Bacterial Genetics Notes

Introduction to Bacterial Genetics

An overview of bacterial genetics, covering genome structure, gene expression regulation, DNA replication mechanisms, mutagenesis and repair, plasmids, and applications in biotechnology.

Basic Genetic Concepts

  • Genome: All the DNA present in a cell or virus.
  • Gene: A polynucleotide sequence coding for a functional product (polypeptide, tRNA, rRNA).
  • Transformation: Change of nonvirulent bacteria into virulent pathogens.
  • Central Dogma: DNA → RNA → Protein (conserved in all life forms).
  • Replication: Synthesis of duplicate DNA using the parental molecule as a template, catalyzed by DNA polymerase.
  • Transcription: DNA-directed RNA synthesis.
  • Translation: Decoding of RNA base sequence in mRNA to synthesize a polypeptide.
  • Histones: Proteins that condense DNA into chromatin.
  • Nucleosome: Histones + DNA (nucleoprotein complex).

Flow of Genetic Information

The central dogma states that genetic information flows unidirectionally: DNA → RNA → protein.

  • Replication: Genetic information transfer from one generation to the next.
  • Gene Expression: Information flow within a single cell.
  • DNA is the storehouse for genetic information.
  • During cell reproduction, DNA replicates and is passed to progeny cells.
  • Cells express genetic information stored in DNA, which is transcribed into mRNA.
  • mRNA is translated into protein.

Central Dogma Processes

  • Replication: Cells synthesize new DNA using original DNA as a template.
  • Transcription: DNA is copied into RNA, which is then used to make proteins.
  • Translation: RNA is translated into a protein.
  • In bacteria, transcription and translation are not physically separated due to the absence of a nuclear membrane, allowing direct translation of RNA into protein.

Nucleic Acid Structure

  • Deoxyribonucleic Acid (DNA):
    • Bases: Adenine, guanine, cytosine, and thymine.
    • Sugar: Deoxyribose.
    • Structure: Usually double-stranded.
  • Ribonucleic Acid (RNA):
    • Bases: Adenine, guanine, cytosine, and uracil (instead of thymine).
    • Sugar: Ribose.
    • Structure: Mostly single-stranded.
    • Types: Messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA) differ in function, synthesis site in eukaryotic cells, and structure.

Bacterial Genome Structure

  • A single, circular DNA molecule containing the cell's genetic information.
  • Located in the nucleoid region of the cytoplasm.
  • Size: Typically 3–5 MB, ranging from 0.7 Mb to almost 10 MB, depending on the species.
  • Structure Characteristics:
    • Circular: DNA molecule is circular without ends.
    • Double-stranded: Composed of two antiparallel strands of deoxynucleotides.
    • Supercoiled: Twisted into a condensed ball.
    • Haploid: Contains a single chromosome.
    • Gel-like: Forms a gel-like mass in the nucleoid.
    • Histones: Basic proteins that package DNA into nucleosomes, forming chromatin and regulating gene expression.

DNA Replication

  • A critical and complex process for all life.
  • Accuracy in copying DNA is essential to prevent lethal errors for the organism.
  • During replication, double helix strands separate, each serving as a template for synthesizing a complementary strand according to base pairing rules.
  • Progeny DNA molecules consist of one new and one old strand (semi-conservative replication).

DNA Replication Patterns

  • Most bacteria have circular DNA.
  • Replication is bidirectional from a single origin.
  • The replication fork is where DNA unwinds, and strands are replicated.
  • A replicon is a genome portion with an origin replicated as a unit (entire genome in bacteria).
  • Replication forms a theta (θ) shaped structure as the forks move around the circle.

Rolling-Circle Replication

  • Occurs during E. coli conjugation and in plasmid replication and some viruses (e.g., phage lambda).
  • Allows rapid, continuous production of many genome copies from a single initiation event, useful for viruses.

DNA Replication Machinery

  • DNA polymerase catalyzes the synthesis of the complementary DNA strand in the 5’ to 3’ direction by forming phosphodiester bonds.
    • Requires:
      • A template read in the 3’ to 5’ direction.
      • A primer (RNA or DNA strand) with a free 3’-hydroxyl group.
      • dNTPs.
  • DNA polymerase III is the major enzyme, assisted by DNA polymerase I.

DNA Replication Steps

  1. Unwinding: Topoisomerase unwinds the coiled DNA strands.
  2. Unzipping: DNA Helicase unzips the DNA strands by breaking hydrogen bonds, creating two template strands.
  3. Stabilization: Single-Strand Binding proteins (SSBs) keep strands separated.
  4. Base Pairing: DNA polymerase III bonds free nucleotides to nucleotides on the template strands using base pairing rules.
  5. Proofreading: DNA Polymerase I proofreads new strands and corrects errors.
  6. Joining: DNA ligase bonds the backbone together.

DNA Replication Mechanisms

  • Helicase: Separates double-stranded DNA into single strands.

  • DNA Polymerase: Creates new DNA strands by assembling nucleotides.

  • DNA Ligase: Binds broken DNA strands together.

  • Single-strand Binding Proteins: Prevent DNA from rewinding around the replication fork.

  • Topoisomerase: Prevents DNA from supercoiling.

  • DNA Gyrase: A topoisomerase that introduces negative supercoiling to compact the bacterial chromosome.

  • Initiator Proteins: Bind to specific sites in the replication origin to form a protein–DNA complex.

  • Primase: Synthesizes short complementary RNA strands (~10 nucleotides) as primers for DNA polymerase.

    • Steps:
      • Initiation: Replication begins at the replication origin, where initiator proteins bind.
      • Unwinding: Helicase opens the DNA at the replication origin.
      • Elongation: DNA polymerases create new DNA strands using a template and primer.
      • Synthesis: The leading strand is synthesized continuously, while the lagging strand is synthesized in small fragments.

  • DNA replication is semi-conservative (each new DNA pair contains one original and one new strand).

DNA Synthesis Details

  • DNA polymerase synthesizes in the 5’ to 3’ direction only.
  • The lagging strand is synthesized in short fragments called Okazaki fragments.
  • A new primer is needed for the synthesis of each Okazaki fragment.

Events at the Replication Fork in E. coli

  • DnaA proteins bind oriC (origin of replication), causing bending and separation of AT-rich strands.
  • DnaB and other helicases separate strands; SSB proteins attach.
  • Primase synthesizes an RNA primer.
  • The Lagging and leading strands are synthesized simultaneously.
  • DNA polymerase I removes RNA primers and fills gaps with DNA.
  • Okazaki fragments are joined by DNA ligase.

Proofreading in DNA Replication

  • Removal of mismatched bases from the 3’ end of the growing strand by exonuclease activity of the enzyme.
  • Carried out by DNA polymerase III.
  • This activity is not 100% efficient.

Termination of Replication in E. coli

  • Replication stops when the replisome (Tus) reaches the termination site (ter) on DNA.

  • Catenanes form when the two circular daughter chromosomes do not separate.

  • Topoisomerases temporarily break the DNA molecules so the strands can separate.

    • TusTus = terminus utilization substance

Gene Structure

  • Gene:
    • Basic unit of genetic information.
    • Nucleic acid sequence coding for a polypeptide, tRNA, or rRNA.
    • Linear sequence of nucleotides with a fixed start and end point.
    • Codons in mRNA code for single amino acids.

Protein-Coding Genes

  • The template strand of DNA directs RNA synthesis and is read in the 3’ to 5’ direction.
  • The complementary DNA strand is the coding strand, with the same nucleotide sequence as mRNA (except thymine).

Gene Elements

  • The promoter is located at the start of the gene and is the recognition/binding site for RNA polymerase; it orients the polymerase.
  • The leader sequence is transcribed into mRNA but is not translated into amino acids.
  • The Shine-Dalgarno sequence is important for the initiation of translation.

Protein-Coding Gene Details

  • Begins with DNA sequence 3’-TAC-5', which produces the codon AUG and codes for N-formylmethionine, a modified amino acid used to initiate protein synthesis in bacteria.
  • The coding region ends with a stop codon, immediately followed by the trailer sequence, which contains a terminator sequence used to stop transcription.

tRNA and rRNA Genes

  • DNA sequences that code for tRNA and rRNA are considered genes.
  • Genes coding for tRNA may code for more than a single tRNA molecule or type.
  • Genes coding for rRNA are transcribed as a single, large precursor.
  • Spacers between the coding regions of both are removed after transcription, some by special ribonucleases called ribozymes.

Regulatory Genes

  • Regulatory sequences encode regulatory genes.
  • Control the expression of one or more other genes.
  • Located 5' or 3' to the start/end site of transcription of the gene they regulate.
  • Inverted repeats play a role in gene regulation, DNA replication, and recombination processes.

Transcription Overview

  • RNA synthesis under the direction of DNA.
  • RNA produced has a complementary sequence to the template DNA.
  • Three types of RNA are produced:
    • mRNA carries the message for protein synthesis.
    • tRNA carries amino acids during protein synthesis.
    • rRNA molecules are components of ribosomes.
  • Steps: Initiation, Elongation, and Termination.

Transcription in Bacteria

  • Polycistronic mRNA is often found in bacteria and archaea; it contains directions for >1 polypeptide catalyzed by a single RNA polymerase.
  • The reaction is similar to that catalyzed by DNA polymerase.

Bacterial RNA Polymerases

  • Most bacterial RNA polymerases have a core enzyme composed of 5 chains (β‘βααω\beta` \beta \alpha \alpha \omega) and catalyzes RNA synthesis.
  • The sigma (σ) factor has no catalytic activity but helps the core enzyme recognize the start of genes.
  • Holoenzyme = core enzyme + sigma factor.
  • Only the holoenzyme can begin transcription.

Transcription: Initiation

  • Only a short DNA segment is transcribed.
  • The promoter is the site where RNA polymerase binds to initiate transcription and is not transcribed.
  • It has a specific sequence before the transcription starting point and a Pribnow box, which contains a consensus sequence.

Transcription: Elongation

  • After binding, RNA polymerase unwinds the DNA.
  • A transcription bubble is produced, which moves with the polymerase as it transcribes mRNA from the template strand.
  • Within the bubble, a temporary RNA:DNA hybrid is formed.
  • Sigma must dissociate from RNA polymerase.

Transcription: Termination

  • Occurs when the core RNA polymerase dissociates from the template DNA.
  • DNA sequences mark the end of the gene in the trailer and the terminator.
  • Some terminators require the aid of the rho factor for termination.

The Genetic Code

  • The final step in the expression of protein-encoding genes.

  • mRNA is translated into the amino acid sequence of a polypeptide chain (process = translation).

  • An understanding of the genetic code is necessary before translation is studied.

    • Reading frame

Codons, Start Codons

  • Codon:
    • Genetic code word, 3 base pairs long.
    • Specifies an amino acid.
    • The anticodon on tRNA is complementary.
  • Start Codon:
    • Start site for translation.
    • Always AUG.

Sense Codons, Stop Codons & Code Degeneracy

  • Sense Codons: The 61 codons that specify amino acids.
  • Stop (nonsense) Codons: The three codons used as translation termination signals (UAA, UGA, UAG); they do not encode amino acids.
  • Code Degeneracy/Redundancy: Up to six different codons can code for a single amino acid.
  • Wobble effect.

Wobble in Genetic Code

  • Loose base pairing.
  • The 3rd position of the codon is less important than the 1st or 2nd.
  • Eliminates the need for a unique tRNA for each codon.

Translation Overview

  • Synthesis of a polypeptide directed by the sequence of nucleotides in mRNA.
  • The direction of synthesis is N terminal → C-terminal.
  • Ribosome = site of translation (protein synthesis).
  • Coupled transcription/translation in Bacteria/Archaea.
  • Polyribosome – complex of mRNA with several ribosomes.

Transfer RNA (tRNA)

  • Tertiary structure due to base pairing within the tRNA molecule.
  • An anticodon is present, which is complementary to the codon, and binds the codon.
  • The 3’ end of tRNA binds the amino acid.
  • Acceptor stem → CCA.

Amino Acid Activation

  • Attachment of an amino acid to tRNA.
  • Catalyzed by aminoacyl-tRNA synthetases.
  • At least 20 different tRNA molecules are needed.
  • Each is specific for a single amino acid and for all the tRNAs to which each may be properly attached (cognate tRNAs).

The Ribosome

  • Site of protein synthesis.
  • Bacterial ribosome:
    • 70S ribosomes = 30S + 50S subunits.
    • A translational domain on both subunits is responsible for translation.
    • 50S exit domain.

tRNA Binding Sites of Ribosome

  • Aminoacyl (acceptor; A) Site: Binds incoming aminoacyl-tRNA.
  • Peptidyl (donor; P) Site: Binds the initiator tRNA or tRNA attached to the growing polypeptide (peptidyl-tRNA).
  • Exit (E) Site: Briefly binds empty tRNA before it leaves the ribosome.

Role of Ribosomal RNA in Translation

  • Contributes to the structure of the ribosome.
  • 16S rRNA:
    • Ribosomal binding site (RBS).
    • The 3’ end binds to the Shine-Dalgarno site (RBS) on mRNA for protein synthesis initiation.
    • Binds protein needed for the initiation of translation and amino acyl-tRNA.
  • 23S rRNA:
    • Ribozyme catalyzes peptide bond formation.
    • Peptidyl transferase-transpeptidation.

Initiation of Protein Synthesis

  • Involves ribosome subunits and numerous additional molecules.
  • N-formylmethionine-tRNA is the bacterial initiator tRNA.
  • Archaea and eukaryotes use methionine-tRNA.

Bacterial Initiation

  • In bacteria, initiation begins when the Shine-Dalgarno sequence of mRNA is aligned with 16S rRNA and the initiator codon binds 16S rRNA in the 30S subunit.
  • 3 initiation factors (IFs) in bacteria:
    • Required for the formation of the initiation complex.
    • GTP catalyzes.

Elongation of the Polypeptide Chain

  • The elongation cycle involves the sequential addition of amino acids to the growing polypeptide.
  • Consists of three phases:
    • Aminoacyl-tRNA binding.
    • Transpeptidation reaction.
    • Translocation.
  • Involves several elongation factors (EFs).

Transpeptidation Reaction

  • Catalyzed by peptidyl transferase of 23S rRNA.
  • The amino group of the A site amino acid reacts with the carboxyl group of the C-terminal amino acid on the P site tRNA.
  • The peptide chain is transferred from the P site to the A site.

Final Phase in Elongation

  • Three simultaneous events:
    • Peptidyl-tRNA moves from the A site to the P site.
    • The ribosome moves down one codon.
    • The empty tRNA leaves the P site.
  • Requires GTP hydrolysis.

Termination of Protein Synthesis

  • Takes place at any one of three codons:

    • Nonsense (stop) codons – UAA, UAG, and UGA.

  • Release factors (RFs):

    • Aid in the recognition of stop codons.
    • 3 RFs function in prokaryotes.
    • Only 1 RF is active in eukaryotes.

  • GTP hydrolysis is required.

    • Peptidyl transferase

Protein Folding and Molecular Chaperones

  • Molecular Chaperones:
    • Proteins that aid in the folding of nascent polypeptides.
    • Protect cells from thermal damage (e.g., heat-shock proteins).
    • Aid in the transport of proteins across membranes.

Protein Splicing

  • Removal of part of the polypeptide before folding.
  • Inteins: Removed portion.
  • Exteins: Portions that remain in the protein.

Gene Expression Regulation

  • The process of controlling which genes are used to make proteins in a cell.
  • Ensures that the correct proteins are made in the right place and time.
  • Why Gene Expression is Regulated:
    * Allows cells to respond to environmental changes.
    * Ensures that cells produce necessary products at the correct time.
  • Examples of Gene Expression Regulation:
    * Transcription factors
    * microRNAs
    * Translational repressors
  • How Gene Expression is Regulated:
    * Transcriptional control- regulates the amount of mRNA transcribed
    * Translation control- regulates translation of mRNA into protein
    * Post-translational control- regulates protein activity and stability
    * Epigenetic regulation- biochemical mechanisms that controls gene expression

Mutagenesis and Repair Mechanisms

  • Mutagenesis:
    • The process of creating mutations in DNA.
    • A mutation in bacteria is a change in the DNA sequence of a bacterial cell.
    • Mutations can be random or caused by external factors, like radiation or chemicals.
    • Caused by errors during DNA replication, exposure to environmental mutagens, or during cellular processes like DNA repair.
  • Types of Mutations:
    • Missense mutations: Change the amino acid sequence of a protein.
    • Nonsense mutations: Create stop codons, which terminate protein synthesis and lead to incomplete proteins.
    • Silent mutations: Change a DNA base sequence, but the new codon still codes for the same amino acid.
  • Repair Mechanisms:
    • Cellular processes that actively correct DNA damage, preventing mutations from becoming permanent and being passed on to future generations.

Major DNA Repair Mechanisms

  • Base Excision Repair (BER): Repairs damaged bases by removing them and replacing them with the correct ones.
  • Nucleotide Excision Repair (NER): Removes larger damaged sections of DNA, including bulky adducts, and replaces them with new nucleotides.
  • Mismatch Repair (MMR): Identifies and corrects mismatched base pairs that occur during DNA replication.
  • Homologous Recombination (HR): Uses a homologous DNA strand as a template to accurately repair double-strand breaks.
  • Non-Homologous End Joining (NHEJ): Joins broken DNA ends together but can be prone to errors and lead to mutations.

Plasmids and Gene Transfer

  • Plasmids:
    * Small, circular DNA molecules in bacteria that act as vectors to carry and transfer genetic information between cells
    * Plasmids enable horizontal gene transfer (HGT)
    * HGT enables genes to be exchanged between multiple bacterial strains
    * Hereditary genes can provide bacteria with necessary capabilities
  • HGT Mechanisms
    * Conjugation- a donor bacterium directly transfers a plasmid to a recipient bacterium through physical contact
    * Transformation- Bacteria can also take up free plasmid DNA from the environment, allowing them to acquire new genetic information from the plasmid
    * Transduction- process of transferring genetic material into a cell using a virus or viral vector
  • Due to their ability to transfer genes, plasmids are extensively used in genetic engineering

Applications of Bacterial Genetics in Biotechnology

  • Bacterial genetics has many applications in biotechnology, including the production of antibiotics, enzymes, and biofuels. It also plays a role in agriculture, medicine, and environmental sustainability.
  • Production:
    • Bacteria are used to produce antibiotics, enzymes, and biofuels.
    • Genetic engineering can improve the efficiency of antibiotic production.
    • Recombinant DNA technology is used to produce safe and therapeutic drugs.
  • Agriculture:
    • Bacteria are used as biofertilizers and biopesticides.
    • Nitrogen-fixing bacteria enrich soil with nitrogen, reducing the need for chemical fertilizers.
    • Genetically modified bacteria can improve plant qualities like pest resistance.
  • Medicine:
    • Bacteria are used to produce therapeutic proteins and vaccines.
    • Genetic engineering can be used to develop new antibiotics to combat drug-resistant pathogens.
    • Genetic advances can be used to develop early diagnostic tests and new treatments for genetic diseases.
  • Environmental Sustainability:
    • Genetically engineered bacteria can degrade pollutants.
    • For example, GM bacteria can be used to clean up mercury pollution and detect arsenic in drinking water.

Research Related to Bacterial Genetics

  • Research in bacterial genetics focuses on the study of bacterial chromosomes, plasmids, and other genetic material; it involves developing tools and techniques to analyze and manipulate bacterial DNA.
  • South African Research Includes:
    • Studies on antibiotic resistance, microbial diversity, and genomics of tuberculosis and Neisseria gonorrhoeae.
    • Antibiotic-resistant bacteria (ARB) are widespread in South Africa, with farm settings being a major contributor.
    • Antibiotic-resistant genes (ARGs) are present in raw and treated water, and their spread can be facilitated by drinking water.
  • Microbial Diversity in the Namib Desert
    • Studying microbial communities
  • Microbial Diversity in South Africa
    • Review of commercial applications of African microbes in pharmaceuticals and advanced plastics.
  • Bacillus dicomae sp. nov.
    • A novel bacterial strain discovered and described by UJ researchers.
      • Isolated from leaves collected in South Africa
    • These discoveries can lead to the development of new products and technologies, driving economic growth.