Presentation4 - Microbio
- 1. Chapter Overview ● “To eat well is to live well” ● 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 1
- 2. Microbial Nutrition Essential nutrients are those that must be supplied from environment. Macronutrients - Major elements in cell macromolecules - C, O, H, N, P, S - Ions necessary for protein function - Mg2+, Ca2+, Fe2+, K+ Micronutrients - Trace elements necessary for enzyme function - Co, Cu, Mn, Zn 2
- 3. Microbial Nutrition Based on its niche, an organism may have evolved to require additional growth factors. - Specific nutrients not required by all cells. - Refer to Table 4.1. A defined minimal medium contains only the compounds needed for an organism to grow. - Refer to Table 4.2. 3
- 4. Some organisms have adapted so well to their natural habitat that we still don’t know how to grow them in the lab. - Rickettsia prowazekii grows only within the cytoplasm of eukaryotic cells. Figure 4.1 Figure 1.1 4
- 5. 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
- 6. How Microbes Obtain Energy 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, while… - Organotrophs use organic molecules. 6
- 7. Figure 4.2 7
- 8. Microbial Nutrition In short, microbes are classified based on their carbon and energy acquisition as follows: - Autotrophs - Photoautotrophs - Chemoautotrophs (or lithotrophs) - Heterotrophs - Photoheterotrophs - Chemoheterotrophs (or organotrophs) 8
- 9. 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. 9
- 10. Figure 4.3 10
- 11. 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 11
- 12. 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 12
- 13. Figure 4.6 13
- 14. Active Transport Requires Energy Coupled transport systems are those in which energy released by moving a driving ion 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. 14
- 15. Figure 4.7 15
- 16. ABC Transporters 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. Are of two main types: - Uptake ABC transporters are critical for transporting nutrients - Efflux ABC transporters are generally used as multidrug efflux pumps 16
- 17. Figure 4.8 17
- 18. Siderophores 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. 18
- 19. Figure 4.9 19
- 20. Group Translocation 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. 20
- 21. Figure 4.10 21
- 22. Culturing Bacteria Microbes in nature exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture. We have succeeded in culturing only 0.1% of the microorganisms around us. Bacteria are grown in culture media, which is of two main types: - Liquid or broth - Solid (usually gelled with agar) 22
- 23. Culturing Bacteria Pure colonies can be isolated via two main techniques: 1) Dilution streaking - Dragging a loop 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. 23
- 24. Figure 4.12 Figure 4.14 24
- 25. Culturing Bacteria Animation: Dilution streaking technique Click box to launch animation 25
- 26. Types of Media Complex media are nutrient-rich but poorly defined. Synthetic media are precisely defined. 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. 26
- 27. Counting Bacteria Microorganisms can be counted directly by placing dilutions on a special microscope slide, called a Petroff-Hausser counting chamber. Figure 4.16 27
- 28. Fluorescence-activated Cell Sorter (FACS) - “Fluorescent” cells are passed through a small orifice and then past a laser. - Detectors measure light scatter in the forward direction (measure of particle size) and to the side (particle shape or granularity). 28
- 29. 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 29
- 30. Exponential Growth Growth rate is the rate of increase in population number or biomass. When growth is unlimited it is called exponential growth because it generates a curve whose slope increases continuously. If each cell produces two cells per generation then the population size at any given time is proportional to 2n 30
- 31. The Mathematics of Growth Generation time is the time it takes for a population to double. For cells undergoing binary fission, Nt = No x 2n where Nt is the final cell number No is the original cell number n is the number of generations 31
- 32. The Bacterial Growth Curve 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. 32
- 33. Figure 4.21 33
- 34. 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 media. Note that the human gastrointestinal tract is engineered much like a chemostat. 34
- 35. Figure 4.22 35
- 36. 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. Figure 4.24 36
- 37. A common pattern emerges in the formation of many kinds of biofilms. Figure 4.25 37
- 38. Biofilms Animation: Biofilm formation Click box to launch animation 38
- 39. Cell Differentiation Bacteria faced with environmental stress undergo complex molecular reprogramming that includes changes in cell structure. Examples include: - Endospores of Gram-positive bacteria - Heterocysts of cyanobacteria - Fruiting bodies of Myxococcus xanthus - Aerial hyphae and arthrospores of Streptomyces 39
- 40. Bacterial 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. 40
- 41. Figure 4.26 41
- 42. Cell Differentiation Animation: Endospore Formation Click box to launch animation 42
- 43. Chapter Summary ● 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 2 main types: - Passive transport does not require energy. - Simple and facilitated diffusion - Active transport requires energy. 43
- 44. Chapter Summary ● 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 44