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
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
Parent
Parent
GFP
GFP
1
of bacteria indicate that GFP expression does not alter the biochemical,
morphological or growth characteristics of the bacterium. However, only anecdotal
0.1
10
20
or limited (i.e., at one temperature) data regarding the effects of GFP expression on
producing strains of Salmonella over a broad range of temperature.
100
Lag Time (h)
To conduct a systematic comparison of the growth kinetics of parent and GFP-
Hypothesis
50
Parent
Parent
GFP
GFP
1
30
100
Lag Time (h)
constructing predictive models with naturally contaminated food.
40
incubated at temperatures from 8 to 48C. Kinetic data were fit to a three phase
0.9
0.6
Parent
Parent
GFP
GFP
1
30
30
40
8
7
6
5
4
3
Parent
Parent
GFP
GFP
2
1
0
10
50
20
E) Salmonella Typhimurium
Parent
Parent
GFP
GFP
20
30
40
40
50
Temperature ( C)
0.9
0.6
0.3
0.0
10
20
30
Temperature ( C)
40
50
50
40
50
40
50
H) Salmonella Typhimurium
8
7
6
5
4
3
Parent
Parent
GFP
GFP
2
1
20
30
Temperature ( C)
10
Parent
Parent
GFP
GFP
40
9
0
10
50
F) Salmonella Dublin
30
Temperature ( C)
10
0.3
1.2
C) Salmonella Dublin
20
20
9
Temperature ( C)
10
0.1
10
Parent
Parent
GFP
GFP
0.3
0.0
10
50
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
0.6
1.2
Temperature ( C)
different from the parent strains and thus, would be suitable marker strains for
Parent strains of Salmonella Typhimurium, Enteritidis and Dublin were
B) Salmonella Typhimurium
20
0.9
G) Salmonella Enteritidis
Temperature ( C)
10
0.1
10
The hypothesis tested was that the GFP strains have growth kinetics that are not
Experimental Approach
40
D) Salmonella Enteritidis
0.0
10
Temperature ( C)
microbial growth are provided in these studies.
Objective
30
10
Maximum Population Density
(log CFU/cm 2)
addition of exogenous substrates. A number of studies with GFP-producing strains
Specific Growth Rate (log CFU/h)
allows the automated counting of large numbers of plates without the need for
10
Specific Growth Rate (log CFU/h)
This is a desirable characteristic for predictive model development because it
Lag Time (h)
detected and counted by illuminating viable cell count plates with ultraviolet light.
1.2
Maximum Population Density
(log CFU/cm 2)
A) Salmonella Enteritidis
Maximum Population Density
(log CFU/cm 2)
100
Specific Growth Rate (log CFU/h)
and eukaryotic cells. Colonies of bacterial cells expressing GFP can be easily
I) Salmonella Dublin
9
8
7
6
5
4
3
Parent
Parent
GFP
GFP
2
1
0
10
20
30
Temperature ( C)
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
constitutively express GFP. Such a high level of marker protein expression
not extended for enough time to detect the stationary phase. Consequently, MPD data were not
could slow growth and decrease maximum population density by creating a
obtained for all incubation temperatures. Nonetheless, GFP decreased MPD on the chicken
competition for and eventual deficiency of essential nutrients.
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).
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
Discussion
strains for developing predictive models in naturally contaminated food, it
The failure of the three GFP strains tested to display similar growth kinetics as the parent strains
should be possible to construct marker strains of Salmonella that do not over-
may have resulted from over-expression of GFP. The plasmid encoding GFP in the current
express GFP and grow in a manner similar to the parent strains. For example,
study was a high copy plasmid in which gfp was under the control of the lacZ promoter for
by placing gfp under the control of a different promoter that requires an inducer
which most Salmonella do not have a lacI gene encoding for the lac repressor protein. It has
not found in food, the expression of GFP could be repressed during the growth
been reported that GFP accounts for up to 75% of total cellular protein in bacteria that
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.
