Transcript 05_Linares - Rutgers University

Cell-specific detection of Alkaline Phosphatase in the
Heterotrophic Bacteria Vibrio alginolyticus
Angelique
1
Linares ,
James
2
Ammerman
RIOS 2005 Rutgers University, New Brunswick, NJ
Abstract
In order to study the effects of inorganic phosphorus on the
microbial ocean community, we focused on the production of
the enzyme alkaline phosphatase in heterotrophic bacteria.
We specifically examined Vibrio alginolyticus and its response
to phosphorus depletion in the environment. It is observed
that V. alginolyticus fluoresces under the ELF stain, which
detects AP activity; however, the number of cells fluorescing is
much smaller than anticipated compared to the total number
of cells.
Vibrio alginolyticus
Introduction
These samples were diluted later to a 1:10 dilution with
regular marine broth and stained with Acridine Orange to
find the total cell counts. The aliquots were allowed to
react 45 minutes in a dark room at room temperature.
After the incubation, the samples were filtered into a 5µm polycarbonate filter. The filters were then removed
and incubated in 10% neutral buffered formalin for 45
minutes in a dark room at room temperature. These
suspensions of colonies in formalin were then drawn onto
another polycarbonate filter (black filters). The filters
were mounted onto glass using the mounting media
provided by the ELF kit. Samples are stable for several
months when kept in a damp container at 4°C.
ELF fluorescence shown in a
marine broth sample
Fluorescing Cell Counts and Overall Cell (ml) Counts
Acridine Orange Count Averages
(Using a 10x10 field of vision)
Average Count Marine Broth /mL
1.00E+12
1.00E+11
Cells ( mL )
Observations were done by adding
nutrients to a media which where
there is little or no phosphorus to
observe Alkaline Phosphatase or (AP)
activity. The species Vibrio
alginolyticus was grown from a
culture for this study. The bacteria
were grown in three different
medias, standard Marine Broth,
filtered Marine Broth, and filtered
Marine Broth with inorganic
phosphate (KH2PO4) added 22 hours
after inoculation.
Acridine Orange Count Averages
(Using a 10x10 field of vision) Filtered
Marine Broth/mL
1.00E+10
Acridine Orange Count Averages
(Using a 10x10 field of vision) Filtered
Marine Broth +P/mL
1.00E+09
1.00E+08
ELF Count Averages (Using a 10x10
field of vision) Marine Broth /mL
1.00E+07
1.00E+06
Ju
ly
Ju 5 2
ly pm
Ju 5 4 (2
ly pm hr
J u 6 10 ( 4 s )
ly
a
h
6 1 m( rs)
Ju 2p 22h
rs
ly m
)
(
Ju 6 2 p 24h
ly m rs
Ju 6 4 (26 )
ly pm hr
s
Ju 7 10 (28 )
ly a m hr
7 1 (4 s)
Ju 2p 6h
ly m rs
)
(
Ju 7 2 p 48h
ly m rs
7 4 (5 )
pm 0hr
(5 s )
2h
rs
)
The bacterial cells were stained using the staining kit the
ELF 97 Endogenous Phosphatase Detection Kit (Molecular
Probes, E6601). The ELF 97 alcohol precipitate shows a
fluorescence emission that is 480-500nm. To assess the
effect of phosphorus stress, the standard ELF staining
procedure was used. First, the ELF stain was prepared by
using the standard protocol, 1:20 ratio. An aliquot of 500
µL of the three cultures were prepared with 25µL of ELF
reagent. Another aliquot of each sample was also taken
and 10% neutral buffered formalin, and placed in a
refrigerator.
Acridine Orange Filtered
Marine Broth Sample
Acridine Orange Marine
Broth Sample
ELF fluorescence shown in a filtered
marine broth sample
ELF Count Averages (Using a 10x10
field of vision) Filtered Marine Broth
/mL
ELF Count Averages (Using a 10x10
field of vision) Filtered Marine Broth
+P/mL
Time/Date (hr/day)
Chart A: The higher numbers are the Acridine Orange Counts, or overall counts of bacterial cells per mL
sample, whereas the lesser counts are thr ELF stain counts, or overall cells expressing AP activity per
mL sample.
Percentage of Overall Cells Fluorescing under ELF Stain
18.00%
Acknowledgements
Special Thanks to my mentor, Dr. James Ammerman, and
Jason Sylvan for all of their help on this study, Costantino
Vetriani for the use of the BX-60 Olympus Microscope, and to
RIOS program for providing me with this opportunity.
Bibliography
14.00%
12.00%
Cells in Marine Broth
10.00%
8.00%
Cells in Filtered Marine Broth
6.00%
Cells in Filtered Marine Broth
+P
4.00%
2.00%
0.00%
Time (hrs)
Acridine Orange
Filtered Marine Broth
+P Sample
The basis of the experiment was to observe if the bacteria
expresses alkaline phosphatase activity, which was observed
through the ELF, and if so, which media had the largest
amount of cells fluorescing and the changes within each
media. Now that this is known, one can try and see why the
percentages were so low. There could have been many errors
within the process limiting the number of fluorescing cells
observed, such as the double filtering of the cells. The
environments could have not been phosphorus limited
enough, and regular phosphatase assays were not done to
confirm the limitation in the medias. Since some
phytoplankton species used in previous ELF experiments it did
not work well with the dye, it is possible that the species used
here also did not work as well. Lastly, the dilution used for
the Acridine Orange slides was too little, making the slides
difficult to count due to the dense and large numbers of cells
per field of vision. Instead of a 1:10 dilution, a 1:100 dilution
would make the slides easier to be viewed and quantification
easier.
16.00%
Ju
ly
5
Ju 2 p
m
ly
(
Ju 5 4 p 2hrs
ly
m
)
61
(4
Ju
0a
h
ly
m rs)
(2
61
2p 2h
rs
Ju
m
)
ly
(
6 2 24h
r
p
Ju
ly m(2 s)
6
Ju 4 p 6hrs
m
ly
)
7 1 (28
0a
hr
Ju
s)
m
ly
(
71
46
2
p m hrs
Ju
)
ly
(
4
72
8h
Ju
p
r
ly m ( s )
7 4 50
h
Ju
ly pm rs )
8 1 (52
h
0a
m rs )
(7
0h
rs
)
The ELF stain has been previously used for alkaline
phosphatase testing on cyanobacteria and phytoplankton,
but has rarely been used on heterotrophic marine bacteria
(Carlsson, Per et. al. 2001; Lomas, M.W et al.2004).
Heterotrophic bacteria have been shown to have a high
phosphate requirement for their overall growth (Cotner, J.B
et. al. 1997). This study aims to find if the heterotrophic
bacteria, Vibrio alginolyticus, produces the enzyme and
the regulation AP activity can be observed as well using the
ELF substrate.
Based on the graphs and calculations, alkaline phosphatase
activity is expressed in the heterotrophic bacteria Vibrio
alginolyticus, in phosphorus-limited environments.
However, the collective percentages of ELF fluoresced cells
based on the overall cell counts are extremely low, being
all less than 14%. This is lower than anticipated since one
of the medias used were phosphorus limited, the filtered
Marine Broth.
Percentage of Elf/Acridine Orange cells per mL
One way scientists have developed to
study phosphorus activity within
plankton is the study of cell surface
enzymes, also known as
"ectoenzymes”, which are essential
catalysts in the break down of
dissolved organic phosphorus and the
surfaces of particulate organic matter.
Alkaline phosphatase is an ectoenzyme
that is induced by a genetic response
to phosphate deficiency.
Aliquots of each
sample were placed
in eppendorf tubes
Conclusion
Samples Viewed under UV Excitation
Materials and Methods
Phosphorus is a vital nutrient for marine life. Phosphorus cycling
includes bacterial uptake of orthophosphates and phytoplankton
assimilate it under suitable lighting conditions. Then the bacteria and
phytoplankton are consumed by filter feeding organisms, and the
food chain continues.
There has been an ongoing debate on which element limits primary
production in aquatic ecosystems. New studies have found that
phosphorus could sometimes be the limiting nutrient. In the past
decades, some work (Lomas, M.W et al.2004; Dyrhrman, S.T et
al.2002) has shown that primary production in marine systems can be
limited by inorganic phosphorus. Studies of phosphorus have taken
place to find out exactly its role is in limiting primary producers.
It is responsible for dephosphorylation, or the removal
of phosphate groups with attached phosphate esters.
Although its regulation is not entirely understood, it is
hypothesized that it is a means for the bacteria or
plankton to produce free phosphate groups for uptake
and use.
One way to observe the activity of this enzyme is by using a
specific method of staining known as Enzyme Labeled
Fluorescence (ELF). It is an enzyme substrate that is used to
label the alkaline phosphatase (AP) activity of a phosphorus
stressed cell. It has been demonstrated that it can
fluorescently label individual phytoplankton cells expressing
AP activity and distinguish single cell AP activity (Carlsson,
P. et al. 2001). It is clearly regulated by phosphate, it is
apparent in -P cells and not in +P cells.
Results
Since this assay has rarely been
used with heterotrophic marine
bacteria, the results could vary
greatly. In past studies, there has
been large variability in the level
of ELF fluorescence due to
differences in cellular AP
expression or specific activity of
the individual enzyme complexes
(e.g. Lomas, M.W et al.2004).
Chart B: The percentages of ELF stained cells over the overall number of cells per mL of each sample. The
filtered marine broth with the added phosphorus percentage stayed lower than the other percentages,
which is what one would assume. The marine broth percentage went from almost 0% to above 16%.
However, the numbers of these percentages were inconsistent with the amount of phosphorus predicted in
each media.
Aguilera, Angeles et al. Detection and quantification of alkaline
phosphatase in single cells of phosphorus starved marine
phytoplankton. MEPS 164:21-35. 1998.
Ammerman, JW. Phosphorus cycling in aquatic environments: Role of
bacteria. Encyclopedia of Microbiology. Rutgers University. New
Brunswick, NJ.
Ammerman, JW et al. Phosphorus Deficiency in the Atlantic: An Emerging
Paradigm in Oceanography. Eos, Transactions, American
Geophysical Union, 84(18): 165-170. 2003
Carlsson, Per and Caron, David A. Seasonal Variation of phophorus
limitation of bacterial growth in a small lake.Limnology &
Oceanography. 46(1): 8- 12. 2001
Cotner, JB et al., Ammerman, James W., Peele, Emily R., and Bentzen,
Ellen. Phosphorus-limited bacterioplankton growth in the Sargasso
Sea.Aquatic Microbial Ecology. 13:141-149. 1997.
Dyrhman, Sonya T et al.,Cell specific detection of phosphorus stress in
Trichodesmium from the Western North Atlantic.
Limnology & Oceanography. 47(6):1832-1836. 2002.
Lomas, et al. Taxonomic variability of phosphorus stress in Sargasso Sea
phytoplankton. Limnology & Oceanography. 49(6): 23052310. 2004.
For more information on this study please contact :
[email protected]