MB 503 Microbial Genetics & Molecular Biology

1.1 Genetics

Genetics is the branch of biology concerned with heredity and variation. In microorganisms, genetics explains how traits are inherited, expressed, altered and transferred between cells.

1.2 Microbial Genetics

Microbial genetics deals with the organization and transmission of hereditary information in microorganisms such as bacteria, archaea, fungi and viruses.

Microorganisms are particularly useful in genetic studies because they have short generation times, relatively simple genomes and can undergo horizontal gene transfer.

1.3 Gene

A gene is a functional unit of hereditary information encoded in nucleic acid. A gene may encode a protein or a functional RNA molecule.

1.4 DNA

Deoxyribonucleic acid (DNA) is the major hereditary material of cellular organisms. DNA is composed of nucleotides containing deoxyribose sugar, phosphate and nitrogenous bases.

The four principal bases in DNA are adenine (A), guanine (G), cytosine (C) and thymine (T).

1.5 Structure of DNA

DNA generally exists as a double-stranded helix. The two strands are antiparallel and are held together by hydrogen bonding between complementary bases.

• Adenine pairs with thymine through two hydrogen bonds.
• Guanine pairs with cytosine through three hydrogen bonds.
• The sugar-phosphate backbone forms the outer part of the helix.
• The nitrogenous bases project toward the interior.

1.6 RNA

RNA is generally single stranded and contains ribose sugar. Uracil replaces thymine. RNA performs structural, catalytic and informational functions in the cell.

1.7 Genome

The genome is the complete genetic material present in an organism or biological entity. A bacterial genome generally consists of a main chromosome and may contain additional extrachromosomal DNA such as plasmids.

1.8 Genotype and Phenotype

2.1 Genetic Recombination

Genetic recombination is the formation of a new genetic combination by rearrangement or exchange of DNA between genetic molecules.

2.2 Horizontal Gene Transfer

Horizontal gene transfer (HGT) is the movement of genetic information between organisms other than by direct parent-to-offspring inheritance.

2.3 Transformation

Transformation is the uptake of naked extracellular DNA by a competent bacterial cell followed by incorporation or maintenance of the incoming DNA.

2.4 Conjugation

Conjugation is the transfer of DNA from one bacterial cell to another through direct cell-to-cell contact. In many Gram-negative bacteria, conjugation involves a conjugative plasmid such as the F plasmid.

Basic mechanism

1. Donor contains conjugative DNA.
2. Donor establishes contact with recipient.
3. Transfer machinery is assembled.
4. One DNA strand is transferred.
5. Complementary strand synthesis occurs.
6. Both cells acquire the transferred DNA.

2.5 Transduction

Transduction is the transfer of bacterial DNA from one cell to another through a bacteriophage.

3.1 Definition

DNA replication is the process by which a DNA molecule produces two daughter DNA molecules. Replication is semiconservative, meaning each daughter DNA molecule contains one parental strand and one newly synthesized strand.

3.2 Important Enzymes

3.3 Replication Fork

DNA polymerases synthesize DNA only in the 5′ to 3′ direction. Because the two DNA strands are antiparallel, synthesis occurs continuously on one strand and discontinuously on the other.

3.4 Steps of DNA Replication

1. Initiation: Replication begins at an origin of replication.
2. Unwinding: Helicase separates the DNA strands.
3. Primer formation: Primase produces RNA primers.
4. Elongation: DNA polymerase adds nucleotides.
5. Primer removal: RNA primers are removed.
6. Gap filling: DNA replaces removed primers.
7. Ligation: DNA ligase seals remaining nicks.
8. Termination: Replication ends when daughter molecules are completed.

3.5 Leading and Lagging Strand

3.6 Prokaryotic vs Eukaryotic Replication

4.1 Definition

Transcription is the synthesis of RNA using one strand of DNA as the template. RNA polymerase catalyzes the process.

4.2 Stages of Transcription

Initiation

RNA polymerase recognizes the promoter region of DNA. In bacteria, sigma factor helps RNA polymerase identify promoter sequences. DNA locally unwinds and transcription begins.

Elongation

RNA polymerase moves along the template DNA and synthesizes RNA in the 5′ to 3′ direction.

Termination

Transcription terminates when RNA polymerase encounters termination signals. Bacteria may use intrinsic termination or factor-dependent termination mechanisms.

4.3 Transcription in Viruses and Bacteriophages

Viral transcription depends on the nature of the viral genome. DNA viruses generally use host or viral RNA polymerases, whereas many RNA viruses require specialized RNA-dependent RNA polymerases.

Retroviruses use reverse transcriptase to synthesize DNA from an RNA template.

4.4 Post-transcriptional Processing

In eukaryotic cells, primary RNA transcripts undergo processing before becoming mature mRNA.

• 5′ capping
• 3′ polyadenylation
• RNA splicing
• Removal of introns
• Joining of exons

5.1 Definition

Translation is the process by which ribosomes decode the nucleotide sequence of mRNA and synthesize a polypeptide according to the genetic code.

5.2 Role of RNA

5.3 Genetic Code

The genetic code is the relationship between nucleotide codons in mRNA and amino acids incorporated into proteins.

• The code is triplet.
• There are 64 possible codons.
• 61 codons specify amino acids.
• Three codons are termination codons.
• AUG commonly functions as the start codon.
• The code is degenerate because several codons can specify the same amino acid.
• The code is largely universal.

5.4 Stages of Translation

1. Activation / aminoacylation of tRNA
2. Initiation
3. Elongation
4. Termination
5. Post-translational processing

Initiation

The small ribosomal subunit associates with mRNA. Initiator tRNA recognizes the start codon. The large ribosomal subunit then joins to form the complete translation complex.

Elongation

Aminoacyl-tRNAs enter the ribosome, peptide bonds are formed, and the ribosome moves along the mRNA.

Termination

When a stop codon enters the ribosomal decoding site, release factors promote release of the newly synthesized polypeptide.

5.5 Post-translational Modification

Newly synthesized proteins may undergo chemical or structural modifications that are necessary for biological activity, localization or stability.

• Proteolytic cleavage
• Phosphorylation
• Glycosylation
• Acetylation
• Methylation
• Disulfide bond formation
• Addition of prosthetic groups

6.1 Importance of Gene Regulation

Gene regulation allows cells to produce gene products only when they are required. This conserves energy and enables adaptation to changing environmental conditions.

6.2 Levels of Regulation

6.3 Lac Operon

The lac operon is an inducible operon responsible for the utilization of lactose in bacteria such as Escherichia coli.

Components

• Regulatory gene lacI
• Promoter
• Operator
• lacZ
• lacY
• lacA

In absence of lactose

The repressor binds the operator and prevents efficient transcription of the structural genes.

In presence of lactose

Lactose-derived inducer interacts with the repressor, reducing its ability to bind the operator. Transcription of the lac structural genes can then occur.

Catabolite repression

The lac operon is also regulated by glucose availability. Low glucose increases cellular cAMP, allowing CAP/CRP-mediated activation of transcription when lactose is available.

6.4 Trp Operon

The trp operon is a repressible operon involved in tryptophan biosynthesis. When tryptophan is abundant, it acts as a corepressor and promotes repression of the operon.

7.1 Definition

A mutation is a heritable change in the nucleotide sequence of genetic material. Mutations may occur spontaneously or be induced by mutagenic agents.

7.2 Types of Mutations

Point mutation

A change involving a single nucleotide pair.

• Silent mutation: codon changes but the same amino acid is specified.
• Missense mutation: a different amino acid is incorporated.
• Nonsense mutation: a codon is changed into a stop codon.

Insertion

Addition of one or more nucleotides.

Deletion

Removal of one or more nucleotides.

Frameshift mutation

Insertion or deletion of nucleotides not in multiples of three can shift the reading frame and alter downstream codons.

7.3 Mutagenic Agents

7.4 Detection of Mutants

Mutants can be detected by identifying changes in phenotype, growth characteristics, nutritional requirements, resistance patterns or molecular markers.

Replica plating

Replica plating allows colonies from a master plate to be transferred to selective media. It is useful for identifying mutants with altered nutritional or resistance phenotypes.

8.1 Definition

Recombinant DNA technology involves the artificial combination, manipulation and propagation of DNA molecules from different sources.

8.2 General Principle

8.3 Sources of DNA for Cloning

• Genomic DNA
• Complementary DNA (cDNA)
• PCR-amplified DNA
• Synthetic DNA
• Plasmid DNA

8.4 Restriction Enzymes

Restriction endonucleases recognize specific nucleotide sequences and cleave DNA at or near those sites.

Many restriction enzymes recognize palindromic sequences. Cleavage may produce sticky ends or blunt ends.

8.5 DNA Ligase

DNA ligase joins compatible DNA fragments by forming phosphodiester bonds between adjacent nucleotides.

8.6 Cloning Vectors

A cloning vector is a DNA molecule capable of carrying a foreign DNA fragment into a host cell and allowing its replication or maintenance.

8.7 Essential Features of a Vector

• Origin of replication
• Selectable marker
• Multiple cloning site
• Suitable size
• Host compatibility
• Sometimes an expression promoter

8.8 Expression Vectors

Expression vectors contain regulatory sequences that permit transcription and translation of a cloned gene in a suitable host.

Important components may include promoters, ribosome-binding sequences, transcription terminators, selectable markers and affinity tags.

8.9 Detection of Recombinant DNA

• Restriction digestion analysis
• Colony PCR
• Gel electrophoresis
• DNA sequencing
• Hybridization
• Reporter gene analysis

8.10 Cloning Eukaryotic Genes in Bacteria

Direct cloning of many eukaryotic genomic genes into bacteria may not produce functional proteins because bacterial cells generally cannot remove eukaryotic introns in the same manner as eukaryotic cells.

Therefore, mature eukaryotic mRNA can be converted into cDNA using reverse transcriptase. The cDNA can then be cloned into an appropriate bacterial expression vector.

9.1 DNA Extraction

DNA extraction involves disruption of cells, removal of proteins and other contaminants, and recovery of purified DNA.

9.2 Plasmid DNA Isolation

Plasmid DNA can be separated from bacterial chromosomal DNA using methods based on differential molecular structure and alkaline lysis.

General alkaline lysis principle

1. Cell resuspension
2. Alkaline lysis
3. Neutralization
4. Removal of precipitated contaminants
5. Recovery of plasmid DNA
6. Purification

9.3 PCR

Polymerase chain reaction (PCR) is an in vitro method for exponential amplification of a selected DNA sequence.

Essential components

• Template DNA
• Forward primer
• Reverse primer
• dNTPs
• Thermostable DNA polymerase
• Mg²⁺
• Reaction buffer

Three fundamental PCR stages

9.4 Real-Time PCR / qPCR

Real-time PCR monitors amplification during the reaction using fluorescence. It can be used for quantitative or relative measurement of nucleic acid targets depending on the assay design.

Important concepts include threshold cycle (Ct/Cq), fluorescent reporter systems and amplification curves.

9.5 RFLP

Restriction fragment length polymorphism (RFLP) detects genetic variation based on differences in restriction enzyme cleavage patterns.

9.6 DNA Fingerprinting

DNA fingerprinting is the analysis of polymorphic DNA markers to generate a genetic profile. It has applications in forensic science, identity testing, population studies and microbial epidemiology.

9.7 Southern Blotting

Southern blotting detects specific DNA sequences after separation of DNA fragments by electrophoresis and transfer to a membrane. A labeled complementary probe is then used for detection.

9.8 Northern Blotting

Northern blotting is used to detect specific RNA molecules. It is useful for studying RNA size and gene expression.

9.9 Western Blotting

Western blotting detects specific proteins using antibodies. Proteins are separated by electrophoresis, transferred to a membrane and detected using specific antibodies.

9.10 PFGE

Pulsed-field gel electrophoresis (PFGE) separates very large DNA fragments by periodically changing the direction of the electric field.

It has historically been important for strain typing and investigation of microbial outbreaks.

9.11 DNA Sequencing

DNA sequencing determines the nucleotide sequence of DNA. Classical Sanger sequencing uses chain-terminating dideoxynucleotides, whereas modern sequencing platforms can generate very large numbers of sequences in parallel.

9.12 DNA Microarray

DNA microarrays allow simultaneous analysis of many nucleic acid targets. They have been used to study gene expression, genetic variation and microbial genomic characteristics.

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