Chapter 4: Bacterial Culture, Growth, and Development | Rutgers University Newark

University:
Rutgers University Newark
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BIO 335 | General Microbiology
Academic year:

2024
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500

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73
Author:

Mahitab S.

CHAPTER 4 Bacterial Culture, Growth, and Development Copyright © 2020 W. W. Norton & Company 1 Chapter Overview § Microbial nutrition 101 § How microbes uptake nutrients § How microbes are cultured § How microbes are counted § The microbial growth cycle § What is a biofilm? § Cell differentiation, and how some prokaryotes “behave” like eukaryotes 2 Introduction § Like all living things, microorganisms need sources of carbon and energy to grow. § Over eons, bacteria have evolved ingenious strategies to find, acquire, and metabolize a wide assortment of food sources. • This owes to the remarkable plasticity of microbial genomes. § Understanding how bacteria use food to increase cell mass and, ultimately, cell number enables us to control their growth. • It even allows us to manipulate them to make useful products. 3 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+ cofactors § Micronutrients • Trace elements necessary for enzyme function – Co, Cu, Mn, Zn 4 How Microbes Build Biomass § 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. 5 How Microbes Obtain Energy – 1 § 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. 6 How Microbes Obtain Energy – 2 § In short, microbes are classified based on their carbon and energy acquisition as follows: • Autotrophs – Photoautotrophs – Chemolithoautotrophs (or lithotrophs) • Heterotrophs – Photoheterotrophs – Chemoheterotrophs (or organotrophs) 7 The Carbon Cycle Glucoser utertotroph 102 Autotroph 8 inarge diff Energy Is Stored for Later Use I.IE as a taitt § A membrane potential is generated when chemical energy is used to pump protons outside of the cell. in § 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. protons move down gradient its.ci 1 isiiaiini target kill Bact if H no imÉ inside tell outside 9 EXanfa.FIm hmg.Eukeaun Bacterial Membrane ATP Synthase 2p I of ATPsynthase embrane F facing astonian ans is no 10 The Nitrogen Cycle – 1 § 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+). Bact have symbioti vfkfgsmhif.fi eamn to fix § Nitrifiers oxidize ammonia to nitrate (NO3–). Nitrate reductaseenzyme § Denitrifiers convert nitrate to N2. N nitrogenFixers nay nitritiers no To penitrifiers P FYI nitrogen µ 11 The Nitrogen Cycle – 2 NO 12 The Nitrogen Cycle – 3 § Nitrogen-fixing bacteria may be freeliving in soil or water, or they may form symbiotic associations with plants. • Rhizobium are symbionts in leguminous plants such as soybeans, chickpeas, and clover. 13 4.2 Nutrient Uptake § Membranes are designed to separate what is outside the cell from what is inside. of plasmamembrane § 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 14 Facilitated Diffusion – 1 not energy throughpare § 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 15 Diffusion Glycerol Facilitated Diffusion – 2 Is why do we studythis n drugto disruptprocess v fac Diff no 919m01 a sincera no lipids him's Bact Die 16 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. Not dependent on ATP for energy • 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. one moleines mumnt allow other molecule to move against gradient 17 Active Transport Requires Energy – 2 Sanfection Diff directions moves aling gradient mg moves against gradient moves Antipan symport Alkaline renditions 18 ABC Transporters Are Powered by ATP – 1 § The largest family of energy-driven transport systems is the ATPbinding cassette superfamily, or ABC transporters. • They are found in all three domains of life. III in § Two main types: • Uptake ABC transporters are critical for transporting nutrients. Nutrients • Efflux ABC transporters are generally used as multidrug efflux pumps. Rid cells of drugs spitsthem out basinally 19 Question altarently ABC Transporters Are Powered by ATP – 2 Thermatog maritima transpt Ague G AB transport Alginate a carbon sayre Biofa m sfumental Alginatefrom EI IIinner 20 Siderophores Are Secreted to Scavenge Iron – 1 § Siderophores are specialized molecules secreted to bind ferric ion (Fe3+) and transport it into the cell. get 2H Is • 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. evers make iron sources unavailable for Bart to use for replication helps stop Bact infection Drug Cefivora siderophore in cephalosporin to sneak 21 Siderophores Are Secreted to Scavenge Iron – 2 ExamA Bust devel resistance wt fflux pump what Ian be it drugmechanism target siderospheres II 22 Group Translocation – 1 § 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. Help w moving due to conc grad 23 Group Translocation – 2 24 Group Translocation – 3 25 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. LAB 26 Bacteria Are Grown in Culture Media – 1 § 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 LAB 27 Bacteria Are Grown in Culture Media – 2 28 Dilution Streaking and Spread Plates – 1 § Pure colonies are isolated via two main techniques: 1. Dilution streaking – A loop is dragged across the surface of an agar plate. 2. Spread plate – Tenfold serial dilutions are performed on a liquid culture. – A small amount of each dilution is then plated. LAB 29 Dilution Streaking and Spread Plates – 2 CAD 30 Dilution Streaking and Spread Plates – 3 UAB 31 Culturing Bacteria 32 Types of Media – 1 § Complex media are nutrient rich but poorly defined. § Minimal defined media contain only those nutrients that are essential for growth of a given microbe. screening mut v wildtype § 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. 33 Types of Media – 2 Table 4.1 Medium Luria Bertani (complex) Ingredients Bacto tryptone, a pancreatic digest of casein (bovine milk protein) Amount per liter 10 grams Organisms cultured Many Gram-negative and Gram-positive organisms (such as Escherichia coli and Staphylococcus aureus, respectively) Luria Bertani (complex) Bacto yeast extract 5 grams Empty cell Luria Bertani (complex) Upper N a upper C l Adjust to pH 7 10 grams Empty cell M 9 medium (defined) Glucose 2.0 grams Gram-negative organisms such as E. coli M 9 medium (defined) Upper N a 2 upper H upper P upper O 4 6.0 grams (42 millimolar) Gram-negative organisms such as E. coli M 9 medium (defined) Upper K upper H 2 upper P upper O 4 3.0 grams (22 millimolar) Gram-negative organisms such as E. coli M 9 medium (defined) Upper N upper H 4 upper C l 1.0 grams (19 millimolar) Gram-negative organisms such as E. coli M 9 medium (defined) Upper N a upper C l 0.5 grams (9 millimolar) Gram-negative organisms such as E. coli M 9 medium (defined) Upper M g upper S upper O 4 2.0 millimolar Gram-negative organisms such as E. coli M 9 medium (defined) Upper C a upper C l 2 Adjust to pH 7 0.1 millimolar Gram-negative organisms such as E. coli Sulfur oxidizers (defined) Upper N upper H 4 upper C l 0.52 grams Acidithiobacillus thiooxidans Sulfur oxidizers (defined) Upper K upper H 2 upper P upper O 4 0.28 grams Acidithiobacillus thiooxidans Sulfur oxidizers (defined) Upper M g upper S upper O 4 times 7 upper H 2 upper O 0.25 grams Acidithiobacillus thiooxidans Sulfur oxidizers (defined) Upper C a upper C l 2 0.07 grams Acidithiobacillus thiooxidans Sulfur oxidizers (defined) Elemental sulfur 1.56 grams Acidithiobacillus thiooxidans Sulfur oxidizers (defined) Upper C upper O 2 Adjust to pH 3 5 percent Acidithiobacillus thiooxidans x 34 Types of Media – 3 § Several media used in clinical microbiology are both selective and differential. • e.g., MacConkey medium 35 Growth Factors and Uncultured Microbes – 1 l specific growth factors depending § Microbes can evolve to require 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. 36 Growth Factors and Uncultured Microbes – 2 Table 4.2 Organism Shigella Diseases Bloody diarrhea Natural habitats Humans Growth factors Nicotinamide, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide Haemophilus Meningitis, chancroid Humans and other animal species, upper respiratory tract Hemin, upper N upper A upper D Staphylococcus Abiotrophia Boils, osteomyelitis Osteomyelitis Widespread Humans and other animal species Complex requirement Vitamin K, cysteine Legionella Legionnaires’ disease Soil, refrigeration cooling towers Cysteine Bordetella Whooping cough Humans and other animal species Glutamate, proline, cysteine Francisella Mycobacterium Tularemia Tuberculosis, leprosy Wild deer, rabbits Humans Complex, cysteine Nicotinic acid, which is derived from upper N upper A upper D, nicotinamide adenine dinucleotide, and alanine (M. leprae is unculturable) Streptococcus pyogenes Pharyngitis, rheumatic fever Humans Glutamate, alanine 37 l Growth Factors and Uncultured Microbes – 3 § 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. 38 Growth Factors and Uncultured Microbes – 4 § 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. 39 Techniques for Counting Bacteria 1 § 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. 40 Direct Counting of Living and Dead Cells – 1 § Microorganisms can be counted directly by placing dilutions on a special microscope slide called a Petroff-Hausser counting chamber. 41 Direct Counting of Living and Dead Cells – 2 § 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. 42 Direct Counting of Living and Dead Cells – 3 § 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). 43 Direct Counting of Living and Dead Cells – 4 l 44 Other Techniques § 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 45 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. asymmetital 46 Q on Eau 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. in 120min – Where n = number of generations 120 20 6 generations as 24 64 • Note: Some cyanobacteria divide by multiple fission. 47 Generation Time § Generation time is the time it takes for a population to double. Doubling time § For cells undergoing binary fission, change A where Nt is the final cell number, N0 is the original cell number, and n is the number of generations. multiply every 20 mini 48 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 no influx of nutrients § The changing conditions in this system greatly affect bacterial physiology and growth. • This illustrates the remarkable ability of bacteria to adapt to their environment. 49 stationary phase acinus be Exam phasestargettime of drugs Bacterial Growth Curves p Iii nowthcurves give us idea as why wetake antibodies every12hr an say on I Logphase isgoodplace totarget wt drug ngw afteraddingcrisisSlimane 50 Longevity Genes, Dormancy, and Persistence § 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. 51 Continuous Culture – 1 § In a continuous culture, all cells in a population achieve a steady state, which allows detailed study of bacterial physiology. opposite of batch culture lontinuous addition of nutrients § The chemostat ensures logarithmic growth by constantly adding and removing equal amounts of culture medium. § Note that the human gastrointestinal tract is engineered much like a chemostat. 52 Continuous Culture – 2 53 Continuous Culture – 3 § The complex relationships among dilution rate, cell mass, and generation time in a chemostat are illustrated here. Highnutrients 54 4.5 Biofilms § In nature, many bacteria form specialized, surface-attached communities called biofilms. Bact secrete film to attach • These can be constructed by one or multiple species and can form on a range of organic or inorganic surfaces. 55 The Biofilm Life Cycle – 1 § 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. 56 The Biofilm Life Cycle – 2 § Chemical signals enable bacteria to communicate (quorum sensing) and in some cases to form biofilms. § Biofilm development involves: • The adherence of cells to a substrate • The formation of microcolonies • Ultimately, the formation of complex channeled communities that generate new planktonic cells 57 Bart make biofilm The Biofilm Life Cycle – 3 Quarumsensingto initiate production of EIS inthe stationary phase ASK T.EE II man more turn namesella There 58 The Biofilm Life Cycle – 4 § 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. Tasa helps keep biofilm together If drugs target TasAfa If if 59 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. 60 Biofilm Formation Animation 61 4.6 Cell Differentiation § Bacteria faced with environmental stress undergo complex molecular reprogramming that includes changes in cell structure. § Examples include: • Endospores of certain Gram-positive bacteria • Heterocysts of cyanobacteria • Fruiting bodies of Myxococcus xanthus Pass through Eirantmental stress • Aerial hyphae and arthrospores of Streptomyces 62 Endospores § 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. 63 Endospore Formation 64 Endospore Formation Animation 65 Cyanobacterial Heterocysts § Anabaena differentiates into specialized cells called heterocysts. § Allows it to fix nitrogen anaerobically while maintaining oxygenic photosynthesis 66 Fruiting Bodies § Myxococcus xanthus uses gliding motility. § Starvation triggers the aggregation of 100,000 cells, which form a fruiting body. 67 Filamentous Structures § Streptomyces bacteria form mycelia and sporangia analogous to those of fungi. § As nutrients decline, aerial hyphae divide into arthrospores that are resistant to drying. 68 Developmental Cycle of Streptomyces coelicolor 69 Review Biofilm Auginite ABC Transpt sederophore Symptar drug Antipart Glycera Mechanism PMF ATPsynthase Chapter Summary – 1 § Microbes require certain essential macronutrients and micronutrients to grow. § Microbes are classified on the basis of their carbon and energy acquisition. § Transport systems can be divided into two main types: • Passive transport does not require energy. – • Simple and facilitated diffusion Active transport requires energy. – Coupled transport – ABC transporters – Group translocation 70 Chapter Summary – 2 § Bacteria can be cultured on solid or liquid media. § Microorganisms in culture may be counted directly or indirectly. § The growth cycle of organisms grown in liquid batch culture consists of four phases: • Lag, logarithmic, stationary, and death § Biofilms are complex, multicellular, surface-attached microbial communities. § Many bacteria can undergo cell differentiation. • Examples: endospores, heterocysts, fruiting bodies, and aerial hyphae 71 Credits This concludes the Lecture PowerPoint presentation for Chapter 4. For more resources, please visit https://stg-iig.wwnorton.com/microb5/full . 92

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