Advances in Microbiology, 2012, 2, 327-331
http://dx.doi.org/10.4236/aim.2012.23039 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Choline Promotes Growth and Tabtoxin Production in a
Pseudomonas syringae Strain
Lucas A. Gallarato, Emiliano D. Primo, Ángela T. Lisa*, Mónica N. Garrido
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales,
Universidad Nacional de Río Cuarto, Córdoba, Argentina
Received May 31, 2012; revised June 29, 2012; accepted July 11, 2012
ABSTRACT
Some Pseudomonas syringae pathovars secrete tabtoxin, a monocyclic β-lactam antibiotic, responsible for chlorosis, the
principal halo blight symptom in susceptible plants as oats, rye, barley, wheat and sorghum, among other. Here, we
demonstrated that the production of tabtoxin in a P. syringae strain increased at least 150%, when choline, betaine or
dimethylglycine were used as nitrogen source, or when choline was added as osmoprotectant in hyperosmolar culture
media. Besides, we investigated the induction of phosphorylcholine phosphatase (PchP) activity when choline or its
metabolites were used as nitrogen sources. PchP is an enzyme involved in Pseudomonas aeruginosa pathogenesis
through its contribution to the breakdown of choline-containing compounds of the host cells. Considering these results
and that the success of a pathogenic microorganism depends on its ability to survive and proliferate in its target tissue,
we propose that choline is one of the plant signals that contribute to establishment of the infection by tabtoxin-produc-
ing strains of P. syringae.
Keywords: Choline; Pseudomonas syringae; Phytotoxins; Phosphorylcholine Phosphatase
1. Introduction
In previous work, we demonstrated that the presence of
choline as carbon and nitrogen sources in a culture me-
dium of Pseudomonas aeruginosa induce at least three
proteins, phosphorylcholine phosphatase (PchP), hemo-
lytic phospholipase C (PlcH) and acetylcholinesterase
(AchE) [1,2]. PchP is involved in the pathogenesis of P.
aeruginosa through the coordinated and sequential action
of PlcH and PchP on phosphatidylcholine or sphyngomye-
lin and phosphorylcholine, respectively [2-5]. In this way,
these enzymes serve to liberate osmoprotective agents
and nutrients that are needed for the growth and sur-
vival of P. aeruginosa in some phosphatidylcholine-rich
environments as occurs in the lungs of cystic fibrosis
patients. In P. aeruginosa PAO1 the gene encoded PchP
(PA5292) was identified and named pchP [6]. A search
for proteins similar to PchP was performed by the Blast
Web interface (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to-
wards the non-redundant protein sequences database
demonstrated the presence of orthologs in other members
of Pseudomonas genus and some mammals and plant
pathogenic organisms. Previous experiments in our labo-
ratory indicated that P. syringae pv. tomato DC3000 was
able to grow in a basal salt medium with choline, betaine
or dimethylglycine as carbon and nitrogen sources [7].
Under any of these conditions the bacteria produced an
acid phosphatase activity with catalytic properties similar
to those described for the P. aeruginosa PchP.
Tabtoxin is a monocyclic β-lactam produced by P. sy-
ringae pv. tabaci, coronafaciens, and garcae, and it had
also been detected in some P. syringae pv. tomato strains
[8-10]. This toxin inhibits the target enzyme glutamine
synthetase, which results in the abnormal accumulation
of ammonia in plant cells, causing the characteristic
chlorosis symptom in susceptible plants [11]. It was re-
ported that both, growth of P. syringae and quantity of
tabtoxin synthesized, were significantly affected by car-
bon source, nitrogen source and amino acid supplements
[12]. Here, we report that choline, a normal constituent
of plant tissues, in addition to inducing a PchP activity in
a phytopathogenic member of Pseudomon as, elevate the
production of tabtoxin by this bacterium in both, iso and
hyperosmolar conditions.
2. Material and Methods
Pseudomonas syringae pv. coronafaciens S5 was isolated
in a field south of Córdoba, Argentina and characterized
in our laboratory. The bacterium was grown aerobically
at 28˚C in a tabtoxin inducer medium (TMM) described
*Corresponding author.
C
opyright © 2012 SciRes. AiM

L. A. GALLARATO ET AL. 328
in [12] and slightly modified in our laboratory. It con-
tained: H2NaPO4 6.52 mM, HK2PO4 4.6 mM, FeSO4 20
mg· L –1, CaCl2 100 mg·L–1 and MgSO4.7H2O 8 mM and
a reducing concentration of sucrose and NH4Cl (20 mM).
When NH4Cl was replaced by choline, betaine, dime-
thylglycine or individual amino acids, they were used at
the same concentration (20 mM). Casamino acids were
used at 0.25% (p/v). When indicated, some of the above
mentioned compounds were added to TMM, as supple-
mentary nitrogen source at final concentration of 1.0
mM.
For tabtoxin determination by an agar diffusion bioas-
say [9,13], agar discs (3 - 4 mm) were removed from M9
minimal medium + glucose + NH4Cl [14] agar plates
before being spread with E. coli as indicator strain. As
source of toxin, 40 µl of a culture filtrate (0.2 µm filter)
of P. syringae pv. coronafaciens S5 grown in TMM with
NH4Cl or the appropiate nitrogen source, was used. The
aliquot culture filtrates were put into the agar holes, and
after 3 hours at 4˚C to allow aliquot diffusion, the plates
were spread with a suspension of the E. coli culture
grown to 0.3 - 0.5 OD600, and then incubated for 16 hours
at 37˚C. The presence of the toxin was revealed by a
growth inhibition halo around the hole containing the
culture filtrate. Glutamine was used to antagonize the
toxin by mixing 25 μl of a 0.1% solution of the amino
acid with cultures filtrates in the agar hole.
In order to quantify results obtained with the agar dif-
fusion test we defined the term: toxigenic activity (TA).
TA was calculated as the relationship between the di-
ameter of the produced inhibition halo (measured in cen-
timeters) and the optical density (OD) of the 72 hours
grown P. syringae S5 cultures used to be tested, since the
final OD values of cultures grown in different conditions
were slightly dissimilar.
To confirm the chlorosis produced by the tabtoxin ac-
tion, seven to ten day old plant leaves of Avena sativa
were cut and put in sterilized Petri dishes with filter pa-
per imbibed with sterile water. Leaves were inoculated
with 10 μl aliquots of a supernatant from a of P. syringae
S5 culture grown for 72 hours using a syringe without a
needle. A positive test for tabtoxin produced chlorosis
after 2 days [15].
PchP and acid phosphatase activities were measured in
whole cells as in [3,6]. One unit of PchP was defined as
the amount of enzyme that released 1 μmol of p-nitro-
phenol from p-nitrophenyl phosphate or 1 μmol of phos-
phate (Pi) from phosphorylcholine per minute at 37˚C.
Protein concentration was determined according to [16],
using bovine serum albumin as the standard.
All our data were analyzed by GraphPad Prism soft-
ware (version 4.00 for Windows GraphPad Software,
San Diego California USA, www.graphpad.com). Differ-
ences were considered significant at p < 0.05.
3. Results
Choline and metabolic derivatives increased tabtoxin
production and PchP activity in P. syringae S5. The
growth of P. syringae S5 in a minimal medium (TMM
medium, see Material and Methods) with choline, betaine
or dimethylglycine as the sole carbon and nitrogen
sources was very low, and scarce tabtoxin production
was detected (data not shown). The use of sucrose and
4
NH

as carbon and nitrogen sources, respectively, en-
hanced the production of tabtoxin as was reported earlier
for a different strain [12]. When NH4
+ was replaced by
20 mM choline, TA increased approximately 55% - 65%
relative to the TA measured from supernatants of su-
crose/ 4
NH

grown cells (control culture, which was con-
sidered the 100%, Figure 1(a)). The derivatives of cho-
line such as betaine and dimethylglycine, also induced
TA with higher values than that found in control culture,
although to a lesser extent compared with choline.
To test the specificity of choline and its derivatives on
tabtoxin production, other nitrogen sources such as
amino acids were used. As shown in Figure 1(a), only
casaminoacids were as effective as 4 in the TA pro-
duced, whereas glycine, alanine and serine only produced
a TA of 85%, 48% and 46% of the TA measured in the
control. The amino acids methionine, threonine and as-
partic and glutamic acids, in spite of supporting growth
of cells, failed to increase production of the toxin. When
the control culture (sucrose/4) was supplemented
with 1 mM of the amino acids, the TA remained similar
to the control (Figure 1(b)). However, the TA increased
50% - 60% over the control when was supple-
mented with 1 mM of choline.
NH
NH
4
NH
NH
PchP activity was measured in the cells grown in the
above culture conditions. The PchP activity determined
in sucrose/4

grown cells was 0.65 ± 0.17 U. mg of
proteins–1 (means ± SEM, n = 6). Only cells grown in the
presence of choline and its derivatives as nitrogen source,
showed a significant increment. Choline and dimethyl-
glycine produced an increased activity of 4 - 5-fold times
and betaine 3 - 4 fold, compared with the activity meas-
ured in control cells.
In P. syringae it was demonstrated that choline pro-
vided consistently better osmoprotection than betaine in
hyperosmolarity [17,18]. Thus, it was determined if tab-
toxin production and PchP activity were affected in cells
cultured under hyperosmotic conditions and if choline
influenced this condition. P. syringae S5 was grown until
stationary phase, in TMM with different osmolarity pro-
duced by raising the sucrose concentration to 0.75 M. A
series of nitrogenous sources were used (20 mM 4
NH

,
20 mM choline, or 20 mM 4 plus 1 mM choline).
At the end of growth, final OD was measured, and cells
were used to measure PchP activity and the culture su-
pernatants to test the production of tabtoxin. When
NH
4
NH


L. A. GALLARATO ET AL. 329
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Toxigenic Activity (TA)
Nitrogen Source
B
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Toxigenic Activity (TA)
A
(a)
(b)
0
0.5
1
1.5
2
00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Final OD
660
Sucrose (M)
A
Ammoni um
Cho
Ammonium/1mM Cho
Figure 1. P. syringae S5 tabtoxin production (TA) in re-
sponse to different nitrogen sources. (a) P. syringae S5 was
grown in TMM with 20 mM sucrose and 20 mM of the cor-
responding nitrogenous compounds during three days at
28˚C. Cell free culture supernatant were used as source of
toxin and assayed by a diffusion bioassay (see Material and
Methods). Tabtoxin production is represented as toxigenic
activity (TA) (see Material and Methods). CasaminoA:
casaminoacids. (b) TA obtained from cells grown with su-
crose/ (Control) and supplemented with 1 mM of the
other tested nitrogen sources mentioned in the figure. Re-
sults are expressed as mean ± SD (n = 4 - 6).
+
4
NH
was the nitrogen source, bacterial growth was impaired
over 0.35 M of sucrose, and the final OD was less than
50% at 0.75 M, compared with the control condition (su-
crose 0.02 M, Figure 2(a)). A lag period of 10 h was
detected with 0.55 and 0.75 M sucrose concentration.
Besides, the production of tabtoxin was practically unde-
tectable over 0.55 M of sucrose (Figure 2(b)), and the
same occurred with the PchP activity. To test the influ-
ence of choline, this compound was added at two differ-
ent concentrations: 1 mM (only as osmoprotectant) or 20
mM (as sole nitrogen source and as osmoprotectant). In
both conditions, it was observed only a reduction of 17%
in the final OD obtained at 0.75 M of sucrose (Figure
1(a)), and cultures presented a minor lag period (4 h). TA
was scarcely altered whenever choline was present in the
culture medium, showing only a 20% of reduction of the
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.10.20.30.40.50.60.70.8
Toxigenic Activity (TA)
Sucrose (M)
(a)
Ammoni um
Cho
Ammonium/1mM Cho
(b)
Figure 2. P. syringae S5 growth and tabtoxin production
(TA) in response to increasing osmolarity. (a) P. syringae S5
was grown in TMM at different osmolarities as a result of
raising sucrose concentration. Sucrose was also the princi-
pal carbon source, and the nitrogen sources tested were 20
mM (○), 20 mM Cho (□) or 20 mM plus 1
mM Cho (■). Bacteria were grown during 72 hours at 28˚C.
The final optical density (OD660) obtained with the different
conditions is represented. (b) Tabtoxin production expre-
ssed as TA (see Material and Methods) was detected by a
diffusion assay as explained in Material and Methods. Re-
sults are expressed as mean ± SD (n = 4 - 6).
+
4
NH +
4
NH
NH
values measured at 0.75 M sucrose with respect to the
control (4

as the only or preferential nitrogenous
source, Figure 2(b)). The PchP activity of the cells
grown in the different conditions indicated that this en-
zyme was induced whenever choline was present in the
culture medium (as an osmoprotectant or as nitrogen
source), with only a 25% - 30% decrease in the activity
measured in cells grown at 0.75 M of sucrose, compared
with the activity of isoosmolar grown cells. The corre-
sponding values of specific activity were: 0.65 ± 0.17 vs
3.30 ± 0.2 and 3.05 ± 0.21 for sucrose 0.02 M/4 NH

20
mM (control), sucrose 0.02 M/choline 20 mM and su-
crose 0.02 M/4 NH

20 mM/choline 1 mM, respectively.
Data are means ± SEM (n = 6).
4. Discussion
In this work we demonstrated that choline, betaine and

L. A. GALLARATO ET AL. 330
dimethylglycine were more efficient for promoting tab-
toxin production as compared to 4 and a series of
amino acids previously used as the best nitrogen sources
for tabtoxin production [12]. The ability of P. syringae
S5 to use choline as an alternative nitrogen source for the
growth and as an osmoprotectant was an expected result.
In this regard, it was reported [17,18] that choline pro-
vided consistently better osmoprotection than betaine
when it was supplied at 1 mM in the culture media, and
that choline was capable of osmoprotectant activity even
as at a concentration as low as 50 µM. Nevertheless, it
was surprising that choline was also able to repress the
inhibitory effect of high osmolarity on tabtoxin produc-
tion. In some other P. syringae pathovars it had been
reported that a variety of nutritional and environmental
factors are involved in phytotoxins production. Thus, it
has been demonstrated that specific plant-released sig-
nals are detected by the toxin-producing bacteria and are
responsible for the expression of coronatine and syrin-
gomycin phytotoxin genes [8,19].
NH
Our observation of the ability of P syringae S5 to in-
duce the PchP activity by choline and its derivatives un-
der iso or hyperosmolar conditions, support the role pro-
posed for the P. aeruginosa enzyme in the metabolism of
phosphorus and nitrogen and in different environmental
conditions [1,2,4]. The P. aeruginosa PchP kinetic prop-
erties studied with its natural substrate phosphorylcholine
suggested the great utility of this enzyme for the estab-
lishment and colonization of the bacteria in different tis-
sues of the host [2,4]. Interestingly, the pchP gene is
highly conserved in the Pseudomonas group, with 18
putative orthologs found so far, including three different
pathovars of P. syringae (P. syringae pv. tomato str.
DC3000, P. syringae pv. syringae B728a and P. syringae
phaseolicola 1448A) and in the multihost P. aeruginosa
PA14 (http://www.pseudomonas.com/).
From the results presented here we propose that cho-
line, and probably its derivatives may be considered one
of the plant-released signals that contribute to an accurate
establishment of the infection provoked by tabtoxin pro-
ducer P. syringae strains. Thus, during the endophytic
phase of P. syringae S5, the action of PchP on apoplast
phosphorylcholine, or the coordinated action of a bacte-
rial lecithinase and PchP on the structural phospholipids
of vegetable cells would enable the hydrolysis of phos-
phorylcholine to obtain choline and phosphorus, which
can be used by the bacterium as nutrients. Choline may
be derived for the synthesis of the osmoprotector betaine,
whose accumulation would allow tolerance to fluctua-
tions in osmolarity in the environment. In the saprophytic
phase, the existence of choline in root exudates just like
its presence in the plant tissues would favor the survival
of the bacterium until it is able to infect again a plant.
Our proposal is in agreement with the studies reported by
[17,18], who suggested that P. syringae has evolved to
survive in relatively choline-rich habitats. Further invest-
tigations would be necessary to determine if choline is
one of the specific plant-released signals contributing to
establishment of the infection by some P. syringae path-
ovars.
5. Acknowledgements
We thank M. Woelker and the Microbiologist student L.
Ruffinatto for their technical assistance, and L. Barberis
for providing the Escherichia coli strain used for tabtoxin
bioassay. ATL is a CareerMember of the Consejo Nacio-
nal de Investigaciones Científicas y Técnicas (CONICET).
LAG and EDP would like to acknowledge fellowship
support from CONICET and the Ministerio de Ciencia y
Tecnología, Córdoba (MinCyT-Cba). This work was su-
pported by grants from CONICET, Agencia Córdoba
Ciencia and SECYT-UNRC of Argentina.
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