Bacterial Genetics

ALL living thing CREATED to PROCREATE!

...to pass down genetic material to the next generation, etc, etc ,etc
[The more diverse the genes in a living thing, the better it is]

Animals, plants and other eucaryotes multiply by SEXUAL REPRODUCTION.

Prokaryotic bacteria do not have the capacity to reproduce sexually.
But they have other mechanisms to avoid genetic uniformity which may be detrimental to
their species, if faced with unfavourable conditions(s)
e.g. antibiotics, antiseptics, etc.

Knowledge on
-Structural characteristics
-Metabolic characteristics
-GENETIC characteristics
 BASIS for understand of many aspects of microbiology, including:

1. Mechanism by which microorganisms cause disease

2. Developing & implementing optimum techniques for microbial
• Detection
• Cultivation
• Identification
• Characterization
3. Understanding antimicrobial action and resistance
4. Developing and implementing tests for antimicrobial resistance detection
5. Designing strategies for disease therapy and control

Genetics:
Study of:
What genes are and how they:
• Carry information
• Replicate
• Pass that information to:
o Subsequent generation
o BETWEEN THEMSELVES (organisms)
And how the expression of their information within an organism determines the
characteristics of that organism

Genes: A segment of DNA on a chromosome, or a sequence of nucleotides in DNA that codes

for a functional product (except in viruses – may be RNA)

DNA: 2 complementary strands wrapped around each other to form a double helix. (like a
twisted ladder)
-each strand consists of many nucleotides
-each nucleotide consists of

Deoxyribose – Purine – Pyrimidine – Deoxyribose

Phosphate Phosphate

Deoxyribose – Pyrimide – Purine – Deoxyribose

-Purine A is always paired with pyrimidine T

-Purine G is always paired with pyrimidine C
...
Genetic information encoded by sequence of bases (A, T, G, C)

Each genetic code (CODON: any 3 of bases) will determine the amino acid synthesized.
Sequence of bases determines sequence of amino acids which in turn determines the type of
protein to be produced.

CENTRAL DOGMA IN GENETICS

dsDNA

duplication/replication

ssDNA

transcription

mRNA

translation

Protein

Genotype: genetic makeup  characteristic of organism

Phenotype: actual/expressed properties

DNA REPLICATION
• One ‘parental’ dsDNA  2 identical DNA strands
• N2 bases – complementary
• 1 strand act as template for the production of the other
• DNA polymerase can attach new nucleotide only at 3’C of pentose, at end of daughter
strands
• Must start replication at 5’C pentose

TRANSLATION: mRNA  AMINO ACIDS

At cytoplasm
5’  3’
5’ end mRNA association with RIBOSOME

Ribosome – 2 subunits  rRNA

↙↘
30s + 50s  70s

-tRNA with anticodon

1 tRNA for each amino acid
3 base sequence (codon)  1 a.m.

Start codon: AUG

Stop codon: UAA, UAG, UGA (nonsense codons)

Protein: polypeptide 50 amino acid  several hundreds

Typical polypeptide = 700 amino acid long

E.coli = about 4000 genes = 4000 polypeptides

MUTATION
● Bacteria reproduce by asexual binary fission
-Genome usually identical in all the progeny
-DNA replication – very accurate
● However, occasionally replication slightly inaccurate
-slightly altered nucleotide sequence, called MUTATION
Which is heritable  subsequent generation
 Genetic diversity
• Can be valuable to bacteria
Frequency
Mutation rate = probability a gene will mutate when a cell divides
Usually as Px10x
If 1:1000 = 1x10-3
For spontaneous mistakes – very rare. 1 x 10-9
In every generation, a few will mutate, but usually removes from gene pool when
-cell dies
-neutral effect
-harmful to organism
However a few useful to organism
e.g. Resistance to AB

A MUTAGEN ↑ rate to 10-6 – 10-3

Identify mutant by
• Positive (direct) selection
1. Media containing antibiotic resistant to
• Negative (indirect) selection
1. Replica planting – for auxotrophicmutants (loss of ability to produce/synthesize)

BACTERIAL CHROMOSOME & DNA

-Single circular chromosome consisting of a single circular ds molecule of DNA, with
associated proteins
-Chromosome – looped & folded & attached at 1 or > points to plasma membrane
e.g. E.coli
DNA :- 4 million base pairs (4x106 bp)
-1 mm long (10-3 m)
E.coli: 1 µm long (10-6 m)

The >1mm long chromosome (1000 x longer) MUST FIT into the 1 µm E.coli!!
How?
DNA – very thin, twisted, coiled = coiled macromolecule
So very tightly packed inside bacterial cell that it only occupies 10% cell volume!

Chromosome contains no histones.

In bacteria: replication : bidirectional.

Chromosome: circular (closed loop) starting point: origin.
Rate of DNA synthesis: 100 nucleotides/second in E.coli at 37°C
Role of DNA ligase.

INTERMICROBIAL DNA TRANSFER & RECOMBINATION

● Bacterial – no sexual reproduction
But DNA transfer btwn bacteria can occur, resulting in NEW STRAIN
Genetic exchanges  genetic diversity, usually beneficial to bacterial (unlike mutation)
e.g. drug resistance
-new nutritional & metabolic capabilities
-increased virulence (toxin production)
-resistance to metabolic poisons, etc
● DNA transfer may involve PLASMIDS
-closed circular extra-chromosomal DNA
-not a necessity for bacterial survival, but can be useful
-↑ versalitity
-↑ adaptability
-Drug resistance
●Interbacterial DNA transfer
-transformation
-conjugation
-transduction

TRANSFORMATION

Streptococcus pneumonia (pneumococcus)

-smooth colonies S (capsulated)
-rough colonies R (non-capsulated)

S – Virulent strain
R – Non-virulent strain

1920s – Fredetick Griffiths

S -> mouse -> died
R -> mouse -> survived
S -> heat killed  mouse  survived
S (heat, killed) + R  mouse  died!!!
TRANSFORMATION
R + DNA of killed S S strain! (virulent & capsulated)

Transformation:-
• Pneumococcus usually late log phase
• H. Influenza
• Bacillus spp. during sporulation
• Artificially induced in E.coli by adding DNA at 4°C, in Ca++ ions, 42°C (briefly), etc

DNA  enter cell  RECOMBINE with host DNA TRANSFORMED DNA

-Usually occurs when there is a high degree of nucleic acid similarity (homology)
-DNA taken up usually short

CONJUGATION
• Genetic material transferred from one bacterium to another through a SEX PILUS.
• Conjugation is mediated by a PLASMID (a circular piece of DNA outside chromosome of
bacteria, which replicates independently)
• Requires cell-to-cell contact (sex pilus)
• Conjugating mates are opposite mating type (F+  F-)
F-factor e.g. plasmid
• Plasmid carry code to synthesize sex pilus
• e.g E.coli (F+  F-)
• In some F+ cells, factor (plasmid) integrate into chromosome – converting F+ cell to an
Hfr (high frequenct of recombination) cell
• Hfr cell  F- cell – also part of chromosome transferred!
• Hfr cells
some (in fact a few)
F plasmid  excised itself from chromosome
In most “excisions” – accurate
A few – not so accurate
Some chromosomal genes “excise” too!  F+  F-  F+
• E.g E.coli : F-lac
• NLF (non-lactose fermenters)  LF (lactose fermenters)

TRANSDUCTION
• Bacterial DNA transferred from a donor a recipient cell via a BACTERIOPHAGE – a virus
that infects bacteria
• 2 types of transduction
 1. Generalised transduction
2. Specialized/restricted transduction

GENERALIZED TRANSDUCTION
• Phage  virulent  LYSIS OF HOST
• Phage  host cell wall  injects DNA into bacterium recombine with chromosome
• Phage DNA – template for new phage DNA & coat
• During phage development inside infected cell, bacterial chromosome is broken apart
by phage enzymes
• Some pieces of host DNA mistakenly packed into phage protein coat
• Phage released  infect another bacteria  + some bacterial DNA (even plasmid
DNA!)
e.g: Phage P1 E.coli
Phage P22 Salmonella

SPECIALIZED TRANSDUCTION
• Temperate phage – infect bacterium without lysing host
-bacteria + phage DNA – LYSOGEN condition – LYSOGENY
-latent phage – PROPHAGE (DNA recombined with host’s DNA)
• Lysogeny not permanent
10-2-10-5/cell generation
prophage – excised from chromosome  cell lysis. (UV ↑ induction)
-excision of prophage usually exact
-BUT occasionally prophage picks up bacterium DNA  another cell
-- SPECIALIZED TRANSDUCTION
• E.g. lysogenic conversion
toxin – C.diphtheriae
- ? Staph
- ? Strept
- ? Clostridia

PLASMIDS
• Tiny, circular, extra-chromosomal strands of DNA which are either
-free (non-conjugative) or
-integrated into the chromosome (conjugative)
• Duplicated
• Passed on to the progeny bacterial cells
• Non-essential for bacterial growth & metabolism
• Often useful to bacteria
-Drug/antibiotic resistance
-Toxin production
-produce enzymes, ↑ pathogenicity
• Useful in modern genetic engineering techniques!

TRANSPOSONS (jumping genes)

• A DNA segment with an insertion sequence at each end – can migrate (transpose) to:
o Another plasmid
o The bacterial chromosome
o Viruses (bacteriophages)
• Insertion sequence: PALINDROME
-- -- G A C G T | C T A C T G A | A C G T C -- --
-- -- C T G C A | G A T G A C T | T G C A G -- --
• Palindrome – can transpose
• Widespread among
o Prokaryotes
o Eucaryotes
o Viruses
 • Barbara McClintock – in corn (maize)
• Size: 700 – 40000 b.p.
• Effects:
o Changes in trait (colony morphology, pigmentation, pili, antigeniticy)
o Transfer of drug resistance
o DNA – rearranged  mutations (deletions, insertions, translocations, inversions,
chromosal breakages)
o Lethal

GENETIC BASIS OF ANTIBIOTIC RESISTANCE

• ANTIBIOTIC RESISTANCE
1. Intrinsic
2. Acquired
ANTIBIOTIC  selective pressure in favour of resistant organisms
• 3 factors:
1. Amount of AB used
2. Frequency bacteria  spontaneous mutations to resistance
3. Prevalence of plasmids able to transfer Resistance
• Control:
1. Control indiscriminate use of AB
-regulatory mechanisms
-hospital
-pharmacy
2. Control AB use in animal husbandry