Microbial life and cell structure

• These notes cover microbial life, cell structures, metabolism, growth requirements, and environmental factors.

Exploring the Microbial World

• Microorganisms, or microbes, are life forms too small for the human eye to see.
• Microbes vary significantly in form and function, inhabiting virtually every environment that supports life.
• Most are unicellular, while some form complex, multicellular structures.
• Microbes live in microbial communities.
• They are the oldest form of life and a major fraction of Earth's biomass.
• Microbes surround plants and animals and affect human life in many ways, from infectious diseases to food production.

Microorganism Structure and Function

• To study microorganisms, microscopy is essential.
• Cultures grow cells in/on a nutrient medium.
• A medium is liquid or solid and contains all required nutrients
• Growth means increase in cell number from cell division.
• A colony contains millions or billions of cells, visible to the naked eye.
• Cells are living compartments that interact with their environment.
• All cells have a cytoplasmic membrane, cytoplasm, ribosomes, and often a cell wall.
• The cytoplasmic membrane separates the cell's interior from the outside.
• Cytoplasm consists of macromolecules, small organic molecules, inorganic ions, and ribosomes.
• Ribosomes are responsible for protein synthesis.
• The cell wall provides structure and protection.
• Prokaryotic cells lack a nucleus and membrane-bound organelles, including Bacteria and Archaea.
• Eukaryotic cells contain organelles, including plants, animals, algae, protozoa, and fungi.
• Eukaryotic DNA is enclosed in a nucleus, while prokaryotic DNA is typically a single circular chromosome in a nucleoid region.
• Prokaryotes may also have plasmids.

Genetic and Metabolic Activities

• The genome is a cell's full set of genes.
• Microbial cells perform activities like metabolism, which includes chemical transformations of nutrients.
• Enzymes are protein catalysts facilitating these transformations.
• Transcription converts DNA information to RNA.
• Translation uses RNA to synthesize proteins via ribosomes.
• DNA replication copies the genome.
• In nature, cells live in microbial communities.

Cell Size, Shape, and Growth

• Morphology refers to cell size and shape.
• A micrometer (µm) is one-millionth of a meter.
• Most prokaryotes range from 0.5 to 10 µm in length; some can be much larger.
• Advantages of small cell size include a higher surface area-to-volume ratio, supporting greater nutrient and waste exchange.

Common Prokaryotic Cell Shapes

• Coccus: Spherical or ovoid.
• Rod/bacillus: Cylindrical.
• Spirillum: Flexible spiral.
• Spirochete: Rigid spiral.
• Some cells have appendages or irregular shapes.
• Cells may stay grouped after division, forming distinctive arrangements.
• Surface-to-volume ratios, growth rates, and evolution are influenced by cell size.
• More efficient than larger cells
• Being small is advantageous.

Domains of Life

• Thee domains of life exist: Bacteria, Archaea, and Eukarya.
• Bacteria are prokaryotes that usually exist as single cells, varying widely in size.
• There are over 80 phylogenetic lineages (phyla) in bacteria.
• Historically, Archaea were associated with extreme environments; however, this is not always the case.
• There are known parasites or pathogens of plants and animals.
• Viruses are obligate parasites that can only replicate within a host cell.
• They are not cells and do not carry out metabolism, instead taking over infected cells; they have small genomes of DNA or RNA.
• Viruses are classified based on structure, genome composition, and host specificity.

Earth's History and Microbes

• The Earth is approximately 4.6 billion years old.
• First cells appeared between 3.8 and 4.3 billion years old.
• The atmosphere was anoxic until ~2.6 billion years ago ago, supporting only anaerobic metabolisms.
• First anoxygenic phototrophs appeared 3.6 billion years ago.
• Cyanobacteria (oxygenic phototrophs) appeared 2.6 billion years ago.
• Plants and animals appeared much later, 0.5 billion years ago
• There are ~2 x 10^30 microbial cells on Earth.

Microbial Ecology and Impact

• Microbes can live in extreme environments too harsh for other life forms.
• Microbial ecology is the study of how microbes affect animals, plants, and the Earth's ecosystem.
• Microbes can be both beneficial and harmful to humans.
• Microbes are agents of disease, play a role in food and agriculture, contribute valuable human products, energy generation, and environmental clean-up.
• Controlling infectious diseases has been a major achievement.
• Most microorganisms are beneficial.
• Microbes contribute to agriculture through nitrogen-fixing and cellulose-degrading processes.
• The gut microbiome digests complex carbohydrates and synthesizes vitamins.

Microbes and Food

• Microbes have negative impacts: they can cause food spoilage and foodborne illness.
• Microbes have positive impacts: harvest, storage, and safety influenced by them.
• They improve food safety and preserve food via dairy products and other food products.

Microscopy

• Antonie van Leeuwenhoek's microscope illuminated samples with visible light.
• Magnification enlarges an image, while resolution distinguishes two objects as distinct.
• The limit of resolution for a light microscope is about 0.2 µm.
• Compound light microscopes use visible light to illuminate cells with two sets of lenses.
• Total magnification = objective magnification × ocular magnification.
• A magnification of 1,000× is needed for 0.2-µm diameter resolution.
• Specimens are visualized through differences in contrast between specimen and surroundings.
• Pigmented microbes add contrast.
• Staining increases contrast for bright-field microscopy.
• Dyes are organic compounds that bind to specific cellular materials. Basic dyes carry a positive charge and bind strongly to negatively charged cell components.
• Gram stain: Bacteria can be divided into two major groups: gram-positive and gram-negative based on cell wall structure
• Phase-contrast microscopy improves the contrast of unstained, live cells. The resulting image shows dark cells on a light background
• Fluorescence microscopy visualizesspecimens that fluoresce, naturally or after staining with fluorescent dyes to glow on a black background.

Advanced Techniques

• Differential interference contrast (DIC) microscopy enhances contrast to yield images with a three-dimensional appearance.
• Confocal scanning laser microscopy (CSLM) uses a computerized fluorescent microscope and laser to generate a three-dimensional image. The computer can focus on single layers of the specimen.
• Electron microscopes use electrons instead of visible light to image cells and structures with greater resolving power (0.2 nm).
• Specimen is coated with metal; an electron beam scans the object which are collected and projected to procure an image.

Pure Cultures

• Aseptic technique prevents contamination of media and solutions with unwanted organisms.
• Pure cultures contain cells from only a single type of microorganism.
• Enrichment culture techniques isolate microbes with specific metabolic characteristics.
• Louis Pasteur disproved the theory of spontaneous generation using the swan-necked Pasteur flask.
• Robert Koch demonstrated the link between microbes and infectious diseases, identifying causative agents of anthrax, tuberculosis, and cholera.
• Solid media is used for obtaining pure cultures of microbes.
• Sergius Winogradsky demonstrated that specific bacteria are involved in specific biogeochemical transformations.

Woese and the Tree of Life

• Carl Woese realized rRNA sequences could infer evolutionary relationships. He discovered rRNA from methanogens, naming them Archaea.
• The phylogenetic tree depicts the evolutionary history of cells, showing three domains.
• The root is LUCA (last universal common ancestor)
• Cultivation-independent methods reveal most microbes have not been cultured yet.
• Ability to grow bacteria rapidly under controlled conditions makes them excellent models for studying life.
• Genetic transfer in bacteria and DNA is genetic material.

Cytoplasmic Membrane Structure

• The cytoplasmic membrane surrounds the cytoplasm, separating it from the environment. Its selectively permeable, transporting nutrients in and waste out.
• Bacterial membranes are 8–10 nm wide and composed of a phospholipid bilayer with embedded proteins.
• The bilayer consists of hydrophobic fatty acid "tails" and hydrophilic "head groups" exposed to both the cytoplasm and the external environment.

Transport Mechanisms

• Active transport enables cells to accumulate solutes against a concentration gradient. Three mechanisms for active transport:
  • Simple transport involves transmembrane transport proteins
  • Group translocation uses a series of proteins.
  • ABC systems consist of a binding protein, transmembrane transporter, and ATP-hydrolyzing protein.
• Energy-driven uptake uses proton motive force, ATP, or other energy-rich compounds.

Cell Wall Composition and Function

• Cell walls withstand osmotic pressure and maintain cell shape.
• Organization and cell wall structure is determined by the Gram stain reaction.
• Peptidoglycan is a rigid polysaccharide layer founded only in Bacteria cell walls
• Glycan tetrapeptide, include Sugar backbone with alternating modified glucose and a short peptide attached.
• Gram-positive cell walls are thick peptidoglycan layers stabilized by peptide interbridges.
• Gram-positive cell walls often contain teichoic acids embedded in the cell wall and covalently linked to peptidoglycan.

Archaea and Diversity

• Archaeal cell walls lack peptidoglycan and do not differ in membrane structure from Bacteria.
• Most lack a polysaccharide wall, instead having an S-layer (protein shell).
• In methanogens, pseudomurein cell wall.
• The outer membrane (OM) in gram-negative cells contains polysaccharides and contains porins.
• Most cells lack cell walls, have tough cytoplasmic membranes (e.g., sterols).

Surface Structures

• Capsules and slime layers are sticky polysaccharide coats outside the cell envelope.
• Fimbriae are thin filaments that assist in surface attachment. · Pili are thin protein structures to stick to surface or forming biofilms, and facilitate conjugation. · Archaea: hami are assist in surface attachment, forming biofilms.

Cell Inclusions Function

• Inclusions function as energy reserves, carbon or phosphorus reservoirs, and/or have special functions.
• Enclosed by thin protein membrane
• Reduces osmotic membrane
• Carbon storage polymers
• Other storage polymers - glycogen: glucose polymer, Sulfur, and Carbonate.

Endospores: Specialized Spores

• Special stains/procedures needed (e.g., malachite green)
• Many layers, core, inner membrane, cortex, outer membrane, endospore coat, exosporium (Figure 2.28)
• Contains dipicolinic acid (DPA), enriched in
• Form of cellular differentiation · Begins with asymmetric cell division and forespore formation · Mother cell engulfs forespore to form outer membrane ■ 200+ sporulation-specific genes in Bacillus subtilis

Defining Life's Requirements

• Catabolic pathways: Generates free energy. - Anabolic pathways: Requires energy. - Metabolism All living cells needs fundamental nutrients such as, - Water
• Carbon and Other Nutrients: All living organisms is made from carbon and the reducing power.

Catabolism

• Catabolism depends on electron flow from electron donor to electron acceptor
• Reduction potential - affinity of substance for electrons
• Redox reactions occur in pairs - half reactions or redox couple
• Aerobic: · Chemoorganotrophs obtain energy and reducing power from organics · 2Aerobic reactions require O2 as electron acceptor

Fermentation

• Enzyme Catalysts: · Free energy calculations do not provide information on reaction rates · Activation energy - minimum energy required for chemical reaction to begin · A catalyst for catalyzing certain reactions

Bacterial Nutrition

• Supply of elements (e.g., carbon, nitrogen, and other nutrients) required for growth. - Macronutrients vs. Micronutrients. - The chemical composition of a bacterial call call require C, O, N, H, P, S are ~96% of dry weight of bacterial cell.

Cultural Methods

• Nutrient solutions used to grow microbes in the laboratory. - Either liquid or solid · Liquid vs solid media. - Different microorganisms may have vastly different nutritional requirements. Need to be pure to culture in laboratory. · Techniques, and Airborne contaminants.

Methods of Counting

• Total cell count. - Microscopic cell count is quick and easy to perform .
• Viable (alive/living) counts: measurement of living, reproducing population. · Great plate anomaly, why is this? Plate count is quick and easy to perform · Turbidimetric measures - quick and easy to perform.

Binary Fission Stages

• Growth: increase in the number of cells: ·Binary fission: cell division following enlargement of a cell to twice original size ·Septum: partition between dividing cells, pinches off between two daughter cells ·Generation (doubling) time: time required for microbial cells to double in number = Differs for microbes and varies depending on conditions

Batch Culture

• Batch culture: a closed-system microbial culture of fixed volume
• Typical growth curve for population of cells grown in a closed system is characterized by four. phases.

Equations

• A relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth The Mathematics of Bacterial Growth: Nt= N0 2

Continuous Lab Culture

• Continuous culture: an open system
• Chemostat: most common type of continuous culture device

Biofilm Formation.

• Planktonic growth: growth in suspension of free-floating/free-swimming cells
• Sessile growth: attached to surface can develop into biofilms · Forms in Stages: Planktonic cell attaches, grows with extracellular polysaccharide, Develops, finally Disperses

Temp Ranges

• Cardinal Temperatures: range of temperature that affects bacteria from hot to cold. · Different temp ranges are low for cold environments, midrange, optimal or high for hot environments

Temp adaptations

• Organisms has adaptations for both cold and hot environments to survive or grow. ·Adaptations for cold includes having:
  • enzymes that function optimally in the cold, membranes that can function a low temperatures, and cryoprotectants. Hot has:
  • heat-stable enzymes, membranes, and production of solutes.

pH balance

:Each microbe has a pH range ~2-3 pH units within which growth is possible :Optimal pH refers to extracellular only The intracellular pH must stay relatively close to neutral (pH 5–9), consistent with macromolecule stability

Controlling growth

• Decontamination: the treatment of an object is to make it safe to handle
• Disinfection: directly targets pathogens, not necessarily all microorganisms

sterilization

• Sterilization: most widely used method of sterilization decimal reduction time; the time required to reduce bacteria viability
• Decimal heat sterilization D amount of the time requires at given temp to reduce bacteria heat
• Autoclave:Uses high pressure and temp to kill endospores
• Pasturization: heats reduce microbial load.