Microbial Growth

Estimation of Microbial Numbers

Accurate quantification of microbial populations is essential for understanding growth dynamics and environmental impacts. Several methods are used to estimate microbial numbers:

• Direct Counting: Microscopic examination and counting of cells, often using a hemocytometer or Petroff-Hausser chamber.

• Spread-Plate Method: Serial dilution and plating of samples on agar plates, followed by colony counting.

• Most Probable Number (MPN): Statistical estimation based on dilution series and growth detection in liquid media.

• Turbidometry (Light Scattering): Measurement of optical density (OD) to estimate cell concentration in a culture.

• Q-RT-PCR of 16S rRNA: Quantitative reverse transcription PCR targeting 16S rRNA genes for precise microbial quantification.

Example: Turbidometry is commonly used in laboratory settings to monitor bacterial growth in broth cultures.

Stages of Growth of Microbial Culture

Microbial cultures exhibit distinct growth phases when cultivated in batch systems:

• Lag Phase: Cells adapt to new environment; little to no cell division.

• Log (Exponential) Phase: Rapid cell division; population increases exponentially.

• Stationary Phase: Growth rate slows; nutrients deplete, waste accumulates; cell division equals cell death.

• Death Phase: Cell death exceeds division; population declines.

Prediction of Cell Numbers: During log phase, cell numbers increase according to:

where is the number of cells at time , is the initial number, and is the number of generations.

Importance of "Persister" Cells

Persister cells are dormant variants of regular cells that survive antibiotic treatment and environmental stress, contributing to chronic infections and biofilm resilience.

Oxygen and Microbes

Microbes exhibit diverse oxygen requirements and possess mechanisms to detoxify reactive oxygen species:

• Toxic Forms of Oxygen: Superoxide (), hydrogen peroxide (), hydroxyl radicals ().

• Enzymes Removing Toxic Oxygen: Superoxide dismutase, catalase, peroxidase.

• Obligate Aerobes: Require oxygen for growth.

• Facultative Anaerobes/Aerotolerant: Can grow with or without oxygen.

• Microaerophilic: Require low levels of oxygen.

• Obligate Anaerobes: Cannot tolerate oxygen.

Temperature Adaptations

Microbes are classified by their optimal growth temperatures:

• Psychrophilic: Grow at low temperatures (0–20°C).

• Mesophilic: Grow at moderate temperatures (20–45°C).

• Thermophilic: Grow at high temperatures (45–80°C).

• Extreme Thermophilic: Grow above 80°C.

Adaptations: Thermophiles possess heat-stable enzymes and membrane lipids.

pH Adaptations

• Acidophiles: Thrive in acidic environments (pH < 5).

• Alkaliphiles: Thrive in alkaline environments (pH > 9).

• Adaptations: Specialized membrane transporters and protein stability mechanisms.

High Osmolarity Adaptations

• Halophiles: Require high salt concentrations for growth.

• Adaptations: Accumulation of compatible solutes and specialized proteins.

Prokaryotic Molecular Biology

Chromosome Size and Structure

Bacterial chromosomes are typically circular and vary in size from 0.5 to 10 Mbp. Archaea may have multiple chromosomes or plasmids.

DNA Synthesis in Lab

In vitro DNA synthesis uses DNA polymerases, primers, and template DNA, as in PCR.

DNA Replication in Cells

• Origin of Replication: Specific site where replication begins (e.g., oriC in E. coli).

• Key Enzymes:

  • Primase: Synthesizes RNA primers.

  • Gyrase: Relieves supercoiling.

  • Helicase: Unwinds DNA.

  • Single-Strand Binding Proteins (ss-BP): Stabilize unwound DNA.

  • DNA Polymerase III: Main enzyme for DNA synthesis.

  • DNA Polymerase I: Removes RNA primers and fills gaps.

  • Ligase: Seals nicks in DNA backbone.

RNA Polymerase (RNAP) and Transcription

• Structure of Bacterial RNAP: Multi-subunit enzyme; core plus sigma factor.

• Sigma Factors: Direct RNAP to specific promoters.

• RNAP in Bacteria and Archaea: Archaea use TBP (TATA-binding protein) and GTFs (general transcription factors).

• Operon Structure: Multiple genes transcribed as a single mRNA.

• Transcription Termination: Rho-dependent and Rho-independent mechanisms.

• RNA Editing in Eukarya: Post-transcriptional modifications alter mRNA sequence.

Translation

• tRNA: Transfers amino acids to ribosome.

• A, P, E Sites: Ribosome sites for tRNA binding and peptide elongation.

• Transcription Initiation: Bacteria use sigma factors; Archaea and Eukarya use TBP and GTFs.

• Chaperone Proteins: Assist in protein folding.

• Bacterial Protein Secretion: Sec and Tat pathways transport proteins across membranes.

Control of Gene Expression

Transcriptional Repressor Proteins and Operators

• trp Operon: Repressed by tryptophan; controls biosynthesis.

• lac Operon: Repressed by lactose absence; controls lactose metabolism.

Transcriptional Activator Proteins

• CRP-cAMP Complex: Activates lac operon in presence of cAMP (low glucose).

Two-Component Regulatory Systems

Consist of a sensor kinase and a response regulator; allow environmental sensing and gene regulation.

Quorum Sensing

Cell-density dependent regulation; important in Staphylococcus aureus virulence.

Alternate Sigma Factors

Enable bacteria to respond to stress (heat shock, nitrogen starvation, sporulation) by changing gene expression.

Attenuation

Regulation of transcription by formation of secondary RNA structures; exemplified in trp operon.

Stringent Response Genes

Triggered by empty tRNAs; alarmones (ppGpp) mediate global gene regulation under nutrient stress.

Riboswitches

RNA elements that bind small molecules and regulate gene expression directly.

Antisense rRNA Genes

Produce RNA molecules that bind mRNA and silence translation.

Mutation, Recombination, and DNA Transfer

Kinds of Mutagenic Agents and Chemicals

• Physical Agents: UV, X-rays.

• Chemical Agents: Base analogs, alkylating agents.

Kinds of Mutations

• Synonymous: No change in amino acid.

• Missense: Changes one amino acid.

• Nonsense: Introduces stop codon.

• Frameshift: Alters reading frame.

Roles of RecA Protein

RecA mediates homologous recombination and DNA repair.

DNA Excision Repair and SOS Repair

• Excision Repair: Removes damaged DNA and replaces with new DNA.

• SOS Repair: Induced by extensive DNA damage; error-prone.

Transformation Competence in Bacteria

Natural and induced competence allow uptake of exogenous DNA.

Conjugation

• Dtr and Mob Proteins: Mediate DNA transfer and mobilization.

• Plasmid Compatibility: Determines coexistence of plasmids.

• Transmissible and Mobilizable Plasmids: Transmissible plasmids encode transfer functions; mobilizable require helper plasmids.

F-Plasmid of E. coli

Encodes conjugation machinery; can integrate into genome for mapping.

Gene Transduction in Bacteria

• Generalized Transduction: Any gene transferred by phage.

• Specialized Transduction: Only specific genes transferred.

Transposons

• Simple Transposons: Carry only transposase gene.

• Compound Transposons: Carry additional genes (e.g., antibiotic resistance).

• Significance: Drive genome evolution and horizontal gene transfer.

Gene Evolution

• Orthologous Genes: Homologs in different species.

• Paralogous Genes: Homologs within same genome.

Viral Diversity

Fates of Virus-Infected Animal Cells

• Lysis: Cell destruction.

• Persistent Infection: Long-term viral presence.

• Transformation: Alteration of cell phenotype.

Bacteriophages with dsDNA Genomes

• T4: Lytic cycle; modifies host RNAP; genome methylation.

• T7: Encodes viral RNAP; used for protein expression.

• Lambda: Switches between lytic and lysogenic cycles.

• Mu: Lytic or lysogenic; transposition activity.

Phages with ssDNA and +RNA Genomes

• M13: ssDNA; persistent infection.

• M2: +RNA genome.

• Tobacco Mosaic Virus: Simple +RNA genome.

• Polio: Simple +RNA genome.

Coronaviruses

• SARS1, MERS, SARS2: Complex +RNA genomes.

• Genome Organization: 5' region directly translated; 3' region transcribed into mRNAs.

• Host Receptor: Virus entry depends on specific host receptors.

• DMVs: Double-membrane vesicles formed during replication.

• Viral Proteases and ORF1 Polyprotein: Proteases cleave polyprotein for functional viral proteins.

Influenza Viruses

• -RNA Genome: 8 segments.

• Antigenic Drift: Gradual mutation.

• Antigenic Shift: Reassortment of genome segments.

Retroviruses

• -RNA Genome: HIV; uses reverse transcriptase and protease.

Hepadnaviruses

• DNA to RNA: Reverse transcriptase activity.

Pox Viruses

• dsDNA Genome: Encode viral RNAP and DNA polymerase.

SV40

• dsDNA Genome: Small; overlapping ORFs.

• Transformation: Can transform host cells.

Summary Table: Microbial Growth Conditions

Summary Table: Types of Mutations

Summary Table: Bacteriophage Types

Additional info: Academic context and explanations have been expanded for clarity and completeness, including summary tables and formulas.