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Growth Kinetics of Parent and Green Fluorescent Protein-Producing Strains of Salmonella Thomas P. Oscar , Agricultural Research Service, USDA, 1124 Trigg Hall, UMES, Princess Anne, MD 21853 410-651-6062; 410-651-6568 (fax); [email protected]. Introduction - PowerPoint PPT Presentation
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Growth Kinetics of Parent and Green Fluorescent Protein-Producing Strains of Salmonella
Thomas P. Oscar, Agricultural Research Service, USDA, 1124 Trigg Hall, UMES, Princess Anne, MD 21853
410-651-6062; 410-651-6568 (fax); [email protected]
The green fluorescent protein (GFP) is a small polypeptide (27 kDa) from the
jellyfish Aequora victoria that has been cloned and expressed in both prokaryotic
and eukaryotic cells. Colonies of bacterial cells expressing GFP can be easily
detected and counted by illuminating viable cell count plates with ultraviolet light.
This is a desirable characteristic for predictive model development because it
allows the automated counting of large numbers of plates without the need for
addition of exogenous substrates. A number of studies with GFP-producing strains
of bacteria indicate that GFP expression does not alter the biochemical,
morphological or growth characteristics of the bacterium. However, only anecdotal
or limited (i.e., at one temperature) data regarding the effects of GFP expression on
microbial growth are provided in these studies.
Objective
To conduct a systematic comparison of the growth kinetics of parent and GFP-
producing strains of Salmonella over a broad range of temperature.
Hypothesis
The hypothesis tested was that the GFP strains have growth kinetics that are not
different from the parent strains and thus, would be suitable marker strains for
constructing predictive models with naturally contaminated food.
Experimental Approach
Parent strains of Salmonella Typhimurium, Enteritidis and Dublin were
transformed with a high copy plasmid encoding wild type GFP under the control of
the lacZ promoter. Growth curves were obtained using cooked chicken burgers
incubated at temperatures from 8 to 48C. Kinetic data were fit to a three phase
linear model to determine lag time (LT), specific growth rate (SGR) and maximum
population density (MPD) at each temperature. Secondary models for the growth
parameters as a function of temperature were generated and compared among the
parent and GFP strain pairs.
Results
The effects of GFP on LT were significant and slightly different among the
serotypes of Salmonella. Whether GFP increased, decreased or did not alter LT
depended on the incubation temperature and serotype (Figure A to C). GFP reduced
SGR in the three serotypes tested. The magnitude of the reduction in SGR was
dependent on the incubation temperature and serotype (Figure D to F). The most
consistent effect was that GFP reduced the optimum SGR by 0.17 to 0.2 log CFU
per h.
Not all of the growth curves exhibited three-phases of growth. In some instances, sampling was
not extended for enough time to detect the stationary phase. Consequently, MPD data were not
obtained for all incubation temperatures. Nonetheless, GFP decreased MPD on the chicken
burgers (Figure G to I). Likewise, GFP reduced MPD by 1 to 1.5 log cycles in the starter
cultures used to inoculate the burgers (results not shown).
Discussion
The failure of the three GFP strains tested to display similar growth kinetics as the parent strains
may have resulted from over-expression of GFP. The plasmid encoding GFP in the current
study was a high copy plasmid in which gfp was under the control of the lacZ promoter for
which most Salmonella do not have a lacI gene encoding for the lac repressor protein. It has
been reported that GFP accounts for up to 75% of total cellular protein in bacteria that
constitutively express GFP. Such a high level of marker protein expression
could slow growth and decrease maximum population density by creating a
competition for and eventual deficiency of essential nutrients.
Although the results of this study indicated that the GFP strains tested displayed
different growth kinetics than the parent strains and thus, would not be good
strains for developing predictive models in naturally contaminated food, it
should be possible to construct marker strains of Salmonella that do not over-
express GFP and grow in a manner similar to the parent strains. For example,
by placing gfp under the control of a different promoter that requires an inducer
not found in food, the expression of GFP could be repressed during the growth
of the pathogen on the food but then induced during growth of the pathogen on
the viable cell count plate by including the inducer in the agar medium.
10 20 30 40 500.1
1
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100ParentParentGFPGFP
A) Salmonella Enteritidis
Temperature (C)
Lag
Tim
e (h
)
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B) Salmonella Typhimurium
Temperature (C)La
g T
ime
(h)
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C) Salmonella Dublin
Temperature (C)
Lag
Tim
e (h
)
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GFP
ParentParent
GFP
G) Salmonella Enteritidis
Temperature (C)
Max
imum
Pop
ulat
ion
Den
sity
(log
CF
U/c
m2 )
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GFPParentParent
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H) Salmonella Typhimurium
Temperature (C)
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imum
Pop
ulat
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Den
sity
(log
CF
U/c
m2 )
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GFPParentParent
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I) Salmonella Dublin
Temperature (C)
Max
imum
Pop
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sity
(log
CF
U/c
m2 )
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0.3
0.6
0.9
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ParentParent
GFPGFP
D) Salmonella Enteritidis
Temperature (C)
Spe
cific
Gro
wth
Rat
e (lo
g C
FU
/h)
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0.3
0.6
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E) Salmonella Typhimurium
Temperature (C)
Spe
cific
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wth
Rat
e (lo
g C
FU
/h)
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0.3
0.6
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ParentParentGFPGFP
F) Salmonella Dublin
Temperature (C)
Spe
cific
Gro
wth
Rat
e (lo
g C
FU
/h)
