Microbial Genetics

1. Microbial Genetics
Ariane Ruby B. Sogo-an
MST Biology

2. Microbial Genetics
• Mutation in Bacteria
• Genetics Exchange in Bacteria
• Recombination and Genetic Engineering

3. Learning Objectives:
1. Define Mutation.
2. Explain the mechanisms involved in Mutation.
3. Familiarize the processes involved on how Genetic
Information are transferred in Bacteria
4. Give the importance of Recombination and Genetic
Engineering

4. Report Outline
• Nucleic Acids
• Central Dogma
• DNA Replication in Bacteria
• RNA Synthesis in Bacteria
• Protein Synthesis in Bacteria
• Changes in the DNA molecule through Mutation
• Transfer of Genetic Information in Bacteria

5. Genetics
• Genetics is a study of Heredity.
• HOW the information contained in Nucleic Acids is expressed?
• HOW this type of molecule is duplicated?
• HOW this duplicated molecules are transmitted to progeny?

6. Nucleic Acids
• Nucleic Acids are large organic molecules that are found in ALL
cells.
• Two Types:
• DNA (Deoxyribonucleic Acid)
• directs protein production
• RNA (Ribonucleic Acid)

7. Nucleic Acids
• Nucleic Acids are large organic molecules that are found in ALL
cells.
• Two Types:
• DNA (Deoxyribonucleic Acid)
• directs protein production
• RNA (Ribonucleic Acid)

8. Nucleic Acids
• Composition:
• Constructed from a string of small molecules called
NUCLEOTIDES.

9. Component of a Nucleotide

10. Nitrogenous Base

11. Primary Structure of RNA

12. Primary Structure of DNA

14. DNA and RNA

15. Ribonucleic Acids
• RNA are normally single stranded molecules.
• Types: (based on their function)
• mRNA (Messenger)
• tRNA (Transfer)
• rRNA (Ribosomal)
• Look for specific example

17. Deoxyribonucleic Acids
• Double Stranded, with each strand wrapped around the other
in a helical fashion forming a double helix.
• Hydrogen bond is specific since A-T (or U in RNA) and G-C
ATCCGGC
TAGGCCG
• Molecule is more stable.

18. Deoxyribonucleic Acids
• Determines the characteristics of an organism and maintains
and controls the vital processes of all cells.
• How is genetic information expressed?
• Transcription (involves formation of RNA molecule using DNA as
a template)
• Translation (consists of the synthesis of a protein using the
genetic information in the RNA)

19. The Central Dogma

21. The Central Dogma
• Gene
• The unit of genetic information or hereditary material contained
in DNA molecule.
• Sequenced nucleotide in the DNA molecule that codes for RNA
molecule and ultimately for the synthesis of a protein.

22. The Central Dogma
• Theory stating that genes guide the synthesis of mRNA and in
turn, directs the order in which amino acids are resembled to
form protein.
• Also postulates that a DNA molecule can direct its own
replication by giving rise of two identical DNA molecule.

23. The Central Dogma
• Reverse Transcription
• Example: Certain cancer causing viruses (retroviruses) are able to
synthesize DNA using RNA as a template.

24. DNA Replication in Bacteria
• Genome – total genetic information in bacteria which consists
of circular DNA molecules found within the cell.
• Most of the genome is contained in a single bacterial
chromosome, although smaller pieces of circular DNA called
plasmids may also carry a few important genes such as those
coding for resistance to microbial drugs.

25. DNA Replication in Bacteria
• The bacterial chromosomes contains most of the genetic
information of bacteria and is attached to the plasma
membrane.
• Size of the chromosomes varies from species to species.
• Example: (per chromosome)
• Mycoplasma – fewer than 1 M nucleotide base pairs and a genome
can code for 1000 proteins.
• E. Coli – 4.5 M nucleotide base pairs that can code for 4500 proteins.

26. DNA Replication in Bacteria
• Both DNA strands are duplicated with each strands functions
as a template that specifies the sequence of bases in the
newly formed complementary strand.
• DNA polymerases
• Process nucleotides from the cytoplasm that are complementary
to the template and fit them into place.
• Parental and New strand = semiconservative.

27. DNA Replication

28. DNA Replication
• 1. The original double helix molecule.
• 2. Helicase enzyme breaks the hydrogen bonds between
complementary base pairs. This unzips the double helix at a
position called the replication fork.
• 3. There is an abundant supply of nucleotides in the nucleus
for the formation of the new polynucleotides.
• 4. Nucleotides base pair to the bases in the original strands.
• 5. DNA polymerase joins together the nucleotides together
with strong covalent phosphodiester bonds To form a new
complementary polynucleotide strand.
• 6. The double strand reforms a double helix under the
influence of an enzyme.
• 7 Two copies of the DNA molecule form behind the replication
fork. These are the new daughter chromosomes.

29. RNA Synthesis in Bacteria
• Transcription
• Involves the assembly of nucleotides by an enzyme called RNA
polymerase that uses a strand of DNA as its template.
• Begins when RNA polymerase binds to the DNA at the
promoter site near the gene to be transcribed.
• RNA polymerase travels along the length of the DNA strand
until it reaches a termination site.

31. RNA Synthesis in Bacteria
• After mRNA is made, it will be used as a guide to make
proteins.
• Ribosomal RNA, after its made, becomes associated with
proteins to form ribosomes.
• tRNA are small RNA molecules that are involved in translating
the information in the mRNA into proteins.

32. MUTATION

34. The Genetic Code
• The start codon is AUG. Methionine is the only amino acid
specified by just one codon, AUG.
• The stop codons are UAA, UAG, and UGA. They encode no
amino acid. The ribosome pauses and falls off the mRNA.

35. Mutation: Base Substitution (Point
Mutations)
G
C
Glu
(d) Run-on mutation
G
C
(a) Silent mutation

37. Mutation: Insertions and Deletions
Figure 8.17a, d
THEBIGCATATETHERAT
THEBIGCBATATETHERAT

38. Summary of Mutation Types
Run-on mutation
Stop codon lost so
protein is extra long
(can also produce
nonsense and run-ons)

39. Spontaneous and Induced Mutation
• Spontaneous mutation rate = 1 in 109 (a billion) replicated base pairs
or 1 in 106 ( a million) replicated genes. Mistakes occur during DNA
Replication just before cell division. This is natural error rate of DNA
polymerase.
• Mutagens increase mistakes to to 10–5 (100 thousand) or 10–3 ( a
thousand) per replicated gene

40. Mutagen
• Mutation relevant
• Cause DNA damage that can be converted to mutations.

41. Physical mutagens
High-energy ionizing radiation: X-rays and g-rays 
strand breaks and base/sugar destruction
Nonionizing radiation : UV light pyrimidine dimers
Chemical mutagens
Base analogs: direct mutagenesis
Nitrous acid: deaminates C to produce U
Alkylating agents
Intercalating agents
Lesions-indirect mutagenesis
1 Mutaagenesis

42. Chemical Mutagens
Base pair altering chemicals (base
modifiers) deaminators like
nitrous acid, nitrosoguanidine,
or alkylating agents like cytoxan
Base analogues “mimic”
certain bases but pair with
others - E.g. 5-fluorouracil,
cytarabine
Acts like a “C”
cytarabine
cytoxan Nitrous acid

43. BASE PAIR ALTERING CHEMICALS

44. Deaminating Agent
• *Deaminating agent - Nitrous acid - removes the anime group
from Adenine and Cytosine
• Nitrous acid is a deaminating agent that converts cytosine to
uracil, adenine to hypoxanthine, and guanine to xanthine. The
hydrogen-bonding potential of the modified base is altered,
resulting in mispairing.

45. BASE ANALOGS

46. Alkylating agents
• Alkylating agents like EMS/MMS(ethyl/methly methyl
sulphonate) add methyl groups to Guanosine . Bulky
attachment to the side groups or bases.

47. Hydroxilating Agents
• Addition of OH (Hydroxyl Group)
hydroxylamine (HA)

48. Intercalating Agents
• Intercalation agents are compounds that can slide between
the nitrogenous bases in a DNA molecule.
• This tends to cause a greater likelihood for slippage during
replication, resulting in an increase in frameshift mutations.
• Example (Sodium Azide)

49. Chemical FrameshiftMutagens Intercalate into DNA
Aflatoxin from
Aspergillus fungus
growing on corn
Benzpyrene in
cigarette smoke
AT
GC
TA
GC
CG
AT
GC
TA
GC
CG
AT
GC
CG
TA
GC
CG
Carboplatin
(anti-cancer drug)
Daunarubicin
(anti-cancer drug)
Bleomycin (anti-cancer
drug produced by
Streptomyces)

50. Mutation: Ionizing Radiation
• Ionizing radiation (X rays, gamma rays, UV light) causes the formation
of ions that can react with nucleotides and the deoxyribose-phosphate
backbone.
• Nucleotide excision repairs mutations

51. X-rays and Gamma Rays Cause
Breaks in DNA

52. Ionizing Radiation: UV
• UV radiation causes
thymine dimers, which
block replication.
• Light-repair separates
thymine dimers
• Sometimes the “repair
job” introduces the
wrong nucleotide,
leading to a point
mutation.
Figure 8.20

53. MDufilho 7/6/11
53
Genetic Transfer
• Horizontal Gene Transfer Among Prokaryotes
• Horizontal gene transfer
• Donor cell contributes part of genome to recipient cell
• Three types
• Transformation
• Transduction
• Bacterial conjugation
© 2012 Pearson Education Inc.

54. Bacterial Sexual Processes
• Eukaryotes have the processes of meiosis to reduce
diploids to haploidy, and fertilization to return the cells to
the diploid state. Bacterial sexual processes are not so
regular. However, they serve the same aim: to mix the
genes from two different organisms together.
• The three bacterial sexual processes:
• 1. conjugation: direct transfer of DNA from one bacterial cell to
another.
• 2. transduction: use of a bacteriophage (bacterial virus) to
transfer DNA between cells.
• 3. transformation: naked DNA is taken up from the environment
by bacterial cells.

55. Transformation
• We aren’t going to speak much of this process, except to
note that it is very important for recombinant DNA work.
The essence of recombinant DNA technology is to
remove DNA from cells, manipulate it in the test tube,
then put it back into living cells. In most cases this is
done by transformation.
• In the case of E. coli, cells are made “competent” to be
transformed by treatment with calcium ions and heat
shock. E. coli cells in this condition readily pick up DNA
from their surroundings and incorporate it into their
genomes.

56. Figure 7.33 Transformation in Streptococcus pneumoniae-overview
MDufilho 7/6/11
56

57. Conjugation
• Conjugation is the closest analogue in
bacteria to eukaryotic sex.
• The ability to conjugate is conferred by
the F plasmid. A plasmid is a small circle
of DNA that replicates independently of
the chromosome. Bacterial cells that
contain an F plasmid are called “F+”.
Bacteria that don’t have an F plasmid are
called “F-”.
• F+ cells grow special tubes called “sex
pilli” from their bodies. When an F+ cell
bumps into an F- cell, the sex pilli hold
them together, and a copy of the F
plasmid is transferred from the F+ to the
F-. Now both cells are F+.
• Why aren’t all E. coli F+, if it spreads like
that? Because the F plasmid can be
spontaneously lost.

58. Figure 7.35 Bacterial conjugation-overview
MDufilho 7/6/11
58
F plasmid Origin of
transfer
Conjugation pilus Chromosome
F+ cell F– cell
Donor cell attaches to a recipient cell with
its pilus.
Pilus may draw cells together.
One strand of F plasmid DNA transfers
to the recipient.
F+ cell F+ cell
Pilus
The recipient synthesizes a complementary
strand to become an F+ cell with a pilus; the
donor synthesizes a complementary strand,
restoring its complete plasmid.

59. Figure 7.36 Conjugation involving an Hfr cell-overview
MDufilho 7/6/11
59
Donor chromosome
Pilus
F+ cell
Hfr cell
Pilus
F+ cell (Hfr)
F plasmid
F– recipient
Donor DNA Part of F plasmid
F plasmid integrates
into chromosome by
recombination.
Cells join via a
conjugation pilus.
Portion of F plasmid partially
moves into recipient cell
trailing a strand of donor’s
DNA.
Conjugation ends with pieces
of F plasmid and donor DNA
in recipient cell; cells synthesize
complementary DNA strands.
Donor DNA and recipient
DNA recombine, making a
recombinant F– cell.
Incomplete F plasmid;
cell remains F–
Recombinant cell (still F–)

60. Hfr Conjugation
• When it exists as a free plasmid,
the F plasmid can only transfer
itself. This isn’t all that useful for
genetics.
• However, sometimes the F
plasmid can become incorporated
into the bacterial chromosome,
by a crossover between the F
plasmid and the chromosome.
The resulting bacterial cell is
called an “Hfr”, which stands for
“High frequency of
recombination”.
• Hfr bacteria conjugate just like F+
do, but they drag a copy of the
entire chromosome into the F-cell.

61. Transduction
• Transduction is the process of moving bacterial DNA from
one cell to another using a bacteriophage.
• Bacteriophage or just “phage” are bacterial viruses. They
consist of a small piece of DNA inside a protein coat. The
protein coat binds to the bacterial surface, then injects
the phage DNA. The phage DNA then takes over the
cell’s machinery and replicates many virus particles.
• Two forms of transduction:
• 1. generalized: any piece of the bacterial genome can be
transferred
• 2. specialized: only specific pieces of the chromosome can be
transferred.

62. Figure 7.34 Transduction-overview
MDufilho 7/6/11
62
Bacteriophage
Phage injects its DNA.
Phage enzymes
degrade host DNA.
Phage
DNA
Host bacterial cell
(donor cell)
Bacterial chromosome
Phage with donor DNA
(transducing phage)
Cell synthesizes new
phages that incorporate
phage DNA and, mistakenly,
some host DNA.
Transducing phage
Transducing phage
injects donor DNA.
Recipient host cell
Donor DNA is incorporated
into recipient’s chromosome
by recombination.
Transduced cell
Inserted
DNA

63. General Phage Life Cycle
• 1. Phage attaches to the cell
and injects its DNA.
• 2. Phage DNA replicates,
and is transcribed into RNA,
then translated into new
phage proteins.
• 3. New phage particles are
assembled.
• 4. Cell is lysed, releasing
about 200 new phage
particles.
• Total time = about 15
minutes.

64. Why do chromosomes undergo
recombination?
Deleterious mutations would accumulate in
each chromosome
Recombination generates genetic diversity

65. Recombination
ABCDEFGHIJKLMNOPQRSTUVWXYZ
abcdefghijklmnopqrstuvwxyz
ABCDEFGhijklmnoPQRSTUVWXYZ
abcdefgHIJKLMNOpqrstuvwxyz

66. Mitotic and meiotic recombination
Recombination can occur both during mitosis
and meiosis
Only meiotic recombination serves the
important role of reassorting genes
Mitotic recombination may be important for
repair of mutations in one of a pair of sister
chromatids

67. Recombination mechanisms
Best studied in yeast, bacteria and phage
Recombination is mediated by the breakage
and joining of DNA strands

68. Benefits of recombination
• Greater variety in offspring: Generates new combinations of
alleles
• Negative selection can remove deleterious alleles from a
population without removing the entire chromosome carrying
that allele
• Essential to the physical process of meiosis, and hence sexual
reproduction
• Yeast and Drosophila mutants that block pairing are also defective
in recombination, and vice versa!!!!

69. Genetic Engineering

70. What is genetic engineering???
Genetic engineering: is the artificial manipulation
or alteration of genes.
Genetic Engineering involves:
• removing a gene (target gene) from one organism
• inserting target gene into DNA of another organism
• ‘cut and paste’ process.

71. Some important terms!!!
Recombinant DNA: the altered DNA is called
recombinant DNA ( recombines after small section of
DNA inserted into it).
Genetically Modified Organism (GMO): is the
organism with the altered DNA.

72. Genetic Engineering breaks the species
barrier!!!
• Genetic engineering allows DNA from different species
to be joined together.
• This often results in combinations of DNA that would
never be possible in nature!!! For this reason genetic
engineering is not a natural process.
• If DNA is transferred from one species to another the
organism that receives the DNA is said to be transgenic.

73. Genetic engineering breaks the species
barrier!!!
• Examples of cross-species transfer of genes:
- a human gene inserted into a bacterium
- a human gene inserted into another animal
- a bacterial gene placed in a plant

74. Alternative names for genetic engineering:
• Genetic Manipulation
• Genetic Modification
• Recombinant DNA Technology
• Gene Splicing
• Gene Cloning

75. Tools used in genetic engineering!!!
• Source of DNA: Target (foreign) DNA – DNA taken from
one organism to be placed into the DNA of a second
organism.
• A cloning vector: Special kind of DNA that can accept
foreign DNA and exactly reproduce itself and the foreign
DNA e.g. Bacterial plasmid (loop of DNA found in
bacteria).

76. Tools Used in Genetic Engineering
Restriction Enzymes:
- are special enzymes used to cut the DNA at specific
places.
- different enzymes cut DNA at specific base sequences
known as a recognition site. For example
i) One restriction enzyme will always cut DNA at
the base sequence: GAATTC.
ii) Another restriction enzyme only cuts at the
sequence: GATC.
- If DNA from two different organisms is cut with the
same restriction enzyme the cut ends from both sources
will be complementary and can easily stick together.

77. Restriction enzymes
DNA 1 DNA 2

78. Tools used in Genetic Engineering
DNA Ligase: enzyme which acts like a glue sticking
foreign DNA to DNA of the cloning vector.
• will only work if DNA from the two DNA sources has
been cut with the same restriction enzyme i.e. sticky ends
of cut DNA will be complementary to each other.
Please note diagram illustrating use of restriction
enzymes and DNA Ligase in production of recombinant
DNA Fig. 19.6 pg. 195

79. Process of Genetic Engineering
Five steps involved in this process:
1. Isolation
2. Cutting
3. Insertion (Ligation)
4. Transformation
5. Expression
Note: The following example will explain how a human
gene is inserted into a bacterium so that the
bacterium can produce human insulin.

80. Process of Genetic Engineering
1. Isolation:
• Removal of human DNA (containing target gene).
• Removal of plasmid (bacterial DNA) from
bacterium.
2. Cutting:
• Both human DNA and plasmid DNA are cut with
the same restriction enzyme.
• Normally plasmid has only one restriction site
while human DNA will have many restriction sites.
Please note diagram 19.7 pg. 196

81. Process of Genetic Engineering
Insertion:
• means that target gene is placed into the DNA of
the plasmid or cloning vector.
• cut plasmids are mixed with human DNA sections
allowing the cut ends to combine.
Transformation
Expression

82. Applications of Genetic Engineering
You must know three applications: one involving a plant,
one animal and one for a micro-organism.
Plants: Weed killer-resistant crops
• many types of crop plants have bacterial genes
added to them.
• these genes make the plants resistant to certain
weed killers (herbicides).
• this means that the weed killers kill the weeds but do
not affect the transgenic plants.

83. Applications of Genetic Engineering
Animals:
There is a growing trend to experiment with inserting
human genes into the DNA of other mammals. The
transgenic animals formed in this way will then produce a
human protein and secrete it into their milk or even into
their eggs.

84. Applications of Genetic Engineering
Animals: Sheep produce human clotting factor
• A human gene has been inserted into the DNA of
sheep.
• This allows the adult sheep to produce a clotting
chemical needed by haemophiliacs to clot their blood
– produced in the milk of the sheep.
Pharming: is the production of pharmaceuticals by
genetically modified animals i.e. sheep, cows, goats etc.

85. Pharming – using animals to make pharmaceuticals

86. Applications of Genetic Engineering
Micro-organisms: Bacteria make insulin
• The human insulin gene has been inserted into a
bacterium (E-coli).
• This allows the bacterium to produce insulin for use
by diabetics.

87. Ethical Issues in Genetic Engineering
GMO’s as a food source:
Outlined below are some fears associated with the use of
GMO’s as a food source:
• Cannibalism:
– eating an animal containing a human gene is a
form of cannibalism.
- feeding GMO’s containing human genes to animals
that would later be eaten by humans.
• Religious reasons: – eating pig genes that are inserted
into sheep would be offensive to Jews and Muslims.
• Offensive to vegetarians/vegans: – eating animal genes
contained in food plants cause concern.

88. Ethical Issues in Genetic Engineering
Animal Welfare:
• There is serious concern that animals will suffer as a
result of being genetically modified.
• use of growth hormones may cause limb deformation
and arthritis as animals grow.

89. Ethical Issues in Genetic Engineering
Genetic Engineering in Humans:
The following issues are a cause for concern:
• If tests are carried out for genetic diseases, who is
entitled to see the results?
• Tests on unborn babies – could this lead to abortion
if a disease is shown to be present?
• Insurance/lending companies – will they insist on
genetic tests before they will insure/lend money to a
person?
• Need for legal controls over the uses to which human
cells can be put.
• Development and expansion of eugenics.

90. END OF REPORT