RESEARCH PROJECTS: MICROBES FROM EXTREME ENVIRONMENTS
Species diversity in extreme environments is only limited to microbes. Extreme
environments under investigation in Dr Bharat Patel's laboratory include:
anaerobic environments (eg insect gastrointestinal tracts), thermal
environments (volcanic hot springs, Great Artesian Basin of Australia, oil
fields) and salin environments (oil fields, salt lakes).
Research projects of variable complexities for
the motivated Advanced studies, 3rd, Honours and PhD student(s) are available.
expose students to a variety of modern & conventional microbiological
cover a number of different research fields:
microbial systematics / molecular microbial ecology
microbial genetics / molecular microbiology
microbial biochemistry / molecular microbiology
microbial biotechnology / molecular microbiology
train students in independent thinking in science.
are relevant to the Australian environments, and include diseases that are of
some concern in Australia.
are offered as collaborative projects with Australian collaborators (CSIRO,
Queensland Health, Water Resources) or with overseas collaborators (France,
New Zealand, India).
A few relevant references are listed at the end of each project in order to
provide more information for intending students. A copy of the reference can be
obtained from the library or by contacting Dr Bharat Patel.
Is Corrosion of Bores in the Artesian Basin Due to Thermophilic
Bores are used to tap the ground water of the Artesian Basin. Some of
the bores fail due to corrosion causing economic loss and hardship to
communities which rely solely on these waters for their activities.
Thermophilic sulfate-reducing bacteria (SRB) are thought to be involved in
the corrosion process. The present study will determine the distribution
numbers, types and the ability of thermophilic SRB to corrode bore casing
the Artesian Basin. Such information may aid in designing and/or
identifying metals which could resist corrosion.
Studies on Thermotolerant Bacterial Human Pathogens
Recently thermotolerant bacterial pathogens (eg. some species of
Listeria, Legionella, Campylobacterium) have been increasingly isolated
from human infections and is currently of great concern. Some of these
species are fastidions and difficult to culture whereas others are
difficult to identify when cultured. Rapid methods of identification
will be investigated to solve these problems.
Microbial Ecology of the Artesian Basin Using 16S rRNA
Oligonucleotide Signature Probes
A unique bacterial mat growing at temperatures of 70 - 72 C has been
observed in a run off channel of an Artesian Basin bore. The uniqueness
has been determined by scanning of methanol extracted pigments and by
electron microscopy of the mat. The aim of the project is to isolate the
total 16S rRNA, prepare a 16S rRNA library and probe the library with
known genus-species 16S rRNA probes in order to determine the community
structure of the thermophilic mat.
rRNA Polymorphism in Thermus and Other Thermophilic Bacteria
Thermus is an extremely thermophilic bacterium isolated from
thermally altered (man-made or naturally) waters. Recently their
ubiquitous presence in such environments has been extended and now also
includes the naturally non-volcanically heated waters of the Artesian Basin.
The validation of four species of Thermus based on numerical taxonomy of
phenotypic characteristics is controversial. The project aims to
investigate the potential of using restriction fragment analysis of 16S
and 23S rRNA genes (on the DNA) of Thermus species in order to verify
Characterisation of Novel Thermophilic Aerobic and Anaerobic Bacteria
Numerous thermophilic bacteria, some of which produce thermostable
enzymes with commercial applications, have been isolated from the Australian
Artesian Basin. The aim is to characterise these isolates using
microbiological procedures such as electron microscopy, gas liquid
chromatography, antibiotic sensitivity studies, G+C content and DNA-DNA
hybridisation so that the isolates can be assigned genus and/or species names.
Studies on Thermostable Restriction Enzymes from Thermophilic
Bacteria Useful in PFGE
Pulse Field Gel Electrophoresis (PFGE) is a new and powerful technique
used to separate large DNA molecules up to 9000Kb). For successful PFGE, all
manipulations including cell lysis to DNA purification and restriction enzyme
digestion has to be done in low melting point agarose "plugs". All
manipulations are therefore time consuming requiring long periods of time
for enzymes to diffuse through the agarose matrix to the site of action.
Reaction times can be reduced if higher temperatures are used which allows
faster diffusion. However, most enzymes including restriction enzymes, used
in the manipulations are thermolabile. The aim of the project is to screen,
isolate and purify thermoactive DNA restriction enzymes from thermophilic
bacteria that are useful in PFGE analysis. The possibility of the project
being undertaken jointly with a Brisbane based Biotechnology company with
the aim of commercialising these enzymes exists.
Phylogeny of Thermophilic Bacterial Isolates of the Artesian Basin
Based on 16S rRNA Sequence Analysis
Several aerobic and anaerobic thermophilic bacterial cultures have been
isolated from the Artesian Basin. Based on conventional microbiological
procedures, several novel isolates have been identified. In other cases,
no conclusions regarding their toxonomic status could be determined. It is
the intention of the project to initially use genus and or species specific
16S rRNA probes (made from 16S rRNA sequence data bank) to probe the
Artesian Basin isolates for an indication of taxonomic relatedness. 16S
rRNA libraries followed by sequence analysis will be pursued for taxonomic
unrelated solates in order to determine their phylogeny.
Isolation of Novel Thermophilic Bacteria from the Heated Waters of the
Australian Artesian Basin
Extremely inhospitable environments (thermal, saline) are colonised by
microorganisms. The diversity of microbes from such niches has shed new
light on the diversity of life and evolution. The identification of
archaeobacteria as a taxon distinct from the eubacteria and eukaryotes
highlights the significance of the last ten years work on extreme
environments. Australia too has an extreme environment, in the form of
the heated waters of the Artesian Basin, the microbiology of which has
not been studied. The aim of the project is to isolate biotechnologically
important groups of novel thermophilic bacteria which include CO2 and H2
utilizers, hydrocarbon degraders, and methane oxidisers.
Cloning of Genes of Commercially Important Enzymes from Thermophilic
Bacteria into Mesophilic Host(s)
Thermostable enzymes such as amylases, cycloglucanotransferases (CGTases),
dextranases, xylanases, cellulases) are of great importance in the food
pharmaceutical and paper-pulp industries. Several novel thermophilic bacteria
capable of producing such thermostable enzymes have been isolated from the
Australian Artesian Basin. The aim of the project is to clone genes of some
of these genes into mesophilic hosts (E. coli and/or B. subtilis) in order
to increase enzyme production and allow easy means of purification. A further
aim is to sequence the cloned genes and undertake site-directed mutagenesis
in order to understand the basis of thermostability of these enzymes with
the view of tailoring such enzymes in future to suit the needs of the
In situ Studies of Thermophilic Bacteria of the Great Artesian Basin
Microbes that are present in the waters of the Great Artesian Basin at
temperatures of 99 C can only be grown in the laboratory at 70 C. Is it
that we do not have the appropriate growth conditions for such microbes
under laboratory conditions or are there very high temperature microbes
eluding cultivation? The project aims to use in situ techniques (eg.
electron microscopy, radio isotope uptake) to obtain an answer to this puzzle.
Bacteriophage of Thermus aquaticus Strains
Thermophilic phage are known to exist in hot springs. Studies of these
phages can open new systems in molecular biology (eg. host-vector systems
for cloning) and will also allow phage typing to be done. The aim of the
project is to isolate and study phage active against Thermus strains.
The Ribosomal Gene Spacer Region(s) in Thermophilic Bacteria
The ribosomal gene spacer region in thermophilic bacteria: The order
of ribosomal genes in bacterial genome is 16S-23S-5S. The regions between
the genes is known as intergenic spacer (IGS) region. Some differences in
IGS has been observed in the mesophilic bacteria and Archael organisms
studied to date. For example, tRNAs from the IGS in Ecoli but not in
Streptomyces and Mycobacterium species indicating what is a typical feature
of the filamentous bacteria. The absence or presence of tRNAs in the IGS
region is also group related in Archeal organisms. The aim of the project
is to identify thermophilic bacteria which contain the tRNA in the IGS region.
This will be achieved using the technique of PCR, followed by cloning and
sequencing of the IGS region. Primers have already been synthesised and
tested for PCR amplification.
Thermostable Restriction Enzymes from Thermophilic Bacteria
With a few exceptions, the commercially produced restriction enzymes
used in molecular biology techniques, are thermolabile. There has always
been a niche for thermostable restriction enzymes but these have not been
investigated for several reasons. We have developed in our laboratory a
rapid protocol for screening of restriction enzymes from the thermophilic
aerobes, Thermus. The aim of the project is to investigate the types of
restriction enzymes present in this group followed by purification of a
few suitable enzymes.
Studies on Recombinant Amylase/Pullnlanase Enzymes from Thermoanaerobacter
A genomic library from the thermoanaerobe Thermoanaerobacter strain
AB10 has been prepared and three genes producing amylase/pullulanase
enzymes identified based on restriction enzyme mapping and Southern Blot
analysis. The aim of the project is to characterise the recombinant enzymes.
Studies will involve enzyme purification by novel methods (eg. heat
denaturation of host E.coli proteins) and enzyme assays.
Pulse-Field Gel Electrophoresis of DNA from Thermoanaerobic Bacteria
Alternating field gel electrophoresis (AFGE) [also known as Pulse-field
gel electrophoresis (PFGE)] is a powerful technique used to separate large
DNA molecules (up to 9,000kb). We currently use Contour-clamped Homogeneous
Electric Field (CHEF) electrophoresis, which is one of nine instruments
which uses the principle of AFGE, to separate large DNA fragments from
thermophilic anaerobic bacteria. Of these, an isolate designated
Thermoanaerobium strain AB10, for which a genomic library and recombinants
containing amylase and pullulanase gene(s) have been identified, is of
considerable interest. The aim of the project is to calculate the genome
size and construct a crude map of the genome of Thermoanaerobacter strain
AB10 using CHEF and other molecular biology techniques.
Molecular Biology of Fervidobacterium nodosum
F.nodosum, an extremely thermophilic anaerobic eubacterium (optimum
temp = 70 C), for first isolated from a New Zealand hot spring and more
recently from the Australian Artesian aquifer, is of considerable interest.
It is (i) one of the most ancient bacterium known based on 16S rRNA phylogeny,
(ii) produces unique morphological features and (iii) appears to possess
unique enzymes. Current research in our laboratory has focussed on its
ecology (using 16S rRNA oligonnucleotide probes) and amylolytic enzymes.
Studies on the Natural Microbial Communities of the Hot Waters of the
Great Artesian Basin (GAB)
Thermophilic bacteria (Thermus species, Glostridium thermohydro sulfurium,
Fervidobacterium species, Bacillus sp and other taxonomically unknown species)
have been isolated from the heated waters of the Great Artesian Basin (GAB).
However, it is generally accepted that only 0.1% to 1% of microorganisms from
any environment can be cultured in the laboratory meaning that approximately
99% of the organisms are never studied. We wish to study microbial
communities of GAB using novel methods (eg. in situ electron microscopy
techniques, ultrafilteration technique to determine phospholipid fatty acid
profiles and DNA concentrations).
Studies on Plasmids from Thermus Species
34 thermus strains have been isolated from the non-volcanically naturally
heated waters of the Australian Artesian Basin. A simple and rapid method
developed for the isolation of plasmids had indicated that considerable
variation in numbers and molecular sizes existed within the 23 strains that
were investigated. This type of plasmid variation may be similar to the
phenotypic variation observed amongst volcanic Thermus strains. It has been
suggested that the extraordinary degree of spontaneous phenotypic variability
in thermophiles may be a genetic property of organisms that are exposed to an
extreme environmental stress not experienced by mesophilic organisms. For
example, loss of plasmids (eg. due to temperature fluctuations) may lead to
the evolution of new phenotypes through loss of plasmid encoded functions such
as thermostability. The aim of the project is to characterise plasmids from
thermus strains in order that the role of plasmids in Thermus can be
undertaken in future, eg. developing vector systems, distribution of
plasmid encoded genes in Thermus chromosomes.
Cryptic Plasmids of Thermus Aquaticus Strains
Recent evidence suggests that a single common plasmid found in various
bacterial species of polluted or oligotrophic environments, produced "survival"
proteins which enabled these species to inhabit such extreme environments.
We have studied plasmids from Thermus aquaticus strains isolated from an
extreme environment viz the thermally heated waters of the Great Artesian
Basin (GAB) but found no correlation between plasmid size and/or numbers.
Studies to determine whether there are common nucleotide sequences in all the
T. aquaticus plasmids, which many produce "survival" proteins that confer
these bacteria the ability to inhabit such a harsh environment, will be
undertaken. The common nucleotide sequence could be used as a probe to
determine a plasmid function(s).
Studies on thermostable Xylanase
Xylanases with particular characteristics are required for the processing
of wheat by-products (a waste product in the form of wheat bran) for making
poultry and pig feed. Wheat bran is currently prohibited from being used as
a feed grain due to poor digestibility and nutrient absorption problems
encountered in the pig and chicken gut. However, if the wheat bran were to
be treated with xylanases the digestibility and absorption could be improved.
The aim of the project is to identify a sylanase which has the following
properties: (i) as the xylanase has to be incorporated into feed grain,
blended in with other ingredients and formed into pellets using a pellet
press at 92 C for at least one minute under 120 kpa pressure, the enzyme has
to be stable to these conditions. These conditions are also critical as they
enable the starch to these conditions. These conditions are also critical as
they enable the starch in the grain to swell for improved digestion or
ingestion, and also enables the elimination of Salmonella typhimurium, an
extremely infective disease of poultry and pigs, (ii) the xylanase will
need to have an optimum activity of around 40 C at pH 6.0 as these are
normal physiological conditions inside the avian and pig gut. This enzyme
would be of importance to the wheat industry.
Characterisation of Novel Thermophilic Aerobic and Anaerobic Bacteria
Numerous thermophilic bacteria, some of which produce thermostable enzymes
with commercial applications, have been isolated from the Australian Artesian
Basin. The aim is to characterise these isolates using microbiological
procedures such as electron microscopy, gas liquid chromatography, antibiotic
sensitivity studies, G+C content and DNA-DNA hybridisation so that the
isolates can be assigned genus and/or species names.
Molecular Analysis of Polysaccharide Hydrolase Genes from Anaerobic
Micro-organisms (with G. Xue, CSIRO)
Gene libraries have been constructed from an anaerobic fungus and an
anaerobic bacterium. A number of genes encoding polysaccharide hydrolases,
such as cellulases and xylanases, have been isolated from the gene libraries.
This project aims to analyse DNA sequences of polysaccharide hydrolase-encoding
genes and to elucidate the structure and function of the enzymes.
Thermostable DNA Polymerases
Thermostable DNA polymerases are used in the amplification, and
sequencing of DNA. Such polymerases can be obtained from thermophilic
microbes. The project aim is to characterise a thermostable DNA polymerase
from a unique thermophilic anaerobic bacterium and compare its properties
with that of other commercially available ones.
Publications and references to research undertaken in my laboratory can
be seen at the URL trishul.sci.gu.edu.au/~bharat/cv/publications
Please contact Dr. Bharat Patel if further details are required
[Created: 13 Sept 1995]
[Modified: 16 Sept 1999]