Transcript P. aeruginosa - York College of Pennsylvania

Utilization of Phenylalanine or Tryptophan as a Single Nutrient Source for
Growth of Pseudomonas aeruginosa
Ronna Thomsen Department of Biological Sciences, York College of Pennsylvania
Obtained from the collection of Dr. Carolyn Mathur
Obtained from the collection of Dr. Carolyn Mathur
Introduction
Methods
Harvest Cells
•P. aeruginosa increased 100 fold over 10 days in the presence of
phenylalanine as the sole nutrient source.
Tryptophan (Figure 1B)
•P. aeruginosa grew logarithmically in the presence of tryptophan.
Negative Control
CaCl2
0.64 mM
(Tubes 2 & 3)
1.28 mM
(Tubes 4 & 5)
Conclusions
•P. aeruginosa increased growth with either phenylalanine or
tryptophan as its only source of nutrient.
Standard Plate Count (SPC)
on Day 0
Incubate @ 30ºC
In a shaking water bath
Phenylalanine
Tryptophan
A.
10
B.
9
Control
0.64mM
1.28mM
Cells/ml
Cells/ml
10 7
10 6
10 8
•Now that we have established this experimental system we can
further test the question about the mesopore protection theory
(Mayer, 1994).
•Future studies may include retesting tryptophan and increasing
the rinse process; if the controls don’t grow under these
conditions then either phenylalanine or tryptophan can be used
to test the hypothesis. Also, it could be beneficial to find a lower
concentration of each of the amino acids to determine when the
growth becomes dependent on the concentration.
Control
0.64mM
1.28mM
10 7
10 6
10 5
10 4
•The phenylalanine control did not grow, but the tryptophan did
to some extent. This growth could be due to the presence of
residual nutrients on the cells caused by inadequate rinsing of
the cells as they were being prepared for the experiment.
•Due to the dramatic increase in growth compared to the control,
phenylalanine is a good experimental system for examining this
theory.
Sampled periodically to measure growth by SPC
10 5
0
•Our hypothesis is that P. aeruginosa will show increased
growth in the presence of phenylalanine or in the presence
of tryptophan as the sole nutrient source.
•P. aeruginosa showed a 10 fold increase over the control after 6
days of growth; however, the control itself also grew, but to a lesser
extent.
Resuspend in CaCl2
to use as innoculum
www.old.jccc.net/~pdecell/biochemistry/aminos.html
•The goal of this study was to develop an experimental
system that could be used to examine the mesopore
hypothesis theory.
•Control showed no growth over the same time period of 10 days.
Wash 3x @ 4000 rpm
with 0.02 M CaCl2
10 8
Objectives
Results
Phenylalanine (Figure 1A)
•P. aeruginosa grew logarithmically in the presence of
phenylalanine.
www.old.jccc.net/~pdecell/biochemistry/aminos.html
Bacteria play an important role at many levels in the
environment. One of the functions of bacteria is the
decomposition of organic matter which is important for the
interactions that take place on the surface of minerals. These
interactions affect the amount of carbon sequestered in various
soils and sediments. They also affect carbon cycles related to
such events as climatic changes and to the formation of fossil
fuels which are produced by continued burial of organic matter
without degradation by the bacterial population (Brantley et al.,
2001). In the soil, these reactions occur on the surface of
minerals within macroscopic divots called mesopores. In 1994,
L. Mayer proposed a hypothesis related to this phenomenon
which stated that organic matter may prevent degradation by
adsorbing into small mesopores found along the surface area of
soils and sediments. According to this theory, organisms such as
bacteria, which can break down organic matter, are too large to fit
into the mesopores and access the organic matter that has been
adsorbed into these spaces.
In order to test the hypothesis that sequestered nutrients in
mesoporous rock are protected from microbial access, one must
first find an organism whose growth is dependent on a single
nutrient that can be adsorbed to mesoporous rock. Zimmerman
has adsorbed both alumina and silica (common rock minerals)
mesoporous samples with the amino acids tryptophan and
phenylalanine. In addition, it has been shown that the bacteria
Pseudomonas aeruginosa, which is a common bacterium in soil,
can grow in minimal media such as tap water (Kooij et al., 1982).
Lanyi (1969) and Kooij et al. (1982) found, through a study that
was somewhat similar to ours, that P. aeruginosa has an
increased growth rate in the presence of amino acids,
particularly, phenylalanine and tryptophan. In order to determine
if P. aeruginosa could be used to examine the Mayer hypothesis,
my study was done to test if P. aeruginosa could grow with
tryptophan or phenylalanine as the sole nutrient.
Prepare overnight nutrient broth
culture of P. aeruginosa at 35°C
1
2
3
4
5
6
Days
7
8
9
10
0
1
2
3
4
5
6
7
Days
Figure 1. Growth pattern of P. aeruginosa in minimal media with different concentrations of phenylalanine
or tryptophan as the sole nutrient source. Increased growth was observed in all concentrations of
phenylalanine and tryptophan compared to the control. Standard plate counts (SPC) were done using nutrient
agar and incubated overnight. Averages of the resulting SPC are shown with error bars indicating 95%
confidence intervals of standard error. (A) Growth curve of P. aeruginosa in the presence of 0.64 mM and
1.28 mM phenylalanine. A total of eight SPC’s were performed over a period of ten days. (B) Growth curve of
P. aeruginosa in the presence of 0.64 mM and 1.28 mM tryptophan. A total of five SPC’s were performed over
a period of six days.
Literature Cited
Lanyi, B. 1969. Amino acid utilization by serologically grouped Pseudomonas aeruginosa strains. Acta Microbiologica Academiae
Scientiarum Hungaricae 6:357-361
Mayer, L.M. 1994. Relationships between mineral surfaces and organic carbon concentrations in soils and sediments. Chemical Geology
114:347-363.
Van der Kooij, D., Oranje, J. P. 1982. Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at
concentrations of a few micrograms per liter. Applied and Environmental Microbiology 44:1086-1095.
Zimmerman, Andrew R., Goyne, Keith W., Chorover, Jon, Komarneni, Sridhar, Brantley, Susan L. 2003. Mineral mesopore effects on
nitrogenous organic mater adsorption. Submitted to Organic Geochemistry.
Acknowledgements:
Carolyn Mathur, PhD, Thesis Mentor; Ron Kaltreider, PhD; Karl Kleiner, PhD