Part I

General Microbiology

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 BACTERIAL GENETICS

• DNA is TRANSCRIBED to messenger RNA

(mRNA)
• mRNA carries the message to the tranfer
RNA (tRNA)
• tRNA is TRANSLATED to an amino acid
chain which makes up proteins
• DNA → RNA → protein
• DNA: Storehouse of information for
protein synthesis

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 DNA Structure

The double helix consists of:

• Two strands of nucleotides wound together
• In ‘right-handed double helix’
• Each chain consists of sugar, phosphate and
nitrogenous bases arranged alternately
• Bases form hydrogen bonds with
complementary bases on the opposite
sugar-phosphate backbone

3
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 DNA Structure

• Attached to each deoxyribose

• One of the four nitrogenous bases,
• Purines: Adenine (A), Guanine (G)
• Pyramidines: Cytosine (C ), Thymine (T)
• The double-stranded nature of the molecule
• Stabilised by hydrogen bonding between the bases:
• Adenine pairs with Thymine (2 H bonds)
• Cytosine pairs with Guanine (3 H bonds)
• A ratio of (A + T)/(G + C) is constant for each species
and varies from one bacterial species to another

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 RIGHT-HANDED COILED DOUBLE HELIX

A schematic drawing of the Watson–Crick

structure of DNA, showing helical sugar-
phosphate backbone of the two strands
held together by hydrogen bonding
between the bases.

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 STRUCTURE OF RNA

• RNA is structurally similar to DNA

• Ribose instead of deoxyribose
• Uracil instead of thymine
• Three different types of RNA:
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
• DNA acts as a template of synthesis of mRNA
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 GENETIC INFORMATION

• Genetic information is stored in DNA as a

code
• Code is a sequence of three bases: triplet
(codon)
• Codons transcribed on mRNA specify single
amino acid
• More than one codon may exist for same
amino acid
• Example: AGG, CGU, CGC, CGA and CGG all
code for the same amino acid

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 DNA: CHEMICAL STRUCTURE

A segment of double-stranded DNA illustrating its chemical structure

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 GENETIC INFORMATION

• Stretches of DNA that do not appear to

function as codons occur between coding
sequences of genes.
• INTRONS: Non-coding intrusions
• EXONS: Coded sequences
• During transcription, the entire genome is
copied
• Introns are excised from RNA copy - intron
splicing
• Leads to a functional coding gene

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 GENE

• Gene is a segment of DNA carrying codes

specifying for a particular polypeptide
• DNA molecule consists of a large numbers
of genes
• Length of DNA expressed as kilobases (1Kb
= 1000 base pairs)
• Bacterial DNA = 4000 Kb
• Human genome = 3 Million Kb long

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 EXTRA CHRMOMSOMAL GENETIC ELEMENTS

• Plasmids are circular DNA molecules

• Autonomous replication (independent
replication) transfer genes from one cell to
another
• May integrate with chromosomal DNA

Plasmid: 1. ampicillin resistance

sequence; 2. origin of replication
site; 3. multiple cloning site

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 GENOTYPIC/PHENOTYPIC VARIATIONS

Genotypic variations: stable, heritable, not

influenced by environment
• Mutations
• Genetic transfer
 Transformation
 Transduction
 Lysogenic conversion
 Conjugation
• Phenotypic variation: Influenced by
environment, temporary, not heritable

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 MUTATION

Random, undirected, heritable variations

Alteration in nucleotide sequence
• Addition, deletion, substitution of one or
more bases
• MISSENSE MUTATION: Triplet code altered
from that normally located at a
particular position
• NON-SENSE MUTATION: Deletion of
nucleotide within a gene

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 MUTATION

• TRANSVERSION: Substitution of a mutant

phenotype by another mutation at a position
distinct from the original mutation
• SUPPRESSOR MUTATION: Reversal of a
mutant phenotype by another mutation at a
portion distinct from the original mutation
• LETHAL MUTATION: Some able to live under
certain conditions (permissive)
Example: temperature sensitive (ts) mutants
live at permissible temperature (35°C); not at
restrictive temperature (39°C)
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 TYPES OF MUTATION

Examples of types of mutations

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 REPLICA PLATING METHOD

• 1943 Luria and

Delbruck
fluctuation test
• 1952 Lederberg
and Lederberg
replica plating

Replica plating method

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 TRANSMISSION OF GENETIC MATERIAL

GENE TRANSFER
• TRANSFORMATION: Transfer of portion of
genetic information through free DNA
• 1928 - Griffith
• 1944 - Avery, Macleod and McCarty

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 TRANSFORMATION

Transformation experiment of Griffith

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 TRANSMISSION OF GENETIC MATERIAL

• TRANSDUCTION: Transfer of portion of DNA

by bacteriophage
• Generalised transduction: Any segment of
donor DNA is transferred
• Restricted transduction: Specific
bacteriophage transduces only a particular
trait. Example: ‘Lamda’ phage of E.coli

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 TRANSDUCTION

Transduction

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 LYSOGENIC CONVERSION

Lysogenic Bacteria
• Process – Lysogenic or phage conversion
• Bacteriophage, two types of life cycles
• Virulent or lytic cycle: Progeny phages
inside host bacterium, ruptures and
release
• Temperate or non-lytic cycles: Host
bacterium unharmed
• Phage DNA integrated in bacterial
chromosome as prophage
• Multiplies synchronously with host DNA

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 CONJUGATION

• ‘Male’ or ‘donor’ bacterium

• Physical contact with ‘female’ or recipient
bacterium
• Leads to transfer of genetic elements
• Bacterial conjugation – Lederberg and
Tatum (1946)
• Sex pilus
• Conjugation tube
• E.coli K12 – role of plasmids in conjugation
first recognised
• Plasmid – ‘sex factor’ or ‘fertility (F) factor’

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 CONJUGATION

Mechanism of DNA transfer during conjugation

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 CONJUGATION

‘F’ FACTOR
• Transfer factor
• Synthesis of sex pilus
• Episome: Integrated into host chromosome
• Hfr: Transfer chromosomal genes to
recipient cells with high frequency
• FI (F Prime): F factor incorporating some
chromosomal genes through FI factor

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 SEXDUCTION

Sexduction. The integrated F factor of an Hfr cell may revert to the

cytoplasmic state. During excision some host genes may be incorporated
in the F’ factor (F). When an F’ cell mates with and F— cell, the host gene
is transferred to the recipient.

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 COLICINOGENIC (Col) FACTOR

• Several strains of coliform - colicins

• Antibiotic-like substances
• Pseudomonas pyocyanea, pyocin
• Corynebacterium diptheriae, diphthericin
• Colicin production – plasmid – Col factor

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 RESISTANCE TRANSFER FACTOR (RTF)

• Multiple drug resistance among bacteria

• Resistance – plasmid mediated
• Transfer – conjugation
• Mechanism: Transferable, episomal or
infectious drug resistance
• Plasmid: Two components
• RTF - Resistance Transfer Factor
• r resistance determinant
• R factor = (RTF + r determinant)

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 RESISTANCE TRANSFER FACTOR (RTF)

Transferable drug resistance. The R+ cell carries the R factor, consisting

of the RTF and r determinants. Its transfer to a sensitive R− bacterium
converts the recipients into a resistant R+ cell.
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 GENETIC MECHANISMS OF DRUG RESISTANCE

Mutation
• Genetic transfer
• Biochemical mechanism
• Decreased permeability to drug
• Development of alternative metabolic pathway
• Production of enzymes inactivating drugs
MUTATIONAL RESISTANCE
• Stepwise mutation: Penicillin
• ‘One-step’ mutation: Streptomycin
• Multiple drug therapy in tuberculosis
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 TRANSFERABLE DRUG RESISTANCE

• Mediated by R factor
• Simultaneous resistance to several drugs
• Prevents dissemination by restricted use of
antibiotics

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 TRANSPOSABLE GENETIC ELEMENTS

• Genetically discrete segments of DNA move

around in ‘cut and paste’ manner
• Chromosomal and extrachromosomal DNA
• Transposons ‘jumping genes’
TRANSPOSITION
• Barbara MacClintock: Nobel Prize for
discovering transposition in maize

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 TRANSPOSONS

• Segment of DNA with one or more genes in

the centre, the two ends carrying ‘inverted
repeat ‘ sequences of nucleotides –
nucleotide sequences complimentary to each
other, but in reverse order
• Each strand of transposon can form a single-
stranded loop carrying the gene and a
double-stranded stem formed by hydrogen
bonding between terminal inverted repeat
sequences

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 TRANSPOSON

Structure of transposon

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 TRANSPOSON

• Small transposons ‘insertion sequences’ or

IS
• Transposons attach to certain regions of
chromosomal, plasmid or phage DNA
• Insertion of transposon leads to acquisition
of new characteristics by the recipient DNA
molecule
• Unlike plasmids, transposons are not self-
replicating and depend on chromosomal or
plasmid DNA for replication
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 MOLECULAR GENETICS

• Analysis and manipulation of DNA using

biochemical and microbiological techniques
• Genetic engineering
• Restriction endonucleases
• DNA probes
• Blotting techniques
• Polymerase Chain Reaction

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 GENETIC ENGINEERING

• Genetic engineering or recombinant DNA

technology

• Consists of isolation of genes coding for a

desired protein from microorganism or cells
of higher forms – their introduction into
suitable microorganisms, in which genes
would be functional – directing production
of specific protein

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 GENETIC ENGINEERING

Uses
• Cloned human insulin
• Interferons
• Somatostatin growth hormone
• Vaccines – Hepatitis B, rabies

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 RESTRICTION ENDONUCLEASES

• Microbial enzymes that cleave double-

stranded DNA
• Examples: Eco RI, Hind III, Taq I
• Restriction enzymes split DNA strands into
fragments of varying length
• These are separated by gel electrophoresis
stained with ethidium bromide and
photographed

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 DNA PROBES

• Specific interaction in base pairing during

DNA or RNA synthesis enables production
of specific probes
• They are radioactive, biotinylated or
otherwise labelled copies of cloned DNA
• Fragments 20-25 nucleotides long –
containing unique sequences that can be
used to detect homologous DNA by
hybridisation

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 DNA Probes
USES
• Diagnosis of infectious diseases
• To detect microbe in cultures, body fluids,
tissues or other material
ADVANTAGES
• High degree of specificity
• Ability to detect minute quantities of DNA
• Capacity to recognise difficult to culture
microbes
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 BLOTTING TECHNIQUES

SOUTHERN BLOTTING
• EM Southern
• DNA fragments obtained by restriction
enzyme digestion - separated on gel
transferred by blotting to nitrocellulose
membrane
• DNA bound to membrane is denatured and
treated with radioactive single-stranded DNA
probes
• These probes hybridise with homologous
DNA to form radioactive double-stranded
segments that can be detected on X-ray film
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 SOUTHERN BLOTTING

Southern blotting
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 NORTHERN BLOTTING

• An analogous procedure used for analysis of

RNA
• RNA mixture separated by gel electrophoresis
• Blotted and identified using DNA or RNA
probes

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 WESTERN BLOTTING

• A similar technique for identification of

proteins
• The protein antigen mixture separated by
SDS-PAGE, blotted on nitrocellulose
membrane and identified by radiolabelled or
enzyme-labelled antibodies as probes
USE
• Ability to separately identify antibodies
against different antigens of a pathogen

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 PCR

• Kary B Mullis – 1983

• Consists of sequential DNA replication,
where products of the first cycle become
template for the next cycle
• Makes available abundant quantities of DNA
from small quantities of the same
USES
• Diagnosis of infections, genetic diseases,
neoplastic diseases and forensic
investigations
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 PCR

Amplification of genomic DNA by PCR (Continues…

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 (Continued…

Amplification of genomic DNA by PCR

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