130 Journal of Basic Microbiology 2008, 48, 130 – 134

Short Communication
Chemotaxis of Pseudomonas sp. to caffeine
and related methylxanthines

Swati Sucharita Dash, Nori Sri Sailaja and Sathyanarayana N. Gummadi

Department of Biotechnology, Indian Institute of Technology – Madras, Chennai Tamil Nadu – 600036, India

Pseudomonas sp. isolated from soil of coffee plantation area has been shown to degrade higher
concentrations of caffeine (~15 g l–1) by N-demethylation at a rate higher than what has been
reported for any strain so far. This strain exhibits positive chemotaxis towards caffeine
(1,3,7-trimethylxanthine) in swarm plate assay and modified capillary assay in a dose
dependant manner. Related methylxanthines and xanthine also act as chemoattractants for the
strain with the highest relative chemotactic response (RCR) seen for xanthine. Chemotaxis in
Pseudomonas sp. is possibly plasmid mediated as indicated by positive chemotaxis of plasmid
transformed E. coli DH5α. The chemotactic abilities of Pseudomonas sp. combined with higher
rates of degradation of caffeine can be used in the development of strategies for biodecaf-
feination of caffeine containing wastes.

Keywords: Chemotaxis / Pseudomonas / Caffeine / Methylxanthines / Plasmid

Received: August 26, 2007; accepted: October 26, 2007

DOI 10.1002/jobm.200700273

*
Introduction
Bacterial chemotaxis is a behavioral response exhibited
by motile bacteria which enables them to detect and
respond to changes in concentration of chemical sub-
stances present in the environment. A wide variety of
organic compounds including sugars and amino acids,
many of which serve as nutritional sources, act as
chemoattractants for motile bacteria such as E. coli and
Salmonella typhimurium [1]. There has been an increased
interest in exploring the role of chemotaxis in biodeg-
radation of pollutants and synthetic chemicals as
chemotaxis has an important role in enhancing the
biodegradation of pollutants by overcoming many of
the limitations in situ bioremediation relating to nutri-
ent availability [2, 3]. Studies indicate that Pseudomo-
nads, the bacterial group well implicated in biodegra-
dation, show positive chemotaxis to an array of aroma-
Correspondence: Sathyanarayana N. Gummadi, Department of Bio-
technology, Indian Institute of Technology – Madras, Chennai – 600036,
India
E-mail: gummadi@iitm.ac.in
Phone: +91-44-2257-4114
Fax: +91-44-2257-4102
tic chemicals, apart from nitrogenous compound such
as amino acids and organic acids [4]. Pseudomonas putida
PRS2000 exhibited positive chemotaxis towards me-
tabolizable and non metabolizable aromatics [5]. Naph-
thalene [6], toluene and benzene [7], nitrobenzoates and
aminobenzoates [8] and chlorinated ethanes [9] also act
as chemoattractants for various Pseudomonas strains.
Caffeine (1,3,7-trimethylxanthine) is one of the major
agro-industrial wastes generated from coffee and tea
processing plants. Caffeine containing wastes are often
released to the surrounding water bodies and it is de-
tected in surface water, ground water and waste water
effluents often at a high concentration [10, 11] making
it an important contaminant of water and soil. Pseudo-
monas sp. isolated from soil of coffee plantation area
[12] and identified by 16s rRNA analysis has been
shown to degrade higher concentrations of caffeine
(~15 g l–1) by N-demethylation at a rate higher than
what has been reported for any strain so far [13]. The
strain was deposited at the National Collection of In-
dustrial Microorganisms, Pune, India with the accession
number NCIM 5235. In the present study, we have
demonstrated for the first time the chemotactic re-

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Journal of Basic Microbiology 2008, 48, 130 – 134
sponse of this strain towards caffeine and related me-
thylxanthines that are the intermediates of caffeine
degradation pathway in this strain.
Materials and methods
Pseudomonas sp. NCIM 5235 was maintained on CAS
(caffeine and sucrose) medium (pH 6.0) the composition
of which is as follows (g l–1): Na2HPO4, 0.12; KH2PO4, 1.3;
CaCl2 ⋅ 2 H2O, 0.3; MgSO4 ⋅ 7 H2O, 0.3; sucrose, 5; caf-
feine, 1.2 and agar, 25. E. coli DH5α strain transformed
with plasmid pCS1182 from Pseudomonas sp. was also
maintained on CAS medium. Non plasmid bearing
E. coli DH5α strain was used as negative control for the
study and was maintained on LB medium with the
following composition (g l–1): tryptone, 10, yeast ex-
tract, 5, NaCl, 10, agar, 15.
Isolation of plasmid DNA from Pseudomonas sp. and
transformation of E. coli DH5α with the isolated plasmid
was carried out as per previously described protocol
[13, 14]. Transformed E. coli colonies were screened by
spread plate technique on minimal medium substituted
with 1.2 g l–1 caffeine and 5 g l–1 sucrose.
For swarm plate assay, cells were inoculated at
the centre of the plates swarm plates consisting of
basal medium (BM) with the following composition
(g l–1): Na2HPO4, 0.12; KH2PO4, 1.3; CaCl2 ⋅ 2 H2O, 0.3;
MgSO4 ⋅ 7 H2O, 0.3 and 0.3% agar followed by incuba-
tion at 30 °C and 37 °C for both Pseudomonas sp and
E. coli. The diameter of the swarm was measured at 12 h
interval for 48 h.
The chemotactic response at various medium con-
ditions was tested on swarm plates substituted with
5 g l–1 sucrose, 1 g l–1 caffeine and 2.2 g l–1 ammonium
sulphate as carbon and nitrogen sources in combina-
tions. The concentration dependent chemotatic re-
sponse for caffeine was studied in swarm plates sub-
Table 1. Chemotaxis of Pseudomonas sp., E. coli DH5α and E. coli DH5α transformed with pCS1182 at various conditions of
medium. The extent of chemotaxis is observed from the swarm diameter. Seven experiments were performed for each set and the
values represented here are the mean of statistically significant measurements (p < 0.05).
S. No. Medium conditions Diameter of swarm (mm)
Pseudomonas sp.
30 °C
–1
1 Minimal medium + 1 g caffeine l 30.8 ± 1.3
+ 5 g sucrose l–1
2 Minimal medium + 1 g caffeine l–1 19.67 ± 0.6
3 Minimal medium + 1 g caffeine l–1 13.17 ± 1.8
+ 2.2 g ammonium sulphate l–1
4 Minimal medium + 5 g sucrose l–1 7.0 ± 1.4
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemotaxis of Pseudomonas sp. to methylxanthines 131
stituted with 5 g l–1 sucrose and caffeine with concen-
trations varying between 1 g l–1 to 20 g l–1. Chemotaxis
towards dimethylxanthines, monomethylxanthine and
xanthine was tested in swarm plates substituted with
5 g l–1 sucrose and methylxanthines at concentration of
0.2 g l–1.
Chemotaxis index for caffeine and other methylxan-
thines was determined by modified capillary assay de-
scribed previously [15]. Relative chemotaxis response
(RCR) was calculated as per the following formula: Rela-
tive chemotaxis response (RCR) = No. of bacteria in test
capillary tube / No. of bacteria in control capillary tube.
RCR ≥ 2 was considered significant [16].
Results and discussion
Positive swarming was observed for Pseudomonas sp. in
swarm plates containing caffeine thus establishing
caffeine as a chemo attractant for this strain. The di-
ameter of the swarm depicted the extent of chemotaxis
in this bacterial strain (Table 1). Extent of swarming
was noted to be highest in medium containing caffeine
and sucrose, which can be attributed to the faster deg-
radation of caffeine in the presence of sucrose. It has
been established from previous studies that although
the strain is not able to utilize sucrose, the sugar acts as
inducer for caffeine degrading enzymes [9].
The swarm diameter was also greater in the medium
with caffeine alone (no sucrose) as compared to where
caffeine was supplemented along with ammonium
sulphate. The lesser extent of swarming can be attrib-
uted to the presence of an additional nitrogen source.
In order to test whether sucrose behaves as a chemoat-
tractant for Pseudomonas sp. used in this study, the cells
were inoculated onto swarm plates containing salts and
sucrose. It has been reported that sugars act as
chemoattractants for many bacterial strains [1]. How-
E. coli DH5α E. coli DH5α transformed
with pCS1182
37 °C 30 °C 37 °C 30 °C 37 °C
25.3 ± 0.6 0 1.75 ± 0.7 2.0 ± 0.004 20.5 ± 0.7
21.0 ± 1.7 0 0.17 ± 0.003 0 4.0 ± 1.4
10.3 ± 0.6 0 1.17 ± 0.4 0 0
5.0 ± 2.0 0 1.5 ± 0.7 0 0
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132 S. S. Dash et al.
ever, negligible swarming was observed in case of su-
crose for Pseudomonas sp. indicating that the strain is
not chemotactic towards the sugar (Table 1).
No swarming was noted for E. coli DH5α strain which
is a non caffeine degrading strain acting as negative
control. However, transformation (with plasmid form
Pseudomonas sp.) conferred chemotactic ability to the
otherwise non-chemotactic E. coli DH5α as indicated by
positive swarming on medium with sucrose and caf-
feine. Previous studies on Pseudomonas sp. have shown
that the caffeine degrading ability of strain was borne
on a 12 kb plasmid which when transformed in to E. coli
DH5α strain, rendered it capable of growing on caf-
feine medium [13]. In fact, for some of the Pseudomonas
strains that are capable of utilizing aromatic and or-
ganic compounds as nutrients (naphthalene), the de-
gradative properties are plasmid borne and so are the
genes for chemotaxis (nitrobenzoates) towards those
compounds [17, 18]. Perhaps the genes for chemorecep-
tor(s) for caffeine are also plasmid borne and these
genes are transferred to E. coli DH5 α upon transforma-
tion. However, chemotaxis for the E. coli DH5α strain
was lower than Pseudomonas strain on the medium with
caffeine as the sole nitrogen and carbon source and was
completely absent on medium with caffeine and am-
monium sulphate (Table 1). This indicates that perhaps,
some of the genes involved in caffeine catabolism, and
not all, are plasmid encoded. These may be the acces-
sory genes necessary for additional characteristics such
as caffeine tolerance and possibly chemotaxis toward
caffeine too. The lesser extent of swarming as com-
pared to the native Pseudomonas strain could also be due
to fact that many of the genes responsible for basic
chemotaxis machinery in Pseudomonas are genome-
encoded [19].
In order to rule out the effect of temperature on
behavior of plasmid and its stability, incubations were
done at both 30 °C and 37 °C for all the above men-
tioned strains. It was observed that even at 37 °C, Pseu-
domonas sp. showed chemotaxis towards caffeine in
the manner similar to that when incubated at 30 °C
(Table 1). This indicates that the plasmid present in
Pseudomonas sp. is stable at 37 °C and is perhaps respon-
sible for chemotaxis as indicated by positive swarming
exhibited by the transformed E. coli DH5α at 37 °C.
Since 30 °C is not a suitable temperature for growth of
E. coli, no swarming was observed in non-transformed
and transformed E. coli incubated at 30 °C.
The swarm diameter of Pseudomonas sp. varied on
medium containing various concentrations of caffeine,
establishing the dose dependant nature of chemotactic
response in this strain (Fig. 1). The extent of swarming,
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Journal of Basic Microbiology 2008, 48, 130 – 134
Figure 1. Chemotaxis of Pseudomonas sp. towards various
concentrations of caffeine. The values represented here are the
mean of statistically significant (p < 0.05) individual measurements
of six measurements.
expressed as the diameter of the swarm, was highest at
caffeine concentration of 2.5 g l–1 caffeine. This was
also in accordance to kinetic studies on the strain
where it was seen that the rate of degradation of caf-
feine is highest at concentration of 2.5 g l–1 caffeine
[20]. This explains the greater swarm size at 2.5 g l–1
caffeine due to faster establishment of caffeine gradient
on the plate. Chemotactic response towards caffeine
was also observed at higher caffeine concentration
(10 g l–1 caffeine) further adding on to the positive as-
pects of this particular caffeine degrading strain. How-
ever chemotaxis was almost negligible at concentra-
tions ≥15 g l–1 caffeine and this can be accounted to the
inability of the strain to utilize caffeine at concentra-
tions ≥15 g l–1.
Pseudomonas sp. exhibited positive chemotaxis to-
wards all the methylxanthine tested and the extent of
chemotaxis was highest for xanthine followed by theo-
bromine and 7-methylxanthine (Fig. 2). Similar results
were obtained when Pseudomonas sp. was incubated at
37 °C further confirming the stable nature of plasmid
at temperature higher than the optimum and its in-
volvement in chemotaxis (Fig. 2). Previous studies have
shown that Pseudomonas sp. metabolises caffeine via
caffeine – theobromine – 7-methylxanthine – xanthine
route [13] and induced cells are also capable of utilizing
the metabolites as substrates, similar to other caffeine
degrading strains reported so far [21, 22]. Also, positive
chemotaxis was exhibited towards theophylline and
paraxanthine which are not identified metabolites of
caffeine degradation by the strain. This is probably due
to similar nature of chemosensory proteins of the
strain. Similar results were obtained with the trans-
formed E. coli DH5α strain when incubated at 37 °C but
no growth was noticed at 30 °C. However discrepancy
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Journal of Basic Microbiology 2008, 48, 130 – 134
Figure 2. Chemotaxis of Pseudomonas sp. at 30 °C (filled blocks),
transformed E. coli DH5α at 37 °C (empty blocks) and Pseudo-
monas sp. at 37 °C (hatched blocks) towards various methylxanthi-
nes and xanthine. The values represented here are the mean of
statistically significant (p < 0.05) individual measurements of six
measurements.
was noticed for paraxanthine and theophylline where
the response was higher for transformed E. coli as com-
pared to Pseudomonas sp. Probably, a separate mecha-
nism of caffeine chemotaxis exists for E. coli. This can
be due to difference in expression of chemotactic pro-
teins towards the compounds or the existence of a
separate chemotactic signaling pathway for paraxan-
thine and theophylline in case of E. coli DH5α strain.
The results of swarm plate assay were reconfirmed
by capillary assay [23]. The two methods resulted in
essentially the same conclusions therefore the data of
the capillary assay are not presented. The relative
chemotactic index was also determined to be signifi-
cantly lower for sucrose as compared to caffeine thus
confirming that sucrose is not a chemoattractant for
Pseudomonas sp.
Bacterial chemotaxis confers additional growth and
survival advantages to motile bacteria as well as con-
tributing to the dissemination of transmissible plasmid
encoded genes for catabolism of various compounds [2].
This is the first report on caffeine chemotaxis and adds
caffeine and methylxanthines on to the list of known
bacterial chemoattractants that are environment pol-
lutants. Further work elucidating the molecular basis
of caffeine chemotaxis can be carried out basing on this
report. Pseudomonas sp. with its chemotactic abilities
combined with higher rates of degradation of caffeine
can be harnessed in the development of strategies for
treatment of caffeine containing wastes.
Acknowledgements
This work is supported by research grant from De-
partment of Science and Technology, Government of
India.
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chemotaxis of Pseudomonas sp. to methylxanthines 133
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