Microbial Genomes: Structure and Function

• A genome comprises all genetic information defining an organism, including all genes whether found on a single chromosome or distributed among multiple genetic elements.
• Microbial genomes, excluding RNA viruses, are encoded by deoxyribonucleic acid (DNA).
• DNA is an ideal information storage molecule due to its stability, mutability, and replicability.

Genomic Features

• Escherichia coli K-12 chromosome 4,639,675 bp.
• Mycoplasma genitalium chromosome is 0.5 Mbp with 484 genes.

Operons

• Operons are genetic regulatory units, are key genetic structures.
• Most genes code for proteins
• Coding regions are functional units called genes.
• Noncoding regions include regulatory sequences like promoters and enhancers

Prokaryotic vs Eukaryotic Genomes

• Less than 15% of prokaryotic genomes consist of noncoding information.
• Eukaryotes: noncoding content exceeds 90%.

DNA Structure and Function

• DNA molecules are nucleic acid polymers composed of nucleotides.
• Nucleotides have three components:
  • Nitrogenous base
    • Purines: adenine (A) and guanine (G)
    • Pyrimidines: cytosine (C) and thymine (T)
  • Deoxyribose sugar
  • Phosphate group

DNA Bonding

• Nucleotides are linked by phosphodiester bonds between the 5' phosphate and 3' OH group.
• Nucleic acids pair via hydrogen bonds.
  • A pairs with T (two hydrogen bonds).
  • G pairs with C (three hydrogen bonds).
• Phosphodiester backbones of complementary molecules are antiparallel
• High temperatures (50°C–90°C) break hydrogen bonds, causing DNA to denature into single strands.

RNA vs DNA

• RNA differs from DNA
  • Usually single-stranded
  • Contains ribose sugar
  • Uracil replaces thymine
  • Folds back on itself to form complex hairpins and other secondary structures.

Bacterial Chromosomes

• E. coli chromosome is a 4.6-million bp molecule.
• When stretched out, 1,500 times longer than the cell itself.
• Constitutes 3–4% of the bacterial cell's dry mass.
• It remains available to DNA binding proteins.
• It is the second-largest molecule after peptidoglycan.
• It features supercoiling.

Bacterial DNA Replication Overview

• DNA replication is semiconservative; each new double helix includes one parental and one newly synthesized strand.
• Replication in bacteria starts at a single origin (oriC).
• Once started, a replication bubble forms which contains two replication forks moving bidirectionally to replicate the chromosome.
• Replication ends at termination (ter) sites located opposite the origin.
• If partially replicated, a chromosome can begin new replication rounds at daughter origins, even before completing the first.

Key Enzymes in DNA Replication

• DnaA: Initiator protein; binds to origin
• DnaB: Helicase; unwinds helix to expose strands of template
• DnaG: Primase; synthesizes RNA primers
• DNA pol III: Major replication enzyme
• DNA pol I: Replaces RNA primer with DNA
• DNA gyrase: Relieves supercoiling

Replication Initiation

• DnaA-ATP melts DNA at the origin and assembles the replisome, a membrane-attached replication machine.
• DNA melting initiates a bubble.
• Within the bubble, the helicase loader(DnaC) loads helicase (DnaB) onto single-stranded templates.
• DnaB recruits primase (DnaG), which synthesizes RNA primers on each template strand.
• Replisome is completed with DNA pol III (polymerase), which is secured by a sliding clamp protein.

Elongation

• DNA synthesis is conducted by DNA-dependent DNA polymerases like DNA pol III.
• DNA polymerases "read" the template nucleotide sequence, building a complementary strand.
• These enzymes can't start "de novo"; they need a free 3' OH group from a primer for nucleotide addition.
• New DNA is synthesized in the 5' to 3' direction.

Leading vs Lagging Strands

• DNA polymerase restrictions create two synthesis modes at each replication fork.
  • Leading strand allows continual synthesis towards the replication fork.
  • Lagging strand: necessitates discontinuous synthesis away from the fork.
    • Here, synthesis involves Okazaki fragments progressively joined to form a continuous strand.

Replisome Activity

• The cell coordinates two DNA pol III enzymes in one replisome.
• Enzymes, primase and helicase, ensure leading and lagging strands are simultaneously synthesized at each fork.
• The lagging strand loops out after passing through its polymerase.
• DNA gyrase functions ahead of each fork, alleviating torsional strain from helicase action.

Primer Removal and Ligation

• RNA primers are removed by RNase H or exonuclease activity by DNA pol I.
• DNA pol I synthesizes a DNA patch utilizing the 3' OH end of a pre-existing DNA fragment as a primer.
• DNA ligase repairs phosphodiester nicks, using energy from NAD (bacteria) or ATP (eukaryotes).

Plasmids

• Genomes of microbes feature extrachromosomal DNA that replicates independently.

  • Includes plasmids and secondary chromosomes

• These genetic complements add to the primary chromosome by introducing new types of genes.

• Plasmids exist in archaea, bacteria, and eukaryotic microbes.

  • They are typically smaller than chromosomes.
  • They are usually circular and negatively supercoiled.
  • These are nonessential genes, often crucial in situations like antibiotic resistance.

• Copy number per cell vary

Plasmid Transfer

• Plasmids are easily transferred among bacterial species.
  • Conjugation: transfer by direct cell-to-cell contact.
  • Transformation: acquisition from dead cells in the environment.
  • Transduction: transmission via replicating bacteriophage.

Microbiomes and Metagenomes

• Microorganisms exist as microbiomes containing numerous species.
• Most members have yet to be grown in a lab.
• DNA sequencing and polymerase chain reaction enable microbiome diversity analysis without direct cultivation.
• Norman Pace and colleagues pioneered DNA isolation and sequencing that sequenced the 16S rRNA gene due to its high conservation.
• Phylogenic studies are done to determine identities of microbiome inhabitants.

Microbiome Composition

• Approximately 100 trillion microbes inhabit the human body.
• The Human Microbiome Project aims to characterize human gut, skin, and oral microbiomes.
• Intestinal microbiota compositions can influence blood chemistry and diseases like IBS.
• Microbiomes affect an individual's susceptibility to diabetes and obesity.

Next-Gen Sequencing

• Characterizing microbiomes on a single gene provides a limited view of microbiome function.
• Metagenomics (acquiring a microbiome's metagenome) enables a deeper understanding.
• Modern "next-generation sequencing" integrates robotics and microfluidics to sequence bacterial genomes in days.

Oceanic Metagenomic Analysis

• Shinichi Sunagawa and colleagues studied oceanic microbes.
• They sequenced 7.2 terabases of data from 68 ocean locations.
  • Earth's oceans have varying microbiome compositions.
  • These assemble as collections of gene functions rather than species collections.
  • Roughly 40% of marine metagenome genes have no known function.