Cell Morphology, Structure and Walls

Cell Morphology and Structure

• Pleomorphic organisms lack a definite shape.
• Bacillus cereus is Gram-positive (+) and a streptobacillus.
• Staphylococcus aureus is Gram-positive (+).
• Escherichia coli is Gram-negative (-) and a random bacillus.

Cell Wall Components: Gram-Positive vs. Gram-Negative Bacteria

• Gram-positive bacteria have a thick peptidoglycan (PGL) layer, teichoic acid, and lipoteichoic acid.
• Gram-negative bacteria have an outer membrane, porins, a thin peptidoglycan layer (PGL), and a periplasmic space.
• Both Gram-positive and Gram-negative bacteria contain hopanoids within their plasma membranes.
• PGL is made up of chains of glycan cross-linked by short peptide chains and glycan contains N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)

Peptidoglycan (PGL) Formation

• Autolysins break β-(1,4)-glycosidic bonds.
• NAG and UDP (uridine diphosphate) form activated monosaccharides in the cytoplasm.
• NAM and UDP form in the cytoplasm and are attached to gaa.
• Bactoprenol picks up NAM to form Lipid I, then NAG to form Lipid II, resulting in a PGL monomer.
• Bactoprenol carries the PGL monomer across the membrane to the periplasmic space.
• NAM-NAG is brought in, and bactoprenol is recycled.
• Glycosylase (PBP) forms β-(1,4)-glycosidic bonds.
• Transpeptidase forms peptide cross-links.

Gram Staining

• Crystal violet stains the cell wall.
• Iodine locks in the color.
• Alcohol acts as a differential agent.
• Safranin stains cells pink.
• Over-decolorization can make Gram-positive appear pink.
• Old cells with broken peptidoglycan can appear pink.

Bacteria without cell walls

• Mycoplasma lacks a cell wall and would stain pink.
• Human cells lack a cell wall and would stain pink or have no color.
• Yeast cells lack peptidoglycan (PGL) and could stain pink or not stain.

Other Cell Wall considerations

• Mycobacterium has a thick cell wall and requires an acid-fast stain.
• The plasma membrane is selectively permeable and forms the outer layer of the cell.
• Hopanoids function as membrane stiffeners and provide protection.

Transport Mechansims

• Passive transport is powered by a concentration gradient, involves no change in the compound, and includes simple diffusion for small, nonpolar molecules.
• Facilitated diffusion is powered by a concentration gradient, involves no change in the compound, and needs carrier proteins or channels for larger molecules.
• Primary active transport is powered by ATP hydrolysis, involves no change in the compound, and uses pump proteins to move molecules against the gradient. Examples include Na+/K+ pumps.
• ABC transport is powered by ATP hydrolysis, involves no change in the compound, and uses substrate-binding proteins.
• Secondary active transport is powered by ATP hydrolysis, involves no change in the compound, and uses ion gradients from primary active transport for symport and antiport.
• Group translocation is powered by phosphoenolpyruvate (PEP) or a high-energy compound and causes a change in the compound (phosphorylation).

External Structures

• Siderophores are proteins secreted to bind iron more tightly than host cells.
• Capsules are tightly bound to the cell wall and composed of polysaccharides, helping bacteria invade the immune system through adhesion.
• Slime layers are loosely attached to the cell wall, are sticky and slimy, forming a thin coating around the cell, made of polysaccharides, and aid in surface adhesion.
• S-layers are protein structures made of repeating protein subunits that form a crystalline pattern. They are found on the PGL surface of Gram-positive bacteria, adhere noncovalently to the outer membrane of Gram-negative bacteria, and help with surface adhesion.

Inclusions and Cell Components

• Inclusions are intracellular storage structures for nutrients.
• Microcompartments are protein-enclosed structures that compartmentalize metabolic processes and hold enzymes and proteins involved in processes.
• Ribosomes are made of proteins and RNA and are involved in protein synthesis. Bacterial subunits are 30S, 50S, and 70S, while eukaryotic subunits are 40S, 60S, and 80S.
• The nucleoid is a dense region of DNA in the cell.
• Chromosomes (bacterial) are singular, circular in shape, and carry essential information.
• Supercoiling is when DNA folds in on itself for protection, and nucleoid-associated proteins assist in packaging DNA.
• Plasmids are small, circular DNA molecules that replicate separately from the chromosome and carry non-essential information, such as antibiotic resistance.
• Pili are small, hair-like structures on the cell surface. Type IV facilitates twitching motility, and sex pili allow cells to exchange DNA.
• Flagella are used for motility, attachment, and act as virulence factors
  • Monotrichous: one flagellum on one end.
  • Amphitrichous: flagella on both ends.
  • Lophotrichous: tuft of flagella on one end.
  • Peritrichous: flagella all around the cell.
  • The flagellum is powered by the proton motive force (PMF), which is powered by an H+ gradient from the electron transport chain (ETC).
  • Normal, straight movement occurs via counter-clockwise rotation; tumbling occurs via clockwise rotation (random movement).
  • Swarming is when cells move together in clumps.
  • Twitching is movement via attaching to a surface and pulling itself along the surface, using Type IV pili.
  • Gliding is smoothly gliding along a surface (e.g., in mycobacteria).
  • Chemotaxis is when a cell moves toward chemical stimuli, usually food. Run = cell goes straight to stimulus
  • Tumble = cell moves randomly until finding stimulus

Endospores

• Endospores are formed primarily by Gram-positive Bacillus species due to nutrient depletion; dehydrated, hard to kill, unable to reproduce, function as a dormant form of life for survival in harsh conditions, replicate via vegetative cell.
  • Dipicolinic acid provides extreme resistance to chemicals and heat.
  • Dipicolinic acid + calcium drags H2O out of cell.
  • SASPs tightly bind to DNA for protection

Cell Division

• MreB prepares the cell for division and elongates the cell.
• FtsZ forms the Z-ring and initiates cell division.
• FtsA & ZipA form a circle of the Z-ring and anchors FtsZ to the cell membrane.
• FtsI forms peptide bonds and is a transpeptidase.
• SlmA covers the nucleoid to prevent DNA from getting split in half and prevents Z-ring formation.
• MinCD inhibits FtsZ at the ends of cells to ensure cell division occurs at the center.
• MinE is bound to the center and pushes MinCD to the ends.
• Septation is the wall that forms in the middle of the cell.
• Divisome is where division occurs.
• Nucleoid occlusion prevents cell division of the bacterial chromosome to avoid errors.

Growth Phases in Bacterial Culture

• Lag Phase: Bacteria are preparing for growth
• Log/Exponential Phase: Rapid Growth
• Stationary Phase: Balancing act of cell growth and cell death due to decline in nutrients and toxin accumulation
• Death Phase: Depleted nutrients and accumulation of waste products
• Long term Stationary phase: Maintenance with lower population and little nutrients

Factors Controlling Bacterial Growth

• Limiting nutrients control bacterial growth
• 1/2x indicates that half the population dies
• Toxins (antibiotics)
• Pigments
• Endospores
• Primary metabolites: are needed for growth and reproduction
• Secondary metabolites: are used for defense and survival

Binary Fission Equation and Example

• Equation: Nt = N0 x 2d
• Where Nt is the number of bacteria after t time, No is the initial number of bacteria, and "d" is the number of doublings
• Example: Initial bacteria = 100, Doubling time = 2 hours, Time elapsed = 6 hours (3 rounds)
  • Calculation: Bacteria = 100 x 23 = 800 bacteria

Media Definitions used to cultivate bacteria

• Defined Media: Exact chemical composition is known.
• Enriched Media: Complex media with added blood components.
• Complex Media: Chemical composition is unknown.
• Differential Media: Allows growth of multiple microbes and displays differences.
• Selective Media: Suppresses unwanted microbes and encourages the growth of wanted ones.

Measuring Bacterial Populations

• Direct Counts: Counting the population size through a sample.
• Flow Cytometry: Counts cells by differentiating electrical conductivity among species.
• Standard Plate Counts: Counts cells through serial dilution.
• Spectrophotometry / Absorbance: Counting by identifying selective absorbance of different wavelengths of light.
• Colony Forming Units (CFU): Number of viable cells that can grow into a colony; distinguishes from dead or non-viable cells.

Environmental Tolerances

• Osmophile: Grows well in sugar.
• Osmotolerant: Can survive in sugar.
• Halophile: Grows well in salt.
• Halotolerant: Can survive in salt.
• Xerophile: Grows well in dry conditions.
• Water Activity: A measure of how much water is available for use. Aw = Psoln / Pw

Water in the Environment

• Capillary Water: Held within cracks and small pores.
• Gravitational Water: Held at low pressures that drains freely out of the soil.
• Hygroscopic Water: Held around soil particles.
• Free Water: Available for use from the soil.
• Bound Water: Held in the soil; can't be used.

pH

• Acidophiles: Grow best below pH 6.5.
• Alkaliphiles: Grow best above pH 8.
• Neutrophiles: Grow best between pH 6.5-8.

Cardinal Temperatures

• Cardinal Temperatures: Minimum, maximum, and optimum temperatures at which an organism grows.
• Psychrophiles: Grow best in cold temperatures (<15°C).
• Psychrotolerant: Grow in cold, but best around 20°C.
• Mesophile: Grows best around 39°C.
• Thermophile: Grows best around 60°C.
• Hyperthermophile: Grows best around 88-106°C.
• Hot Environments (Growth): Long chain, saturated fatty acids, Charged amino acids, Heat shock chaperone proteins, Increased stiffness
• Cold Environments (Growth): Short chain, unsaturated fatty acids, More alpha helices, less beta-sheets, More polar amino acids, Cold shock chaperone proteins bind mRNA, Cryoprotectants lower freezing point

ROS and Sterilization

• Reactive Oxygen Species (ROS): Oxygen molecules with unpaired electrons are highly reactive. Aerobic respiration produces ROS through the ETC. Catalase: Catalyzes the reaction 2H2O2 → 2H2O + O2, protecting cells from oxidative damage caused by hydrogen peroxide. Peroxidase: Utilizes the reaction H2O2 + 2H+ → 2H2O + O, does not usually release oxygen gas as a product, but uses a variety of e- donors for reduction. Superoxide Dismutase: Utilizes the reaction 2O2- + 2H+ → H2O2 + O2, neutralizing superoxide radicals before causing damage.

Sterilization and Disinfection

• Sterilant: Kills all, including endospores (e.g., halogen).
• Disinfectant: Kills most organisms (e.g., halogen, alcohol).
• Sanitizer: Kills many organisms (e.g., halogen, quats, phenolics).
• Antiseptic: Kills many organisms (e.g., halogen, alcohol).
• Chemotherapeutic: Kills some organisms.
• Narrow-Spectrum Drug: Treats only a few pathogens.
• Broad-Spectrum Drug: Treats many different pathogens.

Methods of Controlling Microbial Growth

• Filtration: Liquids only; size-specific.
• Moist Heat: most effective
• Dry Heat: less effective; no water
• Autoclaving: Sterilization using heat and pressure. Pasteurization: heating and cooling measures
• Tyndallization: Heating in increments
• UV radiation: Can be dangerous due to UV rays.
• Phenolics: Tuberculocidal disinfectants that denature proteins (but not effective against all organisms).
• Alcohols: Denature proteins, dissolve lipids. Doesn't work on non-enveloped viruses or endospores.
• Quats: Very effective and nontoxic but doesn't work on endospores or Gram-bacteria.
• Halogens: Bactericidal and works easily.

Factors Influencing Chemical Control

• Population Size: Smaller populations are easier to control.
• Population Composition: Sensitive bacteria are easier to control.
• Temperature: Higher temperatures improve control.
• Local Environment: A clean environment improves control.
• Contact Time: Long exposure improves control.
• Intensity: High potency of agents improves control.
• Growth Arrest: Damage inactivates cell cycle genes, causing cells to go into G0 but not die. Remain viable but not culturable as such. Persister Cells: Microbes with genes allowing for survival against antibiotics

Biofilms

• Biofilm: Defined as a community of microbes that form on a surface.
• Bacterial Adhesion Structures: Made of pili, flagella, capsules, slime layer, S-layers
• Benefits of the biofilm to bacteria:
• Provides protection again antibiotics (abx) and environmental stressors
• Detrimental to humans in that:
• Contributes to persistent infections and is more resistant to treatment
• EPS: helps cells of biofilm attach to their target and each other
• Minimum Inhibitory Concentration (MIC): smallesr concentration of drug that visibly inhibits the growth
• Minimum Bactericidal Concentration (MBC): # of actual living cells

Exploration of cell morphology, cell wall components including Gram-positive and Gram-negative bacteria, and peptidoglycan formation. Key components such as peptidoglycan, teichoic acid, and lipoteichoic acid are discussed. The lesson also covers the structure and function of NAG and NAM.