Bacterial Genetics

dr.Tetty Aman Nasution M Med Sc

- Genetics is the study of what genes are, how they carry information, how their
information is expressed, and how they are replicated and passed to subsequent
generations or other organisms.
- DNA is the genetic material for all living organisms. In bacteria, there is a single
circular (closed) chromosome; which is a polymer of DNA (nucleic acid).
- The DNA in a chromosome exists as one long double helix associated with various
proteins that regulate genetic activity.
- The DNA in a cell is replicated before the cell divides, so each daughter cell receives
the same genetic information.
- Gene is a segment of DNA, a sequence of nucleotides, that codes for a functional
product, usually a protein.
- When a gene is expressed, DNA is transcribed to produce an mRNA; mRNA is then
translated into proteins.
- Clone is a population of cells that are genetically identical.

- Genome: all the genes present in a cell or virus.

- Genotype: the specific set of genes an organism possesses.
- Phenotype: the collection of characteristics of an organism that an investigator can
observe.
• Nucleic Acids Structure:
- DNA made from subunits called nucleotides.
- Each nucleotide contains:
1. Purine (Adenine or A, Guanine or G) or
Pyrimidine (Cytosine or C, Thymine or T)
bases.
2. Deoxyribose sugar.
3. 1, 2, or 3 phosphate groups.
- RNA nucleotides containing ribose sugar.
- Nucleotides are named according to # of
phosphates: e.g., dATP = deoxy adenosine
triphosphate, whereas dAMP = deoxy adenosine
monophosphate.

-Nucleotides in RNA don't have deoxyribose,

don't have prefix "d"; names like ATP, ADP,
AMP refer to RNA nucleotides.
- Purine & Pyrimidine bases are attached to deoxyribose sugar, free to rotate. In DNA,
form specific base pairs: A with T (2 H-bonds), G with C (3 H-bonds).
- Two chains of DNA face in opposite directions, called antiparallel (defined by which
way 3' and 5' sides of sugar molecule are facing).
- In a linear DNA molecule, one strand has free 3'-end, other
(complementary) strand has 5'-end.
5'-CAGCTAGAGTCATCG-3'
3'-GTCGATCTCAGTAGC-5'
• DNA Replication:

- During DNA replication, the two strands of the

double helix separate at the replication fork, and each
strand is used as a template by DNA polymerases to
synthesize two new strands of DNA according to the
rules of nitrogenous base pairing.

- The result of DNA replication is two new strands of

DNA, each having a base sequence complementary
to one of the original strands.
- DNA is synthesized in one chemical direction called 5' to 3' (5' is phosphate end; 3' is
hydroxyl end of deoxyribose).
- Because each double-stranded DNA molecule contains one original and one new
strand, the replication process is called semiconservative.
- DNA polymerase proofreads new molecules of DNA and removes mismatched bases
before continuing DNA synthesis.
- Each daughter bacterium receives a chromosome identical to the parent's.
• Transcription:
- During transcription, the enzyme RNA polymerase synthesizes a strand of RNA
from one strand of double-stranded DNA, which serves as a template.
- RNA is synthesized from nucleotides containing the bases A, C, G, and U, which pair
with the bases of the DNA sense strand.
- The starting point for transcription, where RNA polymerase binds to DNA, is the
promoter site; the region of DNA that is the endpoint of transcription is the terminator
site; RNA is synthesized in the 5' —> 3' direction.
• Translation:
- Translation is the process in which the information in the nucleotide base sequence of
mRNA is used to dictate the amino acid sequence of a protein.
- The mRNA associates with ribosomes, which consist of rRNA and protein.
- Three-base segments of mRNA that specify amino acids are called codons.
- The genetic code refers to the relationship among the nucleotide base sequence of
DNA, the corresponding codons of mRNA, and the amino acids for which the codons
code.
- The genetic code is degenerate; that is, most amino acids are coded for by more than
one codon.
- Of the 64 codons, 61 are sense codons (which code for amino acids), and 3 are
nonsense codons (stop codons) which do not code for amino acids and are stop signals
for translation.
- The start codon, AUG, normally codes for methionine (formylmethionine at the
beginning or a protein).
- Specific amino acids are attached to molecules of tRNA. Another portion of the tRNA
has a base triplet called an anticodon.
- The base pairing of codon and anticodon at the ribosome results in specific amino
acids being brought to the site of protein synthesis.
- The ribosome moves along the mRNA strand as amino acids are joined to form a
growing polypeptide; mRNA is read in the 5' —> 3' direction.
- Translation ends when the ribosome reaches a stop codon on the mRNA.
- In prokaryotes, translation can begin before transcription is complete.
• Regulation of Gene Expression in Bacteria:
- Gene is a linear sequence of nucleotides that is within the genomic nucleic acid
molecule, and that has a fixed start point and end point
- It has controlling elements (e.g., promoters) that regulate expression of a gene.
- It encodes a polypeptide, a tRNA, or an rRNA.
- With some exceptions, genes are not overlapping.
- The segment that encodes a single polypeptide is also called a cistron.
- Procaryotic coding information is normally continuous although some bacterial
genes are interrupted.
- Most Eucaryotic genes have coding sequences (exons) that are interrupted by
noncoding sequences (introns).
• Genetic Transfer and Recombination:
- Genetic recombination is the transfer of DNA from one organism to another. The
transferred donor DNA may then be integrated into the recipient's nucleoid by various
mechanisms.
- Natural mechanisms of genetic recombination in bacteria include transformation,
transduction & conjungation.
 Plasmids:
- A plasmid is an "extra-chromosomal" piece of bacterial DNA.
- Plasmids are stably maintained within bacterial cells, replicating fast enough that they
are passed on to bacterial progeny as the bacteria divide.
- Like bacterial chromosomes, plasmids are circular, double-stranded DNA.
-The major difference between chromosomes and plasmids is that they are much
smaller than chromosomes, and they tend to carry genes that are not essential except in
certain environments.
- There are several types of plasmids, including conjugative plasmids, dissimilation
plasmids, plasmids carrying genes for toxins or bacteriocins, and resistance factors.
- R PLASMID: A plasmid having genes coding for multiple antibiotic resistance and
often a sex pilus.
- F+ PLASMID: A plasmid coding only for a sex pilus.
 Transformation:
- A process during which DNA fragment from a dead, degraded bacterium enters a
competent recipient bacterium.
- The DNA taken up is exchanged for a piece of DNA of the recipient.
- This process was first demonstrated in Streptococcus pneumoniae, and occurs
naturally among a few genera of bacteria.
- A bacterial cell that is capable of being transformed (i.e., of taking up DNA directly
from the environment) is said to be competent.

- Bacteria that are not naturally competent (e.g., E. coli) often can be manipulated in the
laboratory in such a way that they become able to pick up environmental DNA.
 Conjugation:
- This process requires contact between living cells
('bacterial sex').
- One type of genetic donor cell is an F+; recipient cells
are F-. F+ cells contain plasmids called F factors; these
plasmids are transferred to the F- cells during
conjugation.
- Donating bacteria is described as being male, and the recipient then becomes an F+
male and can make a sex pilus. Conjugation serves to convert the recipient bacteria also
to a male.
- When the plasmid becomes incorporated into the chromosome, the cell is called an
Hfr (high-frequency recombinant).

- At the end of Hfr conjugation there was transfer

of some donor chromosomal DNA, but usually not
a complete F+ plasmid, the recipient bacterium
usually remains F-.
 Transduction:
- Transduction is the transfer of fragments of DNA from one bacterium to another
bacterium by a bacteriophage.
- Bacteriophages are viruses that infect bacteria.
- Generalized Transduction by Lytic Bacteriophage is
done following these steps:
1. A lytic bacteriophage adsorbs to a susceptible
bacterium.

2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's
metabolic machinery to manufacture bacteriophage components and enzymes.
3. Occasionally during maturation, a bacteriophage head or capsid assembles around a
fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome
by mistake.
4. The bacteriophages are released.
5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient
bacterium.
6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient
bacterium. DNA is exchanged for some of the recipient's DNA
Bacterial Genetics Overview
 Most bacteria are haploid which means
that there is no such thing as dominance-
recessive relationships among bacterial
alleles.
 Bacteria don’t have sex in the
animal/plant sense of sex (i.e., mating
followed by recombination of whole
genomes).
 Instead, bacteria acquire DNA from other
bacteria through three distinct
mechanisms:
 Transformation
 Transduction
 Conjugation
 Genetic Transfer in Bacteria
• Genetic transfer-results in genetic variation
• Genetic variation-needed for evolution
• Three ways:
– Transformation: genes transferred from one bacterium
to another as “naked” DNA
– Conjugation: plasmids transferred 1 bacteria to another
via a pilus
– Transduction: DNA transferred from 1 bacteria to
another by a virus
 S44-61

Three mechanisms of gene transfer in bacteria

 The Genetic Material
Frederick Griffith, 1928
studied Streptococcus pneumoniae, a
pathogenic bacterium causing pneumonia
there are 2 strains of Streptococcus:
- S strain is virulent
- R strain is nonvirulent
Griffith infected mice with these strains
hoping to understand the difference between
the strains
 The Genetic Material
Griffith’s results:
- live S strain cells killed the mice
- live R strain cells did not kill the mice
- heat-killed S strain cells did not kill the mice
- heat-killed S strain + live R strain cells
killed the mice
 The Genetic Material
Griffith’s conclusion:
- information specifying virulence passed from
the dead S strain cells into the live R strain cells
- Griffith called the transfer of this information
transformation
Transformation
conjugation
Conjugation: Sex or F Pilus
Conjugation: F Plasmid Transfer
Conjugation in E. coli
Conjugation continued…
Conjugation continued…
 S48-65
Using an Hfr for gene transfer
 S53-70

Summary of F
 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.
A second form of gene transfer- transduction
 S55-71
Bacteriophage & the Genetic Code
Phage and its life cycle

Simple techniques
 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.
Transduction by a Bacteriophage
Generalized transduction
Generalized Transduction

Bacteriophages
are viruses that
only infect (and
can kill)
bacteria.
Generalized Transduction
 Generalized Transduction
• Some phages, such as phage P1, break up the bacterial
chromosome into small pieces, and then package it into some
phage particles instead of their own DNA.
• These chromosomal pieces are quite small: about 1 1/2
minutes of the E. coli chromosome, which has a total length of
100 minutes.
• A phage containing E. coli DNA can infect a fresh host,
because the binding to the cell surface and injection of DNA is
caused by the phage proteins.
• After infection by such a phage, the cell contains an exogenote
(linear DNA injected by the phage) and an endogenote
(circular DNA that is the host’s chromosome).
• A double crossover event puts the exogenote’s genes onto the
chromosome, allowing them to be propagated.
Specialized transduction
 Specialized Transduction
• Unlike the F plasmid that can incorporate anywhere in the E. coli
genome, lambda can only incorporate into a specific site, called attλ.
The gal gene is on one side of attλ and the bio gene (biotin synthesis)
is on the other side.
• Sometimes when lambda come out of the chromosome at the end of
the lysogenic phase, it crosses over at the wrong point. This is very
similar to the production of an F’ from an Hfr.
• When this happens, a piece of the E. coli chromosome is incorporated
into the lambda phage chromosome
• These phage that carry an E. coli gene in addition to the lambda genes
are called “specialized transducing phages”. They can carry either the
gal gene or the bio gene to other E. coli.
• Thus it is possible to quickly develop merodiploids (partial diploids)
for any allele you like of gal or bio. Note that this trick can’t be used
with other genes, but only for genes that flank the attachment site for
lambda or another lysogenic phage.
Mutation: Terms & Concepts
 Wild Type refers to the microorganism as
isolated from the wild.
 A mutated microorganism that has lost a
metabolic function, particularly an ability
to synthesize a specific growth factor, is
called an Auxotroph.
 The wild-type parent to an auxotroph is
called a Prototroph.
 A Mutation is found in a gene; a mutant is
an organism harboring a Mutation.
Mutation: Terms & Concepts  We designate mutant phenotypes using
three-letter abbreviations; the phenotype
of a tryptophan-requiring auxotroph would
be described as Trp-.
 A bacterium that has mutated to
resistance to an antibiotic (or other
substance) is given the superscript “R”;
thus, the phenotype ampicillin resistance
is indicated as AmpR.
 Mutants can be spontaneous or induced
by a Mutagen; an agent that causes DNA
to mutate.
  Base Substitution
Types of Mutations
 Point mutation = single base is
substituted.
 Missense mutation = base change
changes single amino acid to different
amino acid.
 Nonsense mutation = base change
changes single amino acid to stop codon.
 Null or Knockout mutation = mutation that
totally inactivates a gene.
  Deletion or insertion mutation = change in
Types of Mutations
number of bases making up a gene.
 Frameshift mutation = insertion or deletion
of something other than multiples of three
bases.
 Frameshifts typically radically change
downstream codons, generating stop
codons, and typically knocking out gene
function.
 Reversion mutation = mutated change
back to that of wild type.
 Artificial Competence
by Electroporation

Competence Most bacteria

Artificially
denotes the are not naturally
induced
ability to take competent but
competence is
up DNA many can be
very important
naked from made artificially
to gene cloning.
the so.
environment.
  F plasmids encode genes that allow both
F and Other Plasmids
their replication and transfer.
 They are thus known as Self-
Transmissible Plasmids.
 There are other plasmids that can take
advantage of conjugation but don’t
encode the the necessary genes. These
are non-self transmissible plasmids.
 Transduction is also capable of
transferring smaller plasmids.
 Plasmids
• What is a plasmid?
• Small circular, self replicating piece of
bacterial DNA
• Episomes = plasmids that can reversibly
incorporate into the bacterial chromosome
• Plasmid genes are advantageous to the
bacteria that has them
• Plasmids that confer resistance to antibiotics
are called R plasmids
Plasmid
• plasmid are genetic elements most
frequently transferred by conjugation.
• Plasmid F (fertility)
• Plasmid R (resistance)
Resistance plasmids = R plasmids made of
RTF(Resistance Transfer Factor) and R
(Resistance) Genes
R plasmids are promiscuous
• R plasmids are promiscuous
• Klebsiella
• E coli andSalmonella
• Serratia
• Shigella
F and Other Plasmids  R plasmids are named not for their mode
of transmission but instead for the
resistance genes that they encode such
as to antibiotics.
 Some plasmids are present in bacteria in
low copy numbers (1 or 2/bacterium)
whereas other plasmids are present in
high copy numbers (such 100s/bact.).
 Plasmids, R and otherwise, can have very
wide host ranges allowing easy transfer of
already evolved genes between bacterial
species.
Self-Transmissible R Plasmid
Note
multiple
resistance
genes.

Resistance Transfer Factor

(conjugation genes)
Transfer of non-R Virulence Factors  Genes that can make bacteria more virulent
(able to cause disease) are called Virulence
Factor genes.
 Virulence factors include fimbriae that allow
attachment to host tissues, exotoxins, etc.
 Virulence factor genes may be transferred by
transformation, transduction, or conjugation.
 Virulence factor genes tend to congregate on
bacterial chromosomes in regions known as
Pathogenicity Islands.
 New bacterial pathogens can emerge via the
uptake of entire pathogenicity islands transferred
intact from unrelated bacteria.
Transfer Protection: R-M Systems
 Not all incoming DNA is necessarily good
for the receiving bacterium (i.e., DNA can
be parasitic).
 Bacteria employ Restriction Enzymes to
protect themselves from the foreign DNA.
 Restriction enzymes recognize specific,
palindromic (same spelling backward and
forward) DNA sequences of 4 to 8 base
pairs in length that are known as
Recognition Sequences.
Transfer Protection: R-M Systems

 Bacteria also employ Modification

Enzymes that modify DNA to protect it
from Restriction Enzymes.
 Together these are called Restriction-
Modification Systems.
 Restriction enzymes are crucial
components of genetic engineering.
Transposons –DNA segments that shift
from one part of the genome to another
 Transposons
• Jumping genes
• Does not depend on complementary base
pairing between homologous regions of the
chromosome.
• Transposons move to regions that the gene
has never been
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