Lecture 11_ Microbial Genetics (1)

Microbial Genetics Overview

• Dogma: Handed down wisdom in microbial genetics.

• Microbial Genetics: Study of chromosome structure, gene structure, heredity, and horizontal transfer in bacteria.

• Many aspects of microbial genetics are substantially different from eukaryotic

  • Typically haploid: Organisms typically have one copy of each chromosome.

  • Genome: All DNA in a cell.

  • Chromosome: Large circular pieces of DNA that is only transferred to daughter cells.

  • Plasmid: Small circular DNA segments.

  • Bacterial Chromosome: Composed of one large loop of DNA.

Learning Objectives

• Introduction to Genetics

  • Mutation and selection.

  • Central Dogma of molecular biology.

  • Operon and Regulon structure, including lack of capping/tailing and alternative splicing.

• Examples of Gene Regulation

  • Regulons, Lac operon, Trp operon.

Key Features of Bacterial Genome

• Many aspects differ from eukaryotic systems.

  • Chromosome:

  • Example: Borrelia possesses a linear chromosome.

  • Example: Streptomyces also has a linear chromosome and is known for antibiotic production.

• Plasmid: Bacterial genomes contain

  • accessory genes and elements - antibiotic resistance genes, virulence genes, and metabolic genes

  • that help diversify populations- allow bacteria to adapt to different environments by acquiring antibiotic resistance making them survive harsh conditions, developing new metabolic pathways.

  • Requirements for Plasmid Growth:

    • Membrane - provides structure & transport

    • ribosome - translate plasmid genes into proteins

    • FtsZ protein- ensures proper plasmid inheritance via cell divison

    • nucleoid- supplies replication factors for plasmid maintenance.

      Chromosome Structure

    • bacterial chromosome- one large loop of DNA molecule that contains all the essential genetic information required for the survival and reproduction of the bacterium.

    • Supercoiled: tightly wound to fit into the volume of the cell. The supercoiling of DNA plays a crucial role in maintaining the structure and accessibility of the genetic material, facilitating both gene expression and DNA replication. Fit inside the small cell while maintaining access to genetic information.

      • Example: E. coli chromosome features:

        • Size: 4.6 million base pairs (4.6 Mbp)

        • Number of genes: Typical genome comprises approximately 4300 genes.

        • Shape: single, circular chromosome

        • supercoiled: highly compacted into the nucleoid

        • If E. coli's chromosome were stretched out, it would be 1000x longer than the single cell.

DNA Replication Process

• Semi-Conservative Replication:

  • Leading Strand: Replicated continuously in the 5' to 3' direction, adding nucleotides to the 3' end. Only one primer is needed to start replication

  • Lagging Strand: Primers are added to allow replication in fragments (Okazaki fragments) moving 5’ to 3’ direction away from the replication fork.

• Key Enzymes in DNA Replication:

  • Topoisomerase: Relieves tension in the DNA strand as it unwinds. Cuts, unwinds, and reseals the DNA strands to prevent tangling.

  • Single-Stranded Binding Proteins: Maintain DNA in a single-stranded form during replication. Prevents rejoining and protects the DNA damage.

  • DNA Polymerase III: Catalyzes phosphodiester bond formation between adjacent nucleotides in the 5’ to 3’ direction.

  • DNA Primase: Places RNA primers for DNA polymerase III attachment. DNA polymerase cannot start replication on its own- it needs a primer!

  • DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides.

    • Utilizes 5' to 3' exonuclease activity for removal and replacement.

  • DNA Ligase: Joins DNA fragments together. seals the gaps between Okazaki fragments on the lagging strand. Forms phosphodiester bonds between adjacent DNA fragments, completing replication.

• Daughter Cells: Each contains one new strand and one parental strand derived from the previous replication round.

• Replication Characteristics:

  • New DNA is synthesized as replication progresses.

  • Single-stranded DNA can bind to itself, forming structures like stem loops without proteins.

Plasmid and Phage DNA Replication

During Binary Fission:

• Bacteriophage: Is a virus that infects bacteria. Functions like plasmids in conjugative transfer.

• Concatemer: Long DNA strands with multiple copies of the same DNA series, produced during replication.

  • Involves the binding of initiator proteins (e.g., RepA protein) to specific origins of replication in circular DNA.

  • Produces circular ssDNA molecules as rolling circle intermediates for synthesis.

• Mechanism of Rolling Circle Replication:

  • Exclusively for plasmid and phage DNA in Gram-positive bacteria.

  • Process involves nicking DNA strands to generate 3' OH free ends, allowing DNA polymerase to synthesize new strands.

  • Results in multiple circular DNA molecules that can be packaged into viral capsids or maintained in bacterial cells.

Mutation and Gene Regulation

• Mutation: Changes in genetic code.

  -When can it happen? 

  • CAN be spontaneous

  • CAN be directed by experimenters

    • ex. UV radiation + site-directed mutagenesis (force mutation in gene)

  • CAN be made by horizontal gene transfer

    • ex. introduce DNA → disrupt function of gene or add new gene

      -What does it affect? affect genotype of microorganism directly

  • Genotype: Sequence of DNA.

    -not always affect phenotype

    • silent mutation= degeneracy in AA code→ 1+ codons for SAME AA

      -How can you used silent mutation to affect phenotype?

    • change folding of RNA + RNA interference

    • change DNA regulatory structure

    • change what tRNA used

    • impact rate of protein synthesis

  • Phenotype: Physical trait expressed by the organism.

  • Examples of mutations include T-T dimers, effects of ethidium bromide, acridine orange, and nucleoside analogues.

• Accuracy of DNA Replication:

  • DNA polymerase's error rate is approximately one mistake per every 10 billion bases.

  • Proofreading capabilities allow removal and correction of incorrectly added nucleotides.

• Causes of Mutations:

  • Exposure to Radiation (UV):

    • T-T dimers

      • instrastrand dimers → dimers between adjacent T bases in SAME strand

      • cause DNA polymerase to inaccurately replicate DNA 

  • Mutagenic Chemicals:

    • Ethidium bromide, Acridine Orange

      • DNA dye → intercalators ⇒ insert in between DNA strands

    • DNA intercalators 

      • compounds → cause mutations in DNA

    • Nucleoside analogues (such as AZT)

      • nucleotide-look alike drugs → cell death

      • used in cancer drugs

  • Error by DNA Polymerase: VERY UNCOMMON

  • Mobile Genetic Elements: introduce itself into coding gene → disrupt function or itself into gene + add extra coding DNA

    • Bacteriophage 

    • Transposon 

    • Retrovirus 

• Induced Mutations: Can result from exposure to radiation, mutagenic chemicals, and mobile genetic elements.

Selection Techniques for Mutants

• Enrichment and Isolation: Identify traits and select for organisms with or without specific traits.

  • Example: Bioremediation using microbes to clean pollutants.

• Direct Selection: Examines populations for mutants displaying desired phenotypes. (positive selection)

  • Can engineer mutants with improved function based on enzymatic activity.

  • study structure-function relationships of proteins

  • elucidate signaling pathways

  • develop novel therapeutics

  • Example: place population in medium-containing penicillin 

  • look for growth of penicillin-resistant mutant strains 

    -Disadvantage: limited to relatively straightforward, harsh selective process

• Indirect Selection: Selects for mutants lacking specific functions under particular conditions.(negative selection)

• idenfity mutants w/ “loss” of replica plating

• ex. auxotrophy- inability to synthesize particular organic compound for growth.

• ex. prototropy- make ALL essential required metabolites for growth

• Use of methods like replica plating to study gene functions.

Ames Test

developed by Bruce Ames (1928-1970s)

• Application of direct selection principles to detect mutagenic chemicals.

• Induces changes in mutation rates through revertant measurements, primarily using bacteria like Salmonella typhimurium→unable to synthesize His.

• The reversion of bacteria back to producing histidine helps measure mutagenicity.

• Controls assess the process and the role of metabolites from liver extracts on mutagen activity.

• test potential mutagens → cause DNA mutations in bacteria ⇒ grow on His-deficient medium

  • IF mutagenic chemical → mutations in bacterial DNA

                                     → revert bacteria to His-synthesizing phenotype  

            → grow on selective medium

• add rat liver extract w/ mutagen 

  • enzymes from extract → affect mutagen + make it MORE reactive

  • How mutagen affects humans? 

    • add rat liver extract simulates processing of the mutagen when ingested (liver processes ingested substances)

• add mixture to culture of salmonella + observe IF ↑ revertant frequency

• create a control (ONLY rat liver extract) + add control to bacteria

Bacterial Gene Expression Control

• Bacterial Genomic Control: Involves regulating polypeptide production from genes.

  • Controls through transcriptional regulation and has significant transcription-level control.

• Key Gene Structures:

  • Promoter: Sequence where RNA polymerase binds to initiate transcription.

  • operator: is a specific DNA sequence in an operon that acts as a regulatory switch for gene expression.

  • polycistronic message: (polycistronic mRNA) is a single mRNA molecule that contains multiple coding regions (cistrons), each encoding a different protein.

  • Operon: Group of genes transcribed as a single unit under a single promoter (polycistronic message).

• Differences from Eukaryotic Genes:

  • Prokaryotic systems lack introns and pre-mRNA.

  • Multiple structural genes can appear on a single polycistronic message, allowing for simultaneous translation and transcription.

Summary of Genetic Structures

• Constitutive Promoter: Active under all conditions.

• Operator: Segment where repressors can bind and inhibit transcription.

• Regulon: Genes controlled by shared regulatory systems.

• Sigma Factors: Help RNA polymerase bind to specific DNA sequences, aiding in the proper initiation of transcription.