Microbial Growth, Genome Biochemistry, and Mechanisms of Microbial Genetics

Biochemistry and Structure of the Genome

• ## Discovery and Historical Foundations of Genetics

  • ### Early Discoveries of Nucleic Acids and Chromosomes

  • Friedrich Miescher (1869): Isolated phosphorus-rich chemical from white blood cell pus on surgical bandages, naming it "nuclein."

  • Richard Altmann (1889): Renamed nuclein to "nucleic acid" upon demonstrating its acidic nature.

  • Albrecht Kossel (1910 Nobel Prize): Isolated and identified the five nucleotide bases: Adenine, Guanine, Cytosine, Thymine, and Uracil.

  • Gregor Mendel (1856--1865): Documented patterns of inheritance using true-breeding garden peas (Pisum sativum). Performed hybridization between $P$ generation, $F_1$ generation (all violet), and $F_2$ generation ($3:1$ ratio, $705$ violet to $224$ white). Published Experiments in Plant Hybridization (1866).

- **Theodor Boveri & Walter Sutton (1902)**: Proposed Chromosomal Theory of Inheritance based on sea urchin development and grasshopper meiotic chromosome segregation.
- **Thomas Hunt Morgan (1915)**: Confirmed Chromosomal Theory of Inheritance using fruit flies (*Drosophila melanogaster*); published *The Mechanism of Mendelian Heredity* (1933 Nobel Prize).
- **Barbara McClintock (1940s--1950s)**: Discovered transposable elements ("jumping genes" / transposons) in maize (*Zea mays*). Identified Dissociation ($Ds$) locus and Activator ($Ac$) locus (1983 Nobel Prize).

• ### Elucidating Genetic Function using Microbial Models

  • Joachim Hämmerling (1930s--1940s): Used single-celled alga Acetabularia ($2\text{--}6\,\text{cm}$ length; containing foot with nucleus, stalk, and cap). Removing foot prevented regeneration, while removing cap allowed cap regeneration. Grafting A. crenulata stalk onto A. mediterranea foot yielded an A. mediterranea cap, proving the nucleus houses hereditary material.

- **George Beadle & Edward Tatum (1941, 1945)**: Irradiated red bread mold *Neurospora crassa* with X-rays. Tested auxotrophic mutants requiring arginine pathway intermediates (Ornithine $\rightarrow$ Citrulline $\rightarrow$ Arginine). Established "one gene--one enzyme" hypothesis (later revised to "one gene--one polypeptide", 1958 Nobel Prize).

• ### Proof of DNA as the Genetic Material

  • Frederick Griffith (1928): Demonstrated horizontal bacterial transformation in Streptococcus pneumoniae. Rough strain ($R$, nonpathogenic, unencapsulated) mixed with heat-killed Smooth strain ($S$, pathogenic, encapsulated) killed mice; live $S$ strain recovered.

- **Oswald Avery, Colin MacLeod, & Maclyn McCarty (1944)**: Used selective enzymatic degradation (proteases, RNases, DNases) on $S$ strain extracts. Only DNase abolished transformation, establishing DNA as the transforming principle.

- **Alfred Hershey & Martha Chase (1952)**: Labeled T2 bacteriophage protein coat with radioactive sulfur ($^{35}\text{S}$) and DNA core with radioactive phosphorus ($^{32}\text{P}$). Infected *E. coli*, blended, and centrifuged. $^{35}\text{S}$ remained in supernatant; $^{32}\text{P}$ entered bacterial pellet, confirming DNA is the hereditary material.

• ## DNA Chemical Structure and Double Helix Architecture

  • ### Nucleotide Chemical Composition

  • Deoxyribonucleotides contain a 5-carbon sugar (deoxyribose, carbons $1'$ to $5'$), a phosphate group, and a nitrogenous base.

  • Purines: Double-ring structure (6-membered ring fused to 5-membered ring): Adenine ($A$), Guanine ($G$).

  • Pyrimidines: Single 6-membered ring structure: Cytosine ($C$), Thymine ($T$, unique to DNA).

- **Phosphodiester Bonds**: Covalent $5'\text{--}3'$ linkages connecting $5'$ phosphate of one nucleotide to $3'$ hydroxyl ($-\text{OH}$) of the next, forming the sugar-phosphate backbone. Polymerization utilizes deoxynucleotide triphosphates ($\text{dNTP}$s), releasing pyrophosphate ($\text{PP}_i$), which is hydrolyzed to drive the endergonic reaction.

• ### Double Helix Discovery and Structural Parameters

  • Erwin Chargaff's Rules: Content of $A = T$ and $G = C$ within a species.

  • Rosalind Franklin & R.G. Gosling: Produced X-ray diffraction Photo 51 demonstrating double-helical structure.

  • James Watson & Francis Crick (1953): Constructed $3\text{D}$ model of DNA double helix (1962 Nobel Prize with Maurice Wilkins).

  • Parameters:

    • Two antiparallel strands ($5'\to 3'$ orientation opposite to $3'\to 5'$ orientation).

    • Right-handed helix with approximately $10\,\text{base pairs (bp)}$ per turn.

    • Asymmetrical spacing creates major grooves and minor grooves for protein binding.

    • Complementary base pairing: $A$ pairs with $T$ via $2\,\text{hydrogen bonds}$; $G$ pairs with $C$ via $3\,\text{hydrogen bonds}$. High \%G+C content increases thermal denaturation resistance ($T_m$).

• ## RNA Structure and Functional Classes

  • ### Chemical Comparison to DNA

  • Ribonucleotides contain ribose sugar (possesses $2'-\text{OH}$ group, making RNA less stable) and Uracil ($U$, pyrimidine pairing with Adenine) instead of Thymine.

  • Typically single-stranded, shorter than DNA, but undergoes extensive intramolecular base pairing to form functional $3\text{D}$ structures.

• ### Three Primary Functional RNA Classes

  • Messenger RNA (mRNA): Short, unstable, single-stranded copy of gene sequence. Serves as intermediary directing polypeptide synthesis at the ribosome (hypothesized by François Jacob & Jacques Monod, 1961).

  • Ribosomal RNA (rRNA): Long, stable RNA making up $\sim 60\%$ of ribosome mass. Aligns mRNA/tRNA and catalyzes peptide bond formation via peptidyl transferase ribozyme activity (crystallized from Haloarcula marismortui by Thomas Steitz & Peter Moore, 2009 Nobel Prize).

  • Transfer RNA (tRNA): Small ($70\text{--}90\,\text{nucleotides}$), stable RNA with cloverleaf $2\text{D}$ / L-shaped $3\text{D}$ structure. Contains $3'$ CCA amino acid binding site and an anticodon loop to match mRNA codons.

• ## Genome Organization, Genotype vs. Phenotype, and Genome Sizes

  • ### Genotype vs. Phenotype

  • Genotype: Full collection of genes contained in a cell.

  • Phenotype: Observable physical and functional characteristics determined by gene expression at a given time.

  • Environmental regulation examples: Streptococcus mutans produces sticky plaque slime layer only in the presence of sucrose; Serratia marcescens produces red pigment prodigiosin at $28\,^\circ\text{C}$ but is white at $37\,^\circ\text{C}$.

• ### Cellular Chromosome Packaging

  • Eukaryotes: Linear, multiple chromosomes; diploid ($2n$). Human genome: $3 \times 10^9\,\text{bp}$ ($3\,\text{billion bp}$ across 23 pairs) measuring $\sim 2\,\text{meters}$. Supercoiling managed by topoisomerases; packaged around histone proteins forming chromatin. Epigenetic modifications (cytosine methylation) regulate expression without changing base sequence.

  • Prokaryotes: Single circular chromosome in nucleoid; haploid ($1n$). Supercoiled domains maintained by DNA gyrase (topoisomerase II) and histone-like proteins.

  • Noncoding DNA: $12\%$ of genome in prokaryotes vs up to $98\%$ in eukaryotes (humans). Regulates transcription/translation, produces small noncoding RNAs, and maintains chromosomal structure.

• ### Extrachromosomal DNA and Lethal Plasmids

  • Extrachromosomal DNA includes organelle genomes (mitochondria, chloroplasts - supporting endosymbiotic theory), viral genomes, and bacterial plasmids.

  • Bacillus anthracis vs. Bacillus cereus: $16S$ rRNA sequences $>99\%$ identical. Virulence of B. anthracis stems from two plasmids: $\text{pX01}$ (encodes 3-part anthrax toxin) and $\text{pX02}$ (encodes protective poly-D-glutamic acid capsule). B. cereus lacks these plasmids.

• ### Comparative Genome Sizes

  • Deltavirus: $1.7 \times 10^3\,\text{bp}$ ($1.7\,\text{kb}$).

  • Mycoplasma genitalium: $5.8 \times 10^5\,\text{bp}$ ($580\,\text{kb}$).

  • Chlamydia trachomatis: $1.0 \times 10^6\,\text{bp}$ ($1.0\,\text{Mb}$).

  • Rickettsia prowazekii: $1.1 \times 10^6\,\text{bp}$ ($1.1\,\text{Mb}$).

  • Treponema pallidum: $1.1 \times 10^6\,\text{bp}$ ($1.1\,\text{Mb}$).

  • Pandoravirus: $2.5 \times 10^6\,\text{bp}$ ($2.5\,\text{Mb}$).

  • Escherichia coli: $4.6\text{--}5.6 \times 10^6\,\text{bp}$ ($4.6\text{--}5.6\,\text{Mb}$).

  • Pseudomonas aeruginosa: $6.3 \times 10^6\,\text{bp}$ ($6.3\,\text{Mb}$).

  • Saccharomyces cerevisiae (yeast): $1.2 \times 10^7\,\text{bp}$ ($12\,\text{Mb}$).

  • Humans: $3 \times 10^9\,\text{bp}$ ($3\,\text{Gb}$).

Mechanisms of Microbial Genetics

• ## DNA Replication

  • ### Semiconservative Replication Mechanism

  • Meselson & Stahl Experiment (1958): Grew E. coli in heavy nitrogen ($^{15}\text{N}$), shifted to light nitrogen ($^{14}\text{N}$). Centrifugation revealed intermediate density band after Generation 1, and two bands (intermediate and $^{14}\text{N}$) after Generation 2, ruling out conservative and dispersive models.

• ### Bacterial DNA Replication Process

  • E. coli genome ($4.6\,\text{Mbp}$) replicates bidirectionally from single origin ($oriC$, $245\,\text{bp}$, AT-rich) at $\sim 1000\,\text{nucleotides/second}$ ($42\,\text{minutes}$ total).

  • Enzymes and Factors:

    • Topoisomerase II (DNA Gyrase): Relaxes supercoiled chromosome ahead of replication fork.

    • Helicase: Unwinds double helix by breaking hydrogen bonds.

    • Single-Stranded Binding Proteins (SSBs): Stabilize unwound ssDNA.

    • RNA Primase: Synthesizes $5\text{--}10\,\text{nt}$ RNA primer providing free $3'-\text{OH}$.

    • DNA Polymerase III: Main elongation enzyme; synthesizes $5'\to 3'$ at $1000\,\text{nt/s}$. Uses dNTPs (cleaving pyrophosphate for energy).

    • Leading Strand: Continuous synthesis toward replication fork.

    • Lagging Strand: Discontinuous synthesis away from replication fork forming Okazaki fragments (discovered by Reiji & Tsuneko Okazaki, 1966).

    • Sliding Clamp: Holds DNA Pol III onto DNA strand.

    • DNA Polymerase I: Exonuclease removes RNA primers and replaces them with DNA.

    • DNA Ligase: Catalyzes phosphodiester bond sealing nicks between fragments.

    • Topoisomerase IV: Resolves concatenated circular chromosomes via double-stranded breaks. Targeted by quinolone antibiotics.

• ### Eukaryotic Replication and Rolling Circle Replication

  • Eukaryotic Replication: Multiple origins per chromosome ($30,000\text{--}50,000$ in humans), replication rate $\sim 100\,\text{nt/s}$. Polymerase $\delta$ synthesizes leading strand; polymerase $\epsilon$ synthesizes lagging strand. RNase H removes RNA primers.

  • Telomeres: Repetitive noncoding $3'$ ends ($\text{TTAGGG}$ repeated $100\text{--}1000\times$ in humans). Extended by Telomerase (contains RNA template, active in germ/stem cells; Elizabeth Blackburn 2009 Nobel Prize).

- **Rolling Circle Replication**: Used by plasmids and viruses. Initiated by nick at double-stranded origin ($dso$). DNA Pol III extends $3'-\text{OH}$ displacing single strand. displaced strand recircularizes, single-stranded origin ($sso$) is primed, and complementary strand synthesized.

RNA Transcription

• ### Bacterial Transcription Mechanism

• Unwound region forms a transcription bubble. Antisense strand acts as template; sense strand is nontemplate.

• RNA Polymerase: Core enzyme ($5\,\text{subunits}$) + $\sigma$ factor = Holoenzyme.

• Promoter Regions: Initiation site ($+1$). Upstream consensus sequences: $-10$ position (TATA box: $\text{TATAAT}$) and $-35$ position (bound by $\sigma$).

• Elongation: $\sigma$ factor dissociates; core enzyme synthesizes RNA $5'\to 3'$ at $40\,\text{nucleotides/second}$.

• Termination: Repeated nucleotide termination signals cause RNA polymerase stalling and dissociation.

• ### Eukaryotic vs. Prokaryotic Transcription Differences

• Eukaryotes utilize three polymerases: RNA Pol I, RNA Pol II (mRNA), RNA Pol III.

• Eukaryotic mRNA is exclusively monocistronic (encodes one polypeptide); prokaryotic mRNA is often polycistronic (encodes multiple polypeptides).

• Pre-mRNA Processing:

  • $5'$ cap: $7$-methylguanosine cap added to prevent degradation and facilitate ribosome binding.

  • $3'$ poly-A tail: String of $\sim 200$ adenine nucleotides added.

  • Splicing: Introns (intervening noncoding sequences) removed, Exons (expressed coding sequences) rejoined by spliceosome containing snRNPs. Alternative splicing allows multiple protein products from a single gene.

• ## Protein Synthesis (Translation)

  • ### The Genetic Code

  • Triplet codons ($64\,\text{total combinations}$ coding for $20\,\text{amino acids}$). Degeneracy/redundancy: multiple codons specify same amino acid, primarily differing at 3rd base (wobble position).

  • $61$ sense codons; $3$ stop/nonsense codons ($\text{UAA}, \text{UAG}, \text{UGA}$). $\text{AUG}$ acts as start codon (methionine).

  • Exceptions: Selenocysteine ($\text{UGA}$ with SECIS element stem-loop structure); Pyrrolysine ($\text{UAG}$ via pylS gene and $\text{CUA}$ anticodon tRNA).

• ### Translation Machinery and Process

  • Ribosomes: Prokaryotic $70S$ ($30S$ subunit with $16S$ rRNA + $50S$ subunit with $5S$ and $23S$ rRNAs); Eukaryotic $80S$ ($40S$ subunit with $18S$ rRNA + $60S$ subunit with $5S, 5.8S, 28S$ rRNAs).

  • Polyribosomes (Polysomes): Concurrent transcription and translation in prokaryote cytoplasm.

- **Initiation**:
  - Prokaryotes: $30S$ subunit binds Shine-Dalgarno sequence ($\text{AGGAGG}$) upstream of $\text{AUG}$. Initiator tRNA carries $N$-formyl-methionine ($\text{fMet-tRNA}_{\text{f}}^{\text{fMet}}$). $50S$ subunit binds to form complex.
  - Eukaryotes: $40S$ subunit recognizes $5'$ cap, tracks to $\text{AUG}$. Initiator tRNA carries unformylated methionine ($\text{Met-tRNA}_{\text{i}}$). $60S$ subunit joins.
- **Elongation**:
  - $A$ (aminoacyl) site: Binds incoming charged tRNA.
  - $P$ (peptidyl) site: Holds tRNA with growing peptide chain (fMet enters $P$ site directly during initiation).
  - $E$ (exit) site: Releases uncharged tRNA.
  - Peptidyl transferase catalyzes peptide bond formation. Translocation moves ribosome by $3\,\text{bases}$ ($0.05\,\text{seconds per amino acid}$ in *E. coli*).
- **Termination**: Release factors recognize stop codons ($\text{UAA}, \text{UAG}, \text{UGA}$), cleave polypeptide from $P$-site tRNA, and dissociate ribosome.

• ## Mutations, Mutagens, and Repair Mechanisms

  • ### Molecular Classification of Mutations

  • Point Mutations: Single base substitution.

    • Silent: Codon change codes for same amino acid (due to degeneracy).

    • Missense: Codon change specifies different amino acid (e.g., conditional mutations).

    • Nonsense: Codon change creates stop codon ($\text{UAA}, \text{UAG}, \text{UGA}$), resulting in truncated protein.

  • Frameshift Mutations: Insertion or deletion of nucleotides not in multiples of 3, shifting the reading frame.

  • CCR5-delta 32: $32\,\text{bp}$ deletion mutation in CCR5 coreceptor gene conferring HIV resistance in $\sim 14\%$ of European descent individuals.

• ### Mutagenic Agents

  • Spontaneous Mutations: DNA polymerase errors ($\sim 1 \text{ in } 10^9\,\text{bp}$).

  • Chemical Mutagens:

    • Nucleoside Analogs: $2$-aminopurine ($2\text{AP}$, pairs with $C$, converting $A\text{--}T$ to $G\text{--}C$); $5$-bromouracil ($5\text{BU}$, pairs with $G$, converting $A\text{--}T$ to $G\text{--}C$).

    • Nucleotide-Modifying Agents: Nitrous acid ($\text{HNO}_2$) deaminates Cytosine to Uracil ($C\text{--}G \to T\text{--}A$) and Adenine to Hypoxanthine ($T\text{--}A \to C\text{--}G$).

    • Intercalating Agents: Ethidium bromide, acridine orange, polycyclic aromatic hydrocarbons slip between base pairs causing insertions/deletions (frameshifts).

- **Radiation Mutagens**:
  - Ionizing Radiation (X-rays, $\gamma$-rays): Forms hydroxyl radicals causing single- and double-strand backbone breaks and base deamination.
  - Nonionizing Radiation (UV light): Causes pyrimidine/thymine dimer formation (covalent linkage between adjacent thymines), stalling replication/transcription.

• ### DNA Repair Pathways

  • Proofreading: 3' to 5' exonuclease activity of DNA polymerases during replication.

  • Mismatch Repair: MutS, MutL, MutH enzymes recognize mismatch in non-methylated newly synthesized strand of hemimethylated DNA in E. coli; cut strand, DNA Pol III fills gap, ligase seals.

  • Dark Repair (Nucleotide Excision Repair): Enzyme complex cuts backbone upstream and downstream of thymine dimer; DNA Pol I fills gap, ligase seals.

  • Light Repair (Photoreactivation): Photolyase enzyme binds thymine dimer and breaks covalent bonds upon activation by visible light (absent in placental mammals).

• ### Mutant Identification Procedures

  • Replica Plating: Identifies auxotrophic mutants (e.g., histidine auxotrophs). Master plate colonies stamped onto velvet and transferred to complete medium and histidine-lacking medium.

- **Ames Test**: Measures reversion rate of *Salmonella typhimurium* histidine auxotroph in the presence of test chemical and rat liver extract to screen for potential carcinogens.

• ## Horizontal Gene Transfer Mechanisms in Prokaryotes

  • ### Overview of Mechanisms

  • Vertical Gene Transfer: Parent to offspring.

  • Horizontal Gene Transfer (HGT): Transfer between organisms of same generation.

  • Three primary modes: Transformation, Transduction, Conjugation.

• ### Transformation and Transduction

  • Transformation: Direct uptake of extracellular naked DNA by competent cells. Recipient processes DNA into single strand to evade nucleases and recombines it into host genome.

  • Transduction: Transfer mediated by bacteriophages.

    • Generalized Transduction: Random host chromosomal DNA fragment mistakenly packaged into phage head during lytic cycle.

    • Specialized Transduction: Imprecise excision of lysogenic prophage carrying adjacent host chromosomal genes (e.g., cholera toxin gene in Vibrio cholerae introduced by CTX phage; diphtheria toxin in Corynebacterium diphtheriae; botulinum toxin in Clostridium botulinum).

• ### Conjugation and Transposition

  • Conjugation: Transfer via direct cell-to-cell contact through conjugation pilus (F pilus).

    • $F^+$ donor cell transfers single strand of $F$ plasmid to $F^-$ recipient cell via rolling circle replication through cytoplasmic bridge; recipient becomes $F^+$.

    • Hfr Cells: High Frequency of Recombination cells created when $F$ plasmid integrates into host chromosome.

    • $F'$ Plasmids: Formed by imprecise excision of integrated $F$ plasmid carrying adjacent chromosomal DNA (e.g., $lac$ genes).

    • Conjugation transfer rate used to map $E. coli$ chromosome ($100\,\text{minutes}$ total for $4.6\,\text{Mbp}$ genome).

    • $\text{R}$ Plasmids: Carry multiple antibiotic resistance genes and conjugation machinery.

- **Transposition**: Transposons ("jumping genes") containing inverted repeats and transposase enzyme move independently within or between DNA molecules. Nonreplicative ("cut-and-paste") or replicative ("copy-and-paste"). Can mobilize antibiotic resistance genes onto $R$ plasmids (e.g., *Shigella* dysentery strains).