Microbial Biotechnology

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 Chapter 5 Introduction

• Microbes (microorganisms) are tiny

organisms that are too small to be seen
individually by the naked eye and must be
viewed with the help of a microscope
• Bacteria, fungi, algae, and protozoa
• Bacteria were the first life forms on earth and
have existed for over 3.5 billion years
• Bacteria are estimated to comprise over 50%
of the earth's living matter

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 5.2 Microorganisms as Tools

• Microbial Enzymes
– Used in applications from food production to molecular
biology research
– Taq DNA polymerase
• Heat stabile, isolated from a thermophile
– Cellulase
• Makes animal food more easily digestible
• Stone-washed jeans

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 5.2 Microorganisms as Tools

• Transformation – the ability of bacteria to take in

DNA from their surrounding environment
– Bacteria will naturally take up DNA from the
environment.
– In biotechnology, cells are treated so that they become
competent and are able to take up DNA more easily.
– Transformation of competent cells is used to
introduced recombinant plasmids into cells so the
bacterial can replicate these plasmids.
– This process is called transformation because one can
"transform" the properties of bacterial cells by
introducing foreign genes.

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 Bacteria use operons to regulate gene
expression
• Bacteria are very important organisms for many applications of
biotechnology, such as the production of human proteins.
• Many initial studies on gene regulation were carried out in bacteria.
Bacteria and other microorganisms can and must rapidly control
gene expression in response to environmental stimuli such as
growth nutrients and changes in temperature and light intensity.
• One interesting aspect of gene expression and regulation in bacteria
is that many bacterial genes are organized in arrangements called
operons.
• Operons are essentially clusters of several related genes located
together and controlled by a single promoter
• The genes of an operon can be regulated in response to changes
within the cell.

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 • Operon: an operator, a promoter, and the genes they control.
• -A group of genes of related function exist in a single transcription
unit, where a single promoter serves all the genes in the unit.
Therefore, the single unit gives rise to multiple protein products of
the functionally related genes in one transcript.
- this is beneficial because a single on-off switch, called an operator,
can control the whole cluster of functionally related genes.
- the operator is located within the promoter (or sometimes between
the promoter and the enzyme coding genes), and controls the access
of RNA polymerase to the gene.

https://www.youtube.com/watch?v=EjRXz1x
Adow

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 5.3 Using Microbes for a Variety of
Everyday Applications
• Therapeutic Proteins
– Bacteria are used to produce medically
important proteins
– Insulin, the first recombinant molecule
expressed in bacteria for use in humans is an
excellent example:

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 5.3 Using Microbes for a Variety of
Everyday Applications

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 5.3 Using Microbes for a Variety of
Everyday Applications

© 2013 Pearson Education, Inc.

 5.3 Using Microbes for a Variety of
Everyday Applications
• Antibiotics
– Produced by microbes that inhibit the growth
of other microbes
– 1928 discovery of penicillin by Alexander
Fleming
– Majority are produced by bacteria, and inhibit
the growth of other bacteria

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 5.3 Using Microbes for a Variety of
Everyday Applications

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 5.3 Using Microbes for a Variety of
Everyday Applications
• Antibiotics
– Improper use of antibiotics has lead to dramatic
increase in antibiotic-resistant bacteria
– Since we can see that known antibiotics attack
a bacterial cell in a limited number of ways,
resistance to one antibiotic often leads to
resistance to many other drugs.
– New antimicrobial drugs that act in unique ways
need to be developed
– Microbiologists are bioprospecting in diverse
habitats to identify sources of new anti microbial
substances
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 5.4 Vaccines

• Vaccines – parts of a pathogen or whole

organisms that can be given to humans or
animals by mouth or by injection to stimulate
the immune system against infection by
those pathogens

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 5.4 Vaccines

• First vaccine developed in 1796 by Edward

Jenner
– Used live cowpox virus to vaccinate against
smallpox

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 5.4 Vaccines

• Three Major Strategies to Make Vaccines

– Subunit vaccines are made by injecting
portions of viral or bacterial structures
– Attenuated vaccines use live bacteria or
viruses that have been weakened through
aging or by altering their growth conditions to
prevent replication
– Inactivated (killed) vaccines are made by
killing the pathogen and using the dead or
inactivated microorganism for the vaccine

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 5.4 Vaccines

• Immune System and Antibodies

– Antigens are foreign substances that stimulate
an immune response
• Whole bacteria, fungi, and viruses
• Proteins, lipids, or carbohydrates
– Immune system responds to antigens by
producing antibodies
• Called antibody-mediated immunity
• B cells, with the help of T cells, recognize and bind
to the antigen
• B cells then develop to form plasma cells that
produce antibodies
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 5.4 Vaccines

• Immune System and Antibodies

– Antibodies are very specific
• Bind to the antigen
• Macrophage can then recognize the antigens coated
with antibodies and "eat" them
– Sometimes our natural production of antibodies
is not enough to protect us from pathogens

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 5.4 Vaccines

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 5.4 Vaccines

• Currently, a majority of subunit vaccines are

made using recombinant DNA approaches in
which the vaccine is produced in microbes
– Hepatitis B
• Genes for proteins on the outer surface of the virus
are cloned into expression plasmids and transformed
into yeast
• Fusion proteins produced by the yeast are purified
– Gardasil, which protects against four strains of
human papillomavirus (HPV)

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 5.4 Vaccines

• Human Immunodeficiency Virus-1 (HIV),

the causative agent for AIDS, is a retrovirus
which infects human immune cells by binding
to a cell and injects its RNA genome, into the
cell
• The viral enzyme, reverse transcriptase,
copies the HIV genome into DNA, which
integrates into the cell's DNA and replicates
along with the host cells until it later is used
to produce viral particles
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 5.4 Vaccines

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 5.4 Vaccines

• Bacterial and Viral Targets for Vaccines

– Biotechnology companies are working on over
50 targets for vaccine development, including
• Influenza
• Tuberculosis
• Malaria
• HIV

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 5.4 Vaccines

• Influenza: Flu viruses mutate rapidly so new flu

vaccines are generated each year.
– Influenza A threatens human health through global
outbreaks called pandemics.
• 1918, influenza killed at least 20 million, future pandemic could
be much worse
• Avian flu (H5N1) caused a pandemic in chickens in Asia in
2003, resulting in the killing of over 200million birds.
– Isolated cases of human infection, but widespread infection did
not occur.
– There is concern that H5N1 may mutate and jump into humans,
so vaccines were developed.
• In 2009, swine flu (H1N1) was effectively controlled by a
vaccine.

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 5.4 Vaccines

• Tuberculosis (TB)
– Caused by Mycobacterium tuberculosis
• 2-3 million deaths per year
• Creates lumpy lesions in lungs
• Concern over resurgence of TB due to drug
resistant lead to WHO declaring a global health
emergency
• The Bill and Melinda Gates Foundation and others
provided $30 million towards research efforts
• The genome has been sequenced, new proteins
were discovered and new vaccines are now in
clinical trials
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 5.4 Vaccines

• Malaria
– Caused by the protozoan parasite Plasmodium
falciparum and transmitted by insects
• Half a billion cases in children each year, nearly 3
million deaths per year
• Plasmodium strains developing resistance to
antimalarial drugs
• Whole-genome microarrays are being used to
identify new gene targets

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 5.5 Microbial Genomes

• 1994 Microbial Genome Program (MGP)

– To sequence the entire genomes of
microorganisms that have potential applications
in environmental biology, research, industry, and
health as well as genomes of protozoan
pathogens

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 5.5 Microbial Genomes

• Why sequence microbial genomes?

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 5.5 Microbial Genomes

• Why sequence microbial genomes?

– Streptococcus pneumoniae, which causes ear
and lung infections, kills 3 million children
worldwide each year
• Many of the vaccines are ineffective in children
• In 2001 the genome was sequenced and many
genes encoding proteins on the surface of the
bacteria were discovered
• Could lead to new treatments, including gene
therapy

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 5.5 Microbial Genomes

• Why sequence microbial genomes?

– Identify genes involved in bacterial cell
metabolism, cell division, and genes that
cause human and animal illnesses
– Find new strains
• For bioremediation or other tasks
• Disease causing organisms

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 5.5 Microbial Genomes

• Viral Genomics

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 5.5 Microbial Genomes

• Creating Synthetic
Genomes: A Functional
Synthetic Genome Is
Produced for a Bacterial
Strain

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 Detection of
Infectious Diseases

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 Before the development of molecular biology techniques,
microbiologists relied on biochemical tests and
bacteria cultured on different growth media to identify
strains of disease-causing bacteria.

For example, when doctors take a throat culture, they use a swab of
bacteria from your throat to check for the presence of Streptococcus
pyogenes, a bacterium that causes strep throat.

Even though these and other similar techniques still

have an important place in microbial diagnosis, techniques
in molecular biology allow for the rapid detection
of bacteria and viruses with great sensitivity.

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 5.7 Microbial Diagnostics

• Microbial Diagnostics –
techniques used to detect and
track microbes
• Bacterial Detection Strategies
– RFLP analysis, PCR and DNA
sequencing
– Databases are available for
comparison of clinical samples
– Used to detect and track bacterial
contamination of food

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 • Many databases of RFLPs, PCR patterns, and bacterial DNA sequences are available
for comparison of clinical samples. For example, if a doctor suspects a bacterial or
viral infection, samples including blood, saliva, feces, and cerebrospinal fluid from
the patient can be used to isolate bacterial and viral pathogens. DNA from the
suspected pathogen is then isolated and subjected to molecular techniques such
as PCR.
• PCR is an important tool for diagnostic testing in clinical microbiology laboratories
and widely used to diagnose infection caused by microbes such as the hepatitis
viruses (A, B, and C), Chlamydia trachomatis and Neisseria gonorrhoeae (both of
which cause sexually transmitted diseases), HIV-1, and many other bacteria and
viruses.

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 5.7 Microbial Diagnostics

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 5.7 Microbial Diagnostics

• Microarrays for Tracking Contagious Diseases

– Microarrays have created new approaches for detecting
and identifying pathogens and for examining host
responses to infectious diseases
– Affymetrics developed the SARS CoV Gene Chip which
can be used to detect SARS
• Contains ~30,000 probes representing the entire viral
genome
– Microarrays also used to find "signature" changes in
gene expression for a particular pathogen

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 5.7 Microbial Diagnostics

© 2013 Pearson Education, Inc.