L1 Intro and Microbial World

Microorganisms - So what?

Dual Roles of Microorganisms on Human Society

• Microorganisms can be both beneficial and harmful to humans

  • most microbes are beneficial (outweigh risks)

  • agents of disease

  • food and agriculture

  • valuable human products, energy generation, environmental clean-up (bioremediation)

Microorganisms - Human Nutrition

gut microbiome - collection of microbes and their metabolites

• digest complex carbohydrates in monogastrics

• prevent colonization by pathogens by competition, synthesize vitamins, etc.

Impact of Microorganisms on Human Society

• Microorganisms and food

  • negative impacts

    • can cause food spoilage and foodborne disease

    • harvest, storage, safety, prevention of spoilage influenced by microbes

  • positive impacts

    • improving food safety, preservation, flavor

      • dairy products (e.g., cheeses, yogurt, buttermilk) → fermentation & chemical processes help ass to flavor/preservation

      • other food products (e.g., saukerkraut, kimchi, pickles, leavened breads, beer, etc.)

      • ATP (adenosine triphosphate), cell’s main energy-carrying molecule

  • Involves controlled microbial metabolism, such as fermentation:

    • Anaerobic microbial conversion of sugars into ATP and end products, such as acids, gases, or alcohol

Fermentation of Glucose By Microbes Are Utilized to Make Different Fermented Foods

Impact of Microorganisms on Human Society - 2

• Microorganisms and industry

  • industrial microbiology: massive growth of naturally-occurring microbes to make low-cost products (e.g., antiobiotics, enzymes, some chemicals)

  • biotechnology: genetically engineered microbes making high-value products in small amounts (like proteins, vaccines, diagnostics)

  • biofilms: growth on submerged surfaces (e.g., pipes, storage tanks, implanted medical devices)

    • good or bad

    • protect bacteria from environmental hazards

    • don’t want on medical devices

  • bioremediation: cleaning up pollutants, wastewater Rx

Biofuel Production

Cleaner/more sustainable way to produce energy

Microorganisms vary greatly in size and morphology!

• Microbial diversity is not just taxonomic; it also includes shape, structure, metabolism, habitat, and interaction with hosts

• Size affects how organisms are visualized, sampled, filtered, and cultured

Microorganisms vary greatly in size and morphology! - 1

Light Microscopy & Discovery of Microbes

• Microbiology began with the microscope

• Robert Hooke: first to describe microbes (mold… spores produced from it) (Micrographia in 1665)

  • illustrated the fruiting structures of molds

Microbiology didn’t exist before the invention of the microscope!

Antoni van Leeuwenhoek

• First to describe bacteria in 1676

• ‘Wee Animalcules’… didn’t have a term for bacteria yet

Microscopy Across Biological Scales

Light Microscopy

• light microscope: illuminated by visible light (most basic)

• Types (ways to improve contrast):

  • bright-field (stained specimens)

  • phase-contrast and dark-field (live unstained cells) → advantage: can see how they move/respond to diff. environments

  • fluorescence (labeled structures and naturally fluorescent cells) → detect organisms

• magnification: the ability to make an object larger

• resolution: the ability to distinguish two adjacent objects as distinct and separate

  • Limit of resolution for light microscope limited by the wavelength of visible light or about 0.2 um at 1000x magnification

Light Microscopy pic

Bright Field Microscopy

• specimens visualized because of differences in contrast (density) between specimen and surroundings

  • distinguish or resolve resolution

• pigmented microbes add contrast

  • not pigmented → staining process to improve contrast

  • helps visualize what organisms are

Improving Contrast in Light Microscopy

• Staining improves contrast

  • Dyes are organic compounds that bind to specific cellular materials

  • Basic dyes: positively charged, bind strongly to negatively-charged cell components (e.g., nucleic acids, acidic polysaccharides, cell surfaces)

  • Examples: methylene blue, crystal violet, and safranin

  • Simple stain uses fixed or dried cells (not live)

Microscopy & Staining Overview

Simple stains answer ‘are cells present and what do they look like?’

Improving Contrast in Light Microscopy - 1

• Differential stains: Different kinds of cells are stained different colors (distinguish cells from one another)

  • Example: Gram stain differences because of cell wall structure; discovered by Danish scientist Hans Christian Gram in 1884 in Berlin

    • Bacteria can be divided into two major groups:

      • Gram-positive bacteria- appear purple

      • Gram-negative bacteria- appear pink/red

        • associated w/ cell wall of a bacteria (uptake of dyes)

Gram stain answer ‘what broad cell wall category is present?’

Improving Contrast in Light Microscopy - 2

Light Microscopy - Phase-contrast

• Improves image contrast of unstained, live cells

• Phase ring amplifies differences in the refractive index of cell and surroundings

• Resulting image- dark cells on a light background

• Especially useful when staining would kill the cells or alter their behavior

• Allows observation of motility, cell division, and general morphology in living preparations

Light Microscopy - Dark-field

• Light reaches the specimen from the sides (rather than through it from below)

• Only light reaching the lens is scattered by specimen

• Image appears light on a dark background

• Useful for very thin or delicate organisms that are difficult to visualize by bright-field microscopy

Comparison

Light Microscopy - Fluorescence

• used to visualize specimens that fluoresce (emit light after illumination with different wavelength)

  • much wider range can distinguish colors

• filters allow the microscope to separate excitation and emission light; cells appear to glow on black background due to these filters

• fluoresce naturally (autofluorescence)

• fluorescent dyes ex. DAPI

• fluorescent proteins ex. Green GFP, Red RFP

• widely used in microbial ecology to enumerate cells, detect specific taxa, or locate molecules within cells

  • tagging specific organelles or structures within cells

Fluorescence microscopy

Imaging Cells in Three Dimensions

• Confocal scanning laser microscopy (CSLM)

  • uses a computerized microscope coupled with a laser source to generate a three-dimensional image

  • focus the laser on single layers of the specimen

  • different layers then compiled to generate 3-D image (rebuild)

Probing Cell Structure: Electron Microscopy

• Electron microscopes use electrons instead of visible light (photons) to image cells and structures

  • much greater resolution than light microscopes

  • electromagnets function as lenses

  • specimens are usually fixed, dehydrated, stained or coated, and imaged in a vacuum

  • camera takes a picture = electron micrograph

• Two types:

  • transmission electron microscopes (TEM) 2-D

  • scanning electron microscopes (SEM) 3-D

Probing Cell Structure: Electron Microscopy -1

• Transmission electron microscopy (TEM): 2-D

  • useful for visualizing internal structure, viruses, macromolecular complexes, and very thin sections of cells (can see entire structure)

  • enables visualization of structures at the molecular level

  • specimen must be very thin (20-60 nm) and stained with high atomic weight substances that scatter electrons well and improve contrast

  • negative staining allows direct observation of intact cells/components

Probing Cell Structure: Electron Microscopy - 2

• Scanning electron microscopy (SEM): 3-D

  • specimen coated with thin film of heavy metal e.g., gold

  • even very large specimens can be observed

  • magnification range of 15-100,000x

  • excellent for surface topology, cell arrangements, and larger specimens, but it does not reveal internal structure unless the specimen is fractured or sectioned

Microbial Cultivation

• Aseptic technique: collection of practices that allow preparation and maintenance of sterile (no living organisms) nutrient and media solutions

  • contamination free

• Pure cultures: cells from only a single type of microorganism; essential for linking traits to a specific microbe

  • confirm it’s not contaminated

• Enrichment culture techniques: select for organisms with desired metabolic capabilities by manipulating nutrients, oxygen, temperature, pH, or other conditions

  • clues on what genus species might be

Microbial Cultivation Expands the Horizon of Microbiology

• Does spontaneous generation occur? - Pasteur

  • Determined that microbes come from existing life and mediate chemical changes

• What is the nature of infectious disease? - Koch

  • Connected specific microbes to specific diseases and formalized causal criteria (directly link diseases to an organism)

• Discovery of Microbial Diversity

  • Enrichment and cultivation methods opened the door to discovering metabolic and ecological diversity

Louis Pasteur

• Louis Pasteur: French chemist and microscopist

  • Discovered optical isomers (tartaric acid crystals)

  • Showed microbes distinguish between isomers

  • Alcohol production with beet juice: Linked yeast to alcohol and bacteria to lactic acid fermentation

  • Proved fermentation is a microbial process, not simply a chemical reaction

  • Connected airborne microbes to contamination (due to something in environment, not spontaneous)

Pasteur and the Defeat of Spontaneous Generation Theory

• Swan-neck, Pasteur flask experiment tested whether life arose spontaneously from broth or entered from the environment

• Emphasize experimental design: the broth was sterilized, air could enter, but dust and microbes were trapped in the curved neck

• Growth occurred only when contamination was allowed

Robert Koch and Infectious Disease

• Koch (1843-1910): German physician and microbiologist

  • experimentally demonstrated the link between microbes and infectious diseases (germ theory of infectious disease)

  • developed solid media for obtaining pure cultures of microbes; made it possible to compare colony morphology

  • identified causative agents of anthrax, tuberculosis, and cholera

  • awarded Nobel Prize in 1905

Koch’s Postulates (4)

Limitations: Some pathogens cannot be cultured easily, some cause disease only in certain hosts or conditions, and asymptomatic carriers complicate interpretation

Discovery of Microbial Diversity

• Entering 20th century, microbiology broadened to studying microbial diversity of soil/water and metabolic processes of microorganism

• Martinus Beijerinck (1851-1931)

  • Developed enrichment culture technique

    • Microbes can be isolated from natural samples in a highly selective fashion by manipulating nutrient and incubation conditions

      • example: nitrogen-fixing rhizobia

Molecular Basis of Life

• Rapid growth of bacteria under controlled conditions - excellent models for studies on fundamental nature of life

• Studies in microbes helped reveal DNA replication, gene expression, mutation, regulation, and metabolism

  • Led to foundations of molecular biology, genetics, and biochemistry

• Metabolic model chemistry: Certain macromolecules and reactions are universal

Molecular Basis of Life - 1

• Cracking the Code of Life

  • Genetic transfer in bacteria and DNA is genetic material (genetic info, one generation → next, one organism → another)

    • Frederick Griffith (1879-1941), Streptococcus pneumoniae - Strain R to Strain S transformation by “genetic material)

      • rough → avirulent, smooth → virulent

      • transformation of genetic material

    • Avery-MacLeod-McCarty experiment (1944) - determined that DNA mediates transformation

    • James Watson, Francis Crick, Rosalind Franklin: double helical structure of DNA

    • Emile Zuckerlandl and Linus Pauling: molecular sequences and evolutionary relationships in DNA and RNA

Frederick Griffith - Transformation

• Hereditary information from heat-killed (IIIS) virulent bacteria changed the phenotype of live nonvirulent (IIR) bacteria

• Key conclusion: Stable material from one cell could alter another cell’s inherited traits, called “transforming principle”

  • suspected some sort of genetic material was being transferred

  • used by bacteria to transfer DNA

Avery-MacLeod-McCarty

• Identified DNA as the “transforming principle” by showing that destruction of DNA eliminated transformation, whereas destroying protein or RNA did not

• Shows how microbiology contributed directly to genetics

Woes and the Tree of Life

• Evolutionary relationships between microorganisms were unclear; however, ribosomal RNA (rRNA) (present in all cells) made it possible to build the first tree of life

• Carl Woese (1929-2012) realized rRNA sequences could be used to infer evolutionary relationships

  • discovered rRNA from methanogens distinct from Bacteria and Eukarya

    • microscopic single-celled organisms; produce methane-gas as a metabolic by-product

    • replicate well in environments that didn’t have tons of oxygen

  • named new group Archaea

  • found new relationships can be deduced by comparing genetic information in the different specimens

Why rRNA?

• A type of non-coding RNA which is the primary component of ribosomes, essential to and present in all cells

• It has the same function in all cells

• Ribosomal RNA sequences differ between species, but do not mutate quickly and are highly conserved otherwise

• Conserved regions allow alignment across distant organisms, while variable regions help distinguish groups

• It is of sufficient length to be significant

Evolutionary relationships and the phylogenetic tree of life, using LUCA

Carl Woese and the Tree of Life

• Phylogenetic tree: depicts phylogeny (evolutionary history) of all cells

  • three domains with LUCA as root

  • evolution along two paths to form Bacteria and Archaea

  • Archaea diverged to distinguish Eukarya

• Cultivation-independent methods show most microbes have not been cultured yet - microbial diversity beyond what culture plates show!!

Culture vs DNA Sequencing

Culture and DNA sequencing answer different questions!

• Culture allows experiments, phenotyping, antimicrobial testing, and recovery of living organisms

• Sequencing can detect uncultured organisms and describe community composition (helps understand relationships between organisms → relatedness)

• But the strongest studies often combine both approaches, using sequence data to guide cultivation or culture data to interpret function

An Introduction to Microbial Life

Review:

• Microorganisms vary dramatically in size, shape, and structure

• All microbial cells fall into one of three major lineages or domains: Bacteria, Archaea, or Eukarya

Don’t forget about viruses! (NOT associated with microbial cells, very small organisms that contain some genomic material)

• However, not all microbes form cells - Viruses lack a cellular structure (have capsule/envelope, no nucleus/cell membrane/cell wall)

• Therefore, all known microorganisms can be classified into one of these four groups

  • No microbial cells → viruses

  • Microbial cells → bacteria, archaea, eukarya (fungi, protozoa)

An Introduction to Microbial Life - 1

• Viruses

  • obligate parasites that replicate within host cell (use host cell membrane)

  • not cells

  • do not carry out metabolism; take over other metabolic systems to replicate

  • have small genomes of double-stranded or single-stranded DNA or RNA

  • very diverse

  • classified based on capsid or envelope structure, genome composition, and host specificity or range

An Introduction to Microbial Life - 2

• Archaea

  • constitute one of the domains of life

  • single-celled organisms

  • these microorganisms lack cell nuclei and are therefore prokaryotes

  • lack of peptidoglycan in their cell walls

  • first discovered were extremophiles, but archaea also inhabit oceans, soils, animals, and other moderate environments

  • ether-linked membrane lipids

  • methanogens are especially relevant to ruminant digestion, anaerobic habitats, and greenhouse gas discussions

An Introduction to Microbial Life - 3

• Fungi (yeasts, molds, and mushrooms)

  • Saprophytic and parasitic eukaryotic organisms

  • Complex cell structure

    • nucleus with membrane, mitochondria, endoplasmic reticulum, cell membrane (ergosterol), cell wall (containing glucan, mannan, chitin), capsule

  • multicellular and filamentous- mold; unicellular- yeast; dimorphic fungi- switch forms (yeast and mold) depending on conditions

  • asexual or sexual reproduction

  • important as decomposers, pathogens, food organisms, and biotechnology platforms

  • eukaryotic nature affects antifungal drug targets and toxicity concerns (targeting similar structures in eukaryotics that are also in fungi)

An Introduction to Microbial Life - 4

• Protozoa

  • Diverse group of unicellular, eukaryotic organisms

  • Many have evolved structural features (organelles) that mimic the organs of multicellular organisms

  • Reproduction is generally asexual by mitotic binary fission or sexual (meiotic) reproduction with several variations

  • Encompass a broad spectrum of organisms belonging to both the kingdoms Protista and Animalia

    • Include subkingdoms Protozoa and Metazoa

      • Metazoa include trematodes, cestodes (Platyhelminthes), and nematodes (Nemathelminthes)

• Opportunists by nature and exploit environmental niches and lifestyles within their hosts that suit their individual needs

Comparison of Archaea, Bacteria, and Eukarya

Review Table

• Ribosomes are not membrane-bound organelles. They are made of rRNA and proteins and consist of large and small subunits. In prokaryotic cells, ribosomes are found freely in the cytoplasm. In eukaryotic cells, they may be free in the cytoplasm or attached to the outside of the rough endoplasmic reticulum.

Microbial Cell Structures