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Gluconate utilization effect towards E. coli fitness in
seawater
Dharmawan, L.M., Lai, Jennifer Y.H., and Lau, Stanley C.K.
The Hong Kong University of Science and Technology, Clear Water Bay
Abstract
• Escherichia coli (E. coli) bacteria respond to
environmental stress by altering their gene expression
• An upregulation in four genes involved in gluconate
metabolic pathway is observed in E.coli strains that
survive better in seawater
• Three variable strains of E.coli are shown to have
different growth and decay rate in medium
supplemented with D-gluconate
Background
Upregulated Genes
Real Time PCR
Maximum OD
Growth Rates
Decay Rate
Conclusion & Discussion
Acknowledgement
Lai et al. (2014) created a model system to study
lysogeny effect towards E.coli survival in seawater.
P2
released
• Phage in E1140
(an environmental
strain) is induced
by mitomycin C
Infecting
E455
• P2 is
used to
infect
E455, a
fecal
strain
E455L
• E455 with
P2
sequence
integrated
in the
genome
The survivability of the three strains are then measured
by producing a decay curve.
Name Protein encoded
gntU Low-affinity gluconate transporter
gntT High-affinity gluconate transporter
gntK Gluconate kinase 2
edd 6-phosphogluconate dehydratase
E455L, showed a decay rate in-between the wild type
fecal strain E455 and the environmental strain E1140.
This implies that the acquisition of P2 virus conferred
environmental fitness to its host.
Fig. 2 Growth and deactivation of the three E.coli strains in autoclaved
seawater at 30 °C overtime
Fig. 1 Method used to generate variable strains of E.coli used in the
experiments
• A transcriptome analysis was conducted in order to
investigate how the addition of the P2 gene in E455
genome could increase survivability in E. coli
• A cluster of gene involved in gluconate metabolic
pathway was observed to be upregulated in E455L
but not in E455
To further confirm the transcriptome analysis result, the
expression level of the candidate genes were measured
through real time PCR.
Fig. 3. Levels of gene expressions across three strains of E. coli with 16s RNA
expression level as internal control. The expression level showed is relative to E455
and RQ stands for relative quantity. RQ was calculated using the formula [(CT gene
of interest – CT internal control)sample – (CT gene of interest – CT internal control)E455]
Fig. 5. Mean growth rate of E. coli at 37oC. No significant difference of growth rate
among the three E.coli strains grown under M9 medium at the p<.05 level [H(2) = 4.526, p
= 0.104]. Significant difference is found in 0.1 M D-gluconate medium at the p<.05 level
[H(2) = 9.871, p = 0.0072]. Post hoc Tukey analysis showed that the mean growth rate of
E455 and E1140 differed significantly at p < .05; E455L was not significantly different from
the other two strains.
16s
rRNA cysG gntU gntT gntK edd
Real time PCR result shows that all four genes are
upregulated in both E455L and E1140, with a much
higher degree of expression in E1440.
At the end of the growth curve, E1140 has the highest
cell concentration amongst all three strains.
Fig. 6 Maximum absorbance measured 15 hours post inoculation in different medium.
There is a significant difference of maximum OD among the three E.coli strains grown
under M9 medium at the p<.05 level [H(2) = 12.117, p = 0.0023]. Post hoc Tukey analysis
showed that the maximum OD of E1140 differed significantly at p < .05. No significant
difference is found in maximum absorbance of strains in 0.1 M D-gluconate medium at the
p<.05 level [H(2) = 2.74, p = 0.2541]. Absorbance value is proportional to cell
concentration.
I would like to extend my gratitude towards Dr. Stanley
Lau and Jennifer Lai who have provided me
with expertise and guidance throughout the project. I
would also like to thank Dr. Samuel Cheung and Yin Ki
Tam for assisting me in administration purposes.
References:
Lai, J. Y., Zhang, H., Chiang, M. H., Yu, M., Zhang, R., & Lau, S. C. (2014). Draft genome sequences of three Escherichia coli strains investigated for the effects of lysogeny on niche
diversification. Genome announcements, 2(5), e00955-14.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods,25(4), 402-408.
Rozen, Y., & Belkin, S. (2001). Survival of enteric bacteria in seawater. FEMS Microbiology Reviews, 25(5), 513-529.
Fig. 4.Real time PCR result in 4% agarose gel, 120V, EtBr. Ladder is NEB 100bp.
The single band observed confirmed the specificity of the primers.
Table 1. List of upregulated genes observed in E455L and E1140
Fig. 3 Gene expression comparison of E455L to E455 from the
transcriptome analysis. T3 and T15 refer to the time point where the RNA
is harvested from the microcosm (in hour)
Fig. 7 Percentage of survival measured within 15 hours interval in different medium. SW
stands for seawater. There is a significant difference of percentage of survival among the
three E.coli strains grown under SW p<.05 level [F(2,8) = 7.2, p = 0.025 and gluconate
supplemented seawater [F(2,8) = 28.46, p = 0.001]. Post hoc Tukey analysis categorizes
the strains into different group as shown.
E.coli grows slower in gluconate supplemented medium
in comparison to M9 medium. E1140, the environmental
strain, has the lowest growth rate among three strains.
E. coli survives better in gluconate supplemented
medium, with E455L showing the highest fitness.
Decay and growth utilizes energy in a different way. A
decay curve is generated to provide more holistic view
on gluconate effect towards fitness.
In order to observe whether the upregulation of
gluconate metabolizing genes confers higher growth
rate in D-gluconate supplemented medium, a growth
curve is generated.
• E. coli strains with higher fitness in seawater, such as
E1140, tends to grow slower but achieve a higher
maximum optical density in the end
• All three strains of E.coli decay at a slower rate in
gluconate supplemented seawater in comparison to
just seawater
• In the presence of D-gluconate, E455L achieved
highest survivability when it is placed in a stressful
condition (decay condition)
• This longer survival time of lysogenic E. coli such as
E455L might be attributed to the increase uptake of
D-gluconate through an upregulation of its
transporter protein