Microbial Genetics.

Microbial Genetics
◼ Genetics is the science of heredity;
it includes the study of what genes
are, how they carry information, how
they are replicated and passed to
subsequent generations of cells or
passed between organisms, and how
the expression of their information
within an organism determines the
particular characteristics of that
organism.
◼ The genetic information in a cell is called the genome.
◼ A bacterial cell's genome includes its chromosome and plasmid.
Chromosomes are structures containing DNA that physically carry
hereditary information.
◼ The chromosomes contain the genes. Genes are segments of DNA
(except in some viruses, in which they are made of RNA) that code
functions.
◼ DNA is a macromolecule composed of repeating units called
nucleotides.
◼ Nucleotide consists of a nucleobase (adenine, thymine, cytosine.
or guanine), deoxyribose (a pentose sugar), and a phosphate
group. The DNA in a cell looks like long strands of nucleotides
twisted together in pairs to form a double helix.

The structure of DNA helps to explain two main features of biological

information storage.
◼ First, the linear sequence of bases provides the actual information.
◼ Second, the complementary structure helps to duplicate DNA
during cell division.
 Genotype and Phenotype
◼ The genotype of an organism is its genetic
information that codes all the specific particular
characteristics of the organism.
◼ The genotype represents potential properties, but
not the properties themselves.
◼ Phenotype refers to actual, expressed properties,
such as the organism's ability to perform a particular
chemical reaction.
◼ So, phenotype is the manifestation of genotype.
◼ Bacteria typically
have a single circular
chromosome
consisting of a single
circular molecule of
DNA with associated
proteins. The
chromosome is
looped and folded Chromosomal

and attached at one

or several points to A prokaryotic chromosome
the plasma
membrane.
 Genetic features of microbes
1. Haploidy – the presence of one
chromosome in microbes.
The gene in prokaryotes works on the
principle of 1 gene – 1 protein
There are:
1 – structural or coding genes,
2 – regulatory genes – determine the
transcription of structural genes,
3 – the operator gene.
2. High reproduction rate:
microbial cells (by transverse division)
3. Offspring cell receives a chromosome
identical to the parent’s
4. Among bacterial cells
there are donor cells and recipient cells

5. The presence of extra-chromosomal

genetic structures in bacteria: plasmids,
transposons, Is sequences
6. Variability (phenotypic and genotypic
The specificity of DNA replication in
a microbial cell
1. it proceeds in a semi-conservative
mode;
2. the reliability of replication is provided
by the link with the CPM;
3. replication starts at a certain DNA locus
and proceeds simultaneously in two
opposite directions;
4. the synthesis of the daughter DNA
chains occur in a graded manner, in short
fragments: 1000-2000 nucleotides, which
are sewn together by ligase enzymes;
5. the replication process is accompanied
by the formation of an intercellular
transverse partition (septum);
6. at the final stage the daughter cells go
apart from one another. Each cell contains
DNA molecules identical to those of the
mother cell.
 RNA and Protein Synthesis
Transcription
is the synthesis of a complementary
strand of RNA from a DNA template. In
the process of transcription, genetic
information in DNA is copied, or
transcribed, into a complementary
base sequence of RNA. The cell then
uses the information encoded in this
RNA to synthesize specific proteins
through the process of translation.
Transcription allows the cell to produce
short-term copies of genes that can be
used as the direct source of
information for protein synthesis.
Messenger RNA acts as an intermediate
between the permanent storage form,
DNA, and the process that uses the
information, translation
Translation
mRNA serves as the source of
information for the synthesis of
proteins. Protein synthesis is called
translation because it involves
decoding the “language” of nucleic
acids and converting it into the
“language” of proteins.
In prokaryotic cells, the translation of
mRNA into protein can begin even
before transcription is complete.
Because mRNA is produced in the
cytoplasm in prokaryotes, the start
of an mRNA being transcribed are
available to ribosomes before the
entire mRNA molecule is even made.
 Types of variability in microbes:
◼ 1- phenotypic or modification - is a temporary,
hereditarily unstable change in the properties of microbes.
They are controlled by the microbial genome but are not
accompanied by changes in the genome and are therefore
rapidly lost (polymorphism, accumulation of spare
substances, spore formation, loss of capsule or
flagella,changes in cultural properties, etc.)

◼ 2- extra-chromosomal (plasmid, transposone, IS)

◼ 3- genotypic: mutations and recombinations.

 Plasmids
Plasmids are extra-chromosomal, double-stranded
super-spiraled DNA molecule. They carry from 40 to
50 genes and, due to this carry definite traits.
Dimensions: from 1,5 to 200 mln Da, the molecular
weight from 3-6 mln Da to 50-70 mln Da.
The larger the molecular weight, the larger and more
complicated the set of genes, the lager number of
the functions, which plasmids perform.
They carry:
1. the genes of self-replication;
2. the genes encoding self-transfer to other microbial
cells (tra-operons);
3. the genes, determining the features of the
plasmid itself.
4. plasmids transferred 1 bacteria to another via a pilus (
the process of conjugation)
 Classification of the plasmids
By localization in a bacterial cell the plasmids may be:
1) autonomous, i.e. not integrated into a bacterial chromosome. They freely move
in the cytoplasm of bacteria and are capable of self-replication;
2) integrated, i.e. incorporated into a bacterial chromosome. Their reproduction is
simultaneous with that of the bacterial chromosome.
By a transmission mode of plasmids in a bacterial population:
1) transmissive, capable of transmission during conjugation (i.e. horizontally);
2) non-transmissive, capable of transmission only during reproduction
(multiplication) (i.e. vertically).
By the functions performed by plasmids:
1) regulatory: participate in compensating the existing metabolic disorders in a
bacterial cell. They incorporate into the damaged genome and restore its
functions;
2) encoding: bring in new genetic information that encodes new extraordinary
features (for ex. resistance to antibiotics).
By a codified feature:
◼ R-plasmids: resistance to multiple drugs;
◼ F-plasmids: fertility, donor functions;
◼ Col-plasmids: production of bacteoricines;
◼ Ent-plasmids: synthesis of enterotoxin;
◼ Hly-plasmids: synthesis of hemolysins.
Transposons possess the following properties:
1) are able to incorporate into various sites
(segments) of a bacterial chromosome;
2) are able to move from one gene to another;
3) if inserted into a bacterial DNA, they can cause
mutations of duplication, and of deletion and
inversion if transposed;
4) are able to incorporate into plasmids, move from
the plasmid to the bacterial chromosome and
backwards;
5) are incapable of self-replication;
6) transposons contain genes that are able to
introduce essential traits into a bacterial cell, for
example, resistance to multiple drugs
 IS-elements (insertion sequences)
1) capacity to move, out only along the replicon into which
they are integrated; they can not move freely;
2) unable to replicate on their own;
3) encode the synthesis of the enzymes for a site-specific
recombination and the enzymes that control transpositions
frequency.
The functions of the IS-sequences:
1) coordinator: the interaction of plasmids, moderate
bacteriophages of transposons and of the genophore to
provide reproduction;
2) regulatory: the genomic activity of a bacterial cell, gene
inactivation, integration with a chromosome to promote its
activity (promoter), switching-in and switching-off of
transcription;
3) induction of the deletion and inversion mutations
 Change in the Genetic
Material
of microbes

Mutation Recombination
 Mutation
◼ Mutation is a stable inherited change in the
properties of a microorganism (morphological,
cultural, biochemical, biological, etc.), which is not
associated with the recombination process. Mutation
which occurs in the nucleoid and is determined by a
definite region (nucleotide) of the DNA molecule, and
cytoplasmic mutation, i. e. inherited changes which
take place in the cytoplasm and are transmitted by
the cytoplasmic structures, are distinguished.

◼ Mutations may be accompanied by loss (deletion) or

addition (duplication) of one base or a small group of
bases in the DNA molecule as well as with a change
in the sequence of the DNA nucleotides.
 Classification of mutations
I. By origin mutations are divided into:
1- spontaneous ("wild“ - arise for no apparent reason);
2- induced (occur as a result of the action of special mutagenic
factors on bacteria: chemical, physical,biological).
II. By place:
1 - point mutations (base substitution)– affect only one nucleotide
per DNA. They arise: a) as a result of the replacement of one
nucleotide by another. Allocate: 1) simple base substitution or
transit: purine is replaced by purine, and pyrimidine by pyrimidine,
2) complex or transversion – when purine is replaced by pyrimidine
and vice versa.
2 – gene mutations affect one gene,
3 – chromosomal mutations affect part of chromosomes.
Gene and chromosomal mutations are based on the transfer of
special genetic structures-transposons - from one part of the
chromosome to another.
 Gene and chromosomal mutations
occur as a result of:
a) rotation of the chromosome section
by 180 – inversion,
b) loss of a large number of
nucleotides – deletion - frameshift
mutation,
c) repetition of the chromosome –
duplication,
d) rearrangement of chromosome
segments - dislocation.
III. By changing the feature
1. direct – lead to the appearance of a new feature,
2. reverse – return the mutated cell to its original
genetic state.
IV. By phenotypic manifestations
1. missense mutations - visible or explicit mutations -
mutations with meaning,
2. nonsense mutations - a terminal codon is formed,
which causes premature termination transcription, and
then - the end of the synthesis of the polypeptide chain
4. frameshift mutations almost always result in a long
stretch ofaltered amino acids and the production of an
inactive protein from the mutated gene.
5. silent mutations
6. lethal mutations
 Genetic Recombinations
◼ Genetic recombination refers to the exchange
of genes between two DNA molecules to form
new combinations of genes on a
chromosome.
◼ This group of variation includes gene
recombinations occurring as the result of
transformation, transduction, and
conjugation. Genetic recombinations of
bacteria and viruses produce recombinants
possessing the properties of both parents: the
main set of the recipient's genes and a small
part of the donor's genes.
 Genetic Transfer in Bacteria
◼ Three ways:
• Transformation: genes transferred from
one bacterium to another as “naked”
DNA
• Conjugation: plasmids transferred from
1 bacteria to another via a pilus
• Transduction: DNA transferred from 1
bacteria to another by a virus.
Transduction-DNA passed from 1
bacteria to another in a bacteriophage
(virus) and then incorporated into the
host DNA.
 TRANSDUCTION
◼ Genetic transfer by transduction is mediated by
bacterial viruses (bacteriophages), which pick up
fragments of DNA and package them into
bacteriophage particles.
◼ The DNA is delivered to infected cells and
becomes incorporated into the bacterial
genomes.
◼ Transduction can be classified as specialized if
the phages in question transfer particular genes
(usually those adjacent to their integration sites
in the genome) or generalized if the selection
of the sequences is random because of
accidental packaging of host DNA into the phage
capsid.
◼ The transfer of genetic elements from one
bacterium to another by a bacteriophage is
termed as transduction. Transduction can be
generalized or specialized.
◼ The generalized transduction is seen in lytic cycle
where segments of bacterial DNA are packaged
inside phage capsid instead of phage DNA. When
such phages infect new bacterial cells, the
bacterial DNA is injected inside. This piece of DNA
may then transfer genes to the host chromosome
by recombination. Any bacterial gene may be
transferred in generalized transduction.
◼ The specialized transduction is usually seen in
temperate phages that undergo lytic cycle. Only
those genes that are adjacent to the prophage
are transferred in specialized transduction.
 Bacteriophage

Definition:
Bacteriophage (phage) are
obligate intracellular
parasites that multiply inside
bacteria by making use of
some or all of the host
biosynthetic machinery (i.e.,
viruses that infect bacteria.)
 What is a Bacteriophage ?
◼ Viruses that attack bacteria
◼ Non-self replicating
◼ Made up of mostly proteins and DNA
◼ Bacterial specific
◼ Able to infect most group of bacteria
◼ Isolated from soil, water, sewage and
most bacterial living zones
◼ Number of progenies in a cell: 50-200
◼ Inject their genome into host cell
• Lytic cycle (virulent)
• Lysogenic cycle (temperate)
 13 Bacteriophage families
Double stranded DNA, Double stranded
Enveloped DNA, Non-enveloped
P2 SIRV 1, 2
Rudiviridae
Myoviridae
T2 Plasmaviridae
Fuselloviridae SSV1

λ Tectiviridae TTV1
PRD1
Siphoviridae
Lipothrixviridae

P22 Corticoviridae PM2

Podoviridae
Single Double
Single-stranded DNA stranded stranded
Inoviridae M13 & fd RNA RNA
MS2 phi666

Microviridae ΦX174 Leviviridae

Cystoviridae
Morphology
 Multiplication of Bacteriophages

Bacteriophages can multiply by two

alternative ways

The lytic cycle ends The lysogenic cycle -

with the lysis and the host cell remains
death of the host alive (specialized
cell transduction)

Virulent bacteriophage Lysogenic or temperate phage

The lytic cycle bacteriophage

The period during viral multiplication when complete, infective virions

aren’t yet present is called the eclipse period
Lytic Life Cycle
 Lysogenic cycle:
◼ Lysogenic or temperate phages are those that can either
multiply via the lytic cycle or enter a dormant state in the
cell. In most cases the phage DNA actually integrates into
the host chromosome and is replicated along with the host
chromosome and passed on to the daughter cells. This
integrated state of phage DNA is termed prophage. This
process is known as lysogeny and the bacteria harboring
prophage are called lysogenic bacteria. Since the
prophage contains genes, it can confer new properties to
the bacteria. These genes can change the properties of the
bacterial cell. This process is known as lysogenic
conversion or phage conversion.
◼ The lysogenic state of a bacterium can get terminated
anytime when it exposed to adverse conditions. The
separated phage DNA then initiates lytic cycle resulting in
cell lysis and releases of phages. Such phages are then
capable of infecting new cells and render them lysogenic.
 The lysogenic cycle of
bacteriophage λ in E. coli.
 Bacteriophage Applications
◼ Bacteriophage therapy
◼ Bacteriophage mediated microbial control
◼ Bacteriophage enzymes
◼ Bacteriophage display
◼ Baceriophage typing
◼ Bacteriophage as biological tracer
◼ Monitoring and validation tool
◼ Bacteriophage based diagnostics
◼ Bacteriophage as cloning vector
◼ Bacteriophage for biodegradation
Thank you for your
attention!