAPI, nucleoid stain.

36 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Chromosome Segregation in Xanthomonas citri ssp. citri A. P. Ucci et al.



Donovan et al. 2010; Ptacin et al. 2010). In order to fur-

ther characterize the asymmetric pattern of chromosome

segregation in Xac, we performed time-lapse microscopy

using Xac parB::pAPU3 (Fig. 7). From t = 0 to

t = 20 min the leftmost dividing rods started new chro-

mosomal replication events that can be seen by the divi-

sion of the ParB-GFP foci (arrows in t = 20 min). Note

that at the beginning of the process (from t = 0 until

t = 20), the origins of replication, and consequently the

ParB-GFP foci associated with them, were situated near

the cellular poles and opposite to the septum. From

t = 20 to t = 40, we see the innermost origins/ParB-GFP

foci moving toward the septum until they reach the

region that will become the new pole after cytokinesis.

Therefore, we conclude that Xac exhibits asymmetric

chromosome segregation.

Discussion

We demonstrated recently that esters of gallic acid per-

turbed the cell division and/or the chromosome segrega-

tion apparatuses of Xac. Thus, these compounds

constitute promising antimicrobial agents against citrus

canker and probably other bacterial diseases (Silva et al.

2013). Here, we further characterized the Xac mutant

strain used by Silva et al. (2013) that is labeled for the

centromere (Xac parB::pAPU3), and extended the studies

with ParB encoded by this plant pathogen. First, we

showed that Xac ParB is stably expressed and operates on

chromosome segregation. Chromosomally encoded ParB-

like proteins have been characterized in several bacteria

(recently reviewed by (Mierzejewska and Jagura-Burdzy

2012)), where the perturbation of their normal function

and/or their cellular dosage with respect to their associ-

ated ParA partners often lead to an increase in the num-

ber of anucleate cells, cell filamentation, and nucleoid

disorganization. Disruption of parB in Xac produced

these classical phenotypes (Figs. 4, 5). In addition, in the

absence of full-length ParB, Xac was not able to colonize

the host citrus (Fig. 3). Second, the subcellular localiza-

tion of ParB-GFP suggests an asymmetric mode of repli-

con segregation in Xac, which resembles the chromosome

segregation patterns observed for C. crescentus, V. chole-

rae, and C. glutamicum (Fogel and Waldor 2006; Dono-

van et al. 2010; Ptacin et al. 2010). This, along with the

fact that lack of ParB produced a remarkable cell filamen-

tation phenotype suggests that ParB may be involved with

cell division in Xac.

The inability to produce disease symptoms in planta

documented for the Xac parB mutant is a novel effect,

which reinforced our previous observations that alkyl gal-

lates that were able to perturb the subcellular localization

of ParB-GFP also precluded the ability of Xac to colonize

citrus (Silva et al. 2013). In the past decade, several stud-

ies gradually unveiled the participation of ParAB proteins

with various cellular processes other than chromosome

segregation. In P. aeruginosa, for example, absence of

ParB altered bacterial growth and affected swarming and

swimming motilities (Lasocki et al. 2007; Bartosik et al.

2009). Slower growth rates were also detected in our

experiments for the parB mutant of Xac. In C. crescentus,

it was demonstrated that ParB operates on cell division

by sequestering the FtsZ inhibitor MipZ to areas away

from the cell center where the divisome should assemble

(Thanbichler and Shapiro 2006b; Kiekebusch et al. 2012).

In the Gram-positive bacterium B. subtilis the ParB-like

Figure 7. Time-lapse microscopy of Xac parB::pAPU3. The

localization of ParB-GFP in Xac parB::pAPU3 evidences an asymmetric

mode of chromosome partitioning. Cells were cultivated in NYG

medium at 30°C until the OD600nm of ~0.3, and photographed at 0,

10, 20, 30, and 40 min as indicated. Arrows mark cells in which the

replication origins have divided near the cellular poles (see text). DIC,

differential interference contrast microscopy. Scale bar corresponds to

4 lm.

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 37

A. P. Ucci et al. Chromosome Segregation in Xanthomonas citri ssp. citri



protein Spo0J recruits structural maintenance of chromo-

somes to the origin of replication where it was proposed

to assist in the organization of the region to enable more

efficient chromosome segregation (Sullivan et al. 2009).

Although Spo0J may help in replicon partition, it is

required for sporulation in B. subtilis, and recently it has

also been implicated with the control of initiation of

DNA replication by modulating the action of the ParA-

like factor Soj (Ireton et al. 1994; Scholefield et al. 2011).

The involvement of ParAB proteins with control of DNA

replication was also reported for V. cholerae (Kadoya

et al. 2011). We are not certain yet whether the pathoge-

nicity shutdown following parB disruption in Xac has any

association with pathogenicity systems/routes encoded by

the bacterium. We have employed yeast two-hybrid analy-

ses in an attempt to identify interactions of ParB with

factors belonging to known pathogenicity systems in Xac

(A. Ucci, S. C. Farah, and H. Ferreira, unpubl. results);

but so far, nothing was detected. Therefore, we believe

that the loss of virulence may well be simply a response

to the perturbation of the vital cellular processes of chro-

mosome segregation and/or cell division.

In this work, we showed evidence for asymmetric chro-

mosome segregation in Xac (Figs. 6, 7). First, we see

newly duplicated origins of replication labeled with ParB-

GFP occupying just one of the cellular poles (Figs. 6B-I,

7). Later, the two ParB-GFP foci are well separated from

each other within the same cellular compartment

(Figs. 6C, 7), which suggests that segregation occurs with

the internally located origins migrating from the point of

duplication toward the opposite pole (Fig. 7). Asymmet-

ric chromosome segregation was documented for C. cres-

centus, V. cholerae, and C. glutamicum as well (Fogel and

Waldor 2006; Donovan et al. 2010; Ptacin et al. 2010).

For the most extensively characterized system, C. crescen-

tus, the chromosome is oriented such that the origin of

replication (designated cori) is close to what is called the

old pole (C. crescentus is known for exhibiting a strict

polar asymmetry, in which diverse protein factors/systems

are arranged in one but not at both cellular poles at

once). Before DNA replication starts, ParB is bound to

parS located around cori; ParB/parS organizes a centro-

mere (Toro et al. 2008) that is kept anchored to the old

pole by PopZ (Bowman et al. 2008; Ebersbach et al.

2008). When replication fires, the centromere is dupli-

cated, and one copy of this region is captured by ParA fil-

aments that come from the opposite cell pole, where they

are anchored to TipN (a factor that marks the new pole)

(Ptacin et al. 2010; Schofield et al. 2010). ParB/parS then

interacts with ParA, ParB stimulates ParA depolymeriza-

tion that shortens the filament, which is followed by sub-

sequent recapture of ParB/parS; orientation of the

centromeric region to the new pole operates chromosome

partitioning. A related segregation mechanism was also

proposed for V. cholerae (Fogel and Waldor 2006), which

presents some differences with that of C. crescentus, one

of which is the use of a distinct polar anchor designated

HubP, which interacts with ParA (Yamaichi et al. 2012).

As for C. glutamicum, DivIVA was demonstrated to be

the factor that tethers the ParB/DNA complex at the cell

pole (Donovan et al. 2012). Considering the similarities

between systems, in particular the ParB-GFP subcellular

localization patterns documented for C. crescentus, V.

cholerae, C. glutamicum, and Xac, we expect to identify

protein factors of equivalent functions in Xac in the near

future.

The localization pattern of ParB-GFP in Xac as well as

the observation that chromosome segregation and cell

division may be interlinked raised the idea that Xac could

control septum placement in a manner similar to C. cres-

centus (Thanbichler and Shapiro 2006b). Curiously, simi-

lar findings and suggestions were recently reported for M.

smegmatis (Ginda et al. 2013), where deletion of parA

produced aberrant phenotypes of chromosome segrega-

tion and septum positioning leading to cell elongation.

The association of chromosome segregation and cell divi-

sion in Xac was first proposed by Silva et al. (2013), who

demonstrated that alkyl gallates able to perturb the locali-

zation of ParB-GFP in this bacterium apparently acted on

septum assembly as well. In C. crescentus, septum site

selection depends on the centromere migration dynamics

(Thanbichler and Shapiro 2006a), where MipZ, an inhibi-

tor of FtsZ polymerization, interacts and localizes with

ParB bound to parS (Thanbichler and Shapiro 2006a;

Kiekebusch et al. 2012). As the centromeric complexes

occupy the poles, the FtsZ inhibitor MipZ is kept away

from the cell center where the septum is assembled.

Therefore, disruption of ParB in C. crescentus perturbs

the control of septation and leads to cell filamentation.

Xac does not have an obvious mipZ homologue on its

genome. MipZ is conserved in a-proteobacteria, and

belongs to the Mrp/MinD family of P-loop ATPases,

being structurally related to the ParA superfamily mem-

bers Soj and MinD (Kiekebusch et al. 2012). However,

Xac carries at least four parA-like ORFs (XAC0192,

XAC1907, XAC2205, and XAC2433) that could have a

mipZ function, which we shall investigate experimentally

soon. Another possibility would be that Xac utilizes other

protein factors for septum site selection. Noteworthy, the

genome of this plant pathogen has MinCDE coding

regions. The MinCD system, which operates division site

selection, has been well studied in B. subtilis and E. coli,

both bacteria displaying symmetrical chromosome segre-

gation (reviewed by Lutkenhaus [2007]). We are in the

process of characterizing if and how Xac coordinates

chromosome segregation with cell division utilizing

38 ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Chromosome Segregation in Xanthomonas citri ssp. citri A. P. Ucci et al.



MinCD. Ultimately, the filamentation observed for the

Xac parB mutant strain could be caused by the action of

a nucleoid occlusion system, analogous to the Noc/SlmA

systems of B. subtilis and E. coli (Wu and Errington 2004;

Bernhardt and de Boer 2005). As illustrated in Fig. 5,

chromosome replication in the Xac parB mutant, followed

by inadequate segregation, apparently produces cells with

several chromosomal copies that if associated with factors

able to inhibit septum assembly/closure would lead to fil-

amentation. However, Xac does not seem to encode any

Noc/SlmA-like protein that could fulfill the function of a

nucleoid occlusion factor (Wu and Errington 2004; Bern-

hardt and de Boer 2005).

Acknowledgments

A. P. U. and P. M. M. M. received scholarships from

FAPESP (2010/02041-8) and CNPq (142293/2009-1),

respectively. We thank C. S. Farah for helping us with the

YTH experiments and valuable discussion. This work was

funded by FAPESP (Grants: 2004/09173-6; 2010/05099-7),

PROPe-UNESP, and PADC-FCF-UNESP. Beneficiário de

auxı́lio financeiro da CAPES, Brasil.

Conflict of Interest

None declared.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. Growth curve of the mutant Xac parB::pAPU2.

Bacteria were cultivated for 36 h in NYG medium at

30°C and 200 rpm. Each point in the graph corresponds

to an average of optical densities (OD600nm) calculated

from three independent experiments; vertical bars indicate

standard deviation values calculated for each average.

Here, we show the measurements done for mutant 3;

however, mutants 1 and 2 exhibited similar growth pat-

terns. Blue, wild-type Xac; red, Xac parB::pAPU2.

Table S1. Oligonucleotides.

ª 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 41

A. P. Ucci et al. Chromosome Segregation in Xanthomonas citri ssp. citri