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. The projects

expose students to a variety of modern & conventional microbiological techniques.

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 Bacteria?
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 speciation.

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 relevant industry.

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 Strain AB10
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 <B.Patel@griffith.edu.au>
[Created: 13 Sept 1995]
[Modified: 16 Sept 1999]