Tate describes
his Australian research experience:
This summer, I conducted a metabolomic study of Mycobacterium
physiology in the laboratory of Dr. Malcolm McConville, an HHMI
International Research Scholar at University
of Melbourne. Working at the University
of Melbourne was an entirely
different experience than my lab work at Kenyon
College.
Although Kenyon is well equipped for a liberal arts college, I was absolutely
amazed at the vast array of analytical equipment available to researchers
at Melbourne Uni. This makes sense when you consider that the
Biochemistry Department alone is composed of many labs with ten plus
PhD students and postdoctorate researchers.
My lab, that of McConville, numbered somewhere in the teens,
with most members of the lab having many years of experience beyond
my education. There were two undergraduates, but they were
honors students, two of the best biochemists in Australia. In Australia
an undergraduate degree consists of three years, with an optional fourth
year to complete honors courses and an intensive research project.
Working with such knowledgeable researchers was an immense privilege,
as there seemed to be no question that someone in the lab could not
answer, nor any problem too difficult to solve.
The drive and dedication of the lab led to many discoveries even
in the few short months that I spent there.
One student completed his PhD thesis, almost missing a lunch
in his honor, as he was already starting postgraduate research projects.
Life in Australia
was also a new experience for me. Melbourne
became a boom town in the mid 1800s after gold was found in the rugged
hills to the northwest. The richness
of that era can still be seen in Melbourne
today, especially in the well preserved terrace houses of the inner
suburbs. The present day city
centers on the banks of the Yarra
River. From Federation square, a ten minute tram ride
up Swanston Street
to Grattan Street
and you are at the University
of Melbourne in the suburb
of Parkville. Once off the tram you can walk east a few blocks
to Lygon Street
in Carlton. The street is known as the little Italy of Melbourne
and features an amazing array of restaurants, cafes, and boutique shopping.
Walking two blocks west on Grattan Street
leads you the Graduate House, where I stayed during my time in Melbourne.
The Graduate House houses approximately 40 of the world’s most brilliant
scholars. The great majority are attending business, law,
or medical school at Melbourne Uni. There I met postdocs and other researchers from
Ireland,
Spain,
Germany,
and Switzerland. Those housemates worked at either the Howard
Florey Institute, or theWalter and Eliza Hall Institute for Medical
Research; each a short walk from the Departments of Biochemistry and
Molecular Biology, and Microbiology and Immunology where I completed
my research.
The Helen Billman Jacobe lab in Microbiology provided me with strains
of Mycobacterium smegmatis, cultures of which
were analyzed using instrumentation in McConville’s lab. M. smegmatis was chosen for my experiments
since it serves as a model organism for M. tuberculosis. Studying the
early stationary phase can lead to clues of how to fight tuberculosis,
as the pathogen stays suspended in the macrophage before turning into
a full on infection.
M. smegmatis is a rod-shaped and gram positive
bacteria. It is non-pathogenic
but has been known to cause soft tissue and bone infections in rare
cases. In addition to being safer than M. tuberculosis, M. smegmatis grows approximately twice as fast, able to utilize glycerol
as the sole carbon source. The
most unique characteristic of M.
smegmatis is its thick, lipid rich, and hydrophobic cell wall. The cell wall is nearly impermeable to polar
molecules, with excellent resistance to acids and bases.
Aerobic cultures of M.
smegmatis were inoculated with frozen seeders at early stationary
phase in Middlebrook 7H9 media with added sodium chloride and dextrose.
Cultures were snap frozen at mid-log phase(ML) and early stationary
phase(ES). Cell extractions were performed with hot ethanol
and prepared for metabolomic analysis by gas chromatography-time of
flight mass spectrometry(GC-TOF MS).
This system runs the sample through a gas chromatographer linked
directly to an extremely powerful and advanced mass spectrometer. Workstation software analyzes the sample runs
creating chromatographs from the GC and a mass spec peak breakdown.
The main purpose of my experiments was to gather whether or not
GC-TOF MS could be used to detect differences between cells grown at
different conditions, or to different densities.
This was proven to be successful as wild type M.
smegmatis(WT) showed a large increase in numerous fermentation acids
and sugars when comparing the concentrations at ES to ML.
The next course of action was to grow mutant strains and see if the
GC-TOF MS system would be able to detect differences falling in line
with expectations. Many of the
mutant strains for M. smegmatis
are lethal, making those cell lines difficult to work with. A PhoR mutant, M. smegmatis 31 was chosen as a model organism due to its stability
and well defined characteristics. PhoR
is part of a two-component signal transduction system with PhoP. PhoR senses low Pi at the outer cell membrane
surface or within the cytoplasm, activating PhoP, a partner response
regulator. PhoP activation turns on gene expression in
the Pho regulon. This in turn
causes the production of PhoA alkaline phosphatase, catalyzing the hydrolysis
of organic and inorganic phosphate monoesters to release Pi. M. smegmatis
31(M31) is known to be glycopeptidolipid(GPL)
deficient, so a known GPL mutant, M.
smegmatis 22(M22) was used as a control for effects from missing
GPLs. As M.
smegmatis was not grown on phosphate regulated media, the experiments
were used only to compare differences between metabolites in cell extracts
of M31, M22 and WT; and not to investigate phosphate levels. The results of my studies on early stationary
phase of WT, M22, and M31 are in the tables and graph below.
Associated with
GPLs Lost in PhoR Mutant Gained in PhoR Mutant
|
D-Alloisoleucine |
Isoleucine |
Cyclopropane
derivative |
| Allonic acid |
Pyridinecarboxylic
acid |
Cadaverine |
| Butanal |
Piperidinecarboxylic
acid |
D-Arabino-Hexonicacid(gamma
Lactone) |
| Ribose |
Bis(2-furfuryl)disulfide |
| Xylitol |
Ornithine |
| Mannose |
Fructose |
| Mannose-6-Phosphate |
Thymol-a-d-glucopyranoside |
| Mannonic acid |
Guaicol-a-d-glucopyranoside |
| 5,6-Dioxoheptanoic
acid |
The GC-TOF MS system is a very complicated analytical
instrument, and I am grateful to the honors and PhD students who were
willing to let me watch and assist them in setting up the brand new
system. This involved standardizing
the system, finding the right column, and tweaking multitudes of settings
for maximal sample analysis. Due to their hard work my samples ran through
smoothly with excellent results.
I also spent much time exploring Melbourne
and the surrounding areas. I
traveled down the Great Ocean Road
which has been compared to the Pacific Coast
Highway in California. A different weekend I went to Phillips
Island and the Nobbies. I also took in the Dadenong range, the Yarra
Valley and Healsville Sanctuary,
and the historic mining town of Ballarat. In metro Melbourne
I spent lots of time wandering around the city; visiting parks, gardens,
monuments, and strolling along the Yarra
River. I was also able to sample a wide variety of
renowned international and local cuisine, shop at one of the world’s
largest markets, and even became a fan of Australian Rules Football. I’m thinking of returning to Melbourne for graduate
school.
|
