Robinson, Tanner

Illuminating novel genes for Escherichia coli growth in varying phosphate conditions using TnSeq and Illumina Sequencing

Faculty Mentor: Dr. William R McCleary, Microbiology and Molecular Biology Department

Introduction

The purpose of my project is to identify novel genes in the genome of Escherichia coli
( E coli) associated with phosphate homeostasis under high phosphate conditions. The
previous efforts that have been employed were great and innovative for their time, but
both lacked the technology currently available, and focused solely on lowphosphate
response. Other labs have used variations of the TnSeq protocol that I have, to
address a variety of questions in many organisms, with great success
(Subashchandrabose et al., 2013).

Methodology

My specific plans have occurred as follows. First, a streptomycin resistant mutant of the
MG1655 strain of E coli was obtained. Next I created a library of ~203,000 mutants by
introducing a Tn5 transposon into this strain (Wiles et al., 2013). I then subjected this
library to growth under three different phosphate conditions; an optimal phosphate
environment (MOPS MedPi), a low phosphate environment (MOPS LoPi), and a high
phosphate environment (MOPS HiPi) for 2030 generations. The idea is that
transposon insertions in genes for phosphate homeostasis will be underrepresented in a
population under a given stress. By sequencing the population of cells which survived a
particular stress (high or low phosphate conditions), I will identify genes with few
insertions in them. These are good candidates for involvement in phosphate
homeostasis. DNA was extracted from each of these libraries, digested and prepared
for sequencing through the addition of specific adapters by PCR.

I submitted these DNA samples for Illumina sequencing. Results were aligned with the
already known gen ome sequence of E coli MG1655. Using this data, we identified
about 20 genes that are potentially important in high phosphate conditions. Since then,
we have obtained knockout mutants for each genes and have been conducting
experiments to confirm the phenotypes. These include overnight growth in optimal
conditions, then diluting and spotting onto plates with varying concentrations of
phosphate. This allows us to see if the phenotype follows what we saw in the TnSeq
data.

Results

Illumina sequencing identified Tn5 insert ions in 4708 genes from E coli MG1655,
between all three libraries. After normalization, 24 of these genes were chosen to
continue working on. These included genes with roles such as phosphoglucomutase,
various phosphatases, and even some y genes. The spotting tests that have been
done on these since that time have yet to yield conclusive results on these genes.
After the prepared libraries had gone through Illumina sequencing, and the genes had
been aligned properly, 24 genes were chosen to continue working with. They were
chosen for one of two reasons; they had a good chance of being related to phosphate
homeostasis, or they were genes known previously to our lab, and could act as good
controls. These were subjected to various tests in order to verify the quantitative
results. The spotting tests are still being worked on right now, because preliminary tests
have been inconclusive.

Discussion and Conclusions

The results of the initial TnSeq experiment have been fruitful. Many genes have been
identified that seem to have a part to play in phosphate homeostasis and response high
and low concentrations. The next step in this project is to continue troubleshooting the
spotting experiment and set up a growth curve experiment. These will establish
whether or not there actually is an advantage the wild type strain has over the knockout
mutants.

The continued exploration of these genes will lead to much greater knowledge about
how phosphate concentrations affect bacterial life, and a more comprehensive
understanding of how E coli is able to respond. Not only that, but the first data showing
genes specifically for high phosphate response have been shown. The continuation of
this project will very likely lead to multiple papers being published in scientific journals.

References

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A.,
Tomita, M., Wanner, B.L., and Mori, H. (2006). Construction of Escherichia coli K12
in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2,
2006.0008.

Subashchandrabose, S., Smith, S.N., Spurbeck, R.R., Kole, M.M., and Mobley, H.L.T.
(2013). Genome-wide detection of fitness genes in uropathogenic Escherichia coli
during systemic infection. PLoS Pathog. 9, e1003788.

Wiles, T.J., Norton, J.P., Russell, C.W., Dalley, B.K., Fischer, K.F., and Mulvey, M.A.
(2013). Combining quantitative genetic footprinting and trait enrichment analysis to
identify fitness determinants of a bacterial pathogen. PLoS Genet. 9, e1003716.