INTRODUCTION
Caffeine (1, 3, 7 trimethylxanthine) is a widely used stimulant of the
central nervous system and is consumed worldwide in form of beverages
and pharmaceutical preparations. It is known to exert numerous physiological
effects on different organisms at micro molar concentrations. The most
significant effects being inhibition of phosphodiesterase resulting in
increase in intracellular cAMP levels; effect on intracellular calcium
levels and antagonism of adenosine receptors (Serafin, 1995). Apart from
that, caffeine has also been reported to be an antimicrobial agent most
effective against E. coli (Ramanaviciene et al., 2003) and
this is attributed to the effect of caffeine on DNA and protein synthesis
in E. coli. Reports also indicate that caffeine enhances the inhibitory
effect of certain antibacterial agents like penicillin and tetracycline
against Staphylococcus aureus and of furazolidone against vibrios
(Banerjee and Chatterjee, 1981).
However numerous bacterial species have been known to degrade this compound
and use it as a source of nutrient for growth, the most prevalent genus
being Pseudomonas (Dash and Gummadi, 2006a). It is therefore interesting
to know the effect of caffeine on such a caffeine degrading strain as
compared to other strains that are incapable of degrading caffeine. The
present study aims to examine the effect of caffeine on Pseudomonas
sp. isolated in our laboratory that is capable of degrading high concentrations
of caffeine (Dash and Gummadi, 2006b; Gokulakrishnan et al., 2007)
and on other bacterial species (both Gram negative and Gram positive bacteria).
This information will be relevant to justify the negative effect that
caffeine, released from byproducts of coffee processing plants, has on
soil and aquatic microflora thereby disturbing ecological balance. The
role of plasmid towards caffeine tolerance in this strain was also investigated.
MATERIALS AND METHODS
Microorganism and Media
This study was carried out at the Department of Biotechnology (Applied
and Industrial Microbiology Laboratory), Indian Institute of Technology
from November 2006 to June 2007. Caffeine degrading strain Pseudomonas
sp. NCIM 5235 previously isolated in our laboratory was maintained
on nutrient agar medium which had the following composition (g L-1):
beef extract 1; yeast extract 2; peptone, 5; NaCl, 5 and agar, 25 and
was sub cultured every two weeks. E. coli DH5α, Enterobacter
aerogenes NCIM 5139, Proteus vulgaris NCIM 2813, Pseudomonas
aeruginosa NCIM 5029, Staphylococcus aureus and Bacillus
subtilis were also maintained on nutrient agar medium and sub cultured
every two weeks.
Flask Culture Experiments
Experiments were carried out in minimal medium (MM) which had the
following composition: (g L-1): Na2HPO4,
0.12; KH2PO4, 1.3; CaCl2, 0.3; MgSO4.
7 H2O, 0.3; glucose, 5 and ammonium sulphate, 2.2 at pH 7.0.
Caffeine medium (CAS) had same composition of MM except in which ammonium
sulfate was substituted with 1.2 g L-1 caffeine. The bacterial
strains were cultured in nutrient broth for 8 h till OD600nm
reached ~ 1.0. Inoculum (4% v/v) for each bacterial strain under study
was then aseptically transferred to two 250 mL Erlenmeyer`s flask containing
50 mL of MM and one 250 mL Erlenmeyer`s flask containing 50 mL of caffeine
medium. Samples were taken every two hours from all the flasks and OD600
nm was measured. At log phase of growth, caffeine was added from
stock solution of 20 g L-1 to one of the two 250 mL Erlenmeyer`s
flask containing 50 mL of MM to achieve a final caffeine concentration
of 2.5 g L-1. Control experiments consisted of strains
grown in MM without addition of caffeine at log phase.
Isolation of Plasmid DNA from Pseudomonas sp. and Transformation
of E. coli DH5α with the Isolated Plasmid
Plasmid DNA from Pseudomonas sp. was isolated using the MiniPrep
plasmid isolation kit (Qiagen). Isolated plasmid was analyzed on 0.8%
agarose gel with a 1 kb DNA ladder (Biolabs Inc.) as marker. Transformation
of E. coli DH5α was carried out by electroporation in a Biorad
Gene Pulser apparatus according to protocol described by Sambrook et
al. (1989). 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. As control, non-transformed cells
were plated onto the same medium. The plates were incubated at 37°C
and growth was observed after 48 h of incubation. The transformed bacterial
strain was maintained on minimal medium substituted with 1.2 g L-1
caffeine and 5 g L-1 sucrose to retain plasmid and was subcultured
every alternate day.
Analysis of Growth of Bacterial Strains
OD600 nm was measured for samples taken at regular intervals.
Cell dry weight for the bacterial strains was calculated from previously
determined values and plotted against time for getting the growth profile.
Cell dry weight for the bacterial strains was calculated from the OD600nm
values as per the following correlation: for E. coli, P. vulgaris,
E. aerogenes and P. aeruginosa OD600nm of 1 corresponds
to 0.25 g L-1 cell dry weight; for Pseudomonas sp. OD600
nm of 1 corresponds to 0.75 g L-1 cell dry weight; for
Bacillus subtilis OD600 nm of 1 corresponds to 0.33
g L-1 cell dry weight and for Staphylococcus aureus OD600
nm of 1 corresponds to 0.30 g L-1 cell dry weight.
Study of Cell Morphology
Gram staining was performed on cells before and after caffeine addition
at log phase and morphology of the strains was observed under microscope
(Carl Zeiss) at 1000X magnification under oil immersion objective.
Cell Viability
Cell viability before and after caffeine addition during growth was
estimated by determining the Colony Forming Units (CFU) of culture using
standard plate count technique. Briefly, samples were serial diluted in
sterile Normal Saline Solution and 100 μL of diluted sample was plated
onto nutrient agar plates. The plates were incubated at 37°C for 18
h and CFU were estimated as per the following formula:
CFU mL-1 = No. of colonies x 1/ Volume
of culture x dilution factor |
Statistical Analysis
All experiments were performed in quadruplicates and the values presented
are the average of four experiments with ±1 to ±5% standard
deviation.
RESULTS
Growth Profile of Bacterial Strains
The growth profile of different bacterial strains was analyzed in
minimal medium (without caffeine), caffeine medium and minimal medium
with caffeine added at the log phase of growth. E. coli DH5α
strain achieved maximum growth in minimal medium with maximum cell dry
weight of 0.75 g L-1 in the 10th hour of growth (Fig. 1a).
No growth was noticed for this strain in caffeine medium. When caffeine
was added to actively growing cultures in minimal medium, there was a
subsequent decrease in OD600 nm values indicating that the
cells failed to grow upon exposure to caffeine (Fig. 1a).
A different scenario was observed for the caffeine degrading Pseudomonas
sp. In this case, maximum cell dry weight obtained in case of minimal
medium was 0.75 g L-1 in 18 h in minimal medium and 0.91 g
L-1 in 12 h in caffeine medium (Fig. 1b).
However, growth was enhanced and the cell dry weight increased to 1.1
g L-1 at 6 h after addition of caffeine (2.5 g L-1
final concentration). The values presented are the average of data obtained
from four experiments with standard deviation of 1-4% about the mean value.
In order to test the effect of caffeine on other bacterial species, experiments
were performed with Gram negative bacteria viz. Enterobacter aerogenes,
Proteus vulgaris and Pseudomonas aeruginosa; and Gram positive
bacteria viz. Bacillus subtilis and Staphylococcus aureus.
All the above mentioned strains failed to grow in caffeine medium and
achieved maximum growth in minimal medium. Upon caffeine addition at log
phase, the growth of all the bacterial strains tested was inhibited as
indicated by non increase of cell dry weight values, the effect more pronounced
in case of Bacillus subtilis (Fig. 1c).
Morphology
Gram staining was performed to study the morphology of the bacterial
strains before and after addition of caffeine to growing cultures. Interesting
results were obtained upon microscopic examination at 1000X magnification.
The prominent morphological change was observed for E. coli DH5α,
which formed long filamentous structures upon addition of caffeine (Fig.
2b) instead of the normal short rods (Fig. 2a) and
this was observed after 3 h of caffeine addition. The length of the filaments
further increased with time and also a decrease in the number of cells
was observed. The
| Fig. 1: |
Growth profile of (a) E. coli DH5α (b) Pseudomonas
sp. and (c) other bacterial strains in minimal medium (MM), minimal
medium with caffeine added at log phase and caffeine medium. Arrow
indicates time point of caffeine addition |
caffeine degrading Pseudomonas sp. shows no such morphological
change and appeared same even after addition of caffeine (Fig.
2c, d). On the other hand, Enterobacter aerogenes,
Proteus vulgaris and Pseudomonas aeruginosa were seen to
be intact before addition of caffeine but undergo complete lysis upon
caffeine addition to growing cells at log phase (Fig. 2e-h).
Similar observation was also found for Gram positive bacterium Bacillus
subtilis (Fig. 2i, j). However,
no distinct change was observed for Staphylococcus aureus apart
from the decrease in cell growth after addition of caffeine (Fig.
2k, l).
Cell Viability
The viability of the bacterial strains after addition of caffeine
at log phase was measured by standard plate count technique. The Gram
negative bacterial species E. coli DH5α, Enterobacter
| Fig. 2: |
Morphology of bacterial strains before and 6 h after
caffeine addition to actively growing cultures. (a) E. coli
DH5α before addition of caffeine and (b) after 6 h of caffeine
addition; (c) Pseudomonas sp., before addition of caffeine
and (d) after 6 h of caffeine addition; (e) Proteus vulgaris
before addition of caffeine and (f) after 6 h of caffeine addition;
(g) Enterobacter aerogenes before addition of caffeine and
(h) after 6 h of caffeine addition; (i) Pseudomonas aeruginosa
before addition of caffeine and (j) after 6 h of caffeine addition
and (k) Staphylococcus aureus before addition of caffeine and
(l) after 6 h of caffeine addition |
| Fig. 3: |
Viability of bacterial strains before and 6 h after
addition of caffeine at log phase of growth determined by standard
plate count technique. Colony counts are expressed as cfu mL-1x108 |
| Fig. 4: |
Effect of caffeine on E. coli DH5α transformed
with plasmid form Pseudomonas sp. (a) Growth profile of transformed
E. coli DH5α in Minimal Medium, Minimal medium with caffeine
added at log phase and Caffeine medium. (b) Morphology of transformed
E. coli DH5α (i) before addition of caffeine and (ii)
6 h after addition of caffeine. (c) Viability of transformed E.
coli DH5α |
aerogenes and Pseudomonas aeruginosa were rendered completely
non viable after exposure to caffeine as shown in Fig. 3.
This was also observed for almost all the dilutions of the bacterial cultures
and can be and regarded as the complete loss of viability. However the
count of the caffeine degrading Pseudomonas sp. was enhanced from
14±0.04x108 to 22±0.1x108 cfu mL-1
after caffeine addition. Proteus vulgaris also showed some resistance
to caffeine as the cells was viable after caffeine addition although the
bacterial count was decreased to 6x108 from 13x108
cfu mL-1. In case of Gram positive bacteria Bacillus subtilis
and Staphylococcus aureus the viability was not much affected
after caffeine addition, the bacterial count only decreased to 14±0.05x108
cfu mL-1 from 16±0.3x108 cfu mL-1
in case of Bacillus subtilis and to 9.8±0.05x108
cfu mL-1 from 11.2±0.2x108 cfu mL-1
in case of Staphylococcus aureus after caffeine exposure. For both
the strains, the bacterial counts were same in minimal medium with caffeine
added at log phase and in minimal medium without caffeine addition, indicating
that caffeine addition does not affect cell viability in Gram positive
bacteria as it does for Gram negative bacterial species. The values presented
are the average of data obtained from four experiments with standard deviation
of 0.3-2% about the mean value.
Effect of Caffeine on E. coli DH5α Transformed with Plasmid
from Pseudomonas sp.
The effect of caffeine on E. coli DH5α transformed with
plasmid (pCS1182) from caffeine Pseudomonas sp. was studied in
minimal medium with and without caffeine addition at log phase and also
in caffeine medium. Unlike the non-transformed E. coli DH5α,
the transformed strain was found to be capable of growing in caffeine
medium upto 10 h after which the growth ceases possibly due to the depletion
of nutrient sources (Fig. 4a). The cell dry weight of
transformed E. coli drops after 10 h in caffeine medium due to
the depletion of nutrients i.e., caffeine and sucrose in the medium. In
the case of MM alone, stationary phase was also attained ~ 10 h (similar
to MM with caffeine) but the biomass yield was more when compared to caffeine
medium. On the other hand in minimal medium substituted with caffeine
at 7 h, the increase in growth can be attributed to the use of caffeine
as an additional nutrient after the original nutrient in the medium has
been exhausted, as seen in case of diauxic growth.
Morphological studies showed that upon caffeine addition the cells became
filamentous, as seen in the case of non transformed E. coli DH5α
(Fig. 4b). To some extent lysis was observed in the cell
population. Viability studies showed that unlike the non transformed E.
coli DH5α, the transformed bacteria retained viability. However
the count dropped to 6x108 cfu mL-1 from 15x108
cfu mL-1.
DISCUSSION
The genus Pseudomonas has been long implicated in utilizing toxic
matter as nutrient for its growth. Most of the time such compounds are
toxic to the growth of other microbial strains and this is an important
feature for the propagation of pseudomonas in the contaminated site. Pseudomonas
sp. isolated in our laboratory from coffee plantation soil is capable
of utilizing caffeine (1, 3, 7-trimethylxanthine) as sole carbon and nitrogen
source and is capable of degrading high concentrations of caffeine (Dash
and Gummadi, 2006b; Gokulakrishnan et al., 2007). It was interesting
therefore to see the effect caffeine has on this strain and other microbial
strains in terms of fundamental aspects such as growth, morphology and
cell viability.
Caffeine when added at log phase of growth retards the growth of E.
coli, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas
aeruginosa and Bacillus subtilis within a very short time,
but the growth of Pseudomonas sp. was enhanced upon addition of
caffeine. This is because of the utilization of caffeine as a source of
nutrient by the caffeine degrading strain and consequent conversion of
caffeine to non toxic intermediates. Previous studies on E. coli
have shown that caffeine inhibits synthesis of DNA (Sandlie et al.,
1980) and impairs RNA and protein synthesis. This can be the reason for
non viability of E. coli cells upon caffeine exposure and lysis
in case of other bacterial species.
The reduction in growth of the bacterial strains is very closely associated
with changes in cell morphology in the various bacterial strains. Both
the transformed and non transformed E. coli DH5α strains form
long filamentous structures after addition of caffeine to growing cultures.
The length of these filamentous forms was noted to increase with further
incubation in the caffeine supplemented medium. Although there are reports
on formation of long filamentous structure upon exposure to caffeine for
various strains of Aerobacter (SundarRaj and Dhala, 1965) till
to date there are no reports on the effect of caffeine on morphology of
E. coli and other bacterial strains. However, previous reports
show that E. coli subjected to stress factor such as nutrient depletion
forms long filaments instead of separating into individual daughter cells
(Koch, 2005). Filamentous form of E. coli has been also reported
in case of exposure of the bacteria to chromate (Ackerley et al.,
2006). Perhaps the same mechanism is adopted by E. coli to overcome
the effect of caffeine on growing culture. Complete or partial lysis of
the cells is noted in case of other Gram negative bacterial species
under study. Similarly cell lysis was noted in the case of Gram positive
bacterium, Bacillus subtilis after caffeine addition. This might
be due to inhibition of important cellular functions such as protein synthesis
or DNA metabolism by caffeine. In contrast, no noticeable change in morphology
or lysis was observed in other Gram positive bacterium Staphylococcus
aureus after addition of caffeine. These results clearly showed that
coccus form is more resistant to caffeine than bacilli form.
Caffeine also affects the viability of the bacterial strains. E. coli
loses viability due to the abnormal elongation of cells. The other gram
negative bacterial strains studied such as Enterobacter aerogenes
and Pseudomonas aeruginosa are non-viable due to complete cell
lysis that occurs after addition of caffeine. On the other hand, Gram
positive bacteria Bacillus subtilis and Staphylococcus aureus
retain cell viability after caffeine exposure. This perhaps can be attributed
to the basic differences in cellular structures of Gram positive and Gram
negative bacteria.
In previous studies it has been shown that the caffeine degrading Pseudomonas
sp. harbors a 12 kb plasmid that is supposed to be involved in caffeine
degradation (Dash and Gummadi, 2006b). An attempt was made to evaluate
the involvement of this plasmid in caffeine tolerance. Results show that
the E. coli DH5α strain is capable of growing in caffeine
medium and upon addition of caffeine to growing cells, the cell growth
achieves constant value instead of decreasing. Also the transformed E.
coli DH5α was viable after caffeine exposure although the viability
is reduced to almost 50%. However, the morphological changes observed
for non transformed E. coli DH5α are also observed for the
transformed one i.e., same filament formation is observed also for the
transformed E. coli DH5α. These findings indicate the possible
role of the plasmid in conferring resistance to caffeine in E. coli
DH5α. However the morphological change due to caffeine appears to
be an integral aspect of E. coli cell which is not eliminated by
the plasmid. Identification of genes on the plasmid can throw light the
subject.
In conclusion, caffeine brings about changes in cell morphology in E.
coli and causes cell lysis in certain other bacterial strains. However
such an effect was not observed in the caffeine degrading Pseudomonas
sp. indicating that the caffeine degrading strain has some intrinsic mechanism
to counteract the effect of caffeine. Further studies on the mechanism
of action of caffeine on bacterial strains will help in understanding
the basics of the evolution and survival of xenobiotic degrading strains
in nature.
ACKNOWLEDGMENT
This study was funded by grant from Department of Science and Technology,
Government of India (Grant No. BIO/06-07/017/DSTX/GSAT).
