Microbial Growth

Estimation of Microbial Numbers

Accurate quantification of microbial populations is essential for microbiological research and applications. Several methods are used to estimate microbial numbers, each with specific advantages and limitations.

• Direct Counting: Involves microscopic examination and counting of cells using a counting chamber (e.g., Petroff-Hausser chamber). Useful for both living and dead cells.

• Spread-Plate Method: A diluted sample is spread on an agar plate, and colonies are counted after incubation. Only viable cells form colonies (colony-forming units, CFUs).

• Most Probable Number (MPN): A statistical method based on serial dilution and observation of growth in liquid media. Used for samples with low cell numbers or when cells do not grow well on solid media.

• Turbidometry (Light Scattering): Measures the cloudiness (turbidity) of a culture using a spectrophotometer. Turbidity is proportional to cell density within a certain range.

• Q-RT-PCR of 16S rRNA: Quantitative reverse transcription PCR targets the 16S rRNA gene, allowing precise estimation of microbial abundance, especially in mixed communities.

Example: Turbidometry is commonly used in industrial fermentation to monitor bacterial growth in real time.

Stages of Growth of Microbial Culture

Microbial populations in batch culture exhibit characteristic growth phases:

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

• Log (Exponential) Phase: Rapid cell division; population doubles at a constant rate. Cell numbers can be predicted using the formula:

• Stationary Phase: Nutrient depletion and waste accumulation halt population growth; cell division rate equals cell death rate.

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

Example: In the log phase, Escherichia coli can double every 20 minutes under optimal conditions.

Importance of "Persister" Cells

Persister cells are dormant variants of regular cells that are highly tolerant to antibiotics and environmental stresses. They are not mutants but phenotypic variants, contributing to chronic infections and antibiotic treatment failure.

• Key Point: Persister cells can survive antibiotic treatment and repopulate once the antibiotic is removed.

Oxygen and Microbes

Microorganisms exhibit diverse oxygen requirements, determined by their metabolic pathways and ability to detoxify reactive oxygen species (ROS).

• Toxic Forms of Oxygen: Include superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (OH•).

• Enzymes Removing ROS: Superoxide dismutase, catalase, and peroxidase neutralize toxic oxygen species.

• Obligate Aerobes: Require oxygen for growth; possess all ROS-detoxifying enzymes.

• Facultative Anaerobes/Aerotolerant: Can grow with or without oxygen; facultative anaerobes use oxygen when available, aerotolerant do not use oxygen but tolerate it.

• Microaerophiles: Require low oxygen concentrations.

• Obligate Anaerobes: Cannot tolerate oxygen; lack ROS-detoxifying enzymes.

Example: Clostridium species are obligate anaerobes and are killed by exposure to oxygen.

Temperature Adaptations

Microbes are classified by their optimal temperature ranges, with specific adaptations for survival at extremes.

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

• Mesophiles: Optimal growth at moderate temperatures (20–45°C).

• Thermophiles: Thrive at high temperatures (45–80°C).

• Extreme Thermophiles: Grow above 80°C; possess heat-stable enzymes and membrane lipids.

• Adaptations to High Temperatures: Include increased GC content in DNA, heat-stable proteins, and saturated membrane lipids.

pH Adaptations

Microbes adapt to acidic or alkaline environments through specialized mechanisms.

• Acidophiles: Grow optimally at low pH (below 5.5); maintain internal pH near neutrality.

• Alkaliphiles: Prefer high pH (above 8); use sodium gradients for energy.

• Adaptations: Include proton pumps and acid/alkali-stable enzymes.

High Osmolarity and Halophiles

High salt concentrations challenge microbial survival; halophiles have evolved to thrive in such environments.

• Halophiles: Require high NaCl concentrations for growth.

• Adaptations: Accumulate compatible solutes (e.g., glycine betaine) to balance osmotic pressure.

Prokaryotic Molecular Biology

Chromosome Size and Structure

Bacterial and archaeal chromosomes are typically circular, double-stranded DNA molecules. Chromosome size varies from about 0.5 to 10 Mbp.

• Key Point: Prokaryotic chromosomes are compacted by supercoiling and DNA-binding proteins.

DNA Synthesis in the Lab

DNA replication can be studied in vitro using purified enzymes and substrates, enabling detailed analysis of the replication process.

DNA Replication in Cells

DNA replication is a highly regulated, semi-conservative process involving multiple enzymes:

• Origin of Replication (oriC): Specific DNA sequence where replication begins.

• Primase: Synthesizes short RNA primers for DNA polymerase.

• Gyrase: Relieves supercoiling ahead of the replication fork.

• Helicase: Unwinds the DNA double helix.

• 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 the DNA backbone.

RNA Polymerase (RNAP) and Transcription

Transcription is the synthesis of RNA from a DNA template, catalyzed by RNA polymerase.

• Structure of Bacterial RNAP: Composed of core enzyme and sigma factor for promoter recognition.

• Sigma Factors: Direct RNAP to specific promoters; different sigma factors recognize different sets of genes.

• RNAP in Bacteria vs. Archaea: Archaeal RNAP is more similar to eukaryotic RNAP II; uses TBP (TATA-binding protein) and general transcription factors (GTFs).

• Operon Structure: In bacteria and archaea, genes are often organized in operons—clusters of genes transcribed as a single mRNA.

• Transcription Termination: Can be intrinsic (rho-independent) or rho-dependent.

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

Translation and Protein Processing

Translation is the process of protein synthesis from mRNA, involving ribosomes, tRNA, and various factors.

• Role of tRNA: Transfers specific amino acids to the growing polypeptide chain.

• A, P, E Sites: Ribosome has three sites for tRNA binding: Aminoacyl (A), Peptidyl (P), and Exit (E).

• Initiation Differences: Bacteria use formylmethionine-tRNA; Archaea and Eukarya use methionine-tRNA.

• Chaperone Proteins: Assist in proper protein folding.

• Bacterial Protein Secretion: Proteins are exported via Sec or Tat pathways.

Control of Gene Expression

Transcriptional Repressors and Operators

Gene expression is regulated by proteins that bind to DNA and influence transcription.

• Repressor Proteins: Bind to operator sequences to block transcription.

• trp Operon: Tryptophan acts as a corepressor; when present, it binds the repressor, which then blocks transcription.

• lac Operon: Lactose inactivates the repressor, allowing transcription of genes for lactose metabolism.

Transcriptional Activators

• CRP-cAMP System: In the absence of glucose, cAMP levels rise, activating CRP, which enhances transcription of the lac operon.

Two-Component Regulatory Systems

These systems allow bacteria to sense and respond to environmental changes.

• Sensor Kinase: Detects environmental signals and autophosphorylates.

• Response Regulator: Receives phosphate group and regulates gene expression.

Quorum Sensing

Quorum sensing is a cell-density-dependent regulatory mechanism, often controlling virulence in pathogens like Staphylococcus aureus.

Alternate Sigma Factors

Bacteria use alternate sigma factors to regulate gene expression in response to stress (e.g., heat shock, nitrogen starvation, sporulation).

Attenuation and Stringent Response

• Attenuation: Regulation of transcription termination based on tryptophan tRNA availability (e.g., trp operon).

• Stringent Response: Triggered by uncharged tRNAs; alarmones (ppGpp) reprogram gene expression during nutrient limitation.

Riboswitches and Antisense RNA

• Riboswitches: Regulatory RNA elements that bind small molecules to control gene expression.

• Antisense RNA: Small RNAs that bind mRNA and inhibit translation.

Mutation, Recombination, and DNA Transfer

Kinds of Mutagenic Agents and Chemicals

Mutagenic agents include physical (UV, X-rays) and chemical (base analogs, alkylating agents) factors that induce mutations.

Kinds of Mutations

• Synonymous (Silent): No change in amino acid sequence.

• Missense: Alters one amino acid.

• Nonsense: Introduces a stop codon, truncating the protein.

• Frameshift: Insertion or deletion shifts the reading frame, often resulting in nonfunctional proteins.

Roles of RecA Protein

RecA mediates homologous recombination and is essential for DNA repair and the SOS response.

DNA Repair Mechanisms

• Excision Repair: Damaged DNA is removed and replaced by DNA polymerase and ligase.

• SOS Repair: Error-prone repair system activated by extensive DNA damage.

DNA Transfer Mechanisms

• Transformation: Uptake of free DNA from the environment; competence can be natural or induced.

• Conjugation: Direct transfer of DNA via cell-to-cell contact; involves Dtr and Mob proteins, plasmid compatibility, and transmissibility.

• F-plasmid: Fertility plasmid in E. coli; can integrate into the chromosome (Hfr strains) and facilitate genome mapping.

• Transduction: Transfer of bacterial genes by bacteriophages; can be generalized (any gene) or specialized (specific genes).

Transposons

• Simple Transposons: Contain only genes for transposition.

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

• Significance: Drive genome evolution and horizontal gene transfer.

Gene Evolution

• Orthologous Genes: Homologous genes in different species derived from a common ancestor.

• Paralogous Genes: Genes related by duplication within a genome.

Viral Diversity

Fates of Virus-Infected Animal Cells

Animal cells infected by viruses may undergo lysis, persistent infection, latent infection, or transformation (cancerous change).

Bacteriophages with dsDNA Genomes

• T4: Lytic cycle; modifies host RNAP and methylates its genome to evade host defenses.

• T7: Encodes its own RNAP; used in biotechnology for protein expression.

• Lambda (λ): Can switch between lytic and lysogenic cycles; regulatory proteins control the switch.

• Mu: Lytic or lysogenic; integrates into host genome via transposition.

Phages with ssDNA and +RNA Genomes

• M13: ssDNA phage; causes persistent infection without lysing the host.

• M2: +RNA phage.

• Tobacco Mosaic Virus (TMV): Simple +RNA genome; infects plants.

• Poliovirus: Simple +RNA genome; infects humans.

Coronaviruses

Coronaviruses (e.g., SARS1, MERS, SARS2) have complex +RNA genomes. The 5' region is directly translated, while the 3' region is transcribed into subgenomic mRNAs. The life cycle involves host receptor binding, formation of double-membrane vesicles (DMVs), and processing of the ORF1 polyprotein by viral proteases.

Influenza Viruses

Influenza viruses have segmented -RNA genomes (8 segments). They undergo antigenic drift (point mutations) and antigenic shift (reassortment of segments), leading to new strains.

Retroviruses and Hepadnaviruses

• Retroviruses (e.g., HIV): -RNA genome; use reverse transcriptase to synthesize DNA from RNA.

• Hepadnaviruses: DNA genome is transcribed to RNA, then reverse transcribed back to DNA.

Poxviruses and SV40

• Poxviruses: Large dsDNA viruses; encode their own RNAP and DNA polymerase.

• SV40: Small dsDNA virus with overlapping open reading frames (ORFs); can transform host cells, leading to cancer.

Table: Comparison of Viral Genomes and Replication Strategies