Bacterial genetics

1. Bacterial genetics &disease
By
Prof. Rasheed Ajani Bakare,
M.B.B.S (Ib), FMCPath, FWACP

2. Bacterial Genetics And Variations

3. The Bacterial Genome
•Is in a single, giant, circular loop of
DNA called a Bacterial chromosome.
•lacks a nuclear membrane
•lies naked in cytoplasm
•located in a region of the cell known
as the nucleoid.
•Is made up of about 3000-6000
genes

4. What is a Gene?
•Genes are distinct DNA
sequences specifying the
sequences of amino acids in a
polypeptide chain

5. Characteristics of a gene
• Each gene determines a particular type of
amino acid assembly.
• The chain of nucleotides that constitute a
gene are composed of groups of bases
called Purines and Pyrimidines
• Gene function can be altered or the entire
gene deleted by genetic mutation

6. Characteristics of a gene (2)
• DNA RNA
Purines
• Adenine Adenine
• Guanine, Guanine
Pyrimidines
•Thymine Uracil
•Cytosine Cytosine

7. Characteristics of a bacterial
genome (2)
 Bacteria are haploid.
 Have unpaired chromosomes so no
heterozygotes occur
 Do not show dominance
 Each bacterium can be regarded as a
clone (genetically identical to
daughter cells)

8. Characteristics of a bacterial
genome (3)
• Each set of three bases is known as a codon
• AAC, GCT, TAG etc.
• Each codon codes for a specific amino acid.
e.g., leucine, valine
• There are over 64 different triplet sequences and
20 amino acids

9. Characteristics of a bacterial
genome (4)
•Undirected spontaneous mutation
occurs
• Associated with rate of growth
• Accentuated by generation time

10. Control of proteins
• The synthesis of proteins is therefore
controlled by DNA.
• The design of each protein is transmitted
from DNA to mRNA (transcription),
• which then instructs the cellular machinery
(tRNA and rRNA etc) (translation) to
assemble a protein

11. Control of proteins (2)
• Proteins
• determine structural and metabolic function
• structural
• Enzymes
•
• Total genetic potential of DNA is the
GENOTYPE
• Part that is manifest or discernable is the
PHENOTYPE.

12. GENETIC VARIATION.
• Variations easily detected because
• Rapid rate of reproduction
• Large bacterial populations) are easily detected.
• Two types-
• Phenotypic
• Genotypic

13. Phenotypic Variation
• a non-heritable variation,
• Temporary adjustment to changes in the environment
• Normally involves all the cells of a culture
• Respond physiologically within the range of potential
of the genotype

14. Phenotypic Variation (2)
• May manifest as changes in
• Morphology (size, shape, staining reactions)
• metabolism and chemical reactions,
• induction of an enzyme, e.g., -lactamase by the presence of
penicillin
• repression of enzymes due to the presence of the end
products in the system.
• Revert back to type when the inducing
circumstance is removed.

15. Genotypic Variation
•Heritable variation
•Result of genetic changes
• mutation
• genetic transfer- acquisition of new
heritable properties from other
organisms

16. Mutation
• Important part of Bacterial evolution.
• Spontaneous mutation is common
• rate between 1 in 107
and 1 in 1010
• Environment not really important
• may favour or select for the variant, which may then grow
and replace the wild type

17. Effect of Mutation
• Morphology
• size of a cell, colonial appearance
• Ability to form spores, flagella or capsules),
• Nutritional requirements,
• Antigenic properties,
• Ability to produce toxins,
• Drug sensitivity/resistance
• Loss of Capsule
• Smooth-Rough (S-R) variation
• Becomes less virulent.
• Best seen in Pneumococci, Salmonellae and Shigellae
• Occurs when bacteria are grown for a long time on artificial
media

18. Types of Mutation
• Base-Pair substitution
• Insertions or deletion

19. Base- Pair Substitution
•E.g., GAA = leucine to
GTA = histidine.
• Called a Missence Mutation
• Results in the substitution of one amino-acid for
another.
• It may or may not affect the function of the
polypeptide.

20. Insertion or deletion
• The insertion or deletion of a base will lead to a
frame shift mutation
• Deletion of a base unless it is compensated for, by
the insertion of a new base very close to the
deleted base, will lead to a new polypeptide being
formed.

21. Insertion and deletion - 2
• DNA = AAC GAA CGC TGA
• RNA = UUG CUU GCG ACU….
= leucine, histidine, alanine, threonine
• If A is deleted there will be a left shift such
that the above DNA code will read starting
from the second A as:
• DNA = ACG AAC GCT GA…..
RNA = UGC UUG CUU CU……
cystine, leucine, arginine, leucine

22. Gene exchange (genetic transfer)
• In eukaryotes
• occurs by sexual reproduction
• haploid gametes fuse to become a diploid zygote
• In Bacteria,
• NO DIPLOID zygotes are formed
• processes exist that allow for the acquisition of foreign DNA

23. Methods of Gene exchange (genetic
transfer)
• Transformation
• gene transfer resulting from the uptake by a recipient
cell of naked DNA from a donor cell.
• Transduction
• Transfer of foreign genes by bacteriophages
• Conjugation
• Direct transfer of DNA from one organism to another
through a modified fimbriae (pilus) called a sex pilus

24. Transferable genetic materials
• Foreign DNA being transferred may be
• Plasmids
• Viral DNA
• Parts of host chromosome
• Transposable Elements
• Transposons
• Insertional Sequences

25. Plasmids
• Closed circular molecules
• Double stranded DNA
• Size from 2-3000 kilobases (100,000-150,000 base
pairs)
• Can replicate independently of the bacterial
chromosome.
• Code for various characteristics
• Fertility (F),
• drug resistance transfer (R),
• toxin production

26. Plasmids (2)
• Two categories
• Transmissible by conjugation (usually Large plasmids)
• Transmissible by transducing phages
• May be lost during growth
• Some cell characteristics they confer are
unstable
• e.g., erythrogenic toxin in some Group A -haemolytic
Streptococci.
• Information is often in the form of
transposable genetic elements called
Transposons

27. Transposons- (Jumping genes)
• Segments of DNA
• Usually containing several gene s
• Easily transferable into the DNA of bacterial
chromosomes, plasmids and infecting
phages.
• incapable of autonomous replication.
• Have special sequences at the end of their
DNA called terminal sequences
• about 15-40 base pairs
• are inverted repeats
• Sticky

28. Insertion sequences
(IS elements)
• Can also be transferred from plasmids to
chromosomes
• Short segments of DNA
• usually about 100-1000 bases (usually less than 1,500)
•
• Contain no instructions for protein synthesis
• Act as gene inactivators when inserted in the middle of
a gene structure
• Presence is noted only when interference with function of the
chromosomal gene occurs.

29. Implication
• Some toxin-, drug-inactivating enzyme can
be on different plasmids or phages.
• E.g., -lactamase of E. coli, N.gonorrhoea and H. influenzae
are on different plasmids which all have the same
transposon.
• heat stable enterotoxin of E.coli is on a transposon that is
also found in Yersinia enterolitica

30. Transformation-
• Initially regarded as a laboratory phenomenon
• Artificial transformation has been achieved in members of the
Enterobacteriaceae (e.g. Escherichia coli, Klebsiella spp.)
• Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria,
Pneumococcus) can take up DNA from the environment
• DNA that is taken up can be incorporated into the recipient's chromosome

31. Transformation
• First detected in the pneumococcus in 1928 by
Griffith
• R (rough, non-virulent) strains converted to the corresponding
parental S (smooth, virulent) type
• Also transformed to a different S-type by heat-killed
cells of that type.

32. Factors affecting transformation
• DNA size state
• Double stranded DNA of at least 5 X 105
daltons works
best.
• Competence of the recipient –
• Some bacteria are able to take up DNA naturally.
• By producing a specific protein called a competence
factor.
• Other bacteria are not able to take up DNA naturally.
• competence can be induced in vitro by treatment with
chemicals (e.g. CaCl2
)

33. Steps in transformation
Uptake of DNA
• Uptake of DNA by Gram+ and Gram- bacteria
differs.
• Gram Positive bacteria
• Take up single stranded DNA
• Complementary strand is made in the recipient.
• Gram Negative bacteria
• Take up double stranded DNA.

34. Significance of transformation
• Transformation occurs in nature and it can lead to
increased
• virulence.
• Drug resistance
• Widely used in recombinant DNA technology

35. Transduction-1
• Transfer of genes by infection with a non-lethal
(temperate) phage.
• Involves transfer of DNA from the host bacteria
to another bacterium.
• First discovered in salmonellae by Lederberg
and Zinder
• Not all phages can mediate transduction.

36. Transduction-2
• Gene transfer - usually between members of
the same bacterial species.
• Range of species determined by Phage host
range.
• if a particular phage has a wide host range then transfer
between species can occur.
• Ability of a phage to mediate transduction is
related to the life cycle of the phage

37. Types of Transduction
• Generalized Transduction
• Potentially any bacterial gene from the donor can be
transferred to the recipient.
• Specialized transduction
• only certain donor genes can be transferred to the
recipient.

38. Significance
• Lysogenic (phage) conversion occurs in nature and is
the source of virulent strains of bacteria
• Responsible for penicillin-resistant strains of
Staphylococcus aureus

39. Conjugation-1
[ A-B-] + [C-D-] = [A+B+C+D+]
a1
a2
a3
• First reported in 1946,
Lederberg while investigating
genetic recombination in E.coli
K12,
• Mated two doubly auxotrophic
strains, (a1
& a2
), each different
in its requirements for two
essential nutrients.
• Got a prototroph that did not
require any of these nutrients

40. Conjugation -2
• It was discovered that transfer of DNA was by
direct contact
• Process called conjugation.
• Conjugation is a quasisexual introduction of
donor DNA into a recipient through a modified
pilus (fimbriae) called a sex pilus.

41. Conjugation-4
• In bacteria two mating types
• donor (male )
• recipient (female)
• Direction of transfer of genetic material is one way
• from a donor to a recipient

42. Mating Types
• Donor
• Has a plasmid called the F factor or fertility factor or sex factor.(F+)
• Recipient
• Lacks the F factor. (F-)

43. F-Factor
• Plasmid
• Has genes needed for its replication and for
its ability to transfer DNA to a recipient.
• codes for the ability to produce a sex pilus
(F pilus)

44. Conjugation
• E.coli undergoing
conjugation.
• Note that the strain
on the left has
fimbriae and the
specialised sex
pilus, i.e., has an
F-factor and is
therefore an F+
F+
F--

45. Effect of Gene transfer
• Never leads to the formation of a zygote as in
eukaryotic cell.
• At best, a merozygote is formed
• part of a donor bacterium’s genome (exogenote is
transferred to an intact DNA of a recipient
• Recombination then takes place
• replacement of resident genes by exogenote genes
• or the addition of exogenote genes to the resident pool.
• exogenote genes may lie separate from the chromosome
or may be integrated into it.

46. Recombinant- DNA Techniques Or
Cloning
Transfer of a gene from one
bacterium,virus or animal cell into DNA
of a living bacterium where it will
replicate and instruct the cell.

47. Recombinant- DNA techniques
 Usually done between closely related
organisms.
 New bacteria are known as recombinant
organisms.
 Applications
 From novel strains of E.coli
 Insulin,
 Growth hormone,
 Components for vaccines (hepatitis B, H.influenzae and
foot and mouth disease.),
 Interferon,
 Bloodclotting factor 8,
 DNA probes for the detection of specific sequences of
particular pathogens