Microbiology: Growth, Genetics, and Control

The Effect of Temperature on Enzyme Function and Bacterial Growth

Enzymes are protein catalysts that are essentially static, but the rate of reaction increases with increasing temperature because molecules move faster and collide more often and more vigorously. However, if the temperature becomes too high, it is fatal to the enzyme's function as they denature. Low temperatures do not necessarily kill the enzymes but rather inhibit their activity because the molecules lack kinetic energy. Every enzyme has an optimal temperature that varies by species. For example, humans have a Glucose-6-phosphatase enzyme from bacteria living inside the body that functions best at approximately $37\,^\circ C$. In contrast, bacteria living in hot springs possess a Glucose-6-phosphatase that functions best at near $75\,^\circ C$.

Bacteria are classified by their growth rates at specific temperature ranges. Psychrophiles grow between $-10\,^\circ C$ and $20\,^\circ C$. Psychrotrophs grow between $0\,^\circ C$ and $30\,^\circ C$. Mesophiles, which include most normal flora and human pathogens, thrive between $10\,^\circ C$ and $50\,^\circ C$. Thermophiles occupy the range of $40\,^\circ C$ to $70\,^\circ C$, while Extreme Thermophiles (mostly archaea) grow from $65\,^\circ C$ to over $110\,^\circ C$. Structural changes allow these adaptations: enzymes in heat-resistant species have more hydrogen bonds to resist denaturation, but they become stiffer at low temperatures. In cold-adapted species, enzymes have fewer hydrogen bonds for flexibility. Additionally, the phospholipid bilayer is temperature sensitive; it can melt if too hot or solidify if too cold. To compensate, organisms adjust the saturation level of fatty acids: more saturated fatty acids provide stability in heat, while more unsaturated fatty acids prevent solidification in the cold.

Osmotic Pressure and Bacterial Adaptations

Osmotic pressure is influenced by solutes such as salts and sugars. A hypertonic environment, where there are more solutes and less water outside the cell, causes water to leave the cell, leading to plasmolysis. This principle allows humans to use high concentrations of salt and sugar to preserve food. Bacterial cells generally have a cell wall that prevents them from bursting in hypotonic environments, though some species lack these walls. Most laboratory media is designed to be isotonic to bacteria, as salts can interact with the positive and negative charges on enzymes and disrupt their function.

Organisms have developed different solutions for high osmotic pressure. Facultative halophiles accumulate organic molecules, such as glycerol, to remain isotonic with their environment. Obligate halophiles, which are often archaea, require high salt concentrations (e.g., $10\%\,NaCl$) and accumulate inorganic ions like $K^+$, $Na^+$, and $Cl^-$ intracellularly to be isotonic. Their enzymes are specifically adapted to function in high-salt conditions. In the lab, Mannitol Salt Agar (MSA) uses high salt concentrations ($7.5\%\,NaCl$) to select for specific microbes.

pH and Oxygen Requirements for Microbial Growth

Most enzymes function optimally at a pH near $7$. If the environment becomes too acidic or too basic, the charges of functional groups in enzyme active sites are altered, leading to denaturation. Optimal pH varies by species; for instance, Chitinase from a bacterium in acidic pools has an optimal activity at pH $2$ to $3$, while Chitinase from soil-dwelling bacteria peaks at pH $6$. Acid preservation is a common method for food, such as pickles, because most microbes cannot survive low pH, and bases are less commonly used because they often taste bad.

Oxygen ($O_2$) serves as an electron acceptor for aerobic respiration, but it can produce toxic byproducts like the superoxide radical ($O_2^-$). To survive, aerobic organisms must produce enzymes like superoxide dismutase, which catalyzes the reaction $2O_2^- + 2H^+ \rightarrow 2H_2O_2 + O_2$, and catalase, which breaks down hydrogen peroxide via $2H_2O_2 \rightarrow 2H_2O + O_2$. Survival in oxygen depends on these protective mechanisms. Thioglycolate medium is used in the lab to determine oxygen requirements. It contains Resazurin, an indicator that turns pink in the presence of $O_2$ and remains colorless without it. Growth patterns include obligate aerobes (top), obligate anaerobes (bottom, killed by $O_2$), facultative anaerobes (mostly top but throughout), microaerophiles (middle, needing low levels), and aerotolerant anaerobes (ferment only, not killed by $O_2$).

Laboratory Methods for Atmospheric Control and Testing

Various techniques allow for the cultivation of bacteria with specific oxygen needs. A Candle Jar is used to create a microaerophilic environment; as the candle burns, it consumes most of the oxygen before going out. To achieve an entirely anaerobic environment, an Anaerobic Chamber is used. This system uses a catalyst and chemical packets (sodium bicarbonate and sodium borohydride) that react with water to remove all oxygen. An anaerobic indicator like Methylene Blue is used, which turns blue when oxygen is present and is colorless in its absence.

A common diagnostic test for oxygen metabolism is the Catalase test. When hydrogen peroxide is added directly to a bacterial culture, the immediate appearance of bubbles indicates the presence of the catalase enzyme, which is converting the peroxide into water and oxygen gas. This correlates to the microbe's ability to handle the toxic byproducts of oxygen metabolism.

Bacterial Reproduction through Binary Fission

Bacteria multiply through a process called binary fission. This process begins with the duplication of the single circular chromosome which is attached to the plasma membrane. DNA replication involves enzymes such as DNA polymerase and helicase. Following replication, the cell undergoes continued growth and enlargement, which serves to separate the two identical chromosomes. Finally, the cell divides into two identical daughter cells.

The speed of this process varies. E. coli can divide every $15$ to $20$ minutes in complex media. On defined media, growth is slower because the cell must synthesize more of its own components. This rapid division allows for fast population growth under optimal conditions.

The Bacterial Growth Curve and Population Dynamics

A bacterial growth curve typically follows four distinct phases. The Lag Phase occurs when bacteria are first introduced to a medium and are busy synthesizing materials but not yet dividing. The Log Phase, or logarithmic phase, is characterized by exponential growth where the rate of division is at its maximum. Taking the log of the population size makes it easier to graph this stage as a linear increase. The Stationary Phase occurs when the rate of death equals the rate of division because food is limited and waste products have accumulated. Finally, the Death Phase is a period of exponential population decline.

To determine the number of bacteria present, several methods are used. Direct counts utilize microscope slides with known volumes. Turbidimetry uses a spectrophotometer to measure cloudiness (higher cloudiness = more bacteria). Flow Cytometry (or a Coulter counter) works by passing cells through a thin tube in single file; a laser beam hits the bacteria, and a detector counts the "shadows." Dilution plating involves spreading samples on plates to find a countable plate ($30$ to $300$ colonies). For example, if $92$ colonies are found on a $1/100$ dilution using $0.1\,mL$, there are $920$ per $mL$ of that dilution, resulting in $920,000\,CFU/mL$ in the original sample.

Genetic Variation and Horizontal Gene Transfer

Variation in eukaryotes is driven by sexual reproduction, involving meiosis, crossing over, and random alignment during Metaphase I. Prokaryotes, being haploid and reproducing by binary fission, create identical cells unless mutation occurs. Mutation is the ultimate source of all variation. Bacteria gain genetic variation through several mechanisms of horizontal gene transfer. Transformation involves the uptake of DNA from the environment. This was demonstrated by Frederick Griffith's experiments with Streptococcus pneumoniae, where heat-killed encapsulated strains transformed live non-encapsulated strains. Treatment with nucleases prevented this, proving DNA is the genetic material. Cells that can take up DNA are termed "competent."

Conjugation is cell-to-cell transfer via a sex pilus. It involves the transfer of an F factor (plasmid). If a plasmid fuses with the main chromosome, it results in an Hfr cell, which can transfer chromosomal DNA during conjugation. Transduction is the exchange of DNA via a bacteriophage (virus). Occasionally, a virus is packaged with bacterial DNA instead of viral DNA, which it then carries to a new recipient. Transposons are "jumping genes" that move around the chromosome. If they move to a plasmid, they can be passed much more readily to other cells.

Control of Microbial Growth and Microbicides

Chemical microbicides are used to kill microbes on surfaces. They target the cell wall, cell membrane, proteins, or nucleic acids. Microbial resistance varies: enveloped viruses, fungi, and vegetative bacterial cells are the easiest to kill. Protozoan cysts and non-enveloped viruses are moderately difficult. Endospores and prions are the most difficult to eradicate. Growth control is defined as death when the organism can no longer grow even if favorable conditions return.

Ideal microbicides should be cheap, broad-spectrum, fast-acting, penetrating, stable, non-corrosive, and non-toxic to humans. Factors affecting efficacy include the length of contact time, temperature (colder temperatures result in slower action), chemical concentration, the number of microbes (especially if in a biofilm), and the presence of organic matter which can deactivate the microbicide or hinder penetration.

Antimicrobial Drugs and Mechanisms of Action

Antibiotics and antimicrobial drugs rely on selective toxicity—killing the microbe without harming the patient. Bacteria are easier to target than viruses, fungi, or helminths because they belong to a different biological domain with distinct enzymes and structures. Common targets include cell wall synthesis (Penicillins, Cephalosporins, Vancomycin, Bacitracin, D-cycloserine), focusing on peptidoglycan or mycolic acids (Isoniazid). Protein synthesis inhibitors target bacterial ribosomes, which are different from eukaryotic ones ($30S$ inhibitors like Tetracyclines and Streptomycin; $50S$ inhibitors like Erythromycin and Chloramphenicol).

Other targets include the cytoplasmic membrane (Polymyxins), though these are less common because bacterial and eukaryotic membranes are more similar. Nucleic acid synthesis can be inhibited by targeting enzymes like DNA gyrase (Quinolones) or RNA polymerase (Rifampicin). Metabolic pathways, such as folate synthesis, are also targeted (Sulfonamides) because humans do not synthesize their own folate and must consume it, whereas bacteria must produce it. These drugs are essential for treating infections, especially those caused by toxin-producing bacteria where inhibiting translation can stop toxin production.

Evolution and Mitigation of Antibiotic Resistance

Antibiotic resistance is a product of evolution through natural selection. Darwin's points apply: there is heritable variation in antibiotic susceptibility within a population due to mutations or bacterial sex. Because antibiotics kill susceptible bacteria, only those with favorable resistance characteristics survive to reproduce. Over time, resistance becomes more common. Mechanisms of resistance include enzymes that break down antibiotics (e.g., beta-lactamase for penicillin), efflux pumps that remove the drug from the cell, or mutations in the drug's target site. While mutations are rare, they become likely in large bacterial populations with short generation times.

To avoid increasing antibiotic resistance, the use of antibiotics in livestock should be reduced, and prescriptions should be handled carefully. Antibiotics should not be used for viral infections. Clinicians should test for susceptibility using methods like the Kirby Bauer test or genetic testing and use the narrowest spectrum antibiotic possible to preserve normal flora. Alternatives to antibiotics include vaccinations, probiotics, and phage therapy.