Microbiology Research

1. Future Studies
Hfq is an RNA chaperone capable of binding
both sRNA and mRNA, which in turn effects
the stability and translation of the mRNA
transcript. This regulatory protein is highly
conserved throughout many virulent bacterial
strains. Research suggests that elimination of
the Hfq gene from the bacterial genome
results in a change in the virulence of the
pathogen. Escherichia albertii is a newly
discovered bacterial strain in the same genus
as Escherichia coli. Using BLAST analysis,
the Hfq gene was concluded to be present in
E. albertii. Therefore we hypothesize that the
removal of the Hfq gene from E. albertii will
result in phenotypic changes that will alter the
virulence of the pathogen.
Biochemical assays, such as catalase tests
and double sugar iron agar tests were
performed to determine if Hfq played a role in
the oxidative stress response as well as how
the strain utilizes glucose and lactose.
Further, the wild type and mutant strains
were exposed to varying pH and temperature
levels to determine if Hfq played a role in
these responses, respectively.
The results obtained concluded that under
the given conditions, Hfq does not play a role
in the oxidative stress response, glucose and
lactose utilization, the pH stress response or
in the response to heat shock.
The Role of Hfq Protein in Heat Shock, Acid Stress and Biochemical Processes
in Escherichia albertii
Andrew Arner, Jim Kavulich, Nicholas Bastian
Department of Biology, Saint Joseph’s University, Philadelphia, PA. 19131
Acknowledgements
Mechanism of Action of Hfq
References
Proximal (Top)
Distal (Bottom)
sRNA/mRNA
mRNA/sRNA
sRNA binds mRNA
The binding of sRNA and
mRNA facilitated by Hfq
ultimately affects the
mRNA stability and its
ability to undergo
translation
Wild Type Hfq-
Round 1 Wild Type Hfq-
1 + +
2 + +
Round 2 Wild Type Hfq-
1 + +
2 + +
Note the similar degree of effervescence outlined by the boxes, suggesting
Hfq does not play a role the oxidative stress response
- A catalase assay was run by emulsifying a smear of both Hfq- and the wild type
strains into 60 microliters of sterile water. The reagent was 3% H2O2.
Catalase Assay
- A DSI agar assay was run by stab streaking the same number of
cells on the DSI agar
- The results between the wild type and Hfq- strains suggest that Hfq
does not play a role under these conditions
- Both strains can utilize glucose but not lactose
The red indicates the use of
the peptone with alkaline
products, increasing the pH
Wild Type Hfq-
The yellow indicates glucose
use with acid production,
dropping the pH
Double Sugar Iron Agar Assay
1.00E+07
1.00E+08
1.00E+09
WT 7 Hfq- 7 Wt 5 Hfq- 5
cfu/mL
Strain & pH Condition
pH Stress Response in cfu/mL
WT 7
Hfq- 7
Wt 5
Hfq- 5
- The pH stress response was determined by exposing the same number
of cells of each strain to LB broth at pH 5 for 10 minutes.
- Control strains were exposed to pH 7 for 10 minutes
The Response to an Environment with pH 5
1.00E+08
1.50E+08
2.00E+08
2.50E+08
3.00E+08
3.50E+08
4.00E+08
4.50E+08
5.00E+08
5.50E+08
6.00E+08
Wt 35 Hfq- 35 Wt 50 Hfq- 50
cfu/mL Strain and Temperature in °C
Heat Shock Response in cfu/mL
Wt 35
Hfq- 35
Wt 50
Hfq- 50
- The response to heat was determined by exposing the same number
of cells of each strain to similar conditions for a period of one hour
- 50 °C (Wild type and Hfq-)
- 35 °C (Wild type and Hfq-)
- The bacteria were diluted in sterile saline by a factor of 10-6
The Response to Heat Shock at 50 °C
Heat Shock Results Competitive Index:
(Hfq-50:Wt50) ÷ (Hfq-35:Wt35) = (1.93 x 108/5.08 x 108) ÷ (2.48 x 108/5.63 x 108)
=
0.860
Acid Stress Response pH 5 Competitive Index:
(Hfq-5:Wt5) ÷ (Hfq-7:Wt7) = (7.93 x 107/6.2 x 108) ÷ (1.36 x 108/7.93 x 108) =
0.744
• Dr. Bhatt for his assistance and supplying of reagents for
the assays
Guang, Yang, Wang Ligui, and Wang Yong. "Hfq Regulates Acid
Tolerance and Virulence by Responding to Acid Stress in Shigella
Flexneri." Research in Microbiology 166.6 (2015): 476-85. Web.
Shakhnovich, Elizabeth, Brigid Davis, and Mathew Waldor. "Hfq
Negatively Regulates Type III Secretion in EHEC and Several Other
Pathogens." Molecular Microbiology 74.2 (2009). Web.
• Increase the temperature and alter the length of
incubation in the heat shock assay to better express the
effects of heat on the Hfq- mutant
– Increase temperature to 60 °C
• Alter the pH and the length of exposure to that
environment to better determine how the Hfq- mutant
responds to an acidic environment in relation to the wild
type
– Decrease the pH to 3 and 4
Abstract
Dilutions, Averages and Standard Deviation
Acid Stress Replicates Heat Shock Replicates
NB: The figures depicted are a representation of average
colony counts. For each strain at each condition, two rounds,
each with two replicates, were plated to best represent the cell
count in cfu/mL.
Wt pH7 9.91 x 107
Hfq- pH7 4.95 x 107
Wt pH 5 2.45 x 107
Hfq- pH 5 1.62 x 107
Wt 35 °C 6.5 x 107
Hfq-3 35 °C 6.99 x 107
Wt 50 °C 6.9 x 107
Hfq- 50 °C 2.99 x 107
Condition SD
NB: Any value at or
close to 1 suggests
no role in the
response to that
condition
Summary and Conclusions
- The similar degree of effervescence in all of the
replicates of the catalase test suggests that Hfq does
not play a role in the response to oxidative stress
under these conditions
- The results of the double sugar iron agar test depict
that both the wild type and mutant strains can utilize
glucose, but not lactose. This suggests that Hfq does
not play a role in the utilization of glucose or lactose.
- The comparative index for the heat shock assay
(0.860) and acid stress assay (0.744) suggest that Hfq
does not play a role in responding to either of these
stresses under the given conditions.