3. Introductory Microbiology

3.1 Monera

The kingdom Monera comprises prokaryotic organisms that are unicellular and lack membrane‑bound organelles. Their genetic material is not enclosed within a nucleus, and they possess a simple cellular organization that enables rapid adaptation to diverse environments.

General Introduction

Monerans are characterized by:

• Prokaryotic cell type – no true nucleus or membrane‑bound organelles.
• Unicellularity – each organism is a single cell, although some form colonies or filaments.
• Absence of membrane‑bound organelles – mitochondria, chloroplasts, endoplasmic reticulum, etc., are missing.

Structure of a Bacterial Cell

A typical bacterial cell consists of several structurally and functionally distinct components:

Mode of Nutrition

Bacteria exhibit diverse nutritional strategies:

1. Autotrophic – synthesize their own organic compounds.
   • Photosynthetic autotrophs (e.g., cyanobacteria) use light energy; pigments include chlorophyll a and phycobilins.
   • Chemosynthetic autotrophs (e.g., Nitrosomonas) obtain energy from inorganic chemical reactions (e.g., oxidation of ammonia).
2. Heterotrophic – obtain carbon from organic sources.
   • Saprophytic – decompose dead organic matter (e.g., Bacillus subtilis).
   • Parasitic – derive nutrients from a living host, often causing disease (e.g., Streptococcus pyogenes).
   • Symbiotic – mutually beneficial association (e.g., Rhizobium in legume root nodules fixing nitrogen).

Bacterial Growth

Under favorable conditions, bacteria reproduce asexually by binary fission. The process can be summarized as:

Parent cell → DNA replication → Cell elongation → Septum formation → Two daughter cells

The typical growth curve of a bacterial population in a closed batch culture consists of four phases:

Factors affecting growth include:

• Temperature – psychrophiles (<15 °C), mesophiles (20‑45 °C), thermophiles (45‑80 °C).
• pH – most bacteria prefer neutral pH (6.5‑7.5); acidophiles and alkaliphiles tolerate extremes.
• Oxygen – obligate aerobes require O₂; facultative anaerobes can grow with or without O₂; obligate anaerobes are killed by O₂.

Cyanobacteria (Blue‑Green Algae)

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis similar to plants. Key features:

• Photosynthetic pigments – chlorophyll a, phycocyanin, phycoerythrin.
• Nitrogen fixation – carried out in specialized cells called heterocysts that protect the oxygen‑sensitive nitrogenase enzyme.
• Bloom formation – rapid proliferation in nutrient‑rich waters can produce harmful algal blooms (HABs) releasing toxins.

Examples:

• Nostoc – forms filaments with heterocysts; symbiotic with fungi in lichens.
• Anabaena – forms heterocysts; used as a biofertilizer in rice paddies.
• Oscillatoria – non‑heterocystous, often found in freshwater; can produce neurotoxins.

3.2 Virus

Viruses are acellular infectious agents that obligately parasitize host cells. They consist of a nucleic acid genome (DNA or RNA) surrounded by a protein coat, and sometimes an outer lipid envelope.

General Introduction

• Acellular – lack cellular structure; cannot carry out metabolism independently.
• Obligate intracellular parasite – must infect a host cell to replicate.
• Genetic material – either DNA or RNA, single‑ or double‑stranded, linear or circular.
• Protein coat (capsid) – composed of repeating protein subunits called capsomeres.

Structure

The basic architecture of a virion includes:

1. Capsid – protective shell made of capsomeres; determines symmetry (icosahedral, helical, complex).
2. Nucleocapsid – the capsid together with the enclosed nucleic acid.
3. Envelope (present in some viruses) – lipid bilayer derived from host membrane, studded with viral glycoproteins.
4. Tail fibers (characteristic of bacteriophages) – protein structures that recognize and bind to specific receptors on bacterial surfaces.

Importance

• Diseases – viruses cause illnesses in plants (e.g., Tobacco mosaic virus), animals (e.g., Foot‑and‑mouth disease virus), and humans (e.g., Influenza, HIV, SARS‑CoV‑2).
• Bacteriophages – viruses that infect bacteria; useful as antibacterial agents and tools in molecular biology.
• Viroids – small, circular RNA pathogens lacking a protein coat; infect plants (e.g., Potato spindle tuber viroid).
• Prions – infectious protein particles causing neurodegenerative diseases (e.g., Creutzfeldt‑Jakob disease).

Bacteriophage: T4 Phage

The T4 bacteriophage infects Escherichia coli and serves as a model for studying viral morphology and life cycles.

Structure:

• Icosahedral head (capsid) containing double‑stranded DNA (~169 kbp).
• Contractile tail with sheath, core tube, base plate, and six long tail fibers.

Life Cycles:

1. Lytic Cycle – virus hijacks host machinery, replicates, assembles progeny, and lyses the host cell to release virions.
   1. Attachment – tail fibers bind to bacterial receptors.
   2. Penetration – sheath contracts, injecting DNA into cytoplasm.
   3. Biosynthesis – viral DNA replication, transcription, translation of early, middle, and late genes.
   4. Maturation – assembly of heads, tails, and fibers.
   5. Lysis – lysozyme degrades peptidoglycan; cell bursts, releasing ~100‑200 progeny phages.
2. Lysogenic Cycle – viral DNA integrates into host chromosome as a prophage; replicates passively with host DNA.
   1. Integration – mediated by phage integrase.
   2. Maintenance – repressor protein prevents lytic gene expression.
   3. Induction – environmental stressors (UV, chemicals) trigger excision and entry into lytic cycle.

Genetic Switch – the decision between lytic and lysogenic pathways is governed by the concentration of the CI repressor and Cro protein; high CI favors lysogeny, high Cro favors lysis.

3.3 Impacts of Biotechnology in Microbiology

Modern biotechnology harnesses microbial systems to produce valuable products, improve health, and address environmental challenges.

Genetic Engineering of Bacteria

Recombinant DNA technology allows insertion of foreign genes into bacterial plasmids, turning bacteria into factories for therapeutic proteins.

• Insulin – human insulin gene inserted into E. coli; yields Humulin® for diabetes treatment.
• Vaccines – antigenic proteins (e.g., Hepatitis B surface antigen) expressed in yeast or bacteria; safe, subunit vaccines.
• Enzymes – proteases, amylases, lipases produced industrially (e.g., Bacillus subtilis alkaline protease for detergents).

PCR Technology

The Polymerase Chain Reaction (PCR) amplifies specific DNA sequences exponentially.

1. Denaturation – heat to ~95 °C separates double‑stranded DNA.
2. Annealing – primers bind to complementary sequences at ~50‑65 °C.
3. Extension – Taq polymerase synthesizes new strand at ~72 °C.

After n cycles, the amount of target DNA ≈ 2ⁿ × initial copies. PCR is essential for cloning, diagnostics (e.g., COVID‑19 RT‑PCR), mutagenesis, and forensic analysis.

Recombinant DNA Technology

Core steps:

1. Isolation of gene of interest.
2. Insertion into a vector (plasmid, bacteriophage, cosmid) using restriction enzymes and DNA ligase.
3. Transformation into host bacterium (e.g., E. coli via heat shock or electroporation).
4. Selection of transformants using antibiotic resistance markers.
5. Expression and purification of the recombinant product.

Key enzymes:

• Restriction endonucleases – cut DNA at specific sequences (e.g., EcoRI cuts GAATTC).
• DNA ligase – joins DNA fragments by forming phosphodiester bonds.
• Reverse transcriptase – synthesizes cDNA from mRNA (used for eukaryotic gene cloning).

Probiotics and Fermentation Technology

Probiotics are live microorganisms that confer health benefits when administered in adequate amounts.

• Common strains: Lactobacillus acidophilus, Bifidobacterium bifidum, Saccharomyces boulardii.
• Mechanisms: competitive exclusion of pathogens, production of bacteriocins, modulation of host immune response.

Fermentation exploits microbial metabolism to produce foods, beverages, and industrial chemicals.

Biofuels

Microorganisms convert biomass into renewable fuels.

• Bioethanol – yeast fermentation of sugars; Saccharomyces cerevisiae engineered for xylose utilization from hemicellulose.
• Biodiesel – transesterification of lipids; oleaginous yeasts (Yarrowia lipolytica) accumulate lipids.
• Biogas – anaerobic digestion by consortia of bacteria and archaea producing methane (CH₄) and CO₂.
• Biohydrogen – photosynthetic bacteria (Rhodobacter sphaeroides) or fermentative clostridia produce H₂.

Overall stoichiometry for ethanol fermentation (simplified):

C₆H₁₂O₆ → 2 C₂H₅OH + 2 CO₂

where glucose (C₆H₁₂O₆) is converted to two molecules of ethanol and two of carbon dioxide.

Bioremediation

Use of microbes to detoxify pollutants.

• Oil spills – hydrocarbon‑degrading bacteria (Pseudomonas putida, Alcanivorax borkumensis) break down alkanes and aromatics.
• Heavy metals – metal‑reducing bacteria (Geobacter sulfurreducens) precipitate metals as insoluble sulfides.
• Pesticides – organophosphate hydrolase‑expressing E. coli degrade pesticides.
• Plastic waste – Ideonella sakaiensis secretes PETase to hydrolyze polyethylene terephthalate (PET).

Key parameters influencing bioremediation efficiency: temperature, pH, nutrient availability (N, P), oxygen levels, and bioavailability of the contaminant.

Summary Table of Biotechnology Applications