MB 501 Advances in Microbiology

1.1 Taxonomy, Systematics and Phylogeny

Taxonomy is the science concerned with the classification, identification and nomenclature of organisms. In microbiology, taxonomy provides a systematic framework for placing microorganisms into groups based on shared characteristics.

Microbial systematics is broader than taxonomy. It includes the study of microbial diversity and evolutionary relationships as well as classification and identification.

Phylogeny refers to the evolutionary history and relationships of organisms. Modern bacterial phylogeny relies heavily on molecular sequence information, especially conserved genes such as the small-subunit ribosomal RNA gene.

1.2 Bacterial Nomenclature

Bacterial nomenclature is governed by internationally accepted principles for assigning scientific names. Bacteria are generally named using the binomial system consisting of genus followed by species.

1.3 Classification of Bacteria

Classification involves placing bacteria into taxonomic groups. Traditional bacterial classification relied heavily on observable phenotypic properties, whereas modern classification integrates molecular and genomic evidence.

Taxonomic ranks commonly used include:

1. Domain
2. Phylum
3. Class
4. Order
5. Family
6. Genus
7. Species

1.4 Identification of Bacteria

Identification is the practical process of determining the identity of an unknown isolate. It may involve examination of morphology, staining characteristics, cultural characteristics, biochemical reactions, antigenic properties and molecular characteristics.

1.5 Classical Characteristics

Classical identification uses observable phenotypic characteristics. These methods remain useful because they are relatively simple, inexpensive and directly related to the physiology of microorganisms.

1.6 Molecular Characteristics

Molecular taxonomy uses nucleic acids, proteins and genomic characteristics to determine relationships among microorganisms. Molecular approaches are particularly useful when phenotypic characteristics are insufficient or highly variable.

Important molecular approaches

• 16S rRNA gene sequencing
• DNA-DNA relatedness
• G+C content analysis
• Restriction fragment analysis
• Multilocus sequence analysis
• Whole-genome sequencing
• Average nucleotide identity (ANI)
• Comparative genomic analysis

1.7 16S rRNA-Based Identification

The 16S rRNA gene is widely used for bacterial phylogenetic studies because it is present in bacteria, performs an essential cellular function and contains both conserved and variable regions.

1.8 Polyphasic Taxonomy

Polyphasic taxonomy integrates several independent types of evidence to establish the taxonomic position of a microorganism. Instead of depending on a single characteristic, it combines phenotypic, genotypic and phylogenetic information.

1.9 Phylogenetic Tree

A phylogenetic tree is a branching diagram representing evolutionary relationships among organisms or sequences. Closely related organisms are represented by branches sharing a more recent common ancestor.

                    ───────── Organism A
               ─────┤
               │    └──────── Organism B
          ─────┤
          │    │
          │    └──────────── Organism C
──────────┤
          │
          │          ─────── Organism D
          └──────────┤
                     └────── Organism E

                    

1.10 Cladogram

A cladogram is a branching diagram that represents relationships based on shared derived characteristics. It emphasizes patterns of common ancestry rather than necessarily representing exact evolutionary time.

1.11 Dichotomous Key

A dichotomous key is an identification tool consisting of a sequence of paired contrasting statements. At each step, one of two alternatives is selected, leading to another pair until identification is achieved.

1.12 Basis for Classification of Bacteria

1.13 Bergey's Manual

Bergey's Manual is an important reference for bacterial systematic classification and identification. It provides information about bacterial groups, characteristics and relationships and is widely used in microbiological taxonomy.

Bacteria synthesize many proteins in the cytoplasm, but proteins involved in nutrient acquisition, adhesion, motility, toxin production, host interaction and extracellular degradation frequently need to be transported across one or more membranes.

2.1 General Secretory Pathway

The general secretory pathway, commonly associated with the Sec system, transports proteins across the bacterial cytoplasmic membrane. Many secreted proteins are synthesized with an N-terminal signal peptide that directs them toward the secretion machinery.

              CYTOPLASM
                  │
                  │ Protein synthesis
                  ▼
          Precursor protein
                  │
                  │ Signal peptide
                  ▼
             Sec pathway
                  │
        ┌─────────┴─────────┐
        │                   │
     SecA/SecB          SecYEG channel
        │                   │
        └─────────→ Protein ─┘
                  │
                  ▼
          PERIPLASM / EXTRACELLULAR
                    

Major components

• Signal peptide: targets the protein to the secretion pathway.
• SecB: assists targeting of some unfolded proteins in Gram-negative bacteria.
• SecA: ATPase that drives protein movement through the membrane channel.
• SecYEG: membrane translocation channel.
• Signal peptidase: removes signal peptides from many exported proteins.

2.2 Tat Pathway

The twin-arginine translocation pathway transports folded proteins across the cytoplasmic membrane. Its name is derived from the conserved twin-arginine motif present in the signal peptide of many substrates.

2.3 Type I Secretion System

The Type I secretion system is a one-step secretion pathway in Gram-negative bacteria. It can transport proteins directly from the cytoplasm to the extracellular environment.

A typical Type I system contains:

• ABC transporter in the inner membrane
• Membrane fusion protein in the periplasm
• Outer membrane channel

2.4 Type II Secretion System

Type II secretion usually involves a two-step process. The protein first crosses the inner membrane, commonly through the Sec or Tat pathway, and is then transported across the outer membrane by the Type II secretion apparatus.

2.5 Type III Secretion System

The Type III secretion system is a specialized apparatus used by several Gram-negative pathogens and symbionts to inject effector proteins directly into host cells.

It is often described as a molecular syringe because the apparatus forms a needle-like structure that connects the bacterial envelope with the host-cell membrane.

        BACTERIAL CELL
        ┌─────────────────────┐
        │ Cytoplasm           │
        │     ↓ Effector      │
        │                     │
        │   ────────────      │
        │      Needle         │
        └─────────┬───────────┘
                  │
                  │
             Host membrane
          ═══════════════════
                  │
                  ▼
          HOST CELL CYTOPLASM
                  │
                  ▼
            Effector action

                    

2.6 Type IV Secretion System

Type IV secretion systems can transfer proteins and, in some organisms, DNA-protein complexes between bacterial cells or from bacteria into eukaryotic host cells.

They are evolutionarily related to conjugation systems and can therefore participate in horizontal genetic transfer as well as delivery of effector molecules.

2.7 Comparison of Protein Secretion Systems

2.8 Bacterial Invasion and Cytoskeletal Function

Several bacterial pathogens manipulate host-cell cytoskeletal components, particularly actin, to facilitate attachment, entry, intracellular movement or dissemination.

Bacterial effector proteins may activate or inhibit host signaling pathways that regulate actin polymerization and membrane rearrangement.

2.9 Disruption of Tight Junctions

Tight junctions are specialized structures that regulate the paracellular passage of substances between epithelial cells. Some pathogenic bacteria produce factors that alter tight-junction proteins and increase epithelial permeability.

Important junction-associated proteins include claudins, occludin and ZO proteins. Disruption can contribute to barrier dysfunction and facilitate bacterial invasion or toxin-mediated effects.

3.1 General Structure of a Prokaryotic Cell

Prokaryotic cells lack a membrane-bound nucleus and membrane-bound organelles characteristic of eukaryotic cells. Bacteria possess a nucleoid containing the chromosome and may also contain plasmids.

                 Capsule / slime layer
              ___________________________
             /                           \
            /     Cell wall              \
           |  _________________________   |
           | |   Cell membrane         |  |
           | |                         |  |
           | |     Nucleoid            |  |
           | |       ~~~~~             |  |
           | |                         |  |
           | |      Ribosomes          |  |
           | |       • • •             |  |
           | |                         |  |
           | |      Plasmid            |  |
           | |        ⭕               |  |
           | |_________________________|  |
            \                           /
             \_________________________/

                    Flagellum → ~~~~~~~~~

                    

3.2 Cytoplasmic Membrane

The bacterial cytoplasmic membrane is primarily composed of a phospholipid bilayer containing proteins. It acts as a selective permeability barrier and participates in transport, energy generation, signal transduction and biosynthetic processes.

Major functions

• Selective transport of molecules
• Maintenance of ion gradients
• Electron transport and ATP synthesis
• Signal transduction
• Secretion of proteins
• Participation in cell-wall biosynthesis

3.3 Bacterial Cell Wall

The bacterial cell wall provides mechanical strength and helps prevent osmotic lysis. The major structural polymer of most bacterial cell walls is peptidoglycan.

Gram-positive bacteria generally possess a thick peptidoglycan layer containing teichoic acids, whereas Gram-negative bacteria possess a thinner peptidoglycan layer located within the periplasmic space and an outer membrane containing lipopolysaccharide.

3.4 Passive Diffusion

Passive diffusion is the movement of molecules from an area of higher concentration to an area of lower concentration without direct expenditure of metabolic energy.

3.5 Facilitated Diffusion

Facilitated diffusion also occurs down an electrochemical gradient, but transport requires membrane proteins such as channels or carrier proteins.

Because transport follows the gradient, direct ATP consumption is not required.

3.6 Active Transport

Active transport moves substances against their concentration or electrochemical gradient. It requires energy and specialized transport proteins.

3.7 Group Translocation

Group translocation is a transport mechanism in which a substance is chemically modified during transport across the membrane. The bacterial phosphotransferase system (PTS) is a classic example.

During PTS-mediated carbohydrate transport, phosphate is transferred through a series of proteins and ultimately transferred to the incoming sugar.

3.8 Iron Transport

Iron is essential for many microbial processes, including electron transport, DNA synthesis and enzyme activity. However, ferric iron is poorly soluble under many environmental conditions. Microorganisms therefore produce specialized iron acquisition systems.

Siderophores

Siderophores are low-molecular-weight iron-chelating compounds secreted by microorganisms. They bind ferric iron with high affinity and facilitate its uptake.

3.9 ABC Transporters

ABC stands for ATP-Binding Cassette. ABC transporters use ATP hydrolysis to drive the transport of specific substrates across biological membranes.

Bacterial growth refers to an increase in cellular components and ultimately an increase in cell number. Under favorable conditions, many bacteria reproduce by binary fission.

4.1 Bacterial Growth Curve

Number
of cells
  │
  │                           ───────────────
  │                       ┌─── Stationary
  │                    ┌───
  │                 ┌──
  │              ┌──
  │           ┌──
  │        ┌──
  │     ┌──
  │   ┌─
  │ ──
  └────────────────────────────────────────────→ Time
      Lag       Log       Stationary      Death

                    

4.2 Lag Phase

During the lag phase, bacteria adapt to the new environment. There is little or no increase in cell number, but cells are metabolically active. Enzymes, ribosomes, nucleic acids and other cellular components may be synthesized in preparation for rapid growth.

4.3 Exponential / Log Phase

During the exponential phase, cells divide at a relatively constant and maximal rate under the given conditions. Cellular components are synthesized in a coordinated manner and the population increases exponentially.

Cells in this phase are often highly metabolically active and are commonly used for physiological and biochemical studies.

4.4 Stationary Phase

The stationary phase occurs when net population increase becomes approximately zero. Nutrient depletion, accumulation of toxic metabolites, oxygen limitation and other environmental changes contribute to this phase.

Some bacteria activate stress-response systems, alter metabolism and produce secondary metabolites during stationary phase.

4.5 Death Phase

During the death phase, viable cell numbers decline because the environmental conditions no longer support survival of the population. Death may occur at different rates depending on the organism and environmental conditions.

4.6 Generation Time

Generation time is the time required for a bacterial population to double under specified conditions.

4.7 Specific Growth Rate

The specific growth rate describes the rate of population increase relative to the existing population.

4.8 Diauxic Growth

Diauxic growth occurs when a microorganism grows in two distinct exponential phases because two carbon sources are available and the organism preferentially utilizes one substrate before switching to the second.

A classical example is growth of Escherichia coli on glucose and lactose. Glucose is preferentially consumed, followed by a lag period during which the cells adapt to lactose utilization.

Cell
number
  │
  │                 _________
  │               /
  │             /
  │           /
  │         /
  │       /
  │     /      ← adaptation lag
  │    /
  │   /
  │  /
  │_/_______________________________ Time
      Glucose       Lactose

                    

Microbial metabolism is the integrated network of chemical reactions by which microorganisms obtain energy, synthesize cellular components, maintain cellular organization and reproduce.

5.1 Nutrients

Microorganisms require nutrients for energy generation, biosynthesis, maintenance and growth. Nutritional requirements vary considerably among microorganisms.

5.2 Macronutrients and Micronutrients

5.3 Carbon, Hydrogen and Oxygen Requirements

Carbon forms the basic framework of cellular macromolecules. Hydrogen participates in proton gradients, reducing equivalents and biosynthetic reactions. Oxygen is present in many cellular compounds and participates in oxidation-reduction reactions.

5.4 Nutritional Types of Microorganisms

5.5 Free Energy and Metabolic Reactions

The direction and energetic feasibility of biochemical reactions can be considered using Gibbs free energy.

5.6 Oxidation-Reduction Reactions

Oxidation-reduction reactions involve transfer of electrons from an electron donor to an electron acceptor.

5.7 Important Electron Carriers

• NAD⁺ / NADH
• NADP⁺ / NADPH
• FAD / FADH₂
• Flavoproteins
• Quinones
• Cytochromes

5.8 Role of ATP

ATP acts as a major energy currency of the cell. Energy released during favorable reactions can be conserved in ATP and subsequently used to drive energetically unfavorable processes such as biosynthesis, active transport and cellular movement.

5.9 Mechanisms of ATP Generation

5.10 Chemiosmotic Theory

The chemiosmotic theory explains ATP synthesis through the movement of protons down an electrochemical gradient across a membrane. Electron transport chains establish the proton gradient, and ATP synthase uses the energy stored in that gradient to synthesize ATP.

       HIGH H⁺ CONCENTRATION
       ─────────────────────────
       │        H⁺ H⁺ H⁺       │
       │          ↓            │
       │       ATP synthase    │
       │          ↓            │
       │        ADP + Pi       │
       │          ↓            │
       │          ATP          │
       ─────────────────────────
       LOW H⁺ CONCENTRATION

       Proton flow → ATP synthesis

                    

5.11 Catabolism and Anabolism

5.12 Embden-Meyerhof-Parnas Pathway

The Embden-Meyerhof-Parnas pathway, commonly called glycolysis, converts glucose into pyruvate through a series of enzyme-catalyzed reactions. It occurs in the cytoplasm and can function under both aerobic and anaerobic conditions.

5.13 Pentose Phosphate Pathway

The pentose phosphate pathway, also known as the hexose monophosphate pathway, performs two major functions: generation of NADPH and production of pentose phosphates such as ribose-5-phosphate.

5.14 Entner-Doudoroff Pathway

The Entner-Doudoroff pathway is an alternative route for glucose catabolism found in several bacteria, particularly many Gram-negative organisms.

It produces pyruvate, glyceraldehyde-3-phosphate and reducing equivalents. Compared with classical glycolysis, the net ATP yield is lower but the pathway can provide metabolic flexibility.

5.15 Krebs Cycle / TCA Cycle

The tricarboxylic acid cycle oxidizes acetyl-CoA and generates reducing equivalents used by respiratory electron transport systems. In bacteria, the cycle occurs in association with the cytoplasmic membrane and cytoplasmic metabolic machinery rather than a mitochondrion.

                  Oxaloacetate
                       │
                       │ + Acetyl-CoA
                       ▼
                    Citrate
                       │
                       ▼
                 Isocitrate
                       │
                       ▼
              α-Ketoglutarate
                       │
                       ▼
                  Succinyl-CoA
                       │
                       ▼
                   Succinate
                       │
                       ▼
                   Fumarate
                       │
                       ▼
                    Malate
                       │
                       ▼
                  Oxaloacetate

                       ↑
                       └──── Cycle continues

                    

Important products per acetyl-CoA

• 3 NADH
• 1 FADH₂
• 1 GTP/ATP equivalent
• 2 CO₂

5.16 Fermentation

Fermentation is an anaerobic metabolic process in which an organic molecule acts as the terminal electron acceptor. It allows regeneration of NAD⁺ so that glycolysis can continue in the absence of an external respiratory electron acceptor.

5.17 Lactic Acid Fermentation

In lactic acid fermentation, pyruvate is reduced to lactate. NADH is oxidized to NAD⁺, allowing continued operation of glycolysis.

5.18 Ethanol Fermentation

In ethanol fermentation, pyruvate is first converted to acetaldehyde and then reduced to ethanol.

5.19 Mixed Acid Fermentation

Mixed acid fermentation produces a mixture of organic acids and other products. The exact product profile depends on the organism and environmental conditions.

Products may include lactate, acetate, succinate, formate, ethanol, CO₂ and H₂.

5.20 2,3-Butanediol Fermentation

In 2,3-butanediol fermentation, pyruvate is converted through acetolactate and acetoin to 2,3-butanediol.

5.21 Anaerobic Respiration

Anaerobic respiration uses an electron transport chain but employs an electron acceptor other than molecular oxygen.

Examples of alternative terminal electron acceptors include nitrate, sulfate, fumarate and carbon dioxide depending on the microorganism.

5.22 Photosynthesis

Photosynthetic microorganisms capture light energy and convert it into chemical energy. Photosynthesis may be oxygenic or anoxygenic.

5.23 Light-Dependent Reactions

In light-dependent reactions, pigments absorb photons and initiate electron-transfer reactions. The resulting electron transport can generate ATP and reducing power.

5.24 Light-Independent Reactions

The light-independent reactions use ATP and reducing power to incorporate inorganic carbon into organic molecules. In oxygenic phototrophs, carbon fixation is associated with the Calvin-Benson cycle.

6.1 General Characteristics of Fungi

Fungi are eukaryotic, non-photosynthetic organisms that obtain nutrients primarily through absorption. They may exist as unicellular yeasts, filamentous molds or dimorphic forms.

• Eukaryotic organization
• Absorptive mode of nutrition
• Cell wall commonly containing chitin and glucans
• Membrane containing ergosterol in many fungi
• Reproduction by sexual and/or asexual mechanisms
• Formation of spores in many groups

6.2 Fungal Cell Structure

6.3 Yeast and Mold

6.4 Fungal Nutrition and Metabolism

Fungi are generally chemoheterotrophic. They secrete extracellular enzymes that degrade complex organic substrates and then absorb the soluble products.

6.5 Asexual Reproduction

Asexual reproduction occurs without fusion of compatible nuclei. It allows rapid production of genetically similar offspring.

Common mechanisms include budding, fragmentation and formation of asexual spores such as conidia or sporangiospores depending on the fungal group.

6.6 Sexual Reproduction

Sexual reproduction generally involves fusion of compatible cells or nuclei followed by nuclear events such as karyogamy and meiosis.

6.7 Yeast Reproduction

Many yeasts reproduce asexually by budding. During budding, a daughter cell develops from the parent cell, receives nuclear material and eventually separates.

Parent cell
   (  ●  )
      │
      └── small bud
           ( ● )

        ↓

Bud enlarges
   (  ● )──( ● )

        ↓

Daughter cell separates

   ( ● )     ( ● )

                    

6.8 Mold Reproduction and Life Cycle

Filamentous fungi produce extensive networks of hyphae called mycelium. Reproductive structures may develop from specialized hyphae and produce asexual or sexual spores.

7.1 General Characteristics of Viruses

Viruses are acellular infectious entities that contain a nucleic acid genome enclosed within a protein capsid, with some viruses possessing an additional lipid envelope. They depend on host cells for replication.

7.2 Structure of a Virion

             Glycoprotein spikes
                ↑  ↑  ↑
          ┌───────────────┐
          │   Envelope    │
          │ ┌───────────┐ │
          │ │  Capsid   │ │
          │ │ ┌───────┐ │ │
          │ │ │ Genome│ │ │
          │ │ └───────┘ │ │
          │ └───────────┘ │
          └───────────────┘

                    

7.3 Viral Genome Types

Viral genomes may consist of DNA or RNA and may be single-stranded or double-stranded. RNA genomes may be positive-sense or negative-sense depending on their relationship to messenger RNA.

7.4 Viral Classification and Nomenclature

Viral classification considers genome organization, replication strategy, virion structure, host range and evolutionary relationships. Modern viral taxonomy increasingly incorporates sequence and phylogenomic information.

7.5 Viral Phylogenetic Analysis

Viral phylogenetic analysis compares homologous nucleotide or protein sequences to infer evolutionary relationships. Sequence alignment followed by phylogenetic reconstruction can help investigate viral diversity, evolution and relationships.

7.6 Bacteriophages

Bacteriophages are viruses that infect bacteria. Their structures vary considerably. Many tailed bacteriophages possess a head containing nucleic acid and a tail apparatus involved in host recognition and genome delivery.

                    _________
                   /         \
                  /   DNA     \
                 |             |
                  \___________/
                       │
                       │
                    Collar
                       │
                 ┌─────┴─────┐
                 │    Tail   │
                 │           │
                 └─────┬─────┘
                       │
                  Tail fibers
                    /  |  \
                   /   |   \

                    

7.7 Propagation of Viruses

Viruses are propagated in suitable host systems because they require living cells for replication. Depending on the virus, propagation may involve bacterial cultures, cell cultures, embryonated eggs, experimental animals or other appropriate systems.

7.8 Identification of Viruses

Viral identification may involve observation of cytopathic effects, antigen detection, nucleic acid amplification, sequencing, electron microscopy or virus isolation.

7.9 Viral Replication

Viral replication generally involves attachment to a susceptible host cell, entry, uncoating, genome expression and replication, assembly and release.

7.10 Lytic Cycle of Bacteriophage

In a lytic infection, the phage replicates inside the bacterial host and ultimately causes cell lysis, releasing newly formed phage particles.

7.11 Lysogenic Cycle

In lysogeny, the phage genome becomes associated with the bacterial genome or persists as a stable genetic element without immediately destroying the host cell. The integrated phage genome is called a prophage.

7.12 Lytic versus Lysogenic Cycle

7.13 Virus Purification

Virus purification aims to separate virus particles from host cellular components while maintaining infectivity and structural integrity where required.

General approaches may include clarification, filtration, differential centrifugation, density-gradient centrifugation, chromatography and concentration techniques.

7.14 Virus Assays

Viral assays quantify infectious virus or detect viral components. Different assays measure different endpoints and therefore should not be interpreted as equivalent measurements.

Plaque Assay

A plaque assay estimates the concentration of infectious virus by counting plaques produced in a susceptible cell monolayer. Each plaque represents localized destruction or inhibition of susceptible cells resulting from viral infection.

TCID₅₀

TCID₅₀ is the tissue-culture infectious dose that produces infection in 50% of inoculated culture units under defined experimental conditions. It is commonly calculated using statistical or endpoint dilution methods.

7.15 Host–Virus Interaction

Host-virus interaction is determined by viral factors and host cellular factors. Important processes include receptor recognition, entry, innate immune sensing, viral immune evasion, intracellular replication and host-cell injury.

1. Explain bacterial taxonomy and discuss the principles of polyphasic taxonomy.
2. Describe the classical and molecular methods used in bacterial identification.
3. Explain 16S rRNA-based bacterial phylogenetic analysis.
4. Describe the General Secretory Pathway and compare Type I, Type II, Type III and Type IV secretion systems.
5. Explain the mechanism of bacterial invasion mediated by manipulation of the host cytoskeleton.
6. Describe bacterial membrane structure and explain different mechanisms of membrane transport.
7. Explain group translocation and the bacterial phosphotransferase system.
8. Describe siderophore-mediated iron acquisition and ABC transporters.
9. Describe the bacterial growth curve and explain the physiological characteristics of each phase.
10. Derive the relationship between generation time and growth rate.
11. Explain diauxic growth and its relationship with catabolite repression.
12. Describe the EMP pathway with enzymes, intermediates and energy yield.
13. Explain the pentose phosphate pathway and discuss its significance.
14. Describe the Entner-Doudoroff pathway and compare it with EMP.
15. Explain the TCA cycle and discuss its role in microbial metabolism.
16. Describe different types of fermentation carried out by microorganisms.
17. Explain oxidative phosphorylation and the chemiosmotic theory.
18. Describe fungal structure, nutrition, reproduction and life cycle.
19. Describe the structure, classification and replication of viruses.
20. Explain lytic and lysogenic cycles of bacteriophages.
21. Describe methods of virus propagation, purification and assay.
22. Explain host-virus interaction and factors determining viral infection.

1. Bergey's Manual of Systematic Bacteriology, Volumes 1–5.
2. Madigan, M. T., Martinko, J. M., and Parker, J. Brock Biology of Microorganisms.
3. Prescott, L. M., Harley, J. P., and Klein, D. A. Microbiology.