Microbiology Fundamentals: From Microbial Ecology to Genomics

Nitrogen Fixation and Symbiosis

Module 3: N₂ + 8e^- + 8H⁺ + 16ATP → 2NH₃ + H₂. Nitrogenase is killed by O₂; leghemoglobin binds O₂ (pink), nodules block O₂, and high respiration lowers free O₂. The plant provides sugars while rhizobia fix N₂. High N levels lead to glutamate, while low N levels lead to glutamine. Plant flavonoids trigger bacterial Nod factors (LPS signals) to initiate symbiosis. Root hair curls (“shepherd’s crook”) trap bacteria and start nodule formation.

Archaea: Biology and Genomics

Archaea: Once thought to be only extremophiles, they are actually ubiquitous. Identified via 16S rRNA (distinct from bacteria and eukaryotes). Methanogens: Only archaea perform methanogenesis, the terminal step of the C-cycle and a source of greenhouse gas (CO₂+H₂ / acetate / methyl → CH₄). They are closer to eukaryotes than bacteria, sharing 70S ribosomes, TATA/TBP + TFB transcription, and RNA polymerase similar to eukaryotic RNA pol II. Asgard archaea (e.g., Lokiarchaeota) possess eukaryotic-like genes: actin, tubulin, and ubiquitin. Unlike bacteria, they have ether-linked isoprenoid membrane lipids (can form monolayers), introns, and no peptidoglycan (resistant to lysozyme and β-lactams).

Groups and Biotechnology

Groups: TACK (many extremophiles), Euryarchaeota (methanogens/thermophiles), Asgard (eukaryotic-like), and DPANN (tiny genomes, symbiotic). Biotech: Heat, salt, and pH stability make them ideal for PCR DNA polymerases and industrial enzymes.

Genomics and Sequencing Methods

Sanger sequencing: Uses primer, DNA polymerase, dNTPs, and ddNTPs; ddNTP stops synthesis, creating fragments of different lengths to reveal the sequence. Whole-genome shotgun: Shreds the genome, sequences tiny fragments, and uses computers to reassemble overlaps; detects SNPs. Hierarchical shotgun: Large DNA is cloned into BACs (library), overlaps are mapped (“tiling path”), and each BAC is shotgun sequenced. Microarray: Hybridization to known DNA probes; requires known sequences; used for SNP genotyping and pathogen ID via fluorescence. RNA-seq: NGS of cDNA from total RNA; no prior sequence knowledge needed; detects all transcripts. Illumina: High accuracy, deep sequencing, best for SNPs/transcriptomics. Nanopore: Fast, long reads, portable, and real-time sequencing.

Microbiome and Metagenomics

Microbiota: Microbes at a body site. Microbiome: Microbes, genes, products, and the environment. Metagenome: Total environmental DNA. Metagenomics: Amplicon/16S identifies “who is there,” while shotgun/functional sequencing identifies “what they can do.” Interactions: Commensalism (host unaffected) and Mutualism (both benefit, e.g., Escherichia coli producing Vitamin K). Prebiotics: Non-digestible fibers feeding beneficial gut bacteria. Probiotics: Live beneficial microbes. Functions: Pathogen protection, vitamin synthesis, immune development, fat storage, and CNS modulation. Most microbes reside in the colon. C. diff treatment: Rebyota (rectal fecal microbiota transplant) and Vowst (oral FDA-approved therapy). Gnotobiotic animals: Germ-free or known-microbe models used to study causality and immune development.

Viruses, Fungi, and Plants

Module 4: -ssRNA: Must package RdRp because the host cannot translate -RNA. +ssRNA: Genome acts as mRNA and can be translated immediately. DNA viruses: Often utilize host polymerases more than RNA viruses. Fungi: Possess chitin and β-glucan cell walls and ergosterol membranes. Drug targets: β-1,3-glucan synthase and chitin synthase. Plants: Fungi provide P, N, and water in exchange for sugars/carbon. HIV/SIV Origin: Primate reservoir (SIV) led to spillover and human-to-human spread. Environmental Cues: High CO₂ promotes yeast form (dissemination/immune evasion), while low CO₂ promotes hyphae (environmental form).

Transcriptional Reporters and Genetics

Module 2: Transcriptional fusion: Measures promoter activity/mRNA production. Translational fusion: Measures transcription and translation efficiency. Promoter Anatomy: -35 (sigma factor recognition), -10 (DNA melting/open complex). Semantide: A molecule carrying evolutionary information. 16S rRNA: A good semantide due to universal presence and conserved function. Antibiotics: Bacteriostatic (stops growth) vs. Bactericidal (kills). Cell Division: MinCDE system ensures FtsZ forms the Z-ring at the midcell. Horizontal Gene Transfer: Conjugation requires tra (machinery), mob (mobilization), and oriT (cis-acting site).

Origins of Earth and Early Microbiology

Module 1: Earth’s age: ~4.54 billion years (meteorite dating). Earliest life: ~3.6–3.8 billion years ago (isotopic fractionation/stromatolites). Early Microbiology: Leeuwenhoek (animalcules) and Pasteur (swan-neck flask disproving spontaneous generation). Cell Structure: Smaller cells have higher surface-to-volume (S/V) ratios for faster diffusion. Gram stain: Pink/red (Gram-negative, thin peptidoglycan, LPS) vs. Purple (Gram-positive, thick peptidoglycan). Growth Dynamics: Batch culture (Lag, Exponential, Stationary, Death) vs. Chemostat (steady-state). DNA Replication: Fidelity ensured by base pairing, proofreading, and mismatch repair. Transcription: Rho-independent and Rho-dependent termination mechanisms.