Microbial Metabolism & Molecular Genetics
Part 1: Microbial Metabolism & Thermodynamics
1. Thermodynamics & Energy
Exergonic Reactions:
Release energy, specifically free energy, in the form of heat (enthalpy) or work.
Spontaneous reactions; they proceed without continuous input of energy.
Gibbs Free Energy (\Delta G) change is negative. If standard conditions are assumed (25^\circ C, 1 atm, pH 7), it is denoted as \Delta G^{\circ'} and its negative value indicates spontaneity and energy release.
Endergonic Reactions:
Absorb free energy from their surroundings.
Gibbs Free Energy (\Delta G^{\circ'}) change is positive.
Non-spontaneous reactions; they require energy input to proceed.
Often coupled with exergonic reactions (e.g., ATP hydrolysis) to make them energetically favorable overall.
Electron Bifurcation:
A unique mechanism found in anaerobic microorganisms, primarily involving flavoenzymes.
A pair of electrons from a single donor is split: one electron is channeled into a thermodynamically favorable (exergonic) pathway, while the other is used to drive an unfavorable (endergonic) reaction.
This coupling allows for energy conservation by driving difficult reductions, such as those necessary for ferredoxin reduction, which is crucial for many anabolic pathways.
2. Redox Reactions (Oxidation-Reduction)
OIL RIG:
A mnemonic: Oxidation Is Loss of electrons, Reduction Is Gain of electrons.
These reactions always occur in pairs; one substance is oxidized while another is reduced.
The Oxidant:
The electron acceptor in a redox reaction.
It gains electrons and, in doing so, becomes reduced.
The Reductant:
The electron donor in a redox reaction.
It loses electrons and, in doing so, becomes oxidized.
Reduction Potential (E_0'):
A quantitative measure, in volts, of a substance's tendency to either gain (be reduced) or lose (be oxidized) electrons.
A more negative E_0' indicates a stronger tendency to donate electrons (a better reductant).
A more positive E_0' indicates a stronger tendency to accept electrons (a better oxidant).
Electron Transport Chain (ETC) Rule: Electrons spontaneously flow from a half-reaction with a more negative reduction potential to a half-reaction with a more positive reduction potential. In an ETC, the first electron carrier typically has the most negative E0', and each successive carrier has a slightly less negative (or more positive) E0' to ensure unidirectional electron flow and energy release.
3. ATP Synthesis
Substrate-Level Phosphorylation (SLP):
Involves the direct transfer of a high-energy phosphate group from an organic substrate molecule to ADP, forming ATP.
This process does not require an electron transport chain or oxygen.
Occurs in metabolic pathways such as Glycolysis (e.g., during the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate and phosphoenolpyruvate to pyruvate) and a few steps in the Krebs cycle (e.g., conversion of succinyl-CoA to succinate).
Oxidative Phosphorylation (OP):
The primary method of ATP production in aerobic respiration.
ATP synthesis is coupled to the establishment of a Proton Motive Force (PMF) across a membrane, which is generated by an Electron Transport Chain (ETC).
Protons flow back across the membrane through an ATP synthase enzyme, driving the phosphorylation of ADP to ATP.
Used in various energy-generating processes: Aerobic respiration (with O_2 as the final electron acceptor), Anaerobic respiration (with other inorganic molecules as final electron acceptors), Chemolithotrophy (using inorganic electron donors), and Phototrophy (light-driven electron transport).
4. Nutritional Classifications
Prefixes:
Energy Source:
Photo-: Organisms that obtain energy from light (e.g., photosynthetic bacteria, algae).
Chemo-: Organisms that obtain energy from chemical reactions (e.g., most bacteria, animals, fungi).
Electron Source (Reductants):
Litho-: Organisms that use inorganic electron donors, such as hydrogen gas (H2), hydrogen sulfide (H2S), ferrous iron (Fe^{2+}), or ammonia (NH_3) (e.g., nitrifying bacteria).
Organo-: Organisms that use organic compounds as electron donors, such as glucose, acetate, or succinate (e.g., E. coli, humans).
Carbon Source:
Auto-: Organisms that fix inorganic carbon, typically carbon dioxide (CO_2), into organic compounds (e.g., plants, cyanobacteria).
Hetero-: Organisms that obtain carbon from pre-formed organic compounds (e.g., animals, fungi, many bacteria).
Example:
Chemolithoheterotroph: An organism that uses chemicals for energy, inorganic compounds as electron sources, but obtains its carbon from organic compounds. This classification is less common but highlights the flexibility of microbial metabolism. A more common example might be a Chemoorganoheterotroph (like humans or E. coli), which uses organic chemicals for energy, organic compounds as electron donors, and organic compounds as a carbon source.
5. Nitrogen Cycle
Nitrification:
A two-step aerobic process primarily carried out by specific groups of chemolithoautotrophic bacteria (nitrifiers).
First step: Ammonia (NH3) is oxidized to Nitrite (NO2^-) by ammonia-oxidizing bacteria (e.g., Nitrosomonas).
Second step: Nitrite (NO2^-) is further oxidized to Nitrate (NO3^-) by nitrite-oxidizing bacteria (e.g., Nitrobacter).
This process is crucial in converting toxic ammonia into less harmful nitrate, which is more readily assimilated by plants.
Denitrification:
The anaerobic reduction of Nitrate (NO3^-) back to gaseous Nitrogen (N2).
This process is carried out by facultative anaerobic bacteria (e.g., Pseudomonas, Paracoccus denitrificans) that use nitrate as a terminal electron acceptor in the absence of oxygen.
Leads to the loss of biologically available nitrogen from ecosystems into the atmosphere.
Nitrogen Fixation:
The conversion of atmospheric Nitrogen gas (N2) into Ammonia (NH3).
This vital process is performed by nitrogen-fixing microorganisms (e.g., Rhizobium in legume root nodules, free-living Azotobacter, cyanobacteria like Anabaena).
The enzyme complex responsible is nitrogenase, which is highly sensitive to oxygen and requires significant energy (ATP) input.
Part 2: Molecular Genetics (The Central Dogma)
1. DNA Replication
Enzymes & Proteins:
DNA Polymerase III (Bacteria) / DNA Polymerase \delta and \epsilon (Eukaryotes): Synthesize new DNA strands by adding nucleotides complementary to the template strand in the 5' to 3' direction.
Helicase: Unwinds and separates the double-stranded DNA helix, breaking hydrogen bonds between complementary base pairs to create a replication fork.
SSBs (Single-Stranded Binding Proteins): Bind to the separated single DNA strands to prevent them from re-annealing and protect them from degradation, keeping them stable for replication.
Primase: An RNA polymerase that synthesizes short RNA primers, which provide a free 3' hydroxyl group for DNA polymerase to initiate DNA synthesis.
DNA Polymerase I (Bacteria) / RNase H (Eukaryotes): Removes RNA primers.
DNA Ligase: Joins Okazaki fragments (on the lagging strand) and other DNA breaks by forming phosphodiester bonds.
DNA Gyrase (Topoisomerase II): Relieves supercoiling ahead of the replication fork by making transient nicks in the DNA strands, especially critical in circular bacterial chromosomes.
Telomerase: (Eukaryotes only) A reverse transcriptase enzyme that adds repetitive DNA sequences (telomeres) to the ends of linear chromosomes, preventing progressive shortening of the DNA during successive rounds of replication.
Genome Structure & Replication Origins:
Bacteria: Typically possess a single, circular chromosome. Replication initiates at a single, well-defined Origin of Replication (oriC) and proceeds bidirectionally around the circle until the two replication forks meet at the termination site.
Eukaryotes: Have multiple, linear chromosomes. To ensure timely replication of their larger genomes, eukaryotic chromosomes contain multiple origins of replication distributed along their length. Replication also proceeds bidirectionally from each origin.
2. Transcription (DNA \rightarrow RNA)
Bacteria:
Occurs entirely in the cytoplasm, as bacteria lack a nucleus.
Prokaryotic RNA Polymerase: A single RNA polymerase enzyme synthesizes all types of RNA (mRNA, tRNA, rRNA). The sigma factor (\sigma subunit) of RNA polymerase recognizes and binds to specific promoter sequences.
Promoter: Key recognition sites include the Pribnow box (a TATAAT sequence at the -10 region) and a -35 region (TTGACA). These sequences position RNA polymerase correctly for transcription initiation.
mRNA: Often polycistronic, meaning a single mRNA molecule can carry coding information for multiple proteins, which are then translated independently (e.g., in operons).
Termination:
Rho-dependent: Requires the Rho protein, an ATP-dependent helicase, which binds to a C-rich region on the mRNA and moves along the transcript to displace the RNA polymerase from the DNA.
Rho-independent: Occurs when the newly synthesized mRNA forms a stable hairpin loop structure (due to inverted repeats) followed by a run of uracils (UUUU). This structure causes RNA polymerase to pause and detach from the DNA template.
Trailer: A non-coding region located downstream of the coding sequence (CDS) but before the transcription terminator. It can contain regulatory elements or ribosome binding sites.
Eukaryotes:
Occurs primarily in the nucleus. Different RNA polymerases (RNA Pol I, II, III) synthesize different types of RNA.
RNA Polymerase II: Synthesizes messenger RNA (mRNA).
Promoter: Eukaryotic promoters are more complex, often containing a TATA box (at approx. -25 to -30 bp upstream) and other regulatory elements that recruit general transcription factors and RNA Polymerase II.
mRNA: Monocistronic; typically, one mRNA molecule codes for only one protein.
mRNA Processing (Post-transcriptional Modifications):
Splicing: Non-coding intervening sequences called introns are precisely removed, and the coding sequences (exons) are ligated together to form a mature mRNA. This process is carried out by the spliceosome.
5' Cap Addition: A modified guanine nucleotide (7-methylguanosine) is added to the 5' end of the mRNA, protecting it from degradation, aiding in ribosome binding, and facilitating transport out of the nucleus.
3' Poly-A Tail Addition: A stretch of 50-250 adenine nucleotides is added to the 3' end of the mRNA, contributing to mRNA stability and promoting translation.
3. Translation (RNA \rightarrow Protein)
Initiation:
Bacteria: The 16S rRNA within the small ribosomal subunit binds to a specific sequence on the mRNA called the Shine-Dalgarno sequence (AGGAGG), which is located a few nucleotides upstream of the start codon (AUG). The first amino acid incorporated is N-formylmethionine (fMet), carried by a special initiator tRNA.
Eukaryotes: The small ribosomal subunit, along with initiator tRNA (carrying methionine, not fMet), binds directly to the 5' Cap of the mRNA. It then scans downstream until it encounters the first AUG start codon, typically within a Kozak sequence.
Elongation:
Transpeptidation: The formation of a peptide bond between the amino acid carried by the tRNA in the A (aminoacyl) site and the growing polypeptide chain held by the tRNA in the P (peptidyl) site. This reaction is catalyzed by peptidyl transferase activity, a ribosomal rRNA enzyme (ribozyme).
tRNA Movement (Translocation): After peptide bond formation, the ribosome translocates along the mRNA. The deacylated tRNA moves from the P site to the E (exit) site, and the peptidyl-tRNA moves from the A site to the P site, making the A site available for the next incoming aminoacyl-tRNA. The sequence of tRNA movement through the ribosome is A site (Acceptor) \rightarrow P site (Peptide) \rightarrow E site (Exit).
Stop Codons:
Also known as nonsense codons, these three codons do not code for any amino acid and signal the termination of translation: UAA, UAG, UGA.
When a stop codon enters the A site, release factors bind to it, causing the hydrolysis of the bond between the polypeptide and the tRNA in the P site, thus releasing the completed protein. The ribosomal subunits then dissociate.
Exceptions: In some organisms, certain stop codons can be repurposed to encode non-standard amino acids like Selenocysteine (UGA) and Pyrrolysine (UAG), indicating a deviation from the universal genetic code.
Part 3: Gene Regulation & Mutations
1. Operons & Regulation
Repressible vs. Inducible Operons:
Inducible Operons: Generally control catabolic (degradative) pathways. They are normally "OFF" (repressed) and are turned "ON" when their specific substrate (an inducer, often the first substrate in the pathway) is present. The inducer typically binds to a repressor protein, causing it to detach from the operator, allowing RNA polymerase to transcribe the genes (e.g., the Lac Operon for lactose metabolism).
Repressible Operons: Typically control anabolic (synthetic) pathways. They are normally "ON" (expressed) and are turned "OFF" (repressed) when the end product of the pathway reaches high intracellular concentrations. The end product acts as a co-repressor, binding to the repressor protein, which then binds to the operator, blocking transcription (e.g., the Tryptophan Operon for tryptophan synthesis).
Constitutive Genes: Also known as "housekeeping genes," these genes encode products that are always required for basic cellular functions and are therefore expressed continuously (e.g., genes for ribosomal proteins, many metabolic enzymes).
The Lac Operon (Glucose vs. Lactose example of Inducible control):
The lac operon encodes enzymes necessary for lactose catabolism.
No Lactose Present: The Lac Repressor protein, encoded by the constitutively expressed lacI gene, is active and binds tightly to the operator region (LacO), physically blocking RNA polymerase from transcribing the lacZYA genes.
Lactose Present: Allolactose, an isomer of lactose, acts as an inducer. It binds to the Lac Repressor, causing a conformational change that prevents the repressor from binding to the operator. This allows RNA polymerase to initiate transcription.
No Glucose (but Lactose Present): When glucose is scarce, intracellular cAMP levels are high. cAMP acts as an allosteric effector, binding to the Catabolite Activator Protein (CAP or CRP). The cAMP-CAP complex then binds to a specific DNA sequence upstream of the lac promoter, enhancing RNA polymerase binding and significantly increasing the rate of transcription (positive control).
Glucose Present (even if Lactose is present): Glucose utilization is preferred. High glucose levels lead to low intracellular cAMP levels. Without sufficient cAMP, the CAP protein does not bind to DNA, and thus, transcriptional activation of the lac operon is significantly decreased, even if lactose is present and the repressor is removed (catabolite repression).
Riboswitches:
Regulatory segments of mRNA molecules that act as genetic switches.
They are RNA domains, typically located in the 5' untranslated region (UTR) of an mRNA, that can bind small metabolites or ions directly without the need for protein mediators.
Binding of the ligand causes a conformational change in the riboswitch, which in turn affects gene expression, primarily at the level of translation initiation in Gram-negative bacteria (by altering ribosome binding site accessibility) and transcription termination in Gram-positive bacteria (by forming alternative secondary structures).
Attenuation:
A regulatory mechanism that couples transcription and translation, primarily found in bacteria for operons encoding amino acid synthesis enzymes (e.g., Tryptophan operon).
Transcription is prematurely terminated based on the availability of the specific amino acid.
The speed of the ribosome translating a leader peptide sequence (which contains codons for the regulated amino acid) dictates the formation of alternative mRNA secondary structures (hairpin loops). One structure signals premature termination of transcription, while another allows transcription to proceed. If the amino acid is abundant, the ribosome translates quickly, leading to terminator hairpin formation. If the amino acid is scarce, the ribosome stalls, allowing an anti-terminator hairpin to form, promoting transcription.
2. Mutations
Point Mutations: Changes affecting a single nucleotide base pair.
Silent Mutation: A change in a single nucleotide within a codon that, due to the redundancy of the genetic code, results in the same original amino acid being incorporated into the protein. No change in protein sequence (e.g., UAU to UAC, both code for Tyrosine).
Missense Mutation: A change in a single nucleotide that alters a codon to specify a different amino acid. The effect on protein function can range from negligible to severe, depending on the nature of the amino acid change and its location.
Nonsense Mutation: A change in a single nucleotide that converts an amino acid-encoding codon into a Stop codon (UAA, UAG, UGA). This leads to premature termination of protein synthesis, typically resulting in a truncated and often non-functional protein.
Frameshift Mutations:
Involve the insertion or deletion of one or more nucleotides (not in multiples of three) within the coding sequence of a gene.
This shifts the entire translational reading frame downstream from the point of mutation, leading to a completely altered sequence of amino acids, often resulting in a premature stop codon and a non-functional protein.
Auxotroph:
A mutant organism that has lost the ability to synthesize an essential nutrient (e.g., an amino acid, vitamin, or nucleotide) and therefore requires that nutrient to be supplied in the growth medium for survival and proliferation.
The wild type counterpart, which can synthesize all necessary nutrients, is called a prototroph.
3. DNA Repair
SOS Response:
A global, inducible DNA repair system primarily found in bacteria, activated in response to extensive DNA damage that severely interferes with DNA replication.
It is a "life-or-death" mechanism due to its highly error-prone nature. While it allows the cell to survive massive damage by replication over lesions, it introduces many mutations (mutagenesis), contributing to genetic variation and antibiotic resistance.
Key proteins include RecA (senses DNA damage) and LexA (repressor of SOS genes).
Photoreactivation (Light Repair):
A direct repair mechanism that specifically targets thymine dimers (or pyrimidine dimers) formed in DNA mainly by UV radiation.
The enzyme photolyase binds to the dimer and, using energy from visible light, breaks the covalent bonds between the adjacent pyrimidines, restoring the DNA to its original state without excising any nucleotides.
Other DNA Repair Mechanisms (Briefly):
Excision Repair (Nucleotide Excision Repair - NER, Base Excision Repair - BER): Removes damaged or incorrect bases/nucleotides and replaces them with correct ones using DNA polymerase and ligase. NER is a broad-specificity repair system, while BER targets specific types of damaged bases.
Mismatch Repair (MMR): Corrects errors that arise during DNA replication (e.g., incorrect base pairing) that were missed by the proofreading activity of DNA polymerase. It distinguishes the newly synthesized strand from the template strand to ensure the correct base is inserted.
4. Horizontal Gene Transfer (HGT)
Transformation:
The process by which competent bacterial cells (cells capable of taking up exogenous DNA) directly uptake "naked" DNA (fragments of DNA released from lysed cells) from their extracellular environment.
This acquired DNA can then be integrated into the recipient cell's chromosome via homologous recombination or maintained as a plasmid.
Discovered by Frederick Griffith in his experiments with Streptococcus pneumoniae.
Conjugation:
The direct transfer of genetic material (usually plasmids) from one bacterial cell to another through cell-to-cell contact, mediated by a specialized pilus (sex pilus).
F^+ Cell: The donor cell, which possesses the F plasmid (fertility plasmid) carrying genes for pilus formation and DNA transfer.
F^- Cell: The recipient cell, which lacks the F plasmid.
Hfr Cell (High Frequency of Recombination): A donor cell where the F plasmid has integrated into the bacterial chromosome. During conjugation with an F^- cell, the Hfr cell can transfer a portion of its chromosome along with parts of the integrated F plasmid. This leads to a higher frequency of recombination of chromosomal genes in the recipient.
Transduction:
The process of genetic transfer mediated by bacteriophages (viruses that infect bacteria).
Generalized Transduction: Occurs during the lytic cycle of a phage. During viral assembly, fragments of the host bacterial DNA are randomly packaged into new phage capsids instead of viral DNA. These "transducing phages" can then inject the bacterial DNA into a new host cell.
Specialized Transduction: Occurs only with lysogenic phages (temperate phages). When a lysogenic prophage excises imperfectly from the host chromosome during induction, it sometimes takes along adjacent host genes. The resulting phage particles can then deliver these specific host genes to new bacterial cells.
Part 4: Cell Structure, Growth & Control
1. Cell Structure
Membranes (Adaptations to Temperature):
The fatty acid composition of the phospholipid bilayer in cell membranes is crucial for maintaining optimal membrane fluidity.
Psychrophiles (Cold-loving organisms): Thrive at low temperatures (0-20^\circ C). Their membranes typically have a higher proportion of unsaturated fatty acids (with double bonds) and shorter fatty acid chains. These properties increase membrane fluidity, preventing the membrane from becoming too rigid in the cold.
Thermophiles (Heat-loving organisms): Thrive at high temperatures (40-70^\circ C). Their membranes often contain a higher proportion of saturated fatty acids (no double bonds) and longer fatty acid chains. This increases the packing density and stability of the membrane, preventing it from becoming too fluid or melting at high temperatures. Archaea living at extremely high temperatures may also incorporate isoprenoids and form lipid monolayers for even greater stability.
Flagella:
Bacteria: Complex protein appendages that rotate like a rigid propeller (driven by a motor embedded in the cell membrane and cell wall) to provide motility. The motor is powered by the Proton Motive Force (PMF), a electrochemical gradient of protons across the membrane. The basal body, hook, and filament are the three main parts.
Eukaryotes: Structurally different from bacterial flagella, being extensions of the cell membrane and containing a 9+2 arrangement of microtubules. They move in a wave-like or whip-like fashion (undulating) and are powered by ATP hydrolysis, which drives the motor protein dynein.
Iron Uptake (Siderophores):
Iron (Fe^{3+}) is an essential micronutrient for nearly all life forms but is often insoluble and unavailable in aerobic environments.
Bacteria secrete high-affinity iron-chelating compounds called Siderophores (e.g., enterobactin, aerobactin).
Siderophores bind to environmental ferric iron (Fe^{3+}) with high specificity, forming a siderophore-iron complex. This complex is then transported back into the bacterial cell via specific receptors and transport systems. Inside the cell, the iron is released and reduced to ferrous iron (Fe^{2+}) for use in metabolic processes.
Cell Envelope (External Layers):
Slime Layer: An unorganized, loosely associated, and easily removable layer of extracellular polysaccharides or glycoproteins. It aids in gliding motility (in some bacteria) and protects against desiccation, but generally provides less protection than a capsule.
Capsule: A highly organized and tightly bound layer of polysaccharides (or sometimes polypeptides) external to the cell wall. It is difficult to remove and is a significant virulence factor, preventing phagocytosis by host immune cells, aiding in adherence to surfaces, and protecting against desiccation (e.g., Streptococcus pneumoniae).
Eukaryotic Organelles:
Rough Endoplasmic Reticulum (RER): A network of membranes studded with ribosomes. Primarily involved in the synthesis, folding, modification (e.g., glycosylation), and quality control of proteins destined for secretion, insertion into membranes, or delivery to other organelles (e.g., Golgi, lysosomes).
Golgi Apparatus: A stack of flattened membrane-bound sacs (cisternae). It receives proteins and lipids from the RER, further processes, sorts, and packages them into vesicles for secretion or delivery to other cellular destinations. It also synthesizes certain polysaccharides.
Lysosome: Membrane-bound organelles containing a diverse array of hydrolytic enzymes (acid hydrolases), which are active at acidic pH. They are involved in the digestion of macromolecules (e.g., proteins, nucleic acids, carbohydrates, lipids), breakdown of cellular debris, and recycling of old or damaged organelles (autophagy).
Endosymbiotic Theory: States that mitochondria (the powerhouse of eukaryotic cells) and chloroplasts (sites of photosynthesis in plants and algae) originated from free-living prokaryotic cells that were engulfed by a host eukaryotic cell.
Evidence Supporting the Theory: Both mitochondria and chloroplasts possess their own circular DNA (similar to bacterial chromosomes), divide by binary fission (like bacteria), have their own 70S ribosomes (similar to prokaryotic ribosomes), and their inner membranes resemble bacterial membranes in composition and transport systems. They also have a double membrane, consistent with an engulfment event.
2. Growth Curve
Lag Phase:
Characterized by little to no increase in cell number. Cells are metabolically active, synthesizing enzymes, RNAs, and other molecules necessary for growth and adapting to the new environment. They are preparing for active division.
Log Phase (Exponential Phase):
Cells are actively growing and dividing at their maximum, constant rate (generation time is shortest and most consistent). The population doubles at regular intervals. This phase is most suitable for physiological studies and is when a population is most uniform.
Stationary Phase:
The net growth rate is zero; the rate of cell division equals the rate of cell death. This occurs due to nutrient depletion, accumulation of toxic waste products, or unfavorable environmental changes (e.g., pH shift). During this phase, some bacteria may undergo morphological changes, such as forming stress-response proteins, endospores (e.g., Bacillus, Clostridium), or secondary metabolites.
Death Phase:
The number of viable cells declines exponentially. Cells are dying at an increasing rate due to prolonged nutrient starvation, severe waste accumulation, and irreversible cellular damage. Some cells may undergo programmed cell death.
Diauxic Growth:
A growth pattern characterized by two distinct growth phases separated by a temporary lag phase. This typically occurs when two different carbon sources are available, and the organism preferentially utilizes one over the other (e.g., E. coli growing on a medium with both glucose and lactose will first consume all the glucose, enter a lag phase while synthesizing enzymes for lactose metabolism, and then begin to grow again on lactose).
3. Measuring Growth
Direct Microscopic Count:
Involves counting individual cells in a known volume using a specialized counting chamber like the Petroff-Hausser chamber or hemocytometer under a microscope.
Advantages: Quick, inexpensive, provides immediate results, and can count cells that don't readily form colonies.
Disadvantages: Cannot distinguish between live and dead cells (unless special stains are used), small cells can be difficult to count accurately, and motile cells require immobilization.
Viable Plate Count (Standard Plate Count):
Measures the number of living (viable) cells capable of forming colonies on a suitable agar medium.
Involves serially diluting a sample and plating aliquots onto agar plates using either the spread plate method (spreading a small volume on the surface) or the pour plate method (mixing the sample with molten agar).
Advantages: Counts only living cells, can be used to isolate individual colonies.
Disadvantages: Time-consuming (requires incubation), selective for culturable organisms (many microbes are non-culturable), and prone to errors from pipetting or dilution.
Turbidity Measurement (Spectrophotometry):
An indirect method that measures the cloudiness or optical density (OD) of a liquid culture using a spectrophotometer.
As microbial cells multiply, the culture becomes more turbid, scattering more light. The spectrophotometer measures the amount of light that passes through the sample; less light transmitted means more cells.
Advantages: Rapid, non-invasive, and useful for monitoring growth over time.
Disadvantages: Doesn't distinguish live/dead cells, not accurate at very low or very high cell densities, and different microbes can have different light-scattering properties.
Other Methods:
Dry Weight: Cells are harvested, washed, and dried to measure their mass. Useful for filamentous organisms.
Measurement of Cell Constituents: Quantifying specific cellular components like protein, DNA, or ATP content.
Flow Cytometry: Rapidly counts and analyzes individual cells in a fluid stream, often using fluorescent markers to distinguish between live/dead cells or different cell types.
4. Control of Microbes
Key Terms:
Sterilization: The most stringent process, aiming to destroy or remove all forms of microbial life, including highly resistant endospores, viruses, and prions, from an object or habitat. A sterile item is completely free of all viable microorganisms.
Disinfection: The process of killing, inhibiting, or removing pathogenic microorganisms from inanimate objects or surfaces. Disinfectants are typically too harsh for living tissues and do not necessarily kill all spores.
Antiseptics: Chemical agents applied to living tissues (e.g., skin, wounds) to kill or inhibit the growth of pathogenic microorganisms. They are generally less toxic than disinfectants and are suitable for topical application.
Sanitization: The process of reducing microbial populations to safe levels as determined by public health standards, typically on inanimate objects like dishes or food preparation surfaces.
Bacteriostasis: A condition where the growth and reproduction of bacteria are inhibited, but the bacteria are not necessarily killed. If the bacteriostatic agent is removed, growth may resume.
Bactericidal: An agent or condition that kills bacteria.
Methods of Microbial Control:
Autoclave (Moist Heat Sterilization):
The most effective and widely used method for sterilizing heat-stable materials.
Uses saturated steam under pressure, typically at 121^\circ C and 15 pounds per square inch (psi) for at least 15-20 minutes (depending on load size).
The high pressure allows steam to reach temperatures above boiling, and the moist heat rapidly denatures proteins and destroys membranes and nucleic acids. This method is effective in killing endospores, which are resistant to boiling at 100^\circ C.
Filtration (Sterilization for Heat-Sensitive Liquids/Gases):
A physical method that removes microorganisms by passing liquids or gases through a filter with pores small enough to retain bacteria, spores, and sometimes viruses.
Suitable for sterilizing heat-sensitive liquids (e.g., antibiotics, vitamins, enzymes, cell culture media) and air (e.g., in biological safety cabinets via HEPA filters).
Radiation:
Gamma Radiation (Ionizing Radiation): High-energy electromagnetic radiation (e.g., from Cobalt-60) that penetrates deep into materials. It causes lethal damage to DNA by forming free radicals and breaking phosphodiester bonds. Used for sterilizing heat-sensitive medical devices, pharmaceuticals, and some foods.
UV Radiation (Non-ionizing Radiation): Shorter wavelength ultraviolet light (typically 260 nm) is absorbed by DNA, causing the formation of pyrimidine dimers (e.g., thymine dimers) which interfere with DNA replication and transcription. It is effective for surface sterilization (e.g., in labs, water treatment) but has poor penetrating power and is easily blocked by glass or surfaces.
D-Value (Decimal Reduction Time):
The time (in minutes) required at a specific temperature or with a specific dose of radiation to kill 90\% of a microbial population (or reduce the population by 1 log cycle).
It is a measure of a microorganism's resistance to a specific treatment, representing the time required for a one-log reduction in viable cells. For example, if the D-value is 10 minutes, then after 10 minutes, 90\% of the initial population will be dead, and 10\% will remain.
Part 5: Virology
1. Viral Structure
Virion:
A complete, extracellular, and infectious viral particle. It consists of a nucleic acid genome (DNA or RNA) enclosed within a protein shell (capsid) and, in some cases, an outer lipid envelope.
The virion is the form in which the virus exists outside the host cell and serves to protect the genome and facilitate its transfer to new host cells.
Nucleocapsid:
The core structure of a virus, composed of the viral nucleic acid genome (either DNA or RNA, single-stranded or double-stranded, linear or circular) tightly associated with its protective protein coat, the capsid.
The capsid provides structural integrity and protects the genome from nucleases and environmental damage.
Capsids can have various symmetries: helical (e.g., tobacco mosaic virus, influenza virus) or icosahedral (e.g., adenovirus, poliovirus).
Envelope:
An outer lipid membrane that surrounds the nucleocapsid in some viruses (enveloped viruses). This membrane is typically derived from the host cell's plasma membrane or other internal membranes (e.g., nuclear envelope, ER, Golgi) during viral budding.
The envelope is studded with viral glycoproteins (spikes) that are crucial for binding to host cells and initiating infection. Enveloped viruses are generally more sensitive to detergents, heat, and desiccation than non-enveloped viruses because the envelope can be easily disrupted.
2. Life Cycle
Entry into Host Cells:
Enveloped Viruses:
Typically gain entry via two main mechanisms: fusion or endocytosis.
Fusion: The viral envelope directly fuses with the host cell's plasma membrane, releasing the nucleocapsid into the cytoplasm (e.g., HIV, measles virus).
Receptor-mediated Endocytosis: The virus binds to a host cell receptor, triggering the cell to engulf the virus in a vesicle. Inside the endosome, a drop in pH often triggers fusion of the viral and endosomal membranes, releasing the nucleocapsid (e.g., influenza virus, SARS-CoV-2).
Non-Enveloped Viruses (Naked Viruses):
Enter the host cell through endocytosis (e.g., poliovirus, adenovirus) or, less commonly, by direct injection of their nucleic acid into the cytoplasm after attachment to the cell surface (e.g., bacteriophages injecting DNA).
Lacking an envelope, they often rely on capsid proteins to interact with cell surface receptors and facilitate entry or uncoating.
Release from Host Cells:
Enveloped Viruses:
Typically exit the host cell via budding. The nucleocapsid associates with areas of the host cell membrane where viral glycoproteins have been inserted. The nucleocapsid then pinches off from the cell, acquiring an envelope derived from the host membrane.
This process often does not immediately kill the host cell, allowing for a sustained infection.
Non-Enveloped Viruses:
Usually cause the host cell to lyse (burst), directly releasing new virions into the extracellular environment. This active lysis leads to the death of the host cell.
Phage Types (Bacteriophages):
Lytic Phages (Virulent Phages):
Phages that always undergo a lytic cycle, meaning they replicate within the host cell and then cause immediate lysis and death of the host bacterium to release progeny phages.
Their life cycle is characterized by adsorption, injection, synthesis (replication of phage DNA/RNA and proteins), assembly, and release by lysis.
Lysogenic Phages (Temperate Phages):
Phages that can choose between a lytic cycle and a lysogenic cycle. In the lysogenic cycle, the phage genome (called a prophage) integrates into the host bacterial chromosome or exists as a stable plasmid.
The prophage remains dormant (latent) and is replicated along with the host chromosome. The host cell, now a lysogen, is immune to superinfection by the same phage and may exhibit new properties (lysogenic conversion). Under certain stress conditions, the prophage can be induced to excise from the chromosome and enter the lytic cycle.
Prions:
Abnormal, infectious proteins that lack nucleic acid (they contain no DNA or RNA).
Prions are misfolded versions of normal cellular proteins (PrP^C^, cellular prion protein) that can induce the normal proteins to misfold into the same abnormal, disease-causing conformation (PrP^Sc^, scrapie prion protein).
This conformational change is autocatalytic and propagates, leading to the aggregation of these misfolded proteins in brain tissue, causing neurodegenerative diseases such as Bovine Spongiform Encephalopathy (Mad Cow Disease) in cattle, scrapie in sheep, and Creutzfeldt-Jakob Disease (CJD) in humans. Prions are extremely resistant to conventional sterilization methods.
Part 6: Microbial Ecology & Host Interactions
1. Interactions
Mutualism:
A symbiotic relationship where both interacting organisms benefit from the association, and often, this relationship is essential for the survival of one or both partners (obligatory).
Example: Lichens (fungus and alga/cyanobacterium); ruminants and their gut microbiota that digest cellulose; nitrogen-fixing bacteria (Rhizobium) in legume root nodules.
Cooperation (Synergism):
A beneficial relationship where both organisms gain from the interaction, but unlike mutualism, the association is not obligatory; both partners can survive independently.
Example: Some microbial consortia where one organism produces a growth factor or nutrient that benefits another, but both could grow alone if necessary.
Commensalism:
An interaction where one organism benefits, and the other organism is neither significantly harmed nor helped (is unaffected).
Example: Many skin bacteria that feed on dead skin cells and excreted substances without affecting the host; some non-pathogenic bacteria in the gut.
Predation:
An interaction where one organism (the predator) consumes another organism (the prey).
Bdellovibrio: A genus of small, Gram-negative, highly motile predatory bacteria. They invade the periplasmic space of other Gram-negative bacteria, where they reproduce and eventually lyse the host cell. This is an obligate predatory relationship.
Vampirococcus: A different type of bacterial predator that attaches to the surface of another bacterium (the prey) and forms a specialized structure (a 'pedicel') to siphon off cellular contents without entering the prey cell.
Parasitism:
An interaction where one organism (the parasite) lives on or in another organism (the host) and benefits by deriving nutrients at the host's expense. The host is always harmed to some degree, though not necessarily killed immediately.
Example: Pathogenic bacteria, viruses, fungi, protozoa (e.g., Mycobacterium tuberculosis causing tuberculosis).
Amensalism:
An interaction where one organism is harmed or inhibited, while the other is unaffected.
Example: Production of antibiotics by fungi or bacteria (e.g., Penicillium producing penicillin) that inhibit the growth of other microorganisms, while the producer is not negatively affected.
Quorum Sensing:
A sophisticated cell-to-cell communication system used by bacteria to monitor their own population density and regulate gene expression in a coordinated manner.
Bacteria release small signaling molecules called autoinducers (e.g., acyl-homoserine lactones in Gram-negatives, peptides in Gram-positives) into the environment.
When the concentration of autoinducers reaches a critical threshold (quorum), it triggers a collective change in gene expression, enabling coordinated behaviors like biofilm formation, virulence factor production, bioluminescence, and antibiotic resistance.
2. Epidemiology
Endemic:
Describes a disease that is constantly or usually present in a particular population or geographic area at a relatively low and predictable level.
Example: Malaria in certain tropical regions, common cold in temperate zones.
Epidemic:
A sudden, often widespread, increase in the incidence of a disease or other health-related event in a specific population or geographic region, significantly exceeding the expected baseline number of cases (often referred to as an