,

TERM PAPER
OF
MICROBIAL PHYSIOLOGY AND METABOLISM
BTY 538
TOPIC: THERMUS aquaticus

SUBMITTED BY:

SHASHI SHARMA

R.NO. P8003B15

REG. NO.-11006142

MSc. Microbiology

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 CONTENTS

 INTRODUCTION
 INDUSTRIAL IMPORTANCE
 NEW RESEARCHES
 FUTURE PROSPECTIVES
 REFERENCES

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INTRODUCTION
Here given introduction by me regarding Thermus aquaticus states the objective for selecting this
microorganism for my term paper work. As Thermus aquaticus is a species of bacterium that
can tolerate high temperatures, one of several thermophilic bacteria that belong to the
Deinococcus-Thermus group. It is the source of the heat-resistant enzyme Taq DNA Polymerase,
one of the most important enzymes in molecular biology because of its use in the polymerase
chain reaction (PCR) DNA amplification technique.

Thermus aquaticus

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 INDUSTRIAL IMPORTANCE

Recently, thermostable polymerases have become important, e.g. Taq DNA

polymerase from Thermus aquaticus. This bacterium has evolved to grow in hot
springs at temperatures which kill most other species. These enzymes allow the
amplification of as little as one molecule of DNA into a large amount by means of
repeated cycles of melting, primer annealing & extension by the enzyme which is
not destroyed by the high temperatures used in this process. This is known as the

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polymerase chain reaction:

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Thermus aquaticus is used in biotechnology to make Taq polymerase, a key constituent of a
process called polymerase chain reaction (PCR). PCR is the process of copying DNA and
amplifying it. PCR uses an enzyme, DNA polymerase, which is active at high temperatures. Thus,
the DNA polymerase of Thermus aquaticus, known as Taq polymerase, is the one used for this
process. The PCR process consists of several rounds of heating the enzyme, each round
doubling the DNA molecules. Taq polymerase is not destroyed in the heating but continues to
work. Thus, Taq polymerase finds wide use in medical diagnosis such as AIDS, and forensics
such as DNA fingerprinting.

Non-homologous recombination mediated by Thermus

aquaticus DNA polymerase I.

RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite
orientations of exon 6, resulted in PCR products containing segments of exons
joined at non-consensus splice sites. Moreover, many of the PCR products
identified were composed of not only a single region containing exonic
segments joined at non-consensus splice sites but, instead, of several repeats
of the non-canonically joined region. To investigate whether these PCR
products represent pre-existing molecules or are generated during the
amplification process, the liver cDNA template was replaced by a plasmid
containing the P450 2C6 cDNA. Surprisingly, PCR products containing repeats
of non-canonically joined exonic segments were again revealed. In some cases
the position of this non-canonical joining was a sequence of one or two
identical nucleotides; however, there were also a number of products lacking
any nucleotide identity at the position of joining. DNA nicking and/or DNA
damage is thought to favour recombination during PCR, probably by
misalignment of incomplete DNA strands; however, the presence of multiple
repeats of the recombined region in the PCR products identified suggests a
certain repetitiveness of the underlying mechanism. It is therefore proposed
that these products result from a template switching event that occurs several
times during a single polymerization step, following a rolling circle model of
DNA synthesis.

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Recombinant Thermus aquaticus RNA polymerase, a new tool for
structure-based analysis of transcription,is another latest research
regarding Thermus aquaticus:

The three-dimensional structure of DNA-dependent RNA polymerase (RNAP) from

thermophilic Thermus aquaticus has recently been determined a. A resolution.
Currently, very little is known about T. aquaticus transcription and no genetic system to
study T. aquaticus RNAP genes is available. To overcome these limitations, we cloned
and over expressed T. aquaticus RNAP genes in Escherichia coli. Overproduced T.
aquaticus RNAP subunits assembled into functional RNAP in vitro and in vivo when co
expressed in E. coli. We used the recombinant T. aquaticus enzyme to demonstrate
that transcription initiation, transcription termination, and transcription cleavage assays
developed for E. coli RNAP can be adapted to study T. aquaticus transcription.
However, T. aquaticus RNAP differs from the prototypical E. coli enzyme in several
important ways: it terminates transcription less efficiently, has exceptionally high rate of
intrinsic transcript cleavage, and is highly resistant to rifampin. Our results, together with
the high-resolution structural information, should now allow a rational analysis of
transcription mechanism by mutation.

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of genomic sequence can be amplified by a factor of more than 10 million(One of DNA
template up to 35 kb could be amplified from a target DNA molecule present only once
in a sample of 105 cells) with high base pair fidelity. This enzyme was soon cloned,
sequenced, and produced in mass quantities for commercial sale. ) Other enzymes with
high optimal temperatures allowing researchers to study them in extreme conditions
are:DNA ligase, alkaline phosphatase, NADH oxidase, isocitrase, dehydrogenase,
amylomaltase and fructose1,6-bisphosphate-dependent L-lactate dehydrogenase.

Besides the revolutionary changes in PCR, ligase chain reaction (LCR), which uses T.
aquaticus ligase, can amplify genetic sequences of stretches of DNA that posses a
desired sequence million or more times within hours. It can amplify and screen in a
single step and screen for mutations simultaneously. LCR is useful in testing for
hereditary diseases, revealing hidden infections and distinguishing between drug
resistant and drug sensitive strains of viruses and bacteria.

Current Research
"THE GENOMOCS OF DISULFIDE BONDING AND PROTEIN
STABILIZATION IN THERMOPHILES"
Recent studies have emphasized the role of disulfide bonds in stabilizing the structure
of intracellular proteins of Thermus aquaticus among some other thermophiles.
Previously the popular belief was disulfide bonds are only present in extracellular
proteins where they stabilize folded proteins against harsh conditions and are rarely
found in the cytosol. The specific protein which seems to be responsible for the
formation of intracellular disulfide bonds seems to be protein disulfide oxido -reductase ,
which functions as a cytoplasmic protein disulfide-isomerase . It has been suggested
that eukaryotic PDI, found in the endoplasmic reticulum where it catalyzes isomerization
of protein disulfide bonds, has evolved from a protein similar to thermophilic PDO. More
research needs to be done on this subject.

Besides thermophiles, elevated intracellular disulfide bonding has been seen in other
extremophiles including; halophiles, alkylophiles, acidophiles, and radiation-tolerant
organisms. This discovery supports the role of intracellular disulfide bonds in stabilizing
proteins in all types of extreme conditions. This study sheds some light on different
`methods used by organisms to stabilize their proteins to adapt to "exotic"
environments. After isolation of Thermus aquaticus, samples of it were deposited in the
American Type Cultures Collection (ATCC), a public repository. Other s cientists had
access to them

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. This experiment was repeated with Taq polymerases from two different commercial sources.
PCR was performed against deionized water sample with or without UV treatment,which ha
sbeen shown to reduce false positive signals). And the clone 3A, 3B and 7C DNA sequences
were added into each group of reaction tubes to serve as positive control, no exogenous DNA
template were added to any of the tubes. Hundred mM tris-HCL buffer with a pH 8.3200 micro
molar deoxynucleoside triphosphates, 2.5mM MgCl2 25pmol of each primer and 1.25 Uof Taq
polymerase and were able to do more research. By the 1980's, it became obvious that
the potential for commercializing the enzymes from this species would prove to be very
high and profitable.

"Presence of Bacterial Phage-Like DNA Sequences in Commercial Taq DNA

Polymerase Reagents, Tamara Newsome,1 Bing-Jie Li,1 Nianxiang Zou,1 and Shyh-
Ching Lo2(2004)"

In a routine study of amplifying the highly conserved gene sequence of the 16S rRNA,
several DNA bands were produced that were different from the expected target sizes.
Characteristics of these PCR products and further PCR study revealed they were
amplified from commercial Taq DNA Polymerase reagents, apparently contaminated
with trace amounts of bacteriophage like dnase. A study USING 16s rrna gene primer
sA, a number of unexpected products with sizes ranging from 100bp more than 20 kb,
in addition to expected 1.5KB. Site products or secondary products are not uncommon,
however this time 3 PCR products, 3a, 3b, 7c, were further studied. They were gel
purified, cloned, sequenced and compared to sequences in genbank database. A small
portion of DNA sequence on 7C, showed 81% homology to tail fiber gene of
pesudemona phage, GH-1 and 88% homology to tail fibers proteins of enterobacterial
phage T7. 3A and 3B showed partial homology to tail tubular protein B(72%) and
putative DNA ligase (55%) of pseudomonas phage GA-1. To confirm findings, four
different primers were designed from phage like DNA sequences of these 3 bands and
were studied by PCR to detect phage like DNA. The PCR band found were of expected
sizes, so all PCR products were confirmed to be phage like DNA sequences by
nucleotide sequencing

Yogurt, a Source of Probiotics

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Though not usually thought of together, both hot springs and yogurt are a source of
bacteria beneficial to humans. In the case of yogurt, these bacteria are  probiotics, also
called “good bacteria.” The National Center for Complementary and Alternative
Medicine (NCCAM) defines probiotics as “live microorganisms, which, when
administered in adequate amounts, confer a health benefit on the host” Probiotics are
similar to the bacteria found in one’s stomach and intestine and may, for the most part,
be classified into two genera, or taxonomic groups:  Lactobacillius and
Bifidobacterium.  Yeasts are another group of microorganisms included in probiotics.

That probiotics are of interest for human health should come as little surprise: the world
is covered with microorganisms, and so are we. Our skin and intestines have a normal
aide in food digestion. For example, the bacterium Bifidobacterium eriksonii, seen
through a microscope in the image below from the Centers generally cause disease
complement of microorganims which protect us against invading microorganisms for
Disease Control and Prevention, lives in the oral cavity and does not and.

Furthermore, each individual’s complement of microorganisms is unique and can be

disrupted by invading microorganisms or medication. Food poisoning is an example of this
situation. The common side effects of antibiotics provide another example: while being used
to treat infections caused by invading microorganisms, antibiotics can also kill normal
microorganisms in the digestive tract, causing nausea and diarrhea. Given that the
interactions among the various microorganisms have implications for human health, the

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ability to beneficially influence these interactions through probiotics would provide another
avenue for disease treatment.

A joint conference between NCCAM and the American Microbiology Society found evidence
supporting the use of probiotics as treatments for diarrhea and irritable bowell syndrome,
among other ailments. However, when studying probiotics as cures for ailments such as
these, the conference found low benefits and a strong placebo effect. Therefore, they
encouraged larger, controlled trials before any conclusive benefits be ascribed to probiotics.
To support such efforts, NCCAM funds research on the role of probiotics in disease and
normal human physiology. For instance, researchers at Virginia Polytechnic Institute are
studying how probiotics affect the human immune system 

Thermus Aquaticus role in human welfare

While the potential benefits of probiotics are straightforward, the benefit gained from a
certain bacterium found in hot springs only became apparent after years of research.
Biomedical researchers today employ a very useful and versatile technique called
polymerase chain reaction, abbreviated as PCR. Researchers use PCR to amplify, or make
copies of, an original portion of DNA. Developed in the 1980s by Kary Mullis, PCR has
dramatically altered the way scientists perform molecular biology. Initially, Mullis used an
enzyme (or biological catalyst) called polymerase. However, PCR requires repeated
incubations at near-boiling temperatures which destroy the polymerase enzyme. Therefore,
Mullis needed to add more polymerase enzyme after each incubation, making the technique
expensive and labor intensive.

The widespread use of PCR for research, disease diagnosis, and forensic analysis was made
possible by a bacterium, Thermus aquaticus, isolated from Octopus Hot Spring (see photo
from the National Parks Service, below) in the Yellowstone Basin.Isolating its heat-stable
polymerase, named Taq polymerase, allowed researchers to explain T. aquaticus’ ability to
survive its high-temperature native environment. Recognizing that Taq polymerase could
withstand the heat of the PCR’s repeated incubations, Mullis tried substituting it for the
original polymerase enzyme. As hoped, Taq polymerase only needed to be added at the
beginning of PCR, decreasing the time and cost of the procedure. For his invention of PCR,
Mullis won the Nobel Prize in Chemistry in 1993. In the years following the invention of
PCR, scientists have established many variations tailored to specific DNA targets and

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biomedical applications, making PCR a prime example of biotechnology. However, this
technique is not just useful for researchers. As mentioned above, PCR is used for disease
diagnosis.

While the utility of T. aquaticus and of the biotechnology stemming from PCR are well
characterized, researchers continue to discover more hot spring bacteria, opening the door
to the development of more bacteria-based biotechnology. For instance, researchers
recently described a new bacterium, Chloracidobacterium thermophilum, isolated from
Yellowstone’s hot springs. Cab. Thermophilum, as it is abbreviated, is the only member of
phylum Acidobacteria known to undergo photosynthesis, making sugar, used as food, from
sunlight and carbon dioxide. However, the potential uses of Cab. thermophilum for human
benefit remain unknown.

Though the potential to derive biomedical benefits from bacteria—whether probiotics, Cab.
Thermophilum, or others—is high, more research into the biological function and
environmental interactions of these bacteria is needed to understand their role in human
health and medicine. company B. Since company also provided MgCl2 solutions and buffers,
they were also tested by swapping them with other samples of buffer and MgCl2. Still same
results were observed. Presence of DNA contamination in Taq Polymerases were recorded
before but most were exogenous bacterial DNA. This study is the first to show bacteriophage
like DNA present. Scientist and Researchers were made aware of these findings. Using tainted
amplification reagents can lead to misleading and confusing results.

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 FUTURE ASPECTS OF THERMUS aquaticus
Potentials and limitations of molecular diagnostic methods
in food safety
Molecular methods allow the detection of pathogen nucleic acids (DNA and RNA) and therefore,
the detection of contamination in food is carried out with high selectivity and rapidity.In the last
2 decades molecular methods have accompanied traditional diagnosticin routine pathogen
detection, and might replace them in the upcoming future. The implementation indiagnostics of
four of the most used molecular techniques(PCR, NASBA, microarray, LDR) are described and
com-pared, highlighting advantages and limitations of each of them. Drawbacks of molecular
methods with regard to traditional ones and the difficulties encountered in pathogen detection
from food or clinical specimen are also stated Moreover, criteria for the choice of the target
sequence for a secure detection and classification of pathogens and possible developments in
molecular diagnostics are also proposed. Molecular biology Á Food safety Á Diagnostic
ÁPCRÁ Microarray Á NASBAÁ LDR. Zoonoses and food borne pathogens are health
threatening agents that cause disease and death in humans, even in developed countries. In
Europe as well as in the USA,pathogens such as Listeria, Escherichia
coli,Salmonella,Campylobacterand Mycobacterium bovisare widespreadand are occasionally the
cause of disease outbreaks .Traditional diagnostic methods identify a pathogen based on its
phenotype: e.g. classification according to the ability to grow on a certain media, to metabolize a
given chemical compound, etc. The exact classification of a serotype is achieved with the use of
antibodies, generally directed against membrane proteins, or with serotype specific
bacteriophages. The correct assessment of a clinical isolate cantake 2–3 days or longer.
Therefore, the development ofrapid and secure methods to detect and trace the origin
ofpathogens and contaminants is urgently needed . Faster and simpler methods would be a great
advantage for many diagnostic purposes. Food safety could be greatly enhanced
by the use of fast diagnostic methods allowing the immediate detection of pathogens Fast
diagnostic methods include those based on the recognition and amplification of nucleic acids. As
the same detection technique can be applied to identify nucleic acids from all organisms, the
same strategies can be used in clinical diagnosis as for the detection of food-borne pathogens
and GMOs. Methods for the amplification and detection of very small quantities of nucleic acids
have been available for many years, but only in the last 10–15 years have been employed in
diagnostics. Furthermore,in the last decade the amount of nucleic acid sequence data available
for many organisms, including the whole genome sequence of a large number of pathogens has
provided more support for DNA/RNA-based test. The Polymerase chain reaction (PCR) was the
mostimportant development for research in molecular biolology.It is now the basic technique for
the developmentof most molecular diagnostic methods for food safety and other fields . In
diagnostic PCR, specific primers directed against the DNA of the organism to be detected are
used. The homology between primers and the target DNA confers specificity to theamplification.
The presence of the amplification product at given reaction conditions reveals the presence of the
organism in the tested sample. The traditional method of visualizing the amplified product by
ethidium bromide (EtBr) on an agarose gel has more recently been replaced by the less toxic and
more sensitive SYBR GREEN, a dye that emits fluorescence upon intercalating into the double
stranded DNA. SYBR GREEN can also be conveniently used in a real-time PCR machine.

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The real-time PCR machine is a thermal cycler able to stimulate the fluorescent dye with a laser
and to quantify the fluorescence of the reaction mix, and so the

amplification product, after each cycle. The measurement of the amplified product in real-time
allows to be quantified while the reaction is in the exponential phase and before.During the
exponential phase, differences between samples are a simple function of the initial concentration
of the target DNA and can be, therefore, immediately assessed. Moreover, the comparison with
reference samples of known concentration allows the quantification of the initial concentration of
the target DNA. Nevertheless, the implementation of SYBR GREEN in real-time amplification
experiments does not allow discriminating between specific target amplifications and

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co-produced PCR artefacts, such as non-specific amplifi- cations or primer dimmers.This could
interfere with the detection and quantification of the target DNA, especially at low
concentrations. PCR reliability, in terms of specificity of pathogen detection and
quantification,has been improved by the use of dye quenched probes.TaqMan probes, which are
the most commonly used dye quenched probes in diagnostics, are short DNA oligonucleotides
(normally 10 bp long–10mer) specific to the target sequence between the two primers used in the
PCR. TaqMan probes carry a fluorophore at one end and a quencher at the other, which prevent
the fluorophore from being visible. During PCR cycling, the TaqMan probe specifically anneals
to the single strand DNA target sequence and is degraded by the 30–50exonuclease activity of
the DNA polymerase. The fluorophore, separated from the quencher, then becomes visible . The
fluorescence, measured after each cycle in a real-time PCR machine, is proportional to the
amount of the specific target amplification product and does not include PCR artefacts.
Moreover, the use of labelled probes allows the use of an internal positive control (IPC), to
reveal the presence of PCR inhibitors. This feature is essential to assess the absence of
amplification from a suitable sample, and to dispel the suspicion of false negative results. The
IPC comprises a second TaqMan probe and of an artificial oligo carrying the annealing site for
the IPC probe. The site is flanked by the two annealing sites of the same primers used in the
amplification of the test sample, so that the artificial oligo can be amplified in the reaction. The
IPC is added in the reaction mix and the reaction is performed in a real- time PCR machine
equipped with two lasers capable of revealing and distinguishing the IPC and the sample sig-
nals. The IPC amplification demonstrates the absence of PCR inhibitors. Therefore, the
concomitant absence of sample amplification can be considered as a valid result.
PCR implementation in diagnostics The TaqMan PCR system has been recently used to detect
bacterial, fungal, viral and nematode contamination from different matrices including food,
human and animal specimens, environmental samples, etc.. PCR is the most widely used
molecular diagnostic technique due to its fast and easy to use protocol. Originally, 2–3 h were
needed to complete a PCR, but nowadays more advanced PCR systems can deliver a result in a
matter of minutes . The high sensitivity of the PCR can be further Taq and PCR. Primers and
TaqMan probe anneal to the target DNA. TheTaq polymerase synthesises the new strand starting
from the primer. The exonuclease activity of theTaq polymersase degrades

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the

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improved with the application of reverse-transcription-PCR.

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