The Earth is 4.

6 billion years old and microbial life is thought to have first appeared
between 3.8 and 3.9 billion years ago; in fact, 80% of Earth's history was exclusively
microbial life. Microbial life is still the dominant life form on Earth. It has been estimated
that the total number of microbial cells on Earth on the order of 2.5 X 10 30 cells, making
it the major fraction of biomass on the planet.

Phylogeny refers to the evolutionary relationships between organisms. The Three

Domain System, proposed by Woese and others, is an evolutionary model of phylogeny
based on differences in the sequences of nucleotides in the cell's ribosomal RNAs
(rRNA), as well as the cell's membrane lipid structure and its sensitivity to antibiotics.
Comparing rRNA structure is especially useful. Because rRNA molecules throughout
nature carry out the same function, their structure changes very little over time.
Therefore similarities and dissimilarities in rRNA nucleotide sequences are a good
indication of how related or unrelated different cells and organisms are.

There are various hypotheses as to the origin of prokaryotic and eukaryotic cells.
Because all cells are similar in nature, it is generally thought that all cells came from a
common ancestor cell termed the last universal common ancestor (LUCA). These
LUCAs eventually evolved into three different cell types, each representing a domain.
The three domains are the Archaea, the Bacteria, and the Eukarya.

Figure 1.
3.11.3.1: A phylogenetic tree based on rRNA data, showing the separation of bacteria, archaea, and
eukaryota domains.
More recently various fusion hypotheses have begun to dominate the literature. One
proposes that the diploid or 2N nature of the eukaryotic genome occurred after the
fusion of two haploid or 1N prokaryotic cells. Others propose that the
domains Archaea and Eukarya emerged from a common archaeal-eukaryotic ancestor
that itself emerged from a member of the domain Bacteria. Some of the evidence
behind this hypothesis is based on a "superphylum" of bacteria called PVC, members of
which share some characteristics with both archaea and eukaryotes. There is growing
evidence that eukaryotes may have originated within a subset of archaea. In any event,
it is accepted today that there are three distinct domains of organisms in
nature: Bacteria, Archaea, and Eukarya. A description of the three domains follows.

INTERMEDIARIES BETWEEN BACTERIA, ARCHAEAE,
AND EUKARYA DOMAINS?
There is a "superphylum" of bacteria called PVC, referring to the three members of that
superphylum: the Planctomycetes, the Verrucomicrobia, and the Chlamydiae. Members
of the PVC, while belonging to the domain Bacteria, show some features of the
domains Archaea and Eukarya.

Some of these bacteria show cell compartmentalization wherein membranes surround

portions of the cell interior, such as groups of ribosomes or DNA, similar to eukaryotic
cells. Some divide by budding or contain sterols in their membranes, again similar to
eukaryotes. Some lack peptidoglycan, similar to eukaryotes and archaea. It has been
surmised that these bacteria migh be an intermediate step between an ancestor that
emerged from a bacterium (domain Bacteria) and an archael-eukaryotic ancestor prior
to its split into the domains Archaea and Eukarya.
Figure 1.3.21.3.2: Electron micrograph of the bacterium Gemmata obscuriglobus, a
planctomycete noted for its highly complex membrane morphology, illustrating representative
morphologies. Scale bar = 500nm. Santarella-Mellwig R, Franke J, Jaedicke A, Gorjanacz M,
Bauer U, Budd A, et al. (2010) The Compartmentalized Bacteria of the Planctomycetes-
Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins. PLoS Biol 8(1):
e1000281. doi:10.1371/journal.pbio.1000281

The Archaea (archaebacteria)

The Archaea possess the following characteristics:

a. Archaea are prokaryotic cells.
b. Unlike the Bacteria and the Eukarya, the Archaea have membranes composed of
branched hydrocarbon chains (many also containing rings within the hydrocarbon
chains) attached to glycerol by ether linkages (Figure 1.3.31.3.3).
c. The cell walls of Archaea contain no peptidoglycan.
 d. Archaea are not sensitive to some antibiotics that affect the Bacteria, but are sensitive to
some antibiotics that affect the Eukarya.
e. Archaea contain rRNA that is unique to the Archaea as indicated by the presence
molecular regions distinctly different from the rRNA of Bacteria and Eukarya.

Figure 1.3.3
1.3.3: Membrane Lipids of Archaea, Bacteria, and Eukarya. The Bacteria and the Eukarya have
membranes composed of unbranched fatty acid chains attached to glycerol by ester
linkages. The Archaea have membranes composed of branched hydrocarbon chains attached
to glycerol by ether linkages.

Archaea often live in extreme environments and include methanogens, extreme

halophiles, and hyperthermophiles. One reason for this is that the ether-containing
linkages in the Archaea membranes is more stabile than the ester-containing linkages in
the Bacteria and Eukarya and are better able to withstand higher temperatures and
stronger acid concentrations.

The Bacteria (eubacteria)
Bacteria (also known as eubacteria or "true bacteria") are prokaryotic cells that are
common in human daily life, encounter many more times than the archaebacteria.
Eubacteria can be found almost everywhere and kill thousands upon thousands of
people each year, but also serve as antibiotics producers and food digesters in our
stomachs. The Bacteria possess the following characteristics:

a. Bacteria are prokaryotic cells.

b. Like the Eukarya, they have membranes composed of unbranched fatty acid chains
attached to glycerol by ester linkages (Figure 1.3.31.3.3).
c. The cell walls of Bacteria, unlike the Archaea and the Eukarya, contain peptidoglycan.
d. Bacteria are sensitive to traditional antibacterial antibiotics but are resistant to most
antibiotics that affect Eukarya.
e. Bacteria contain rRNA that is unique to the Bacteria as indicated by the presence
molecular regions distinctly different from the rRNA of Archaea and Eukarya.
Bacteria include mycoplasmas, cyanobacteria, Gram-positive bacteria, and Gram-
negative bacteria.

The Eukarya (eukaryotes)
The Eukarya (also spelled Eucarya) possess the following characteristics:

a. Eukarya have eukaryotic cells.

b. Like the Bacteria, they have membranes composed of unbranched fatty acid chains
attached to glycerol by ester linkages (Figure 1.3.31.3.3).
c. Not all Eukarya possess cells with a cell wall, but for those Eukarya having a cell wall,
that wall contains no peptidoglycan.
d. Eukarya are resistant to traditional antibacterial antibiotics but are sensitive to most
antibiotics that affect eukaryotic cells.
e. Eukarya contain rRNA that is unique to the Eukarya as indicated by the presence
molecular regions distinctly different from the rRNA of Archaea and Bacteria.

The Eukarya are subdivided into the following four kingdoms:

1. Protista Kingdom: Protista are simple, predominately unicellular eukaryotic organisms.

Examples includes slime molds, euglenoids, algae, and protozoans.
2. Fungi Kingdom: Fungi are unicellular or multicellular organisms with eukaryotic cell
types. The cells have cell walls but are not organized into tissues. They do not carry out
photosynthesis and obtain nutrients through absorption. Examples include sac fungi,
club fungi, yeasts, and molds.
3. Plantae Kingdom: Plants are multicellular organisms composed of eukaryotic cells. The
cells are organized into tissues and have cell walls. They obtain nutrients by
photosynthesis and absorption. Examples include mosses, ferns, conifers, and flowering
plants.
4. Animalia Kingdom: Animals are multicellular organisms composed of eukaryotic cells.
The cells are organized into tissues and lack cell walls. They do not carry out
photosynthesis and obtain nutrients primarily by ingestion. Examples include sponges,
worms, insects, and vertebrates.

It used to be thought that the changes that allow microorganisms to adapt to new
environments or alter their virulence capabilities was a relatively slow process occurring
within an organism primarily through mutations, chromosomal rearrangements, gene
deletions and gene duplications. Those changes would then be passed on to that
microbe's progeny and natural selection would occur. This gene transfer from a parent
organism to its offspring is called vertical gene transmission.

It is now known that microbial genes are transferred not only vertically from a parent
organism to its progeny, but also horizontally to relatives that are only distantly related,
e.g., other species and other genera. This latter process is known as horizontal gene
transfer. Through mechanisms such as transformation, transduction, and conjugation,
genetic elements such as plasmids, transposons, integrons, and even chromosomal
DNA can readily be spread from one microorganism to another. As a result, the old
three-branched "tree of life" in regard to microorganisms (Figure 1.3.11.3.1) now
appears to be more of a "net of life."

Microbes are known to live in remarkably diverse environments, many of which are
extremely harsh. This amazing and rapid adaptability is a result of their ability to quickly
modify their repertoire of protein functions by modifying, gaining, or losing their genes.
This gene expansion predominantly takes place by horizontal transfer.

Summary
1. Phylogeny refers to the evolutionary relationships between organisms.
2. Organisms can be classified into one of three domains based on differences in the
sequences of nucleotides in the cell's ribosomal RNAs (rRNA), the cell's membrane lipid
structure, and its sensitivity to antibiotics.
3. The three domains are the Archaea, the Bacteria, and the Eukarya.
4. Prokaryotic organisms belong either to the domain Archaea or the domain Bacteria;
organisms with eukaryotic cells belong to the domain Eukarya.
5. Microorganism transfer genes to other microorganisms through horizontal gene transfer
- the transfer of DNA to an organism that is not its offspring.

Contributors
 Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY,
CATONSVILLE CAMPUS)
O MANY SPECIES!
The collage above shows a single species in each of the six kingdoms into which all of
Earth's living things are commonly classified. How many species are there in each
kingdom? In a word, millions. A total of almost 2 million living species have already
been identified, and new species are being discovered all the time. Scientists estimate
that there may be as many as 30 million different species alive on Earth today! Clearly,
there is a tremendous variety of life on Earth.

Figure 2.3.12.3.1:
Archaebacteria (NASA; public domain via wikimedia); Bacteria (De Wood and Chris
Pooley/Agricultural Research Service, USDA; public domain via wikimedia); Protist
(MichaelTaylor, 2014; via shutterstock.com); Fungus (Tony Hisgett; CC BY 2.0 via flickr.com);
Animal (úlfhams_víkingur; CC BY 2.0 via flickr.com).

What Is Biodiversity?
Biological diversity, or biodiversity, refers to all of the variety of life that exists on
Earth. Biodiversity can be described and measured at three different levels: species,
genetic, and ecosystem diversity.

 Species diversity refers to the number of different species in an ecosystem or on Earth

as a whole. This is the commonest way to measure biodiversity. Current estimates for
Earth's total number of living species range from 5 to 30 million species.
  Genetic diversity refers to the variation in genes within all these species.
 Ecosystem diversity refers to the variety of ecosystems on Earth. An ecosystem is a
system formed by populations of many different species interacting with each other and
their environment.

Defining a Species
Biodiversity is most often measured by counting species, but what is a species? The
answer to that question is not as straightforward as you might think. The formal
biological definition of species is a group of actually or potentially interbreeding
organisms. This means that members of the same species are similar enough to each
other to produce fertile offspring together. By this definition of species, all human
beings alive today belong to one species, Homo sapiens. All humans can potentially
interbreed with each other but not with members of any other species.

In the real world, it isn't always possible to make the observations needed to determine
whether different organisms can interbreed. For one thing, many species reproduce
asexually, so individuals never interbreed even with members of their own species.
When studying extinct species represented by fossils, it is usually impossible to know
whether different organisms could interbreed. Therefore, in practice, many biologists
and virtually all paleontologists generally define species on the basis of morphology,
rather than breeding behavior. Morphology refers to the form and structure of
organisms. For classification purposes, it generally refers to relatively obvious physical
traits. Typically, the more similar to one another different organisms appear, the
greater the chance that they will be classified in the same species.

Classifying Living Things

People have been trying to classify the tremendous diversity of life on Earth for more
than two thousand years. The science of classifying organisms is
called taxonomy. Classification is an important step in understanding the present
diversity and past evolutionary history of life on Earth. It helps make sense of the
overwhelming diversity of living things.

Linnaean Classification

All modern classification systems have their roots in the Linnaean classification system.
It was developed by Swedish botanist Carolus Linnaeus in the 1700s. He tried to
classify all living things that were known at his time. He grouped together organisms
that shared obvious morphological traits, such as the number of legs or shape of
leaves. For his contribution, Linnaeus is known as the “father of taxonomy.”
 Figure 2.3.22.3.2: The hierarchy of taxa in the original Linnaean system
of taxonomy included taxa from the species to the kingdom. The domain was added later.
(Public Domain; Pengo via wikipedia.org).

The Linnaean system of classification consists of a hierarchy of groupings, called taxa

(singular, taxon). Figure 2.3.22.3.2 shows an expanded version of Linnaeus's original
classification system. In the original system, taxa range from the kingdom to the
species. The kingdom is the largest and most inclusive grouping. It consists of
organisms that share just a few basic similarities. Examples are the plant and animal
kingdoms. The species is the smallest and most exclusive grouping. Ideally, it consists
of organisms that are similar enough to interbreed, as discussed above. Similar species
are classified together in the same genus (plural, genera), similar genera are classified
together in the same family, and so on all the way up to the kingdom.

Binomial Nomenclature

Perhaps the single greatest contribution Linnaeus made to science was his method of
naming species. This method, called binomial nomenclature, gives each species a
unique, two-word Latin name consisting of the genus name followed by a specific
species identifier. An example is Homo sapiens, the two-word Latin name for humans.
It literally means “wise human.” This is a reference to our big brains.

Why is having two names so important? It is similar to people having a first and a last
name. You may know several people with the first name Michael, but adding Michael’s
last name usually pins down exactly who you mean. In the same way, having two
names uniquely identifies a species.

Revisions in the Linnaean Classification

Linnaeus published his classification system in the 1700s. Since then, many new species
have been discovered. Scientists can also now classify organisms on the basis of their
biochemical and genetic similarities and differences rather than just their outward
morphology. These changes have led to revisions in the original Linnaean system of
classification.

A major change to the Linnaean system is the addition of a new taxon called the
domain. The domain is a taxon that is larger and more inclusive than the kingdom, as
shown in Figure 2.3.22.3.2. Most biologists agree that there are three domains of life on
Earth: Bacteria, Archaea, and Eukarya (Figure 2.3.32.3.3). Both the Bacteria and the
Archaea domains consist of single-celled organisms that lack a nucleus. This means that
their genetic material is not enclosed within a membrane inside the cell. The Eukarya
domain, in contrast, consists of all organisms whose cells have a nucleus. In other
words, their genetic material is enclosed within a membrane inside the cell. The
Eukarya domain is made up of both single-celled and multicellular organisms. This
domain includes several kingdoms, including the animal, plant, fungus, and protist
kingdoms.

Figure 2.3.32.3.3: Three domains

of life: Bacteria, Archaea, and Eukarya. (CC BY-SA 4.0; Crion).

Phylogenetic Classification
Linnaeus classified organisms based on morphology. Basically, organisms were grouped
together if they looked alike. After Darwin published his theory of evolution in the
1800s, scientists looked for a way to classify organisms that took into account
phylogeny. Phylogeny is the evolutionary history of a group of related organisms. It is
represented by a phylogenetic tree, or some other tree-like diagram, like the one in
Figure 2.3.32.3.3 for the three domains. A phylogenetic tree shows how closely
related different groups of organisms are to one another. Each branching point
represents a common ancestor of the branching groups. Figure 2.3.32.3.3, for example,
shows that the Eukarya shared a more recent common ancestor with the Archaea than
they did with the Bacteria. This is based on comparisons of important similarities and
differences between the three domains.

Summary
 Biodiversity refers to the variety of life that exists on Earth. It includes species diversity,
genetic diversity within species, and ecosystem diversity.
 The formal biological definition of species is a group of actually or potentially
interbreeding organisms. Our own species, Homo sapiens, is an example. In reality,
organisms are often classified into species on the basis of morphology.
 A system for classifying living things was introduced by Linnaeus in the 1700s. It
includes taxa from the species (least inclusive) to the kingdom (most inclusive).
Linnaeus also introduced a system of naming species, called binomial nomenclature.
 The domain, a taxon higher than the kingdom, was later added to the Linnaean system.
Living things are generally grouped into three domains: Bacteria, Archaea, and Eukarya.
The human species and other animal species are placed in the Eukarya domain.
 Modern systems of classification take into account phylogenies, or evolutionary histories
of related organisms, rather than just morphological similarities and differences. These
relationships are often represented by phylogenetic trees or other tree-like diagrams.

Review
1. What is biodiversity? Identify three ways that biodiversity may be measured.
2. Define biological species. Why is this definition often difficult to apply?
3. Explain why it is important to classify living things and outline the Linnaean system of
classification.
4. What is binomial nomenclature? Give an example.
5. Contrast Linnaean and phylogenetic systems of classification.
6. Describe the taxon called the domain, and compare the three widely recognized
domains of living things.
7. True or False. Humans have identified all of the species on Earth.
8. True or False. In the binomial nomenclature for humans, Homo is the genus
and sapiens refers to the specific species.
9. A kingdom is a:
A. domain
B. taxon
C. genera
D. phylogeny
10. In Linnaean classification, similar classes together make up a ___________ .
11. Based on the phylogenetic tree for the three domains of life above, explain whether you
think Bacteria are more closely related to Archaea or Eukarya.
 12. A scientist discovers a new single-celled organism. Answer the following questions about
this discovery.
A. If this is all you know, can you place the organism into a particular
domain? If so, what is the domain and if not, why not?
B. What is one type of information that could help the scientist classify the
organism?
13. Define morphology. Give an example of a morphological trait in humans.
14. Which type of biodiversity is represented by the differences between humans?

15. Why do you think it is important for the definition of a species

that members of a species can produce fertile offspring?

Chapter 1 Main Themes of Microbiology

What is Microbiology ?

Microbiology is a study of microorganisms (microbes, germs) and their activities

What is a microorganism?
An organism too small to be seen without a microscope (Smaller than 0.5 mm) (Bacteria,
Fungi, Protozoa, Algae, Viruses)
 
Branches of Microbiology
Medical Microbiology
Industrial Microbiology
Environmental Microbiology
Agricultural Microbiology

 Microbiology is part of some other disciplines

•         Biotechnology
 •         Food production (Dairy products (yoghurt, cheese), Sauerkraut, Pickles,
Sausages, Bread, Beer, Wine, Etc.)
•         Drug production (penicillin)
•         Bioremediation – detoxification by use of microorganisms
•         Genetic Engineering (Manipulation of genes of microbes in order to create new
products)

 The Impact of Microbes on Earth

•         The first organisms on the planet Earth
–        Ubiquitous – can be found everywhere
•         Photosynthesis
–        Oxygen production
•         Primary production
–        Synthesis of organic mater
•         Decomposition

 The Impact of Microorganisms on Human Health

•         Microorganisms as disease agents
•         Microorganisms live inside our bodies,
–        normal microflora,
–        pathogenic
•         Our body resists the invasion of pathogenic microbes by
–        skin,
–        mucous membrane,
–        stomach acid
–        antimicrobial chemicals - interferons
•         Introduction of antibiotics changed the rates for the leading causes of death
 
CELL TYPES
There are two basic cell types:
                        - Prokaryotic
                                    Bacteria
                                    Archea
                        - Eukaryotic
                                    Fungi
                                    Protozoa
                                    Algae
                                    (helimints, plants, animals)
            Major difference - organization of nuclear structure
                         ۞Viruses

CELLULAR ORGANIZATION IN MICROBES

Prokaryotic
•         Cell envelope  
•         Nucleoid
 Eukaryotic
•         Cell envelope
•         Organelles
•         Nucleus

Viruses
•         Do not have cellular structure
•         One type of  nucleic acid DNA or RNA
•         Can reproduce only when inside a host cell - parasite

Microbial Dimensions
•         Prokaryotic cells: 1-10 mm
•         Eukaryotic cells: 10-100 mm
•         Viruses : 10-100 nm

Lifestyle of Microbes
•         Autotrophs
•         Heterotrophs
•         Parasites

   Naming and Classifying Microorganisms

•         Classification of organisms is called Taxonomy
•         Based on the system introduced by Carl Linnaeus (1735)

             Binomial system of nomenclature

Genus: Bacillus           Species:Bacillus subtilis  

•         Bacterial taxonomy has relied on phenotypic analysis (Morphology, cell

structures, storage products, Gr- staining, biochemical characteristics)

•          Molecular taxonomy - Nucleic acid analysis                                                           

All organisms belong to three groups: The Three Domain System

–        Bacteria
–        Archaea
–        Eukarya

 History of Microbiology

Who Started Microbiology? 

Antoni van Leeuwenhoek (1673-1723), a Dutch merchant

•         The first person to see microbes as being ALIVE!
•         Father of bacteriology.
 What Leeuwenhoek Could See?
•         Different shapes of bacteria                                       
•         Red blood cells, spermatozoa, plant and animal cells
•         Magnification of his microscope was 32X

Robert Hooke’s Microscope Compared to Leeuwenhoek’s

•         Robert Hook introduced the compound microscope (objective lens + ocular lens)
•         Robert Hook (1665) reported that the smallest  biological structural unites were
“little boxes” - cells.

•         CELL THEORY – all living things are composed of  cells

Golden Age of Microbiology - 1830-1900

Scientists searched for answers to the following questions:

–        Is spontaneous generation of microbial life   possible?
–        What causes fermentation?
–        What causes disease?
–        How can we prevent infection and disease?

Is Spontaneous Generation of Microbial Life Possible?

•         Aristotle proposed spontaneous generation – living things can arise from non-living
matter
•         Toads and snakes could be born of moist soil
•         Maggots (the larvae of flies) could arise from corpses

Finally!
•         Arguments about spontaneous generation resolved by the French scientist Louis
Pasteur – 1850’s

How he really did it?

•         Pasteur’s S-shaped flask kept microbes out but let air in.

Pasteur’s discoveries
•         Performed the most convincing experiments disproving the theory of spontaneous
generation
•         Role of yeast in alcohol fermentation
•         Devised the process of pasteurization and basis of aseptic techniques
•         Disease of vine - could be prevented by heating the wine for a short time to a temp. 
55-600C
•         The first preventive treatment for rabies

What Causes Disease?

•         Pasteur developed germ theory of disease, but had no proof…began working on
Anthrax, but could not get a pure culture of the disease producing bacterium
1876 Robert Koch     

German bacteriologist, studied causative agents of disease and PROVED the Germ Theory
of Disease.
•         Gave the first proof that bacteria cause disease (by isolating the anthrax bacillus in
pure culture) - germ theory of disease
•         Perfected the technique of isolating bacteria in pure culture - solid media -boiled
potato
•         Discovered Mycobacterium tuberculosis  - the organism causing the tuberculosis.
Tuberculin - substance used for diagnosis of tuberculosis.
•         Introduced the staining procedure for M. tuberculosis (acid fast staining)
•         First to stain bacterial smears
•         Discovered the causative agent of cholera
•         1905 received the Nobel Prize for Medicine

Koch’s postulates - Identify criteria for proving that a specific type of a microorganism
causes a specific disease:

    1. The microorganism should be constantly present in a diseased animal

    2. The microorganism must be cultivated in pure culture

    3. When inoculated into a healthy animal, such microorganism should cause
characteristic disease symptoms

    4. The microorganism should be re-isolated from the experimental animal, and it should
have the same characteristics as the original microorganism.

Joseph Lister
•         Father of modern surgery
•         Applied antiseptic (carbolic acid) treatment for prevention and care of wound
infection      
•         Introduced the procedure of disinfection of operating rooms

Chemotherapy - treatment of disease by using chemical substances

•         The first antibiotic was discovered by accident
•         Alexander Fleming 1928 discovered penicillin
•         Produced by mold: Penicillim chrysogenum
•         Commercial production started in 1945 in the US

Problems with antibiotics

•         Can be toxic to humans
•         Spread of new varieties of resistant microorganisms

Development of techniques that facilitated further progress of microbiology

•         Pure cultures (clone) is a culture consisting of only one type of microorganisms
 •         Use of Petri dishes
•         Aseptic techniques
•         Bacteria as distinct species

Developments of microbiology in the twentieth century

•         Bacteriology
–        Study of Bacteria. New species of bacteria are discovered regularly
•         Mycology
–        Study of fungi. Fungal infections rising
•         Parasitology
–        Study of protozoa and parasitic worms
•         Immunology

            - Study of immunity. Vaccines available for many diseases

•         Virology
–        1892 Dimitri Iwanowski, reported on the presence of infectious agents much
smaller than bacteria. He discovered the tobacco mosaic virus.
•         Recombinant DNA technology
–        Fragments of foreign DNA can be incorporated into bacterial genom

The Establishment of the Scientific Method

•         Scientific method – based on an experimental system
•         It includes:
–        1. Observation and description of a phenomenon
–        2. Formulation of an hypothesis to explain the phenomena
–        3. Performance of experimental tests (to confirm or disprove the hypothesis).
•         If supported – Hypothesis becomes a theory

The Chemistry of Biology

Atoms
•       Building blocks of molecules
•       Subatomic particles
–      Shell
•       Electrons (-e) rotate about the nucleus in their orbitals
–      Nucleus
•       Protons (+ charged)
•       Neutrons
Electron Orbitals and Shells
•       Electrons rotate around the nucleus  in orbitals (pathways)
•       Low level energy electrons – nearest to the nucleus
•       1st shell – 1 orbital (2e-)
•       2nd shell – 4 orbitals (up to 8e-)
•       3rd shell – 9 orbitals (up to 18e-)
Elements
Each element contains characteristic number of protons and electrons – position in the table
•       There are 118 known
•       Elements in groups have some similar properties to each other
 
Isotopes
•       Have a different number of neutrons; same physical properties
•       Some isotopes have unstable nuclei – emit energy – radioactivity
•       Significance in Biology
–      Tracing atoms and molecules in metabolic reactions,
–      Diagnostic purposes
–      Sterilization (gamma irradiation)
Molecules
•       Two or more atoms combined form a molecule – a new compound has new
characteristics
•       Compounds are formed when atoms share, donate, or lose electrons
•       Number of electron in the outer shell – valence
•       Determines the readiness of an element to react with other element
How the atoms bind together?
•       Through the chemical bonds
–      Covalent bonds
–      Ionic bonds
–      Hydrogen bonds
Covalent bonds
•       Bonds between atoms that share electrons
•       Electrons are not shared equally – change of polarity
Ionic Bonds
•       Electrons are transferred from one atom to another
•       When valences complement each other
•       Ex. - NaCl
Ionization
•       Dissociation of molecules (atoms) into charged particles:
            Cations (+)
            Anions (-)
•       When added to water, crystals of NaCl get ionized – Na+ Cl-
•       Electrolytes – conduct electricity: acids, bases, salts
•       Each ion becomes hydrated –surrounded by water molecules
•       Hydrophilic molecule attracts water (NaCl)
•       Hydrophobic molecules repel water (benzene – a non-polar molecule)
Hydrogen bonds
•       Attractive forces between nearby molecules
•       Ex. Water molecules; proteins and DNA
•       Represented with dotted line
Oxidation-Reduction Reactions
•       Oxidation – losing electron
•       Reduction – receiving electrons
Chemical formulas
 •       Atomic symbols and number of atoms in a molecule: H2O
•       Do not provide information on position of bonds between atoms
Reactions
•       Synthesis reaction
–      Equation must be balanced     2H2 + O2     2H2O
•       Decomposition reactions      Larger molecule is broken up in two smaller
units  2H2O2     2H2O + O2
Solutions
•       Mixture of substances solute – (solid, gaseous, liquid) in the solvent (liquid)
•       Water is the most common solvent
Acidity, Alkalinity, pH
Solutions can be acidic or basic
H2O       H+  + OH-   ionization  of water
H+ H+  + OH-   access of H+ acidic pH
H+ + OH- OH- OH-  access OH- basic pH
pH is a measure of concentration of H+ and OH- ions
pH=-log [H+]
Inorganic and Organic Compounds
Compounds can be:
•       Inorganic ( do not have C and H combined)
            NaCl, CaCO3…
•       Organic (CH4) – complex compounds with C bonded to other atoms
The Chemistry of Carbon
•       Carbon is the fundamental element of life -Why?
•       Molecular skeleton
•       Have 4 electrons in outer orbital – can form 4 bonds
•       Most often forms stable bonds with C, H, O, N, S, and P
Functional Groups
•       Carbon binds to other atoms via molecular groups – functional groups
•       Functional groups determine the characteristics of a molecule
Macromolecules
•       Smaller molecules (monomers) are assembled into larger compounds – macromolecules
(polymers)
Classes of carbohydrates
Monosaccharides
–      Have 3-7 C atoms
•       Pentose (5 carbons)
•       Hexose (6 carbons)
–      Glucose
Disaccharides
Sucrose
Polysaccharides
Glycosidic bonds
•       Bonds between two sugars
•       Carbons from two molecules are bonded via oxygen with release of  H2O molecule
Function of Polysaccharides
 •       Provide structural support (cell wall)
•       Nutrient and energy storage
•       Some examples
–      Cellulose
–      Agar
–      Chitin
–      Peptidoglycan
–      Glycocalyx
Lipids
•       Not soluble in water
•       Classes of lipids:
–      Triglycerides
–      Phospholipids
–      Steroids
–      Waxes
Biological Significance of Lipids
•       Storage material (triglycerides)
•       Membrane lipids
–      Phospholipids
•       hydrophilic and hydrophobic portion
–      Cholesterols
•       Provides support to the cell wall of some bacteria
Proteins
•       Composed of amino acids
•       Assembled together through peptide bonds
•       Peptide: short chain of amino acids
•       Polypeptide (proteins): long chains of amino acids
•       Protein structure determines its functionality:
–      Primary
–      Secondary
–      Tertiary
–      Quaternary
Peptide bonds
•       Bond between amino group of one AA and carboxyl group of another AA
The Nucleic Acids
•       DNA and RNA; informational molecules
•       Contain genetic information
•       Composed of nucleotides
•       Nucleotides composed of:
–      Bases
–      Sugars
–      Phosphate
ATP: The energy molecule of cells
•       Adenosine triphosphate contains:
–      Adenine
–      Ribose
 –      Three phosphates
•       Gives off energy when the bond is broken and one phosphate group removed

Chapter 3
Tools in Microbiology
Inoculation
•          Introducing a sample (the inoculum) into a container with a nutrient medium
•          The medium contains appropriate nutrients that sustains the growth of microorganisms
•          Some microbes have to be inoculated into a living organism
Isolation: Separating one species from another
•          Obtaining Pure Culture
•          Cultures composed of cells arising from a single cell - PURE CULTURES
Types of media
•          1. Physical state
–        Liquid
–        Solid (agar)
•          2. Chemical composition
–        Synthetic
–        Nonsynthetic (complex)
•          3. Functional type
–        General purpose
–        Enrichment
–        Selective
–        Differential
Measures to be taken when working with microbiological media
-          Needs to be sterilized
-          Prevent contamination
         Synthetic media
-          Known chemical composition (NaNO3 – 3g/l; glucose 2g/l…)
         Non-synthetic (complex) media
-          Contains chemically undefined components (Pepton, beef extract..)
         Enrichment medium – supports the growth of a specific group of microorganisms (Ex. N2-
fixing)
         Selective media - favor specific microorganisms and inhibits the others (methylene blue
inhibits the growth of Gram+ bacteria)
         Differential media - contain substances that permit detection of microorganisms with
specific metabolic activity
Incubation
•          Microbiological cultures are placed in temperature-controlled chambers – incubators
•          Temperature: 20-400C
–        Pathogenic:  370C
MICROSCOPE – The Instrument
•          Microscopes are the instruments that magnify the cell (object) to extent at which the cell
details become visible
•          Leeuwenhoek’s microscope had one lens
 •          Robert Hooke invented the compound microscope - multiple lenses
Microscope – The basic principle
•          The specimen is magnified with the objective lens (real image)
•          This image is magnified by ocular lens (virtual image)
•          An enlarged and inverted image is received by retina
Basic features of microscopy
•          Magnification
•          Resolution
•          Contrast
Magnification
•          Magnification is the result of light refraction
•          Mag =  Objective Power x Ocular Lens Power
•          Ex:          Objective lens = 10X
Ocular Lens Power = 10X
Mag = 10 x 10 = 100X
Use of immersion oil with high power objectives
Immersion oil has the same refractive index as the glass. Refractive index is a measure of
relative velocity at which light passes through a material
Resolution
Resolution (resolving power) is the ability of a lens to distinguish two adjacent points as two
separate objects. In light microscopes resolution is 0.2 m (limit - 2000X)
How does the resolution depend on the wavelength?
•          Resolving distance =  Wavelength of light /2 x NA (numerical aperture)
•          The shorter the wavelength - the greater the resolution
Contrast
•          Specimen must contrast with their background
•          This can be achieved by:
–        Changing the refractive index of specimen
•          Stain the specimen
Types of Microscopes
Light Microscopes
1. Bright field
2. Phase contrast
3. Fluorescent
4. Dark filed
5. Differential Interference
6.Confocal
Electron Microscope
1. Transmission
2. Scanning
Light Microscopy - Compound Microscope
Optical microscope parts:
•          Illuminator,
•          Condenser,
•          Objective lens
•          Ocular lens (eyepiece)
Dark-Field Microscopes
•          Best for observing pale objects
 •          Only those light rays scattered by specimen enter objective lens
•          Specimen appears light against dark background
•          Increases contrast and enables observation of more details
Fluorescent Microscopy
•          Fluorescence is the ability of certain substances to absorb short wavelengths of light and emit
light at a longer wavelength
Immunofluorescence
Diagnostic procedure:                                                                                   
•          Antibody produced against a specific bacterium
•          Conjugate antibody and fluorochrome
•          Treat the unknown bacterium
•          If suspected bacteria are indeed present they will bind the tagged antibodies
•          Ultraviolet (or near) light is used as a light source
Phase Microscopes
•          Provides better contrast and more details in the cell.
•          The light rays that hit the specimen travel a different path than the rays, which do not hit
the specimen
Differential Interference Microscopy (Nomarsky)
•          Uses two beams of light
•          Higher resolution
•          3-D images
Confocal Microscopy
•          Uses fluorescent dyes and UV lasers to illuminate the sample
•          An image is taken in a single plane that is not thicker than 1.0 μm
•          Resolution increased by up to 40% because emitted light passes through pinhole aperture
•          Computer constructed 3-D images
Electron Microscopy
Two types of electron microscopes:
–        Transmission (TEM)
–        Scanning (SEM)
Source of illumination is an electron beam
Advantage of using EM
•          Resolving distance = Wavelength of light :2
Wavelength of visible light= 4000A
–        Resolution (light microscopy): 2000 A (0.2 mm)
                                                1 angstrom = 1.0 × 10-10 meters
                                                1 mm = 10-6 meters
•          E.M. uses an electron beam as a source of illumination (100 000 times shorter wavelength than
visible light)
–        Resolution (EM): 2 A
–        Magnification 10,000X to 100,000X
Transmission electron microscope (TEM)
•          Image formed by the electrons transmitted  through a specimen
•          A specimen is a thin section of material (fixed, embedded, and sliced – never alive)
•          TEM is used for objects smaller than 0.2 mm
Scanning electron microscope
•          Used to study the surface of the cell / tissue
•          Image formed by the  electrons reflected from the surface 3-D view
PREPARATION OF SPECIMENS FOR OPTICAL MICROSCOPES
•          Wet mount (living) preparation
–        Unstained
–        Stained (methylene blue)
•          Heat fixed smear
–        Thin film of material containing microorganisms is spread over the surface of the slide
–        Air dried
–        Heat fix (kill and fix bacteria to the slide)
Staining microbial cells
1. Fresh living preparations
2. Fixed, stained smears
Fixed smears:
•          Simple stains
•          Differential stains
–        Gram Stain
–        Acid-Fast Stain
•          Special stains
–        Negative (Capsule) Stain
–        Flagellar Stain
–        Fluorescent Stains
–        Endospore Stain
 
 

Prokaryotic Cells

Chapter 4 Prokaryotic Cell

Prokaryotic Cells - Bacteria

•         Genetic material not enclosed with a membrane

•         Absence of other membrane-enclosed organelles
•         Cell division by binary fission
•         Small; <1.0-3.0 μm in diameter

External Structures

•         Glycocalyx – Capsule or Slime

•         Flagella

•         Fimbriae and Pili

Internal Structures
 •         Cytoplasm
•         Nucleoid
•         Ribosomes
•         Inclusion granules

 Flagella

•         Long, whip-like structures that extend beyond surface of cell

•         Responsible for movement
•         Present in some bacteria

 Arrangements of Flagella

•         Monotrichous - Single polar flagellum

•         Lophotrichous - Two or more flagella at one end of the cell
•         Amphitrichous  - a single flagellum or a tuft of flagella on each end of the cell
•         Peritrichous -   flagella distributed over the entire cell

Structure of Flagella:

•         basal body - anchors the flagellum to the cell wall and plasma membrane
•         hook anchors the flagellum to the basal body
•         filament - outermost region of the flagellum, composed of the protein –
flagellin

Attachment of flagella

In Gram positive bacteria

•         1 pair of rings

•         Only inner pair is present

In Gram negative bacteria

•         2 pairs of rings

•         Outer pair of rings is attached to the cell wall
•         Inner pair anchored to the plasma membrane

Bacterial Movement

Rotation of flagella
 -          Counter-clockwise – swims forward
-          Clockwise– tumble

Chemotaxis in bacteria

Motility of bacteria enables them to move towards or away from the environmental stimuli -
taxis

-          chemotaxis - chemical stimulus

-          phototaxis - light as a stimulus

Bacteria contain receptors in the cell wall to pick up chemical stimuli

Attractant – towards the stimulus – many runs

Repellent – many tumbles

Appendages for attachment – Fimbriae

•         Sticky, proteinaceous, projections

•         Used by bacteria to adhere to each other a host, surfaces in the environment
•         May be hundreds per cell and; shorter than flagella
•         Serve an important function in biofilms

Appendages for mating –sex pili

Long hollow tubules composed of pilin

•         Bacteria typically only have one or two per cell

•         Join two bacterial cells and mediate the transfer of DNA from one cell to
another (conjugation)
•         Also known as conjugation pili or sex pili

Bacterial Surface Coating – Glycocalix - Protects cells from drying

o   Slime layer – loosely attached to the cell surface; Water soluble

o   Capsule – Tightly bound to the cell surface
  Higher pathogenicity - May prevent bacteria from being recognized and
destroyed by host phagocytes
  Enables attachment to surfaces (plant roots, water pipes, surface of teeth etc.)

The Cell Envelopes

 •         Cell wall
•         Cell membrane
•         The outer membrane (some bacteria)

Cell wall

•         Surrounds the whole cell

•         Protects the cell from adverse effects of the outside environment
•         Withstands the pressure of the cell
•         Maintains the shape of bacterium
•         Main component: peptidoglycan

Peptidoglycan is composed of:

  Sugars:
         N-acetylglucosamin NAG
         N-acetylmuramic acid NAM
  Amino acids

Chains of NAG and NAM attached to other chains by tetrapeptide crossbridges

Gram + and Gram – bacteria

1884 Hans Christian Gram introduced Gram staining

Cell wall of Gr+ bacteria

Staining depends on the structure of the cell wall

•         Thick layer of peptidoglycan - (prevents the rinse out of a blue dye  complex

during the Gram-staining - cell appear violet)
•         Contains also teichoic acid
•         Lipoteichoic acids anchor peptidoglycan to cell membrane

Cell wall of Gr- bacteria

o   Multilayered structure composed of:

  Thin layer of peptidoglycan 
  Outer membrane:
         bilayer - phospholipids
         channel proteins (porins)
          lipopolysaccharide (LPS)

During Gram-staining procedure, the blue dye complex is washed out (by acetone), the second
dye (Safranin – red) stains the cell - Cell appears red      

Outer membrane

Function:

•         Evading phagocytosis
•         Barrier to some antibiotics, lyzozyme, heavy metals…

Porins - Proteins - Act as a pore that permits the passage of certain molecules

LPS – Lipopolysaccharide

•         LPS also known as endotoxin

•         Released from dead cells when cell wall disintegrates
•         May trigger fever, vasodilation, inflammation, shock, and blood clotting – it is
TOXIC
•         Can be released when antimicrobial drugs kill bacteria

Damage to the Cell Wall

Can be caused by:

•         Penicillin - interferes with the formation of the cell wall

•         Lysozyme - enzyme that ruptures the cell wall

Spheroplast -spherical cell without cell wall

Nontypical Cell Wall

Mycobacterium – most of their cell wall composed of waxy lipid - mycolic acid

Can be detected by acid-fast staining

Mycoplasmas - lack of cell wall – pleomorphic morphology

Structures Internal To the Cell Wall

Plasma (cytoplasmic) membrane – A thin structure surrounding the cell composed of:

         Phospholipid bilayer
          Proteins

Phospholipid bilayer: One layer consists of

                        - hydrophylic (waterloving)  heads

                        - hydrophobic (waterfearing) tails

Proteins

•         Integral proteins (channels)

•         Peripheral proteins (enzymes)
•         Glycoproteins (receptors)

Functions of cytoplasmic membrane:

•      Controls passage of substances into and out of the cell; selectively permeable

•      Site of energy production (ATP synthesis)

•      Harvests light energy in photosynthetic prokaryotes

•      Site of Flagella Insertion

•      Site of Pili, Fimbriae Insertion

•      DNA attached to one point on cytoplasmic membrane

Cytoplasm

A substance of the cell inside the plasma membrane

Consists of

80% of water, Proteins, Carbohydrates, Lipids, Minerals

Nuclear Area

•         Bacterial chromosome – a single circular molecule of DNA

•         Not surrounded by nuclear membrane

•         Plasmids – extrachromosomal genetic elements – small circular DNA molecules, carry genes
for antibiotic resistance
Ribosomes

•         Small globular structures in cytoplasm

•         Composed of 2 subunits  - 70S (30S+50S)

•         Sites of protein synthesis

•         Consists of rRNA and proteins

•         Targeted by some antibiotics

Inclusions

•         Metachromatic granules – reserve of phosphate

•         Polysaccharide granules

•         Lipid inclusions

•         Sulfur granules

•         Gas vacuoles

•         Polyhidroxybutirate

Endospores

•         Resting cells – dehydrated cells with a thick cell wall

•         Formed in unfavorable conditions

•         Layers of spore coats provide resistance to dehydration, high temperatures, toxic chemicals,
radiation

•         25-million-year-old spore germinated

•         Spores germinate into vegetative cells

Basic morphological shapes of bacteria

  Cocci
o   Diplococci –n pairs, Streptococci – in chains; Tetrad - four, Sarcinae - eigth,
Staphylococci – irregular clusters
  Bacilli
 o   Bacillus - single rods; Diplobacilli - in pairs; Streptobacilli in chains;
Coccobacilli – short rods
  Spiral Bacteria
o   Vibrio - curved rods; Spirilla - corkscrew – rigid; Spirochetes - helical – flexible

Dimensions of Bacteria

Average: 1.0-3.0 μm in diameter

Very small: Nanobacteria  -- 0.05 – 0.2 μm

Very big: 300 μm

Chapter 5
The Eukaryotic Microorganisms
 
•         Membrane surrounding DNA -  nucleus
•         Internal membrane-bound organelles
•         Dimensions: 10-100 μm in diameter
•         More complex structure
•         Comprised of algae, protozoa, fungi, animals, and plants
 
The History of Eukaryotes - Endosymbiotic Theory
•         Eukaryotic cell evolved from an association between
–        large anaerobic prokaryote
–        oxygen requiring heterotroph (mitochondria)
–        photosynthetic prokaryote (cyanobacteria)
•         At the begging, they were undigested pray or internal parasites
•         Retained portion of DNA, ribosomes (70S), and cytoplasmic membranes
 
External Structures of Eukaryotic Cells
•         Flagella and Cilia - Projections used for cellular locomotion
•         Few and long
•         Filaments anchored to cell by basal body; no hook
•         May be single or multiple; generally found at one pole of cell
•         Do not rotate, but undulate rhythmically
Cilia
•      Shorter and more numerous than flagella
•      Coordinated beating propels cells through their environment
•      Also used to move substances past the surface of the cell
Internal structure of a flagellum or cilium
•         Cytoplasm containing  microtubules
•         Nine pairs of microtubules arranged in a ring  (2x9 +2)
 •         Microtubules are composed of tubulin
•         Surrounded by the plasma membrane
Cell wall
Various polysaccharides
•         Algae – cellulose, silicate, agar
•         Fungi - chitin (insects)
•         Yeast - glucan and mannan
•         Protozoa - do not have a typical cell wall - flexible outer covering - pellicle
Cytoplasmic membrane
•         Similar to the plasma membrane of prokaryotes (phospholipid bilayer)
–        Functions as a selective permeable barrier
•         Differences:
–        Contain carbohydrates which serve as receptor sites in the “cell to cell”
communication
–        Sterols – provide stability of the membrane
Organelles
•         Functional structures inside the cytoplasm
–        Nucleus
–        Endoplasmic reticulum
–        Golgi complex
–        Lysosomes
–        Vacuoles
–        Mitochondria
–        Chloroplasts
•         Not all the organelles are present in all cells
The Nucleus
•         The largest structure in the cell, spherical or oval
•         Contains DNA + proteins – histones
•         In non-reproducing phase DNA appears as a  threadlike mass – chromatin
•         In reproducing phase chromatin threads become shorter and thicker -  chromosomes
The Nucleus
•         Nuclear envelope - double layered membrane
•         Nuclear pores enable communication of nucleus with the cytoplasm
•         Contains nucleolus (nucleoli) - site of RNA synthesis
Endoplasmic reticulum (ER)
•         Network of flattened membranous sacks
•         Continuous to nuclear envelope
•         Transportation of substances from the nucleus to cytoplasm
Two types of ER:
•         Rough - with ribosomes
–        Synthesis and transport of proteins and phospholipids
•         Smooth - without ribosomes
–        Synthesis of phospholipids, fats, steroids
Golgi Complex
•         Complex of flattened sacks composed of phospholipid bilayer
•         Found close to the ER
 •         Receives proteins packaged in transitional vesicles (budded off the ER)
•         Function: modifies, packages and delivers proteins  by secretory vesicles
•         within the cell
•         outside of cell (vesicle is fused with a cytoplasmic membrane and its content
released - exocytosis)
Lysosomes
•         Membrane enclosed spheres
•         Formed from Golgi complex
•         Contain the catabolic enzymes (including lysozyme)
•         Fuses with the food vesicle - phagolysosome
•         Digest macromolecules, old cell parts, and microorganisms
Mitochondria
•         Rod-shaped or spherical structures
•         Double membrane
–        outer membrane smooth
–        inner membrane folded - forming cristea
•         Matrix - central part of a       mitochondrium
•         Cristae are the sites for many chemical reactions
•         Main role is in the ATP production (“powerhouse of the cell”)
•         Contain their own DNA, replicates independently
•         Contain 70S ribosomes
Chloroplasts
•         Membrane enclosed structure which is the site of photosynthesis
•         Inside the chloroplast there are flattened membrane sacs - thylakoids (stacked together
- grana) – contain chlorophyll
•         Replication by simple division
•         Have 70S ribosomes
Contain their own DNA
Cytoplasm
•         The substance inside the plasma membrane
•         Internal structure – cytoskeleton
–        microfilaments – rods
–        microtubules - cylinders
•         Provide:
–        support and shape of the cell
–        transportation of substances throughout the cell
•         Cytoplasmic streaming - the movement of the cytoplasm
Ribosomes
•         Granular structures - sites of protein synthesis
•         They are either
–        attached to the ER or nuclear membrane
–        free in the cytoplasm
•         Larger than prokaryotic ribosomes (80S - subunites - 60S + 40S)
 
Survey of Eukaryotic Microorganisms
•         Fungi
 •         Algae
•         Protozoa
The Kingdom of the Fungi
•         Morphology:
–        Yeast - unicellular
–        Molds - multicellular 
–        Mushrooms - macroscopic
•         Found in: water, soil, on animal or plant hosts (parasitic)
Molds
•         Body consists of filaments – hyphae
–        Septate hyphae – cross walls (septa) divide hyphae into cell-like units
–        Nonseptate hyphae –the whole hypha is one cell with many nuclei
•         Hyphae make up  mycelium
Fungal Nutrition
•         Fungi are heterotrophs – require organic compounds for their growth
–        Saprobes – on dead plants and animals
–        Parasites – on living organism
•         Fungal infection - mycosis
•         Most fungi are aerobic with exception of yeast (facultative anaerobes)
•         More resistant to osmotic pressure than bacteria
•         Can grow with a very low moisture
•         Can metabolize complex carbohydrates (cellulose, lignin)
Reproduction – formation of spores
Asexual spores
•         Formation of spores by fragmentation of hyphae
–        Conidiospores (spores not enclosed in a sac)
–        Sporangiospores – spores enclosed within a sac – sporangium
Algae
•         Eukaryotic phototrophs
•         Morphology:
–        Microscopic: unicellular, filamentous, colonial  
–        Macroscopic multicellular (seaweed) body is called thallus
•         Ecology
–        Marine and freshwater environments
–        Primary producers
–        Some algae are toxic
Protozoa (first animal)
•         Morphology:
–        Unicellular
–        Lack of cell wall (ectoplasma)
–        Some have a mouth-like opening
•         Nutrition: Heterotrotrophic or parasitic
•         Habitat: water and soil, some are parasitic
•         Reproduction:          
–        Asexual - by mitotic division
–        Sexual - conjugation
 –        Encystment – Cyst enables parasitic protozoa to survive outside a host.
Medically important representatives of Protozoa
Amoebas
•         Entamoeba histolytica causes dysentery when in human intestines.
•         Transmitted between humans through ingestion of the cyst
Flagellates
•         Trichomonas vaginalis – a parasite, found in vagina and in the male urinary tract.
•         Transmitted by sexual intercourse. It does not have cysts – it is sensitive to
desiccation
•         Trypanosoma brucei causes African sleeping sickness
•          Transmitted by tsetse fly
 
 
Chapter 6
 
Viruses
 
General Characteristics of Viruses
•         Are they living organisms?
•         Obligate intracellular parasites
•         Contain either RNA or DNA
•         Use the metabolic machinery of host cell to synthesize their own nucleic acids
•         They have no or just few enzymes of their own
•         Can infect: animals, plants, algae, fungi, and bacteria
•         The size of viruses ranges from 20 to 14,000 nm
Viral components
Naked viruses
•         Virus is composed of
–        nucleic acid
–        protein coat - capsid
•         Capsid is composed of protein subunits called capsomeres
Enveloped viruses
•         Some viruses have the capsid covered with an envelope (lipids, proteins, and
carbohydrates)
Morphology of viruses
•         Based on capsid structure there are three different morphological types:
–        Helical
–        Polyhedral
–        Complex
Helical viruses
Naked helical viruses
•         Long rods
•         The capsid is cylindrical with helical structure (Tobacco mosaic virus)
Enveloped helical viruses
•         Helical nucleocapsid placed within an envelope (influenza)
Polyhedral viruses
 •         The capsid is a regular polyhedron with 20 triangular faces (Poliovirus)
•         Nucleic acid packed in the center
 
The Viral Envelope
•         Roughly spherical
•         Sometimes the envelope is covered with spikes (carbohydrates)
•         Function of capsid/envelope:
–        Protection
–        Attachment of viruses to the host cells
–        Introduction of nucleic acid into host cell
Complex viruses
•         Bacterial viruses – bacteriophages
–        head - polyhedral
–        tail- helical
–        tail fibers
At the Core of a Virus
•         Either DNA or RNA
–        Double/single stranded DNA
–        Double/single stranded RNA
•         Single stranded RNA
–        Positive-sense (directly translated into proteins)
–        Negative sense (need to be converted into a proper form)
•         Genom is small
–        Hepatitis B virus: 4 genes
–        Human genom: 30,000 genes
•         Besides nucleic acids core may contain enzymes required for replication
Adsorption
•         Viruses use their attachment sites (glycoprotein) to attach themselves to receptor sites on
the plasma membrane of animal cell
•         The attachment sites can be:
–        small fibers or spikes on the virus envelope (Influenza)
–        Capsid spike (naked viruses)
•         The receptor for a particular virus is specific – host range
–        Virus can Infect only the specific cells (Hepatitis B – liver cells)
–        Can vary from one person to another
Penetration
Two ways to penetrate the cell
•         1. Endocytosis - it is an active process by which nutrients are brought into a cell
–        Cell membrane folds inwards forming a vesicle.
–        Vesicle is transported inside the cell.
•         2. Fusion with the cell membrane
–        Viral envelope fuses with the host cell membrane
Uncoating
•         The vesicle, viral envelope and capsid are destroyed and the nucleic acid is released into
the cytoplasm
–        Enzymes (of the host cell) degrade the proteins of the capsid
Biosynthesis of DNA viruses
•         The viral DNA takes over the genetic expression of the host
•         Viral DNA is synthesized in the host nucleus by viral enzymes
•         Capsid is synthesized in the cytoplasm by using host enzymes and amino acids
•         Assembly of coat proteins and DNA takes place in the nucleus
•         Newly formed virus particle is transported along the endoplasmic reticulum
Maturation and Release
In enveloped viruses
•         Process is called budding or exocytosis
•         The assembled capsid pushes through the plasma membrane
•         A portion of the plasma membrane becomes the viral envelope
•         The release of viral particles is gradual – there is no sudden death of the host cell
In the nonenveloped viruses
•         Host cell plasma membrane raptures
•         Causes death of the host cell
The Biosynthesis of RNA Viruses
•         Different groups of RNA viruses have different mechanism of mRNA formation
•         An example: Retroviridae – HIV
•         Has reverse transcriptase – to produce double stranded DNA
•         Integrated into host DNA – provirus
•         It remains latent or produces new viruses
Viruses and cancer
•         Some viruses can cause cancer
•         Human and animal genomes normally contain oncogenes
–        Activation of these genes causes cancer.
–        They can be activated by mutagenic chemicals, radiation, and viruses
•         When the oncogenic virus infects the cell, its genetic material is integrated into the host
cell’s DNA.
•         Such infected cells show different characteristic – uncontrollable growth
Latent viral infection
•         Some viruses can remain in host for long period of time without causing any symptoms.
•         Ex: Herpex simplex virus – infection of skin, lives in nerve cells.
•         Large portion of human population carries this virus, only 10-15% exhibits the disease.
Viruses That Infect Bacteria
•         Bacteriophages
•         Most widely studied bacteriophage is T4
•         They can have two types of life cycle:
–        Lytic
–        Lysogenic
Morphology of T4
–        Capsid
–        Tail
–        Fibers
–        Baseplate
Lytic cycle - (in T4 bacteriophage)
Attachment
 •         Tail fibers used as attachment sites,
–        the complementary receptor sites are located on the bacterial cell wall
Penetration
•         The tail sheath contracts and the tail penetrates the cell wall.
–        DNA from the head is injected into bacterial cell. The head remains outside.
Biosynthesis
•         The virus DNA triggers host DNA degradation, stops host protein synthesis
•         The virus uses the host nucleotides and enzymes to:
–        synthesize its own DNA
–        synthesize its own proteins
Maturation
•         Viral DNA and capsid is assembled into a mature viral particle
Release
•         Lysozyme is synthesized within the cell – this causes bacterial cell to break and release
the virus particles
Lysogenic cycle (in bacteriophage lambda)
         After penetration, the viral DNA is integrated into bacterial DNA (prophage)
Lysogenic cycle
•         Action of UV light or some chemicals initiates the lytic cycle
•         The phages that have both of these cycles are called lysogenic phages or temperate
phages
•         The bacterial cell containing a lysogenic phage is called lysogenic cell
Characteristics of Lysogenic Cell
•         It is immune to new viral infection
•         Can exhibit new properties – lysogenic conversion (toxicity of Clostridium botulinum)
•         Can transfer genes from one bacterium     to another - transduction
Cultivation of viruses
Viruses must be cultivated within living cells
Cultivation of Bacteriophages
•         Plaque method
–        Melted agar + host bacterial cells + virus
–        A lawn of bacteria is formed
–        A virus infects the bacterial cell and lyses occurs
–        New infection will result in formation of a clear zone of lysed bacteria – plaque
Growing Animal Viruses
Can be grown in:     
•         Living animals (mice, rabbits, guinea pigs)
–        Some human viruses can not grow in animals
•         Bird Embryos
–        Virus injected in the embryonated chicken egg
–        The death or damage of the embryo indicates the presence of viruses
–        Some virus vaccines are produced by this method
 
 

hapter 7
 Microbial Nutrition, Ecology, and Growth

Microbial Nutrition
•          Nutrition is a process of acquiring chemical substances from the environment
•          The absorbed nutrients are used
–        for energy yielding processes
–        growth
•          The chemical elements absolutely needed - essential nutrients
–        Macronutrients: C, H, O…
–        Micronutrients: Mn, Zn, Ni…

Sources of Essential Nutrients

Carbon
•          Structural backbone of living matter
–        50% of microbial dry weight is C
–        Autotrophs derive C from CO2
–        Heterotrophs derive C from organic matter

Nitrogen
•          14% of microbial dry weight is N
–        required for protein, DNA, RNA, ATP synthesis
•          Microorganisms derive N by:
–        Breaking down proteins into amino acids (reuse of amino acids)
–        NH4, – ammonium ions
–        NO3 – nitrate
–        N2 – nitrogen fixers
•          Free-living
•          Symbionts with plants

Other Elements
•          Sulfur - synthesis of sulfur-containing amino acids
•          Phosphorus - synthesis of DNA, RNA, ATP and phospholipids of cell membrane
•          Trace elements – minerals needed as enzyme cofactors
•          Growth factors – organic chemicals that cannot be synthesized by certain organisms
(vitamins, certain amino acids…)

Nutritional Types

                Energy source Carbon source

Photoauthtrophs Light CO2
Photoheterotrophs Light Organic
Chemoautotrophs Chemical CO2
Chemoorganotrophs Chemical Organic

 Heterotrophs
 •          Chemoheterotrophs
–        Energy and Carbon source from organic molecules
•          Saprobes derive nutrients from dead organic material
–        Opportunistic pathogene – a saprobe infecting  the compromised
host
•          Parasites derive nutrients from living organisms
–        Pathogenes – harm the host (Streptococcus)
–        Obligate intracellular parasites (Rickettsias, Chlamydias, Viruses)

 How microbes eat?

•          Absorb nutrients that are dissolved

•          The molecules need to be small
•          The big molecules are degraded by extracellular enzymes
•          Diffusion of water molecules through a selectively permeable membrane
•          Water molecules will move from the side that has more water to the side with less water
•          Until equilibrium is reached

 Osmotic variations

•          Depending on the concentration of water and solutes on either side of cell membrane, the
cell can be subjected to: isotonic, hypotonic, and hypertonic osmotic conditions
•          Isotonic – water concentration is equal inside and outside
•          Hypotonic solutions have lower solute concentrations; cells placed in these solutions will
swell and burst
•          In hypertonic solution the cellular water passes out of the cell -  Plasmolysis – shrinking
of the cell content inside the plasma membrane
•          Used in food preservation.
•          High concentration of salt or sugar draws the water out of microbial cell.

 The Movement of molecules across the cell membranes

•          Diffusion – movement of molecules from an area of higher concentration to the area of
lower concentration
•          Used for transport of small molecules (O2)

 Facilitated diffusion
•          Substance to be transported -combines with the plasma membrane protein – transporter
•          This changes the shape of the transporter – substance is moved across the membrane and
released
•          No energy needed

Active transport
•          Brings in molecules against a gradient
•          Involves
–        Membrane proteins – permeases
–        Pumps (transport of H+, K+, Na+)
 •          Expenditure of energy (ATP)
•          Group translocation ( a type of active transport)
–        the substance is chemically altered while being transported into the cell

 Eating and drinking by eukaryotic cells

Endocytosis
•          Engulfing particles and molecules from the outside with the cell membrane

Pinocytosis
•          Absorbing liquids (oils)

Phagocytosis
•          White blood cells can ingest whole cells - bacteria

 Environmental Factors that Influence Microbes

•          Physical
–        Temperature    
–        pH
–        Osmotic pressure
•          Chemical
–        Elements (C, N,S,P)
–        Trace elements
–        Oxygen
–        Growth factors

 Temperature
•          Microorganisms have minimum, optimum and maximum growth temperatures

Categories of Microbes Based on Temperature Range

Ranges                 Optimum
•          Psychrophiles                  -100 to 200C         120C
•          Psychotrophs                       00 to 300C          220C
•          Mesophiles                          100 to 500C        370C
•          Thermophiles                     400 to 700C        620C
•          Hyperthermophiles         650 to 1100C      9400C

 Is it possible to make money on unusual microbes?

•          Thomas Brock (1965) isolated the bacterium Thermus aquaticus from the hot springs in 
Yellowstone Park.
•          An unusual microorganism that grows at high temperatures
•          Later on, it was discovered that this organism possess an enzyme (DNA polymerase)
involved in DNA synthesis that is active at 720C
•          With the development of the PCR technology production of this enzyme became a
multimillion-dollar business
About the hot springs
 •          Hot spring – a natural discharge of groundwater with elevated temperatures
•          Geothermal energy- an alternative source of energy
•          Used for different purposes:
•          Bathing, heating, generating electrical energy
•          Hot springs are found all over the world
•          Volcanic areas

Oxygen requirement
•          Obligate aerobs – require O2 to live
•          Facultative anaerobs – can grow in absence of O2
•          Obligate anaerobs – killed by O2
•          Microearophiles – require O2 at concentrations lower than those in air

Oxygen is deadly for obligate anaerobes

•          How can this be true?
•          Superoxide free radical – O2- and H2O2 is formed during the normal metabolism
•          Aerobes produce superoxide dismutase to detoxify O2-  by reducing it to H2O2

                2H2O2                              2H2O + O2 

Anaerobes lack superoxide dismutase

 Effect of pH
•          Most bacteria grow in pH range 6.5-7.5
•          Low and high pH inhibits growth of bacteria
•          Mild acids can help preserve foods by preventing further microbial growth (sauerkraut,
pickles)
•          Acidophiles tolerant to acidity (bacteria and fungi)
•          Alkalinophiles live in alkaline soils and water up to pH 11.5

 Effect of Osmotic Pressure

•          Microorganisms that live in high salt concentration are called Halophyles
•          Obligate halophyles – require high salt concentrations to survive, grow in up to 30% salt
•          Facultative halophyles - tolerate high salt concentrations (up to 2%)

 Microbial Growth
•          There are two aspects of microbial growth:
•          Increase in the cell size
•          Increase in the cell number –The growth of bacterial culture

The growth of bacterial culture

•          Bacterial culture grows by doubling of individual cells (binary fission)
•          Growth of bacterial culture is defined by  “generation time”
•          Generation time (G.T.) is the time required for a cell to divide.
•          For most bacteria G.T. is  30 – 60 min 
•          E. coli -  20 min (1 cell after 20 generations will produce 1 million cells)
•          Logarithmic graphing is used to describe the growth of bacterial culture
 Stages in the Normal Growth Curve
•          Lag phase – little or no change in cell number. Cells are metabolically active (enzyme,
DNA  synthesis)
•          Log phase – exponential growth phase – active reproduction, high metabolic activity
•          Stationary phase - Number of microbial deaths = number of new cells. Metabolic
activity slow.
•          Death phase The number of deaths greater than number of new cells

 Measurement of microbial growth

•          Direct Methods
–        Viable Plate Counts
–        Direct Microscopic Count
•          Indirect Methods
–        Turbidity (not covered in this lecture)
–        Metabolic activity (not covered in this lecture)

Plate Count
•          Suspension of cells (water, milk, urine) is inoculated onto agarized medium

•          Usually serial dilutions are required

•          One single cell is transformed into a visible single colony          
•          Only viable cells are detected
•          Plates that have between  30-300 colonies are counted

 Direct Microscopic Count

•          Specially designed slide - cytometer
•          Slide contains a well with inscribed squares of known area and volume
•          The cells are counted under the microscope, multiplied with the factor that gives the
count per ml
•          Disadvantages of the method:
•          All cells are counted - including dead cells
•          Motile cells are difficult to count
•          High concentrations of the cell are required
•          Advantage of the method
•          It is fast - no need for incubation
 
 

Chapter 8

Microbial Metabolism

Metabolism is
•         Collection of ALL biochemical reactions that take place within cells of an organism
•         The ultimate function of metabolism is to reproduce the organism
Two classes of chemical reactions:
•         ANABOLISM
–        Building of complex molecules from simpler ones
–        Process by which a cell is built up
–        Energy requiring process (endergonic)
•         CATABOLISM
–        Process by which complex molecules are broken down into simpler ones - energy
released (exergonic)

Metabolism includes the following processes:

•         Nutrient uptake
•         Conversion of nutrients into cell components
•         Conversion of nutrients into energy
•         Excretion of waste products

Chemical reactions within the cell are directed by ENZYMES

•         Proteins that catalyze (accelerate) chemical reactions
•         They are specific; they catalyze a single type of chemical reaction
•         After the reaction, they can be reused
•         The enzyme molecule is bigger than a substrate molecule and serves as physical site for
reaction

 Enzymes Structure

Apoenzyme (protein) + Cofactor - nonprotein (metal ion) or

Coenzyme - organic molecule = Holoenzyme

 Active site of the Enzyme

•         Tertiary and quaternary structure of a protein molecule provide the active site – the site
where the substrate binds (groove)

Enzyme-Substrate Interaction
•         Substrate and enzyme make a temporary union
•         Substrate is inserted into the active site
•         The process is reversible

Role of Coenzymes
•         Removes or donates atoms from or to a substrate
•         Electron carriers (remove electrons from the substrate; transfer them to other molecules)
•         Many coenzymes are vitamins
•         Most important coenzymes:
–        Nicotinamid adenin dinucleotide (NAD+)- catabolic reaction
–        Nicotinamid adenin dinucleotide phosphate (NADP+) anabolic reactions
–        Derivatives of B vitamin
The Mechanism of Enzymatic Action
•         Enzyme attaches to the substrate at the active site
•         Enzyme-substrate complex is formed
•         Substrate molecule is transformed

            -products of the reaction are released

•         The unchanged enzyme can start a new reaction

Synthesis and Hydrolysis Reactions

•         Synthesis
–        Anabolic reactions in which two molecules are united into a new bigger molecule
–        This is dehydration reaction (ATP used and H2O molecule released
•         Hydrolysis
–        Digestion of macromolecules
–        Breaking the bonds require an input of H2O

Transfer Reactions: Oxidation-Reduction Reactions

•         Oxidation - removal of electrons (+energy)
•         Reduction - gain of electrons
•         These reactions are always coupled

The Sensitivity of Enzymes to Their Environment

•         Environmental factors affect the activity of the enzymes
•         The enzymes are unstable in extreme conditions (high temperatures, extreme pH,
osmotic pressure)
•         These conditions cause – Denaturation – the shape of the enzyme molecule is changed

Ribozymes- Unconventional Enzymes

•         A form of RNA that can act as catalyst
•         Their only substrate is RNA
•         Removes sections of RNA and joining together the remaining pieces

Types of Enzyme Control

There are two types of enzymes:

•         Constitutive - Present in constant amounts
•         Regulated -Their concentration is regulated
–        Direct Control
•         Competitive inhibition
•         Noncompetitive inhibition
–        Control of Enzyme Synthesis
•         Enzyme repression
•         Enzyme induction

Competitive inhibitors
•         Substances with similar structure as the real substrate
Noncompetitive inhibitors
•         Interact with another part of the enzyme – Allosteric site
•         Causes changing the shape of the active site

Control of Enzyme Synthesis

•         Repression
–        The excess of the end product suppresses the synthesis of the enzyme
•         Induction
–        Presence of a particular substrate induces the synthesis of the relevant enzyme

Energy in Cells

Two types of energy transaction processes

•         Exergonic – release of energy
            X + Y          Z + Energy
•         Endergonic – consumption of energy
            Energy + A + B            C

Generation of ATP (adenosine triphosphate):

Phosphorilation – addition of P to a compound (ADP + P = ATP)

 Catabolism: Getting Materials and Energy

•         Glucose is the most frequent nutrient used for obtaining energy
•         There are three metabolic pathways in which glucose is transformed:
•          Aerobic respiration
•         Anaerobic respiration
•         Fermentation

Respiration of glucose occurs in three stages:

•         Glycolysis
•         The Krebs Cycle
•         Electron transport chain system

Glycolysis (splitting of glucose)

•         Occurs in cytoplasm of most cells
•         Involves splitting of a six-carbon glucose into two three-carbon sugar molecules
•         Direct transfer of phosphate between two substrates– substrate level phosphorylation
•         Net gain:
–        2 ATP molecules,
–        2 molecules of NADH
–        pyruvic acid

Substrate Level Phosphorylation

•         high-energy P directly transferred from a substrate to  ADP
 Glycolysis
•         Three-carbon molecules are oxidized  to
–        2 molecules of pyruvic acid
–        2 NAD 2 NADH+
–        4 ATP formed
•         Energy balance of Glycolysis = 2 ATP

Aerobic respiration

Krebs cycle (Tricarboxylic Acid Cycle)

•         A series of biochemical reactions in which chemical energy is released step by step
•         Starting compound is pyruvic acid (from glycolysis) which is transformed into Acetyl
CoA
•         Acetyl CoA is decarboxilated (loss of CO2 molecule) and entered into tricarboxylic acid
cycle
•         The reduced coenzymes are formed NADH+ and FADH2 (energy stored) – enter the
Respiratory Chain
•         In this cycle ATP, NADH and FADH are formed
•         NADH and FADH enter the electron transport chain
•         Total (Glycolysis + Krebs cycle + electron transport chain) =  38 ATP/glucose

Electron transport chain

•         Sequence of carrier molecules that can be oxidized and reduced
•         Located in plasma membrane of prokaryotic cells or in mitochondria in eukaryotic cells
•         Energy is released by transfer of electrons from high-energy to lower energy compounds
•         Protons (H+) are actively pumped out

ATP synthesis – Chemiosmosis

•         Accumulated protons (H+) from outer compartment diffuse through the ATP synthase to
inner compartment
•          Rotation of the ATP synthase causes bonding of ADP + Pi         ATP

Anaerobic Respiration

         Final electron acceptor is an inorganic molecule, other than O2

o   NO3-  +  NADH       NO2-   +   H2O   +   NAD+ (process called
denitrification)

Fermentation
•         Further oxidation of Pyruvic acid (obtained in glycolysis) without the presence of O2
•         Partial oxidation without the presence of O2
•         Final product is organic molecule (not H2O)
•         Only small amounts of ATP is recovered
•         Different bacteria perform different types of fermentation
•         Final product: lactic acid, ethanol, propionic acid, CO2, H2, acetic acid, etc.
Photosynthesis
•         Present in plants and photosynthetic bacteria and algae – Photoautotrophs
•         Conversion of light energy into chemical energy
•         Chemical energy used for conversion of CO2 into reduced carbon compounds (sugars).
 
 
 

Chapter 9

Microbial Genetics

What is Genetics?
•         The science of heredity
•         Research in Genetics takes place on several levels
–        Organismal
–        Cell
–        Chromosome
–        Molecular

Where genetic material can be found?

•         Genetic information is contained within genetic material – DNA and RNA
•         It can be found in:
•         Prokaryotic organisms
–        Nucleoid (bacterial chromosom)
–        Plasmids
•         Eukaryotic organisms
–        Chromosome
–        Mitochondria
–        Chloroplasts
–        Plasmids
•         Viruses
–        DNA
–        RNA

Some definitions
•         Chromosome – Cellular structure -packaged DNA molecule
•         Gene – A segment of DNA that codes for functional product (protein)
•         Genotype – The sum of all genes -Genetic make-up
•         Phenotype - Manifestation of genotype (ability to perform particular chemical
reaction)

The size and packaging of genomes

•         E.coli 4,288 genes
•         Human cell: 20,000 – 25,000 genes
 •         Size of the cell of E. coli – 1 m (diameter)
•         Size of the stretched out DNA – 1mm (1000 times longer)

DNA molecule is tightly packed in the form of chromosome

Structure of DNA
•         DNA molecule – double helix
•         Composed of nucleotides
•         Nucleotides composed of
–        Bases
•         Purine (adenine, guanine)
•         Pyrimidine (cytosine, thymine)
–        Deoxyribose sugar
–        Phosphate group
•         Single strands of DNA have opposite orientation (3’ and 5’)
•         G-C held together with 3 hydrogen bonds
•         A-T held by 2 hydrogen bonds

DNA Replication – overall process

       Topoisomerase unwinds the double helix of DNA
       Helicases break hydrogen bonds
       Bases of single stranded DNA exposed
       Replication fork is formed
       Synthesis of the new strand by attachment of complementary nucleotides

Other enzymes involved in DNA replication

•         Helicase – Untwists the DNA helix by braking the H bonds
•         Primase - Synthesis of RNA primer
•         DNA polymerase III – adding bases to the new DNA
•         Ligase - joining of the of DNA fragments
•         Topoisomerase I, II  - Supercoiling

Details of replication
•         Helicase opens the double helix
•         Complementary nucleotides attach themselves to the exposed
bases                                          
A-T                        C-G
•         DNA polymerase III joins the added nucleotide into a growing DNA strand
•         Semiconservative replication (double stranded DNA contains one old and one new
strand)

DNA Replication
•         Leading strand is synthesized continuously
•         Lagging strand is synthesized in pieces (Okazaki fragments)
•         The strands are synthesized in the 5’ to 3’ direction
•         DNA polymerase I removes RNA primers
 •         Lygase joins the fragments

The Flow of Genetic Information

•         DNA Replication – reproduction of cells
•         Transcription – information contained within DNA is copied  RNA
•         Translation – polypeptides synthesized from RNA nucleotide sequences  amino acid
sequence of protein

The difference between DNA and RNA

                                    DNA               RNA

Sugar component:    desoxyribose     ribose

                                    G -----------C

                                    C------------G

                                    T-------------A

                                    A------------ U  (uracyl)

Transcription
•         Synthesis of RNA from DNA as a template
•         Messenger RNA (mRNA) is formed by transcription of a portion of DNA (gene)
•         Sequence of nucleotides from DNA are rewritten in RNA

Process of transcription
•         RNA polymerase binds to the promoter    
•         Unwinds the double helix of DNA
•         One DNA strand acts as a template for synthesis  of RNA
•         RNA polymerase puts free nucloetides together forming RNA chain
•         As the new RNA grows, polymerase  moves along the DNA
•         When the RNA polymerase reaches the sites called terminator - the   transcription ends
- RNA polymerase and new RNA strand are released

Translation
•         Is the process of translating nucleic acid language into the language of proteins
(sequence of amino acids)
•         Codons are groups of three nucleotides (mRNA)
•         The sequence in the codon determines which amino acid will be incorporated into a
protein
•         Translation is taking place in ribosomes

The beginning of protein synthesis

•         Ribosom moves along mRNA and reads the codons
 •         Transfer RNA (tRNA) has a sequence of three bases – anticodons complementary to
codons
•         For each amino acid there is a specific tRNA
•         The first tRNA recognizes the start codon and brings methionine

Elongation and termination

•         Peptide bonds are formed between amino acids -polypeptide is formed
•         Blank tRNA discharged
•         When nonsense codon is reached, translation is terminated
•         Ribosome is separated into its subunits

Simultaneous transcription and translation

•         This is a fast process
•         More than one ribosome reads one mRNA molecule
•         Most genes (75%) are constitutive – their products are produced constantly
•         Other genes are regulated so that they are transcribed and translated only when needed

Transcription and translation in Eukaryotes

•         The introns – non-functional segments of DNA) are transcribed but not translated
•         The genes need to be processed
•         The introns are cut out
Mechanisms of Genetic Control
•         Repression
–        Inhibition of gene expression
•         Mediated by a protein – repressor
•         Induction
–        Induction of gene expression (transcription)
•         Inducer – a substance that induces transcription
Mechanisms of Genetic Control
•         Repression
–        Inhibition of gene expression
•         Mediated by a protein – repressor
•         Induction
–        Induction of gene expression (transcription)
•         Inducer – a substance that induces transcription
Operon model of gene expression
Inducible operon
Lactose operon (regulates lactose metabolism) - must be activated by inducers
Functional gene consists of:
            Regulatory gene
            Operon
                   Control region
                        Promoter - RNA polymerase initiates transcription
                        Operator – go or stop signal
                   Structural genes
                        - b-galactosidase
                        - lac permease
                        - transacetylase
Changes in the Genetic Material
•         Mutation – change of the genetic code
•         Horizontal gene transfer
–        Transformation
–        Conjugation
–        Transduction

Mutation
•         During the replication of DNA - an error in base sequence may occur - mutation
•         It can be:
–        Spontaneous
–        Induced
•         Mutation can have beneficial or deleterious (lethal) effect
–        beneficial - mutant enzyme - enhanced activity (rare)
–        lethal - lost activity of the enzyme (almost always)

Categories of mutation
•         Point mutation – substitution, insertions, and deletions of a single base in the DNA
•         Missense mutation – substitution of one amino acid (Inactive or reduced activity
protein)
•         Nonsense mutation - creating a stop codon in the middle of protein
•         Frameshift mutation -deletion or insertion of a DNA fragment; causes changes in
many amino acids

Mutagens
•         Chemicals - nitrous acid (converts adenine into form that pairs with C instead of T)
•         Nucleoside analogs  (molecules that are structurally similar to normal bases)
•         Radiation-
–        X-rays, gamma rays (errors in replication; physical damage of DNA)

Transformation
•         Fragments of the DNA are transferred from one microorganism to another (competent)
– incorporated into genom
•         Experimentally shown (but not understood) by Frederick Griffith in England 1928   

Conjugation
•         “Sexual contact” between two mating cells
•         Donor cell has sex pilli and F+ (fertility factor) a gene located on the plasmid
•         DNA is transferred from one cell to another through sex pillus

Plasmids
•         Small circular self-replicating DNA elements
–        Conjugative plasmids
 –        Enable survival under challenging conditions (exotic substrates –
hydrocarbons, etc)
–        Production of toxins (kill other bacteria)
–        Resistance factors (antibiotics)
 
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Ch. 10 Genetic Engineering

 
•         Biotechnology - Application of biological systems (microorganisms) to obtain a product (food,
antibiotics, vitamins)

•         Recombinant DNA technology – procedures by which a fragment of DNA (gene) of one organism is
incorporated into the genom of a different organism
 
Goals of Genetic Engineering
•         Create organisms that synthesize products humans need (insulin)
•         Eliminate undesirable phenotypic traits (e.g. supression of ripening in tomatoes)
 
Tools and Techniques of Genetic Engineering
•         Restriction enzymes – major tool
•         Analysis of DNA – gel electrophoresis
•         Nucleic acid hybridization
•         DNA sequencing
•         Polymerase Chain Reaction - PCR
 
Restriction Enzymes
•         DNA cutting enzymes
•         They recognize and cut specific fragments of DNA  (sequences of nucleotides in DNA)
•         They leave single stranded sticky ends of DNA
•         DNA from different sources cut with the same restriction enzyme will produce the same type of
sticky end
•         DNA is cut on a specific  Palindromic sequence
•         Palindromes are sequences that are identical when read in opposite directions in two strands
 
DNA Gel electrophoresis
•         Separation of DNA fragments based on their size
•         In agarose gel, DNA fragments are subjected to an electrical current
•         DNA molecule has a negative charge – moves toward the positive pole
•         Smaller fragments move faster
 
Nucleic Acid Hybridization
•         A fragment of a single-stranded nucleic acid (DNA, RNA) can hybridize (unite) with another
fragment that has a complementary sequence of nucleotides
 
Hybridization with a probe
•         The method used to detect specific nucleotide sequence in an unknown sample by using a
gene probe
•         Gene probe is a short segments of DNA of a known sequence
•         A probe carries a radioactive label 
 
DNA sequencing
•         A process in which exact sequence of nucleotides in a DNA segment is determined
 
Polymerase chain reaction - PCR
•         Technique by which small amount of specific DNA fragment can be amplified in vitro
•         What is needed?
–        PCR machine - thermal cycler
–        Target DNA that serves as a template
–        Supply of 4 nucleotides
–        DNA polymerase
–        Primers
•         Primers are short fragments of DNA that are complementary to the target DNA
One PCR cycle has 3 basic steps:
•         Denaturation: 940C – separation of DNA strands
•         Priming: 50–65 0C, primer attached to complementary strand of DNA
•         Extension: 720C
     DNA polymerase extends the molecule by adding nucleotides
•         Typically we use 20-40 cycles – millions of copies of DNA
 
Recombinant DNA Technology – The procedure
•         A selected gene is removed from the genetic donor
•         This gene is incorporated into a vector (plasmid or virus)
•         The vector is inserted into the cloning host (bacteria, yeast)
 
Vectors
•         Vectors are DNA molecules into which a segment of foreign DNA can be incorporated
(plasmids, transposons and viruses)
•         Vector’s characteristics:
–        self-replicating
–        circular shape
–        proper size – to be able to accept foreign DNA
–        must have a promoter
–        must have a gene for antibacterial resistance
 
Inserting foreign DNA into cells
Transformation
•         Plasmid from the surrounding environment is taken up by a cell
•         Cells have to be made competent - by soaking them in calcium chloride
 
Screening of bacteria that contain foreign DNA
•         The vector (plasmid) contains the gene for ampicilin resistance
•         Just those cells that have been transformed can grow on medium containing ampicilin
 
Synthetic DNA
•         DNA synthesis machine
•         Short fragments of DNA (120 nucleotides) can be synthesized
•         We must know the sequence of the DNA fragment that we want to synthesize
 
Applications of Recombinant DNA Technology
•         Pharmaceutical and Therapeutic Applications
–        Protein synthesis
–        Vaccines, DNA vaccines
–        Genetic screening
–        DNA fingerprinting
–        Gene therapy
 
Some examples
•         Hormone insulin, needed by diabetics
•         The gene for insulin was cloned into E. coli
•         Before it was obtained from the pancreases of slaughtered  animals
•         The first commercial success of recombinant DNA technology
 
•         Somatostatin - Human hormon used for treatment of giantism (an excessive secretion of growth
hormone)
•         Before - 500,000 sheep brains were needed to produce 5 mg of somatostatin.
•         Today - 8 l culture of genetically engineered bacteria to obtain the equivalent amount
 
Subunit vaccines
•         A protein portion of the virus is cloned
•         Hepatitis B vaccine (Saccharomyces cerevisiae carries the virus gene on a plasmid)
•         Advantage - there is no chance of becoming infected during vaccination
 
DNA vaccines
•         A single gene from that pathogen is artificially copied and multiplied.
•         That gene is then injected into a muscle. Muscle cells tend to take up this gene and use it as
one of their own genes, making the product
•         The immune system will recognize that product as foreign, and will start producing antibodies
•         A single gene from that pathogen is artificially copied and multiplied.
•         That gene is then injected into a muscle. Muscle cells tend to take up this gene and use it as
one of their own genes, making the product
•         The immune system will recognize that product as foreign, and will start producing antibodies
 
Agricultural applications
•         Transgenic organisms – recombinant plants and animals altered by addition of genes from
other organisms
•         Improving Crops
–        Herbicide resistance
–        Salt tolerance
–        Freeze resistance
–        Pest resistance
–        Improvements in nutritional value and yield
 
Creating transgenic plants
•         Agrobacterium tumefaciens - plant parasite that can incorporate its DNA into plant’s genom by
using Ti plasmid
 •         Ti plasmid can be engineered to contain a new gene
 
Some examples
Pest resistance - Tomato Plant with Bacillus thuringiensis toxin
Resistance to herbicides
•      Glyphosate (Roundup) kills all plants
•      Gene for resistance to glyphosate incorporated into   crop plants
•      Now farmers can kill weeds without killing crop
•         MacGregor tomatoes- Gene for pectin degradation suppressed, so they have a longer shelf life
 
Transgenic Animals
•         Why to create a transgenic animals?
•         The product (protein) can be collected in milk or semen
•         Many human genes have better expression in animals than in bacteria
•         Foreign genes are inserted into an embryo by using a virus or an injection
 
Gene Therapy
•         Mostly preliminary work
•         Missing or defective genes replaced with normal copies
•         Possible treatment for: cystic fibrosis, sickle cell anemia, some types of hemophilia, some types
of diabetes
 
Antisense DNA and RNA
•         Antisense strand of DNA recognizes and binds to the complementary mRNA fragment
•         This results in blocking the expression (translation) of the harmful gene
•         Antisense drugs are being researched to treat cancers and other diseases
 
Genetic screening
•         Many genetic diseases can be detected by genetic engineering techniques
•         Technique: Southern blotting (Ed Southern 1975)
•         Inherited forms of breast cancer can be detected
 
HOME

hapter 11

Physical and Chemical Control of Microbes

The purpose of controlling microbial growth – To stop spreading the diseases

or food spoilage

Methods:
•          Physical agents
–        Heat
–        Radiation
 •          Chemical Agents
–        Gases
–        Liquids
•          Mechanical removal
–        Filtration
•          Air
•          Liquids

Methods of Microbial Control

•          Sterilization - Destruction of all forms of microbes including endospores
(by steam under pressure or ethylene oxide)
•          Disinfection -Destruction of vegetative cells of pathogenic
microorganisms (by chemicals or physical methods)
•          Pasteurization - Application of high temperature (720 C) for short period
of time (15 sec) with the purpose of reducing the number of microbes
•          Antiseptic - Antimicrobial agent that is sufficiently non-toxic to be
applied on living tissue
•          Sanitization - Lowering the number of microbes on eating and drinking
utensils (by heat or chemical disinfectant)
•          Decontamination – Mechanical removal of microbes from organisms or
non-living objects

Terminology
•          Bactericidal (germicidal, microbicidal)- agent that destroys or kills
bacteria (suffix  cide - kill)
•          Bacteriostatic - agent that inhibits bacterial growth  (stasis - to stop)

What is Microbial Death?

•          Permanent loss of reproductive capabilities
•          The cell structures become dysfunctional
•          Antimicrobial treatment leads to killing of microbial population at the
constant rate

Factors that affect death rate:

•          Time of exposure (lower temp. can be compensated with longer
exposure)
•          The number of microbes
•          Microbial characteristics (endospore, vegetative cells)
•          Agent used
•          Environmental influences (suspending medium, pH)

The Mode of Action of Antimicrobial Agents

 •          Plasma membrane - when damaged, cell content leaks into the
surrounding medium
•          Proteins- enzyme active sites inactivated
–        Complete denaturation
–        Different shape
–        Blocking the active sites
•          Nucleic Acid - radiation or some chemicals lethally damage the DNA or
RNA (microbes can no longer replicate)
–        UV radiation causes formation of dimmers between two thymine
bases

Physical methods of microbial control

Heat
•          Moist heat and dry heat
•          Mechanism: denaturing the enzymes
•          Most commonly used method of killing the microbes
•          Thermal death point - the lowest temp at which all the microbes are
killed in 10 min
•          Thermal death time – the minimal length of time needed to kill all
bacteria at given temperature

Moist heat – nonpressurized steam

•          Mechanism: coagulation of proteins
•          Boiling (1000C) for 10 min kills vegetative cells of bacteria, viruses, and
fungi
•          Hepatitis virus can survive up to 30 min of boiling; some bacterial
spores can survive more than 20 h.

Tyndalization – boiling the medium for 60 min repeatedly for 3 day

Autoclaves – steam under pressure

•          Provide high temp. and high pressure (Pressure: 1 atm, temp.: 1210 C)
•          All microbes are killed in 15 min
•          Steam should contact all surfaces
•          Time is different for larger volumes
•          Used for sterilization of:
–        Culture media
–        Equipment
–        Biological waste

Pasteurization
 •          Original pasteurization: 630 C for 30 min
•          Today’s pasteurization – high temperature short-time pasteurization:
720 C for 15 sec. or
•          Ultra-high-temperature treatment - Exposure to 1340 C for 3 sec. then
rapidly cooled

Dry heat sterilization

•          Mechanism: oxidation
•          Flaming – inoculating loops
•          Hot-air sterilization
–        Oven - 1700 C for 2h

Desiccation
•          In the absence of water microbes cannot grow but can survive
•          Bacterial spores can survive for centuries           
•          Survival depends on microbial type and organism’s environment
(embedded in mucus - better survival)
–        Mycobacterium tuberculosis – long survival
–        Neisseria gonorrhoeae – dies after a few hours of air drying

Low temperatures
•          Effect depends on the microbial type
•          Ordinary refrigeration (0-70 C) - bacteriostatic effect
•          Psychotrophs grow slowly
•          Pathogenic bacteria will not grow
•          Rapid freezing – microbes become dormant
–        Lyophilization – frozen samples (bacterial cultures) dried in
vacuum

Slow freezing – more harmful

Can Microbes Survive Millions of Years Traveling in Space? Experts Say

"Yes"

Radiation
         Ionizing radiation (gamma rays, X rays) -radiation ejects electrons –
ions are formed
         Non-ionizing radiation (UV light)

Ionizing radiation
•          Short wavelength, high energy
•          Emitted by radioactive elements (Co)
 •          Mechanism of action: ionization of water which forms hydroxyl radicals
which react with DNA
•          Used for sterilization of
–        Medical supplies (plastic syringes, Petri plates etc.)
–        Certain food (spices, meat, vegetables)

Nonionizing radiation
•          UV light, germicidal light – 260 nm – used for disinfection
•          Mechanism of action:
–        damage of DNA – formation of thymine dimmers
–        Toxic free radicals are formed
•          Sterilization of the air (hospital rooms, operating rooms, cafeteria)
•          Disadvantage
–        Poor penetration
–        Harmful for human eyes, skin

Filtration
•          Removal of microbes from a solution
•          Membrane filters (pore size 0.2 or 0.45 um)

Osmotic pressure
•          High concentration of salt causes water to leave the cell
•          Used in preservation of food (high sugar concentration - fruit preserve)

Chemical methods of microbial control

•          Effectiveness of the disinfectant depends on:
•          Type of the chemical agent
•          Type of microbes
•          Concentration of a disinfectant
•          Time of contact
•          pH of the medium
•          Temperature

Types of Disinfectants

Halogens
•          Fluorine, bromine chlorine, and iodine
•          Iodine is the oldest antiseptic
•          Iodine tincture – skin disinfection
•          Chlorine - gas (Ca-hypochlorite; Na-hypochlorite- bleach)
•          Mode of action: oxidizing agent - alters cellular components
•          Disinfection of drinking water, swimming pools, household (bleach)
Phenolics (derivatives of phenol)
•          Used first time by Lister – carbolic acid
•          Mechanism of action: damages the plasma membrane, enzyme
inactivation
•          Advantage: active even in the presence of          organic compounds
•          Hexachlorophene (bisphenol) used in antimicrobial soaps        

Is antibacterial soap any better than regular soap?

•          The antibacterial components of soaps need to be left on a surface for
about two minutes in order to work.

Alcohols
•          Ethanol or isopropanol 60% - 95%
•          Kills vegetative cells of bacteria and fungi (not spores and nonenvelope
viruses)
•          Mechanism of action: protein denaturation

Is pure ethanol a better disinfectant than 70% ethanol? Why?

•          100% ethanol coagulates proteins in the cell wall
•          70% ethanol penetrates the cell wall and coagulates the proteins inside
the cell

Hydrogen Peroxide
•          3% solution used as an antiseptic
•          Skin and wound cleansing
•          Mouthwash
•          Contact lens
•          Surgical implants
•          Endoscopes

Chemicals with surface action: Detergents / Soaps

•          Detergents are polar molecules - surfactants
•          Decrease the surface tension among molecules and water
•          Soaps and Detergents are not antiseptics – they break the oily film on
the surface of skin
•          They have microbicidal power when mixed with quaternery ammonium
compounds

Heavy metals
•          Silver, mercury, copper, gold, arsenic
•          Only mercury and silver have germicidal significance
 •          Mechanism of action: ions combine with sulfhydril groups - protein
denaturation
•          1% Silver nitrate - antiseptic
•          Copper sulfate - controls algal growth
•          Can be toxic to humans

Evaluation of a disinfectant

         Filter paper method

•          Paper disks are soaked in a solution of disinfectant and placed  on a
agar previously inoculated with a test organism
•          Observe the inhibition zone around the disk

Aldehydes (formaldehyde, glutaraldehyde)

•          Most effective antimicrobials
•          Formalin - used for preservation of biological specimens
–        High level disinfectant
–        Toxic - carcinogenic
•          Glutaraldehyde –
–        Used for disinfection of hospital instruments
–        Mode of action: forms covalent cross-links with functional groups
of proteins
–        Kills bacterial spores, fungal spores and viruses
 
HOME

Chapter 12

 Antimicrobial Drugs

 
•         Chemical substances used for treatment of infectious diseases - chemotherapy
•         Antimicrobial drugs have selective toxicity (harmful against microbes and not the host)
•         Antibiotic – substance produced by one microorganism that is inhibitory or toxic against
other microorganism
•         Penicillin was the first antibiotic discovered by Alexander Fleming in 1928. 
•         Mold – Penicillium notatum. Its commercial use started in 1945
•         New antibiotics are being discovered by screening large number of microbes (400,000
screened; 3 useful drugs)

Spectrum of Antibacterial Activity

•         An antibiotic can be effective against a
–        narrow group of microbes (Penicillin G – against Gram positive bacteria)
 –        broad-spectrum antibiotic
•         disadvantage- eliminates also the normal microflora

Action of Antimicrobial Drugs

•         Bactericidal – kills the microbes
•         Bacteriostatic – inhibits the growth

Modes of antimicrobial action:

•         Inhibition of
–        Cell wall synthesis
–        Protein synthesis
–        Cytoplasmic membrane synthesis
–        Synthesis of essential metabolites
–        DNA synthesis

Inhibitors of cell wall synthesis

•         Penicillins and cephalosporins interfere with the formation of peptidoglycan layer in the
cell wall of bacteria (not present in eucaryotic cells). Bactericidal.
•         Block the enzymes that cross-link N-acetyl glucosamin and N-acetyl muramic acid
•         Less effective against Gr negative bacteria – do not penetrate the outer membrane

Penicillins
•         Natural penicillin (Penicillin G)
•         It has a narrow spectrum (staphylococci, streptococci, and spirochetes)
•         It is rapidly excreted from the body
•         More efficient when injected than when taken orally.
•         It is susceptible to penicillinases (enzymes that cleave the penicillin molecule)

Semisynthetic penicillins
•         Partially produced by the mold Penicillium and partially by a chemical process
•         The change of the shape of the molecule makes it more difficult to break up by
penicillinase.

β-Lactamase (penicillinase) action

•         Bacteria resistant to penicillins have the enzyme  b-lactamase
•         It breaks the Lactam ring

Some other cell wall inhibitors

•         Cephalosporins
–        Similar to penicillin, resistant to b-lactamase
–        Oral administration possible
•         Bacitracin
–        Polypeptide antibiotic effective against Gr + bacteria
–        Major ingredient of Neosporin – used for skin infections

Protein Synthesis Inhibitors

 •         Aminoglycosides (Streptomycin)
–        Change in the 30S ribosome shape; mRNA misread
–        Isolated from Streptomyces
•         Chloramphenicol
–        Inhibits formation of polypeptide chain
–        Bacteriostatic
–        Use restricted – suppresses formation of blood cells
Tetracyclin
–        Interferes with attachment of tRNA to mRNA
–        Effective against Gr+ and Gr-
–        Bactriostatic
•         Erythromycin
–        Binds to ribosome and prevents its movement along the mRNA
–        Not effective against Gr negative bacteria
–        Bacteriostatic

Injury to Plasma Membrane

•         Polymixin B
–        changes the permeability of plasma membrane
–        Effective against Gr – bacteria -  Pseudomonas
–        Bactericidal effect

Inhibition of nucleic acid synthesis

•         Rifampin –
–        Inhibits the synthesis of mRNA
–        Used for treatment of mycobacterial infections (tuberculosis)
–        Good penetration of tissues
–        Cannot penetrate the cell envelope of Gr negative bacteria
–        Bactericidal

Inhibitors of the Synthesis of Essential Metabolites

Sulfanamides
•         Bacteria require PABA  (para-aminobenozoic acid) for synthesis of folic acid (essential
for synthesis of nucleic bases)
•         A sulfanil amid drug mimics the PABA and acts as competitive inhibitor

Antifungal drugs
•         Restricted use - target the same mechanisms as those found in higher animals (protein
and nucleic acid synthesis)
•         Amphotericin B is the most commonly used antifungal antibiotic
•         Produced by Streptomyces
•         It combines with sterols - components of fungal plasma membrane causing an increase of
its permeability
•         The drug is toxic to kidneys
Griseofulvin
 •         Produced by Penicillium
•         Effective against fungi infecting hair and nails
•         Interferes with mitosis (reproduction)
•         Binds to keratin

Antiviral drugs
•         Viruses cause 60% of infectious diseases
•         There is a limited number of antiviral drugs because viruses are endocellular pathogens

Actions of Antiviral Drugs

•         Inhibition of virus entry
–        Fuzeon – prevents binding of viral (HIV) receptor to cell receptor
–        Amantadine – prevents entry of influenza virus into cell
•         Inhibition of nucleic acid synthesis
–        Acyclovir – Purine analog – terminates DNA replication
–        Zidovudin – reverse transcriptase inhibitor (HIV)
•         Inhibition of viral assembly
–        Indinavir – protease inhibitor – prevents maturation of virus particles

Analogs of Viral DNA and RNA components

Acyclovir
•         Effective against herpesvirus (genital herpes)
•         The drug has a structure similar to guanosine nucleoside (component of DNA).

How bacteria evade the action of antibiotics?

•         Inactivation of the drug (b-lactamase)
•         Prevention of penetration
•         Efflux – pumping the drug out of the cell    video
•         Alteration of the drug’s target sites (mutation in a single amino acid in the ribosome)

Natural selection and Drug Resistance  Where does it come from?

•         Any microbial population contains individual cells with a prior mutation (drug
resistance)
•         If the population is exposed to a drug – the resistant cells will eventually become
dominant

The Rise of Drug Resistance

The reasons:
•         Use of antibiotics for cold or influenza
•         Patient’s failure to follow the prescribed treatment
•         Long-term, low-dose treatment of acne
•         Use of antibiotics in animal feed (antibiotics promote growth and weight gain in farm
animals)
MRSA - (Methicilin Resistant Staphylococcus aureus)
•         Methicilin – the first semisynthetic penicillin
•         Designed to evade the action of penicillinase
•         Staphylococci developed resistance
•         Methicillin discontinued in the US

New Approaches to Antimicrobial Therapy

•         Probiotic approach - desirable bacteria are included in animal’s feed; this could prevent
development of harmful bacteria

Side Effects of Antibiotics

•         Hearing impairment
•         Liver and kidney damage
•         Toxic effect when taken with other drugs

Other Side Effects

•         Diarrhea
•         Allergies
•         Candida albicans vaginal infections
•         Clostridium difficile large intestine infections (pseudomembranous colitis).

Suppression of Normal Microbiota

•         Beneficial bacteria colonize human body
•         Broad-spectrum antibiotics targets the infectious agent but also the normal microflora

Testing of antibiotic effectiveness Complication called superinfection

•         The treatment of bacterial infection is based on estimation which antibiotic is the most
effective
•         If the case of bacterial resistance, the isolated strain should be tested against different
antibiotics

The diffusion method

•         The surface of agar medium is uniformly inoculated with a test organism
•         The filter paper discs impregnated with different antibiotics
•         The effective antibiotic will inhibit the growth of bacteria - clear zone will occur.
•         The diameter of zone indicates the effectiveness of the antibiotic

Broth dilution test

•         Broth containing different antibiotic concentrations is inoculated with the test bacterium
•         A minimal concentration of antibiotic that prevents a visible bacterial growth is
called minimal inhibitory concentration (MIC)
•         Minimal bactericidal concentration (MBC) is the minimal concentration of an antibiotic
that kills bacteria (not only inhibits)
 
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 Chapter 13

Microbe – Human Interactions

Three types of microorganisms are associated with human body

•         Normal microbiota – microorganisms colonizing the human body without harming it
•         Transient microbes – rapidly lost
•         Pathogenic microbes – invade and damage the cells and tissue

Acquiring Resident Biota

•         The uterus is germ-free
•         First exposure to microbes occurs during the passage through the birth canal
•         Feeding
•         Contact with the environment and family members

The Progress of Infection

•         Infection starts with an invasion of a pathogen
•         How successful this invasion will be, depends on:
–        Pathogenicity – the ability of a microbe to cause disease in  another organism
–        Virulence – the degree of pathogenicity; relative ability of a microbe to cause a
disease
•         Virulence factors (enzymes, toxins, capsule…)

Establishing the Infection

•         The microbes enter the body through -Portal of entry
–        Attaching to the host
–        Surviving host defenses
–        Causing disease

How Microorganisms Enter a Host?

Portals of entry:
•         Mucous membrane (Lines the body cavities that are open to the environment)
•         Skin (openings or cuts)

Infectious agents that enter the skin

•         Skin is a tough barrier
•         Broken skin enables penetration of microbes
•         The common infectious agents:
–        Staphyloccus aureus (boils)
–        Streptococcus pyogenes (impetigo)
–        Clostridium (gangrene, tetanus)
•         Other ways of penetrating the skin
–        Digestive enzyme of microbes
–        Insect bites
–        Hypodermic needles
 •         Mucous membrane
–        Respiratory tract is the most common portal of entry - through inhalation
(common cold, influenza, pneumonia, tuberculosis …)
–        Gastrointestinal tract - through food, water and contaminated hands (hepatatis
A, typhoid fever, amoebic disentery …)
–        Genitourinary tract - through sexual contact (STD’s)

Is the number of invading microbes important?

•         A certain (minimal) number of microbes have to enter the body in order to cause an
infection – infectious dose
•         Infectious Dose for:
–        Q fever (Rickettsia) – 1 cell
–        Tuberculosis – 10 cells
–        Gonorrhea – 1000 cells
–        Cholera – 1,000,000,000

Preferred portal of entry

•         Pathogens have preferred portal of entry
Ex: Streptococcus that is inhaled can cause pneumonia, when swallowed  it does not
show symptoms

Attaching to the Host

•         Microorganisms attach themselves to the host cell through:
•         Fimbriae (bacteria)
•         Capsules (bacteria)
•         Spikes (viruses)

Adherence
•         Attachment is based on binding of specific molecules on both host and pathogen
•         Surface molecules of the pathogens (ligands) bind to the specific molecules of host
tissue cells (receptors)
•         Ligands are located on glycocalyx or on fimbriae

Surviving Host Defense

•         Pathogenic microbes are recognized by white blood cells – phagocytes
•         Phagocytes engulf and destroy the pathogen
•         Some microbes know how to evade phagocytosis
–        Produce toxins that kill phagocytes (leukocidins)
–        Produce capsule
–        Can survive inside the phygocyte

Causing Disease

How Virulence Factors Contribute to Tissue Damage?

•         Virulence factors:
1. Exoenzymes
 2. Toxins   
      •Exotoxins
      •Endotoxins
3. Blocked phagocytic response 

  

Exoenzymes
•         Exoenzymes can dissolve structural chemicals in the body
–        Hyaluronidase - hydrolyses hyaluronic acid  (a substance that cements animal
cells together)
–        Coagulases – coagulate (clot) the fibrinogen (protection against phagocytes)
–        Kinases – digest the blood clots - formed to isolate the infection

Bacterial Toxins
•         Exotoxin
–        Actively excreted by a living bacterial cell
•         Endotoxins
–        Part of the cell wall of Gr negative bacteria

Exotoxins
•         Released from bacterial cells (Gr+ or Gr-)
•         They are proteins, some are enzymes

Naming exotoxins:
–        Hepatotoxins
–        Cytotoxins
–        Neurotoxins
–        Enterotoxins

Representative Exotoxins
•         Botulinum toxin – Clostridium botulinum
–        Neurotoxin that prevents the transmission  of impulses from the nerve cell to
the muscle
–        Flacid paralysis
•         Tetanus toxin – Clostridium tetani
–        Blocks the relaxation pathway of muscles; uncontrollable muscle contraction.

Endotoxins
•         Is part of the outer layer of the cell wall of Gram negative bacteria
•         Endotoxins are lipopolysaccharides
•         Endotoxin has to be released from the cell wall in order to cause the symptoms
•         The symptoms are: chills, fever, weakness aches, shock and even death
•         Microbial toxins can cause:
•         Fever, diarrhea, cardiovascular disturbance, shock, inhibition of protein
synthesis, disrupt the nervous system
The Patterns of Infection
•         Localized infection -infection that is limited to a small area of body (abscesses)
•         Systemic infection -microbes are spread throughout the body (measles)
•         Focal infection – Infectious agent migrates from a local infection to other tissues
•         Mixed (polymicrobial) infection – more than one infectious agent is involved
•         Primary infection - infection that causes initial illness
•         Secondary infection - infection by opportunistic pathogen
•         Acute infection - develops rapidly but lasts a short time (influenza)
•         Chronic infection - develops slowly but lasts for long period o time (tuberculosis,
hepatitis B

Stages of Clinical Infections

•         Incubation period - time period between initial infection and first symptoms of disease
(several hours in pneumonic plague; several years leprosy)
•         Prodromal stage – feeling of discomfort (1-2 days)
•         Period of invasion -  expression of all symptoms
•         Convalescent period – decline of symptoms - recovery

Signs and Symptoms

•         Inflammation – swelling of the tissue
–        Edema – accumulation of fluid
–        Granulomas and abscesses – walled-off cells of damaged cells and microbes
–        Rashes, skin eruption
•         Signs of infection in the blood
–        Leukocytosis – increased level of blood cells
–        Bacteremia – bacteria present in the blood; do not multiply
–        Septicemia – microorganisms multiply in the blood
•         Unnoticed infections
–        Asymptomatic, subclinical

The Portal of Exit

The discharged microorganisms are the source of a new infection

•         Respiratory or salivary portals
•         Moist secretion (mucus, nasal discharge) – coughing, sneezing, laughing,
talking
•         Fecal
•         Intestinal infection (damage of intestinal mucus) causes rapid peristalsis
(movement of intestines) – diarrhea
•         Urogenital tract
•         vaginal discharge, semen, urine (STDs, tuberculosis of kidneys)
•         Blood
•         When removed for testing purposes or when released during tissue injury
•         AIDS, hepatitis

Persistence of Microbes
 •         Microbes start an infection from the reservoirs
•         Reservoir:
–        Living reservoir
•         Human body
•         Animal body
–        Non-living reservoir
•         Soil
•         Water
•         Plants

Living reservoir

Source of infection:
•         Diseased person or animal
•         Carrier - people that harbor pathogens without any signs of illness (AIDS, hepatitis,
gonorrhea, streptococcal infection)
–        Incubation carriers – spreads the disease during the incubation period
–        Convalescent carriers – recuperating patients
–        Chronic carrier – carry the agent for long period of time

Passive carriers – medical personnel

Animals as Reservoirs
•         Animals are Vectors – they transmit the infectious agent from one organism to another
•         Biological vectors
–        The agent multiplies within the vector (Plasmodium - Mosquito - Malaria)
•         Mechanical vectors
–        Mechanically transmits the agent to food or directly to humans

Transmission of Infectious Agents

•         Communicable diseases – infected host transmits the agent to another host
•         Noncommunicable diseases – infection acquired through
–        contact with an agent from its own body (opportunistic pathogens)
–        from nonliving reservoir (Clostridium tetanus)

Nosocomial Infections  -Hospital acquired

•         5-15% of hospital patient acquire nosocomial infection
•         Factors responsible for nosocomial infections:
–        The characteristics of microbes in the Hospital (opportunistic, antibiotic
resistant)
•         Patients are compromised by:
–        Broken skin or mucous membranes
–        Suppressed immune system
•         Chain of transmission
–        Transmission from hospital staff to patient
–        From patient to patient
 –        From fomites (non-living objects) to patients
–        Through ventilation system

Epidemiology

Study of frequency and distribution of disease in human population

•         Frequency of cases:
–        Incidence of a disease –Number of new cases of a disease during a given time
interval, usually one year
•         Used to determine probability of developing a specific disease 
–        Prevalence of a disease – Total number of current cases in the entire population
regardless of when it first appeared

Frequency of Occurrence
•         Endemic disease – constantly present in a population (common cold)
•         Sporadic disease – occurs occasionally (typhoid fever in the U.S.)
•         Epidemic disease – many people in a given area acquire a disease in short period of
time (influenza)
•         Pandemic disease – epidemic disease that occurs in a large geographic region
 
HOME
Chapter 14
 
Host Defenses I
Nonspecific Defenses
 
Defense Mechanisms of the Host
•       Innate, nonspecific
–      1st Line of defense (anatomical and physiological barriers)
–      2nd line of defense (cellular and chemical systems)
•       Acquired, specific
–      3rd line of defense
•       Naturally acquired
–      Active (Infection)
–      Passive (Maternal antibodies)
•       Artificially acquired
–      Active (Vaccination)
–      Passive (Immune serum)
First Line of Defense
•       Physical factors
–      Skin
–      Mucous membrane
•       Chemical factors
–      Sebum
–      Gastric acid
–      Lysozyme
 
Skin
Skin consists of two layers:
•       epidermis - outer thinner portion made of     tightly packed cells (upper layer -
dead cells)
•       dermis - inner thicker portion; Gives skin strength  
Infection can develop when the epithelial surface is broken
 
Mucous membranes
•       Mucous membranes line the gastrointestinal, respiratory and genitourinary tracts
•       Epithelial layer secrets the mucus that maintains the surface of the membrane
always moist
•       Mucous membranes are more susceptible to infections than skin
Other physical barriers
•       Lacrimal apparatus
–      provides washing action; removal of microbes
•       Ciliary escalator
–      The mucus membrane of the lower respiratory tract is covered with cilia; propel
mucus containing microorganisms upward
Chemical Factors
•       Sebum – oily substance produced by skin; contains unsaturated fatty acids – inhibit
the growth of certain pathogenic bacteria
•       Gastric juice – mixture of hydrochloric acid, enzymes, and mucus; kills most of
bacteria except Clostridium botulinum and Staphylococcus aureus
•       Lysozyme in saliva and tears – enzyme that hydrolyzes the peptodoglycan
 
Second and Third Line of Defense: Immune system
Responsible for:
•       Surveillance of the body - white blood cells
•       They recognize foreign material – distinguish between “self” and “nonself”
•       Destruction of foreign entities
Systems involved in immune defenses
•       Reticuloendothelial system
•       Extracellular fluid
•       Bloodstream
•       Lymphatic system
Reticuloendothelial system
•       Consists of:
–      Network of connective tissue fibers that surround all organs
–      Phagocytic cells located in reticular connective tissue
–      Interconnects neighboring cells
•       Provides a passageway between tissues and organs
Blood
•       A liquid connective tissue, consists of plasma and blood cells
–      Plasma - a fluid containing: water (92%); proteins (antibodies); fibrinogen,
hormones, nutrients, O2 and CO2
–      Blood cells:
•       Erythrocytes – red blood cells
•       Leukocytes – white blood cells
•       Thrombocytes – platelets
Formed Elements in Blood
•       Erythrocytes- carry oxygen and carbon dioxide in the blood
•       Platelets- involved in blood clotting
•       Leukocytes- involved in defending the body against invaders
•       Granulocytes
•       Agranulocytes
Agranulocytes
•       Cytoplasm appears uniform under a light microscope
•       2 types
–      Monocytes
–      Lymphocytes- involved in specific immunity
•       B lymphocytes
•       T lymphocytes
Monocytes
•       Produced in bone marrow – discharged into the bloodstream and transformed into
macrophages
•       Macrophages are responsible for:
–      Phagocytosis
–      Processing foreign molecules – presenting them to lymphocytes
–      Secreting compounds involved in immune response
Lymphatic system
•       Consists of lymphatic fluid, vessels, and organs
•       Lymphatic fluid is  plasma that moved out of the blood vessels and circulates in
the space between the tissue cells
•       Lymphatic vessels collect the lymph and return it to the circulatory system
Lymphoid Organs
•       Lymph nodes
–      Aggregated in armpit, groin, and neck area
–      Filters lymph
•       Spleen
–      Abdominal cavity
–      Filters blood – worn-out erythrocytes
•       Thymus
–      Pharyngeal region
–      T-cell maturation
Second Line of Defense: Inflammation
Inflammation is a body’s response to microbial infection
•       Three stages of inflammation:
–      Vasodilation
–      Edema and pus formation
–      Tissue repair
Vasodilation and increased permeability of blood vessels
•       An increase of diameter of blood vessel – enables increased blood flow to the
damaged area
•       Caused by the chemicals released from damaged tissue
–      Histamin - vasodilation
–      Chemical mediators – increase permeability of blood vessels
Edema
•       Accumulation of fluid in the tissue
•       White blood cells (phagocytes) migrate from the blood vessels – diapedesis – they
squeeze themselves between the endotelial cells
•       Chemotactic migration towards the site of injury
•       Phagocytosis is followed by formation of pus (cellular debris, bacteria)
Fever
•       Abnormally elevated body temperature in response to an infection
•       Body thermostat (hypothalamus) normally set at 370C
•       Substances called Pyrogens can reset the body thermostat to higher setting
•       Pyrogens
–      Exogenous – products of infectious agents
–      Endogenous (liberated from white blood cells)
•       Benefits of fever
–      Inhibits the growth of temperature sensitive microorganisms
–      Increased production of transferins (decreased availability of iron)
–      Faster tissue repair
•       Complications of fever
–      Tachycardia (accelerated heart rate)
–      Dehydration
–      Electrolyte imbalance
–      Coma
Phagocytosis (eat, cell)
•       Certain types of white blood cells eliminate the microbes by phagocytosis
•       The most important phagocytic cells are macrophages
•       Macrophages either reside in a specific organ or they wander throughout the
tissues 
 
Mechanism of Phagocytosis
There are five phases of phagocytosis
•       Chemotaxis - Phagocytes are attracted by:
–      microbial products
–      damaged tissue cells
•       The plasma membrane of the phagocyte attaches to the microbe and identifies it as
“nonself”
•       Ingestion
–      A phagocyte extends the pseudopds that engulf the microbe
–      Inside the phagocyte, the microbe is located within a sac called phagosome
•       Formation of phagolysosyomes
–      Phagosome fuses with lysosome forming a single structure – phagolysosome
•       Digestion
–      Bacteria are killed by reactive oxygen species (H2O2, singlet oxygen) and
lysozyme
•       Excretion
–      Bacteria are digested. The waste products discharged outside the cell.
 
Microbial Evasion of Phagocytosis
•       Some microbes resist the attachment of phagocyte by producing large capsules
•       Microbial toxins can kill a phagocyte
•       Microbial enzyme can lyse phagolysosome
•       Some microbes enter the phagocyte – they either multiply or remain dormant
 
Antimicrobial substances
The body produces antimicrobial substances:
•       Interferon
–      Interferon alpha and beta (produced by lymphocytes, macrophages, fibroblasts)
–      Interferon gamma (produced by T-cells)
•       The complement system
–      Serum proteins that contribute to destruction of microbes
 
Interferons
•       Proteins that interfere with viral multiplication.
•       Produced and released by a virus-infected cell 
•       Interferon enters now the neighboring non-infected cell
•       This triggers the cell to produce antiviral proteins (AVPs)
•       Other effects:
–      Defense against other, non-viral microbes
–      Play role in maturation B and T lymphocytes
–      Inhibits cancer cells
 
Complement proteins
•       The complement system consists of about 30 proteins found in the serum
•       Designated by the letter “C”
•       Act in a cascade (one reaction triggers another)
•       Complement activation occurs in 3 pathways
–      Classical
–      Lectin
–      Alternative
 
Steps in classical complement pathway
•       Initiation –
–      C1 component binds to antibodies that are bound to a pathogen
•       Amplification
–      C1 activates other components
•       Polymerization
–      The components aggregate and integrate into pathogen membrane
•       Membrane attack
–      The final product is an enzyme that punctures pores in the membrane
 
 
 
 HOME
 Figure 1.
3.11.3.1: A phylogenetic tree based on rRNA data, showing the separation of bacteria, archaea, and
eukaryota domains.

More recently various fusion hypotheses have begun to dominate the literature. One
proposes that the diploid or 2N nature of the eukaryotic genome occurred after the
fusion of two haploid or 1N prokaryotic cells. Others propose that the
domains Archaea and Eukarya emerged from a common archaeal-eukaryotic ancestor
that itself emerged from a member of the domain Bacteria. Some of the evidence
behind this hypothesis is based on a "superphylum" of bacteria called PVC, members of
which share some characteristics with both archaea and eukaryotes. There is growing
evidence that eukaryotes may have originated within a subset of archaea. In any event,
it is accepted today that there are three distinct domains of organisms in
nature: Bacteria, Archaea, and Eukarya. A description of the three domains follows.

INTERMEDIARIES BETWEEN BACTERIA, ARCHAEAE,
AND EUKARYA DOMAINS?
There is a "superphylum" of bacteria called PVC, referring to the three members of that
superphylum: the Planctomycetes, the Verrucomicrobia, and the Chlamydiae. Members
of the PVC, while belonging to the domain Bacteria, show some features of the
domains Archaea and Eukarya.

Some of these bacteria show cell compartmentalization wherein membranes surround

portions of the cell interior, such as groups of ribosomes or DNA, similar to eukaryotic
cells. Some divide by budding or contain sterols in their membranes, again similar to
eukaryotes. Some lack peptidoglycan, similar to eukaryotes and archaea. It has been
surmised that these bacteria migh be an intermediate step between an ancestor that
emerged from a bacterium (domain Bacteria) and an archael-eukaryotic ancestor prior
to its split into the domains Archaea and Eukarya.

Figure 1.3.21.3.2: Electron micrograph of the bacterium Gemmata obscuriglobus, a

planctomycete noted for its highly complex membrane morphology, illustrating representative
morphologies. Scale bar = 500nm. Santarella-Mellwig R, Franke J, Jaedicke A, Gorjanacz M,
Bauer U, Budd A, et al. (2010) The Compartmentalized Bacteria of the Planctomycetes-
Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins. PLoS Biol 8(1):
e1000281. doi:10.1371/journal.pbio.1000281

The Archaea (archaebacteria)

The Archaea possess the following characteristics:

a. Archaea are prokaryotic cells.
 b. Unlike the Bacteria and the Eukarya, the Archaea have membranes composed of
branched hydrocarbon chains (many also containing rings within the hydrocarbon
chains) attached to glycerol by ether linkages (Figure 1.3.31.3.3).
c. The cell walls of Archaea contain no peptidoglycan.
d. Archaea are not sensitive to some antibiotics that affect the Bacteria, but are sensitive to
some antibiotics that affect the Eukarya.
e. Archaea contain rRNA that is unique to the Archaea as indicated by the presence
molecular regions distinctly different from the rRNA of Bacteria and Eukarya.

Figure 1.3.3
1.3.3: Membrane Lipids of Archaea, Bacteria, and Eukarya. The Bacteria and the Eukarya have
membranes composed of unbranched fatty acid chains attached to glycerol by ester
linkages. The Archaea have membranes composed of branched hydrocarbon chains attached
to glycerol by ether linkages.

Archaea often live in extreme environments and include methanogens, extreme

halophiles, and hyperthermophiles. One reason for this is that the ether-containing
linkages in the Archaea membranes is more stabile than the ester-containing linkages in
the Bacteria and Eukarya and are better able to withstand higher temperatures and
stronger acid concentrations.

The Bacteria (eubacteria)
Bacteria (also known as eubacteria or "true bacteria") are prokaryotic cells that are
common in human daily life, encounter many more times than the archaebacteria.
Eubacteria can be found almost everywhere and kill thousands upon thousands of
people each year, but also serve as antibiotics producers and food digesters in our
stomachs. The Bacteria possess the following characteristics:

a. Bacteria are prokaryotic cells.

b. Like the Eukarya, they have membranes composed of unbranched fatty acid chains
attached to glycerol by ester linkages (Figure 1.3.31.3.3).
c. The cell walls of Bacteria, unlike the Archaea and the Eukarya, contain peptidoglycan.
 d. Bacteria are sensitive to traditional antibacterial antibiotics but are resistant to most
antibiotics that affect Eukarya.
e. Bacteria contain rRNA that is unique to the Bacteria as indicated by the presence
molecular regions distinctly different from the rRNA of Archaea and Eukarya.

Bacteria include mycoplasmas, cyanobacteria, Gram-positive bacteria, and Gram-

negative bacteria.

The Eukarya (eukaryotes)
The Eukarya (also spelled Eucarya) possess the following characteristics:

a. Eukarya have eukaryotic cells.

b. Like the Bacteria, they have membranes composed of unbranched fatty acid chains
attached to glycerol by ester linkages (Figure 1.3.31.3.3).
c. Not all Eukarya possess cells with a cell wall, but for those Eukarya having a cell wall,
that wall contains no peptidoglycan.
d. Eukarya are resistant to traditional antibacterial antibiotics but are sensitive to most
antibiotics that affect eukaryotic cells.
e. Eukarya contain rRNA that is unique to the Eukarya as indicated by the presence
molecular regions distinctly different from the rRNA of Archaea and Bacteria.

The Eukarya are subdivided into the following four kingdoms:

1. Protista Kingdom: Protista are simple, predominately unicellular eukaryotic organisms.

Examples includes slime molds, euglenoids, algae, and protozoans.
2. Fungi Kingdom: Fungi are unicellular or multicellular organisms with eukaryotic cell
types. The cells have cell walls but are not organized into tissues. They do not carry out
photosynthesis and obtain nutrients through absorption. Examples include sac fungi,
club fungi, yeasts, and molds.
3. Plantae Kingdom: Plants are multicellular organisms composed of eukaryotic cells. The
cells are organized into tissues and have cell walls. They obtain nutrients by
photosynthesis and absorption. Examples include mosses, ferns, conifers, and flowering
plants.
4. Animalia Kingdom: Animals are multicellular organisms composed of eukaryotic cells.
The cells are organized into tissues and lack cell walls. They do not carry out
photosynthesis and obtain nutrients primarily by ingestion. Examples include sponges,
worms, insects, and vertebrates.

It used to be thought that the changes that allow microorganisms to adapt to new
environments or alter their virulence capabilities was a relatively slow process occurring
within an organism primarily through mutations, chromosomal rearrangements, gene
deletions and gene duplications. Those changes would then be passed on to that
microbe's progeny and natural selection would occur. This gene transfer from a parent
organism to its offspring is called vertical gene transmission.
It is now known that microbial genes are transferred not only vertically from a parent
organism to its progeny, but also horizontally to relatives that are only distantly related,
e.g., other species and other genera. This latter process is known as horizontal gene
transfer. Through mechanisms such as transformation, transduction, and conjugation,
genetic elements such as plasmids, transposons, integrons, and even chromosomal
DNA can readily be spread from one microorganism to another. As a result, the old
three-branched "tree of life" in regard to microorganisms (Figure 1.3.11.3.1) now
appears to be more of a "net of life."

Microbes are known to live in remarkably diverse environments, many of which are
extremely harsh. This amazing and rapid adaptability is a result of their ability to quickly
modify their repertoire of protein functions by modifying, gaining, or losing their genes.
This gene expansion predominantly takes place by horizontal transfer.

Summary
1. Phylogeny refers to the evolutionary relationships between organisms.
2. Organisms can be classified into one of three domains based on differences in the
sequences of nucleotides in the cell's ribosomal RNAs (rRNA), the cell's membrane lipid
structure, and its sensitivity to antibiotics.
3. The three domains are the Archaea, the Bacteria, and the Eukarya.
4. Prokaryotic organisms belong either to the domain Archaea or the domain Bacteria;
organisms with eukaryotic cells belong to the domain Eukarya.
5. Microorganism transfer genes to other microorganisms through horizontal gene transfer
- the transfer of DNA to an organism that is not its offspring.

Contributors
 Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY,
CATONSVILLE CAMPUS)