MicroBio6 Lecture PPT Ch04 HP PDF

CHAPTER 4 Lecture Slides Bacterial Culture, Growth, and Development Copyright © 2024 by W. W. Norton & Company, Inc.  CHAPTER OVERVIEW 4.1 MICROBIAL NUTRITION 4.2 NUTRIENT UPTAKE 4.3 CULTURING AND COUNTING BACTERIA 4.4 THE GROWTH CYCLE 4.5 BIOFILMS 4.6 CELL DIFFERENTIATIO...

CHAPTER 4 Lecture Slides Bacterial Culture, Growth, and Development Copyright © 2024 by W. W. Norton & Company, Inc.  CHAPTER OVERVIEW 4.1 MICROBIAL NUTRITION 4.2 NUTRIENT UPTAKE 4.3 CULTURING AND COUNTING BACTERIA 4.4 THE GROWTH CYCLE 4.5 BIOFILMS 4.6 CELL DIFFERENTIATION  4.1 MICROBIAL NUTRITION  Essential nutrients are those that must be supplied from the environment.  Macronutrients Major elements in cell macromolecules – C, O, H, N, P, S Cations necessary for protein function – Mg2+, Ca2+, Fe2+, K+  Micronutrients Trace elements necessary for enzyme function – Co, Cu, Mn, Zn Nutrient Supplies Limit Microbial Growth Microbes Build Biomass through Autotrophy or Heterotrophy  All of Earth’s life-forms are based on carbon, which they acquire in different ways. Autotrophs fix CO2 and assemble into organic molecules (mainly sugars). Heterotrophs use preformed organic molecules. Microbes Build Biomass through Autotrophy or Heterotrophy  All organisms require an energy source. Phototrophs obtain energy from chemical reactions triggered by light. Chemotrophs obtain energy from oxidation- reduction reactions.  Lithotrophs use inorganic molecules as a source of electrons.  Organotrophs use organic molecules. Microbes Build Biomass through Autotrophy or Heterotrophy  In short, microbes are classified based on their carbon and energy acquisition as follows: Autotrophs – Photoautotrophs – Chemolithoautotrophs (or lithotrophs) Heterotrophs – Photoheterotrophs – Chemoheterotrophs (or organotrophs) Microbes Build Biomass through Autotrophy or Heterotrophy The Carbon Cycle Energy Is Stored for Later Use  A membrane potential is generated when chemical energy is used to pump protons outside of the cell.  The H+ gradient plus the charge difference form an electrochemical potential called the proton motive force.  The potential energy stored can be used to transport nutrients, drive flagellar rotation, and make ATP by the F1FO ATP synthase. Energy Is Stored for Later Use The Nitrogen Cycle  N2 makes up nearly 79% of Earth’s atmosphere but is unavailable for use by most organisms.  Nitrogen fixers possess nitrogenase, which converts N2 to ammonium ions (NH4+).  Nitrifiers oxidize ammonia to nitrate (NO3–).  Denitrifiers convert nitrate to N2. The Nitrogen Cycle The Nitrogen Cycle  Nitrogen-fixing bacteria may be free-living in soil or water, or they may form symbiotic associations with plants. Rhizobium are symbionts in leguminous plants such as soybeans, chickpeas, and clover.  4.2 NUTRIENT UPTAKE  Membranes are designed to separate what is outside the cell from what is inside.  Selective permeability is achieved in three ways: Substrate-specific carrier proteins, or permeases Dedicated nutrient-binding proteins that patrol the periplasmic space Membrane-spanning protein channels or pores Facilitated Diffusion  Facilitated diffusion helps solutes move across a membrane from a region of high concentration to one of lower concentration. It does not use energy and cannot move a molecule against its gradient.  Example: the aquaporin family that transports water and small polar molecules such as glycerol Facilitated Diffusion Active Transport Requires Energy 1. Coupled transport systems are those in which energy released by a driving ion moving down its gradient is used to move a solute up its gradient. In symport, the two molecules travel in the same direction. In antiport, the actively transported molecule moves in the direction opposite to the driving ion. Active Transport Requires Energy Active Transport Requires Energy Active Transport Requires Energy ABC Transporters Are Powered by ATP 2. The largest family of energy-driven transport systems is the ATP-binding cassette superfamily, or ABC transporters. They are found in all three domains of life. Three components: solute-binding protein, membrane- spanning protein and ATPases.  Two main types: Uptake ABC transporters are critical for transporting nutrients. Efflux ABC transporters are generally used as multidrug efflux pumps and do not need solute-binding proteins. ABC Transporters Are Powered by ATP Siderophores Are Secreted to Scavenge Iron  Siderophores are specialized molecules secreted to bind ferric ion (Fe3+) and transport it into the cell. The iron is released into the cytoplasm and reduced to the more useful ferrous (Fe2+) form.  Note: Neisseria gonorrhoeae employs receptors on its surface that bind human iron complexes and wrest the iron from them. Siderophores Are Secreted to Scavenge Iron Group Translocation Avoids “Uphill” Battles 3. Group translocation is a process that uses energy to chemically alter the substrate during its transport. The phosphotransferase system (PTS) is an example present in all bacteria. It uses energy from phosphoenolpyruvate (PEP) to attach a phosphate to specific sugars. The system has a modular design that accommodates different substrates. Group Translocation Avoids “Uphill” Battles  4.3 CULTURING AND COUNTING BACTERIA  Microbes in nature exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture.  After almost 140 years of trying, we have succeeded in culturing less than 1% of the microorganisms around us.  The vast majority has yet to be tamed. Bacteria Are Grown in Culture Media  Bacteria are grown in culture media, which are of two main types: 1. Liquid or broth – Useful for studying the growth characteristics of a pure culture 2. Solid (usually gelled with agar) – Useful for trying to separate mixed cultures from clinical specimens or natural environments Bacteria Are Grown in Culture Media Dilution Streaking and Spread Plates  Pure colonies are isolated via two main techniques: 1. Dilution streaking (= Streak plate) – A loop is dragged across the surface of an agar plate. – Best to obtain single colonies. 2. Spread plate – Tenfold serial dilutions are performed on a liquid culture. – A small amount of each dilution is then plated. – Best to count colonies. – Only viable cells will grow. Dilution Streaking and Spread Plates Dilution Streaking and Spread Plates Dilution Streaking and Spread Plates Complex versus Minimal Defined Media  Complex media are nutrient rich but poorly defined.  Minimal defined media contain only those nutrients that are essential for growth of a given microbe.  Best for checking metabolism of a microbe. Complex versus Minimal Defined Media TABLE 4.1 Composition of Commonly Used Media Medium Ingredients per liter Organisms cultured Lysogeny broth; also Bacto tryptonea 10 g Many Gram-negative and Gram-positive called Luria Bertani Bacto yeast extract 5g organisms (such as Escherichia coli and broth (complex) NaCl 10 g Staphylococcus aureus, respectively) Adjust to pH 7 M9 medium (defined) Glucose 2.0 g Gram-negative organisms such as E. coli Na2HPO4 6.0 g (42 mM) KH2PO4 3.0 g (22 mM) NH4Cl 1.0 g (19 mM) NaCl 0.5 g (9 mM) MgSO4 2.0 mM caC12 0.1 mM Adjust to pH 7 Sulfur oxidizers NH4Cl 0.52 g Acidithiobacillus thiooxidans (defined) KH2PO4 0.28 g MgSO4 7H2O 0.25 g CaCl2 0.07 g Elemental sulfur 1.56 g grown in an atmosphere of 5% CO2 Adjust to pH 3 a Bacto tryptone is a pancreatic digest of casein (bovine milk protein). Selective, Differential, and Enrichment Media  Enriched media are complex media to which specific blood components are added.  Selective media favor the growth of one organism over another.  Differential media exploit differences between two species that grow equally well. Selective, Differential, and Enrichment Media  Several media used in clinical microbiology are both selective and differential. e.g., MacConkey medium Growth Factors and Uncultured Microbes  Microbes can evolve to require specific growth factors depending on the nutrient richness of their natural ecological niche.  Growth factors are specific nutrients not required by other species. A microbe needs them in order to be able to grow in laboratory media. e.g., Streptococcus pyogenes requires glutamic acid and alanine because it can no longer synthesize them. Growth Factors and Uncultured Microbes TABLE Growth Factors and Natural Habitats of 4.2 Organisms Associated with Disease Organism Diseases Natural habitats Growth factors Abiotrophia Osteomyelitis Humans and other animal Vitamin K, cysteine species Bordetella Whooping cough Humans and other animal Glutamate, proline, cysteine species Francisella Tularemia Wild deer, rabbits Complex, cysteine Haemophilus Meningitis, chancroid Humans and other animal Hemin, NAD species, upper respiratory tract Legionella Legionnaires' disease Soil, refrigeration cooling Cysteine towers Mycobacterium Tuberculosis, leprosy Humans Nicotinic acid (NAD),a alanine (M. leprae is unculturable) Shigella Bloody diarrhea Humans Nicotinamide (NAD)a Staphylococcus Boils, osteomyelitis Widespread Complex requirement Streptococcus Pharyngitis, rheumatic fever Humans Glutamate, alanine pyogenes Both nicotinamide and nicotinic acid are derived from NAD, nicotinamide adenine dinucleotide. Growth Factors and Uncultured Microbes  Some species have adapted so well to their natural habitats that we still do not know how to grow them in the lab. Some of these uncultured organisms depend on factors provided by other species that cohabit their niche. Growth Factors and Uncultured Microbes  Growth Factors and Uncultured Microbes  Obligate intracellular bacteria are also unculturable. e.g., Rickettsia prowazekii, the cause of epidemic typhus fever, has adapted to grow within the cytoplasm of eukaryotic cells and nowhere else. Techniques for Counting Bacteria  There are many reasons why it is important to know the number of organisms in a sample.  Counting or quantifying organisms invisible to the naked eye is surprisingly difficult. Each of the available techniques measures a different physical or biochemical aspect of growth. – Thus, cell density values derived from these techniques may not necessarily agree with one another. Techniques for Counting Bacteria  Microorganisms can be counted directly by placing dilutions on a special microscope slide called a Petroff- Hausser counting chamber. Techniques for Counting Bacteria  Living cells may be distinguished from dead cells by fluorescence microscopy using fluorescent chemical dyes. Dead bacterial cells fluoresce orange or yellow because propidium (red) can enter the cells and intercalate the base pairs of DNA. Live cells fluoresce green because Syto-9 (green) enters the cell. Techniques for Counting Bacteria  Direct counting without microscopy can be done using an electronic technique that not only counts but also separates populations of bacterial cells according to their distinguishing properties.  The instrument is called a fluorescence-activated cell sorter (FACS) or flow cytometer. Fluorescent cells are passed through a small orifice and then past a laser. Detectors measure light scattered in the forward direction (measure of particle size) and to the side (particle shape or granularity). Techniques for Counting Bacteria Techniques for Counting Bacteria Techniques for Counting Bacteria  A viable bacterium is defined as being capable of replicating and forming a colony on a solid medium. Viable cells can be counted via the pour plate method.  Microorganisms can be counted indirectly via biochemical assays of cell mass, protein content, or metabolic rate. Also, by measuring optical density (OD) by which dead cells also are counted.  4.4 THE GROWTH CYCLE  Most bacteria divide by binary fission, where one parent cell splits into two equal daughter cells.  However, some divide asymmetrically.  Hyphomicrobium divides by budding. Exponential Growth  The growth rate, or rate of increase in cell numbers or biomass, is proportional to the population size at a given time. Such a growth rate is called “exponential” because it generates an exponential curve, a curve whose slope increases continually. If a cell divides by binary fission, the number of cells is proportional to 2n. – Where n = number of generations Note: Some cyanobacteria divide by multiple fission. Generation Time  Generation time is the time it takes for a population to double.  For cells undergoing binary fission, Nt = N0 x 2n where Nt is the final cell number, N0 is the original cell number, and n is the number of generations. Stages of Growth in a Batch Culture  Exponential growth never lasts indefinitely.  The simplest way to model the effects of a changing environment is to culture bacteria in a batch culture. A liquid medium within a closed system  The changing conditions in this system greatly affect bacterial physiology and growth. This illustrates the remarkable ability of bacteria to adapt to their environment. For some bacteria, quorum sensing begins at the late log phase. Generation Time Stages of Growth in Batch Culture Stages of Growth in Batch Culture Stages of Growth in Batch Culture  Rhodopseudomonas palustris, a phototrophic, aquatic Gram- negative bacterium, can enter a growth- arrested state for months during carbon or nitrogen restriction, but only in the presence of light. Stages of Growth in Batch Culture Continuous Culture  In a continuous culture, all cells in a population achieve a steady state, which allows detailed study of bacterial physiology.  The chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture medium. A liquid medium within an open system  Note that the human gastrointestinal tract is engineered much like a chemostat. Continuous Culture Continuous Culture  4.5 BIOFILMS  In nature, many bacteria form specialized, surface- attached communities called biofilms. These can be constructed by one or multiple species and can form on a range of organic or inorganic surfaces. The Biofilm Life Cycle The Biofilm Life Cycle The Biofilm Life Cycle  Bacterial biofilms form when nutrients are plentiful. Once nutrients become scarce, individuals detach from the community to forage for new sources of nutrients.  Biofilms in nature can take many different forms and serve different functions for different species.  The formation of biofilms can be cued by different environmental signals in different species. The Biofilm Life Cycle  Chemical signals enable both aerobic and anaerobic bacteria to communicate (quorum sensing) and in some cases to form biofilms when population reaches a certain number.  Biofilm development involves: The adherence of cells to a substrate The formation of microcolonies Exopolysaccharide (EPS) production by bacteria Ultimately, the formation of complex channeled communities that generate new planktonic cells The Biofilm Life Cycle The Biofilm Life Cycle The Biofilm Life Cycle  For many bacteria, sessile (nonmoving) cells in a biofilm chemically “talk” to each other in order to build microcolonies and keep water channels open. Bacillus subtilis also spins out a fibril-like amyloid protein called TasA, which tethers cells and strengthens biofilms.  Biofilm Differentiation and Communication  Bacteria growing in biofilms also exhibit a type of cell differentiation initiated by physiological conditions that develop in different layers of the biofilm. Escherichia coli biofilms exhibit two-layer differentiation vis- à-vis oxygen availability.  4.6 CELL DIFFERENTIATION  Bacteria faced with environmental stress undergo complex molecular reprogramming that includes changes in cell structure.  Examples include: 1. Endospores of certain Gram-positive bacteria 2. Heterocysts of cyanobacteria 3. Fruiting bodies of Myxococcus xanthus 4. Aerial hyphae and arthrospores of Streptomyces Endospores Are Bacteria in Suspended Animation  Clostridium and Bacillus species can produce dormant spores that are heat resistant.  Starvation initiates an elaborate 8-hour genetic program that involves: An asymmetrical cell division process that produces a forespore and ultimately an endospore  Sporulation can be divided into discrete stages based primarily on morphological appearance. Endospores Are Bacteria in Suspended Animation Endospores Are Bacteria in Suspended Animation Cyanobacteria Differentiate to Fix Nitrogen  Anabaena differentiates into specialized cells called heterocysts.  Allows it to fix nitrogen anaerobically while maintaining oxygenic photosynthesis Myxococcus Differentiation Is a “Family” Gathering  Myxococcus xanthus uses gliding motility.  Starvation triggers the aggregation of 100,000 cells, which form a fruiting body. Actinomycetes: The Fungus-like Bacteria  Streptomyces bacteria form mycelia and sporangia analogous to those of fungi.  As nutrients decline, aerial hyphae divide into arthrospores that are resistant to drying. Actinomycetes: The Fungus-like Bacteria