Transcript Escherichia coli - Biology - University of Central Oklahoma

Refined Description of a Gene Locus in Escherichia coli Encoding for Bile Salt
Resistance and Sensitivity
Eden Bernstein, Anna Graves, Jonna Whetsel, and James Bidlack
Department of Biology, University of Central Oklahoma, Edmond, OK 73034
Abstract
This project is a continued investigation of a chromosomal mutation that causes
bile salt sensitivity in Escherichia coli. Previous attempts to narrow down the
location of the mutation suggest that it affects the yciS and yciM genes. Our new
focus is to conclusively demonstrate that the mutation confers bile salt sensitivity
using several strains of E. coli, including the wild type and mutant strains of the
bacterium as well as two genetically-engineered strains that lack the yciS and
yciM genes. Primers for the yciS and yciM genes have been used to amplify DNA
segments from the wild type and mutant strains of E. coli and we are now in the
process of cloning and sequencing target DNA. Once cloned, genes for bile salt
resistance and sensitivity will be inserted into counterpart bacterial strains to
determine if resistant strains can be made sensitive to bile salts and if sensitive
strains can be made resistant to bile salts. Results will provide details for a new
gene locus description that includes the location, sequence, and phenotype for bile
salt resistance and sensitivity in E. coli.
Introduction
Multi-drug resistant bacteria are a prominent concern among the scientific and
medical community. Drug resistance in bacteria is known to proliferate through
horizontal gene transfer. Genes conferring resistance through a variety of
mechanisms (including efflux pumps among others) may be encoded in plasmids
and transferred among cells through conjugation mediated by a pillus.
In 2002, a strain of Escherichia coli displayed impaired growth on a bile salt agar
plate, suggesting a bile salt sensitive phenotype distinct from other Gram-negative
bacteria. Bile salt sensitivity in E. coli (Strain JC3272I) was initially thought to be
associated with a type IV secretion system coded by the F plasmid, conferring
sensitivity to F+ strains (Bidlack and Silverman, 2004). It was later discovered
that the sensitivity was the result of a chromosomal mutation involving the genes
yciS and yciM, and not coded by the F plasmid (unpublished results). yciS is a
conserved inner membrane protein and yciM is a putative heat shock protein. The
mechanism of sensitivity is not yet known. Knock out experiments of these genes
have demonstrated a correlation with not only bile salt, but antibiotic sensitivity
(Baba et al., 2004). Hence, the biomedical implications of investigating the
phenotypic properties of the mutation through gene transfer include possible
insight to methods of antibiotic resistance. This preliminary study will
expectantly serve as the foundation for future clinical trials that will apply these
concepts to the betterment of human lives.
Methods
Cultures: Five E. coli strains were utilized in this experiment: BW25113, JW1271,
JW1272, JC3272F, and JC3272I. They were stored in a -80˚ freezer and streaked on
LB agar plates (Figure 1).
DNA Extraction: Using an inoculating loop, randomly selected colonies were
placed into microcentrifuge tubes containing 100 µL of deionized H2O.
PCR mixture: PCR mixture included: DI H2O, Mg2+, Primers, Extracted DNA,
Taq polymerase, and dNTP’s.
Figure 6: E. coli growth on 1.5% bile
salt LB plates
Thermal Cycler: A pre-set program was used on the Thermo PCR Sprint Thermal
Cycler to amplify the targeted locus (Figure 2).
Results and Discussion
SDS Polyacrylamide Electrophoresis: A gel was prepared using a mixture of 1.5 g
agarose and 150 mL of TE Buffer. This mixture was heated until clear, with 5 µL of
EtBr then added. Carolina blue loading dye was added to the amplified PCR
samples for electrophoresis. The gel and amplified PCR mixtures were ran on a
FisherBiotech Model H5 Horizontal Gel Electrophoresis System.
Gel Imaging: The Kodak Gel Logic 100 gel imager was used to identify and
document the results from electrophoresis.
Bile salt plate: 15 grams of bile salt were mixed with 40 grams of LB agar and
1000 mL DI H2O to form bile salt plates. 2 µL of various dilutions of the colonies
were plated onto respective sections.
The PCR reactions must result in an abundant quantity of DNA products such that
the amplified DNA from a single isolated colony is sufficient for several
procedures. These include gel electrophoresis for verification of the desired 1.8
Kbp length, sequencing, and cloning through pUC19 (plasmid). Thus, PCR
techniques are currently being refined to allow for optimal results. Previous trials
(Figure 3) have not yielded the desired quantity of products. We are planning to run
bulk PCR reactions involving several reactions from each individual colony to
circumvent this issue.
Other members of the group are investigating the growth of the strains in LB agar
plates and 1.5% bile salt LB agar plates (Figures 5 and 6). The appropriate plasmid
and restriction enzymes have already been designated and ordered, such that the
PCR products can be cloned once the new PCR procedures are implemented and
the PCR products are verified.
Future research could investigate the specific mechanism which confers the mutated
strain sensitivity to bile salt. Furthermore, a continuation of this project could
consider alternative phenotypic properties of the mutation, such as results of
exposure to other substrates, including antibiotics.
Literature Cited
Figure 1: Anna streaking E. coli on LB agar
Figure 3: Gel amplification at the
desired gene locus
Acknowledgements
Funding for this project was provided by the University of Central Oklahoma
CURE-STEM program and Office of Research & Grants. We thank Dr. Philip
Silverman of the Oklahoma Medical Research Foundation for his research and
guidance necessary for the current progress of this experiment. We also thank our
Research Group for much needed assistance and support (Figure 4).
Figure 5: E. coli growth on LB agar
Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko,
M. Tomita, B. Wanner, and H. Mori. 2006. Construction of Escherichia coli K-12
in-frame, single-gene knockout mutants: the Keio collection. Molec. Syst. Biol.
2:2006.0008.
Bidlack, J.E., and P.M. Silverman. 2004. An active type IV secretion system
encoded by the F plasmid sensitizes Escherichia coli to bile salts. Journal of
Bacteriology 186:5202-5209.
Figure 2: Jonna loading samples into
thermal cycler
Figure 4: Research group