Comprehensive Study Notes on Microbial Genetics, Growth, and Immunology
Bacterial Anabolism and the Biofilm State
Anabolism represents the metabolic process of building complex biomolecules. This energy-consuming series of reactions allows bacteria to synthesize essential components including proteins, exopolysaccharides, and cell wall structures. In their natural environments, bacteria do not typically exist in isolation; instead, approximately of all bacterial infections are caused by biofilms. Biofilms are complex, multi-layered communities that can be polymicrobial, containing more than one distinct strain or species of microorganism. While many biofilms are associated with pathogenic infection, some polymicrobial biofilms comprise the human microbiota, which is essential for healthy physiological function.
Analytical Techniques for Microbial Quantification
To determine the number of colony-forming units (CFUs) in either a pure isolation or a complex mixture, researchers utilize serial dilutions. This process involves taking a set volume, such as a undiluted sample, and adding it to of broth to create a total volume for successive dilution steps. After plating the diluted samples, colonies are counted. For a count to be statistically valid, the plate should ideally contain between or colonies. Another method for assessing growth is turbidity, which is the measurement of light scattering or cloudiness in a liquid culture. This is measured using a spectrophotometer or a microplate reader. It is important to note that turbidity does not measure viability, as both living and dead cells contribute to the scattering of light.
The Mechanics of DNA Replication and Antibiotic Interference
DNA replication is the process of making an exact copy of the genome before cell division, though mutants can occur during this process. Replication is semiconservative, meaning each new DNA molecule consists of one original strand and one newly synthesized strand. DNA helicase is responsible for opening the double helix by breaking the hydrogen bonds between bases. Approximately 30 different proteins are involved in the replication process. DNA polymerase 3 synthesizes the new strand but specifically requires a primer, which is a short strand of RNA laid down by an enzyme in the direction to provide a free group for nucleotide attachment. Topoisomerases are used to alleviate the stress caused by DNA supercoiling. The leading strand is synthesized continuously as the replication forks move, whereas the lagging strand is synthesized discontinuously in segments known as Okazaki fragments because synthesis must occur in the direction. DNA ligase is ultimately required to link these discontinuous strands together by forming phosphodiester bonds.
Quinolone antibiotics, specifically fluoroquinolones such as Ciprofloxacin, interfere with DNA replication by binding to DNA gyrase and topoisomerase. By inhibiting these enzymes, the drugs prevent the alleviation of supercoiling stress and the separation of chromosomes at the end of the replication cycle. Bacteria such as Enterohemorrhagic Escherichia coli (EHEC) react strongly to this inhibition; the induced stress leads to an increase in toxin production.
Bacterial Growth Phases and Quorum Sensing
Bacterial populations progress through four distinct growth phases. The first is the lag phase, where cells are adjusting to their new environmental conditions. This is followed by the log phase, characterized by rapid planktonic growth and exponential expansion; this is the period when cells are "happiest" and replication is at its peak. As nutrients run out and waste products accumulate, the population enters the stationary phase. In this state, secondary metabolites are produced, including bacteriocins and other antimicrobials. Finally, the death phase occurs when the rate of cell death exceeds the rate of growth. Throughout these phases, bacteria use quorum sensing for chemical communication. Gram-negative bacteria utilize acyl-homoserine lactones (AHLs), which are autoinducers that build up in the media over time. This chemical signaling allows the population to coordinate activities once a certain population density is reached.
Genetic Flow and Gene Expression
The central dogma of molecular biology dictates the flow of information from DNA to RNA and finally to protein. Transcription is the process where a gene in the DNA is copied into messenger RNA (mRNA) by the enzyme RNA polymerase. In prokaryotes, genes are often organized together into an operon, which is a group of genes controlled by a single regulatory sequence. An example is the Lac operon, which enables bacteria to utilize lactose. This operon encodes three enzymes: LacZ, which encodes -galactosidase, as well as LacY and LacA. The use of the Lac operon often results in a diauxic growth effect, where the bacteria consume glucose first and then switch to lactose.
Translation is the stage where ribosomes read the mRNA to build proteins. Ribosomes recognize a specific sequence called the ribosome binding site (RBS) or the Shine-Dalgarno sequence, which is a series of adenine and guanine bases upstream of the start codon. The presence of the RBS and the start codon () sets the reading frame for protein synthesis. Nucleotides are read as codons, where one codon (three nucleotides) corresponds to one amino acid. Translation continues until a stop codon is reached, causing the ribosome to dissociate and fall off the mRNA. Certain antibiotics, such as tetracycline and erythromycin, work by interfering with this protein synthesis process.
Mechanisms of Horizontal Gene Transfer
Horizontal Gene Transfer (HGT) allows for the quick evolution of bacteria by moving genes between cells. There are three primary mechanisms for this transfer. Transformation involves the uptake of naked DNA from the environment; bacteria like Bacillus, Neisseria, Streptococcus, and Acinetobacter are naturally capable of becoming competent for this process. Researchers can also induce competence using an electroporator. Transduction occurs when viruses (bacteriophages) provide new genes to a bacterial cell after infection. Conjugation is an analogue to bacterial sex, involving the use of a pilus to exchange DNA directly between cells. Additionally, transposons serve as mobile genetic elements that can move between different locations within a genome or between cells.
Virology: Bacteriophages and Viral Classification
Viruses are composed of nucleic acids encased in capsid proteins. Bacteriophages, which often have a distinct space-ship shape, inject their nucleic acids into a bacterial host while leaving the protein capsule on the exterior. These phages can be lytic, causing the lysis and death of the bacteria, or lysogenic. In the lysogenic cycle, viral nucleic acid is inserted into the bacterial genome to create a prophage. If the prophage detects damage to the host cell, it can activate and initiate the lytic phase. Viral pathogenesis is often linked to bacteriophages that carry genes for toxins, such as Streptococcus pyogenes (T12 phage) which causes Scarlet fever via erythrogenic toxin, Corynebacterium diphtheriae (corynephage) which causes Diphtheria via diphtheria toxin, Clostridium botulinum (phages) causing Botulism via botulinum toxin, Vibrio cholerae (CTX phage) resulting in Cholera via cholera toxin, and Escherichia coli (Lambda phage) producing Shiga toxin which leads to HUS and bloody diarrhea.
Animal viruses differ from bacteriophages as the whole capsule often enters the cell, and many possess an envelope made of phospholipids and proteins obtained from a previously infected cell. Viruses are classified using the Baltimore system into seven groups: 1. dsDNA (such as Pox and Herpes), 2. ssDNA (such as Parvovirus), 3. dsRNA, 4. ssRNA (such as SARS-CoV-2), and 6. ssRNA-RT (such as HIV).
Immunology: Innate and Adaptive Systems
The immune response is divided into innate and adaptive systems. The innate response is inborn, fast, and relatively nonspecific. Its first line of defense includes physical barriers like intact skin, mucociliary action, peristalsis, and papillary action; chemical barriers like mucus, lysozyme in tears and saliva, and the low pH of the stomach; and microbiological barriers like the microbiota. The second line of defense involves innate immune cells like neutrophils, macrophages, NK cells, and dendritic cells. These cells use pathogen recognition receptors (PRRs) to detect Pathogen Associated Molecular Patterns (PAMPs), initiating signal transduction cascades that lead to changes in gene expression and phenotype. Some receptors trigger phagocytosis, where degraded pathogens are presented on the cell surface via MHC class II (Major Histocompatibility Complex). Chemical signals like cytokines and chemokines coordinate the inflammatory response, characterized by redness, heat, swelling, and pain.
The non-cell component of the second line of defense is the complement system, a series of enzymes in the blood. It can be activated via the classical pathway (antibodies), the alternative pathway (spontaneous), or the MBL binding lectin pathway (binding to carbohydrates). All pathways converge at the protein, leading to effector functions: opsonization (tagging for phagocytosis), inflammation, and the formation of a membrane attack complex (MAC) that creates holes in pathogen membranes. The adaptive immune system is slow, very specific, and develops over time. It involves primary lymphoid tissues where B and T cells are made (bone marrow and thymus) and secondary lymphoid tissues (lymph nodes and spleen) where they are activated. B cells produce antibodies, while T cells include (Helper T cells) and (Killer T cells).