1. 1 Chapter 4: Bacterial Culture, Growth, and Development We are only 10% human the rest is pure microbes

2. 2 Chapter Overview ● 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

3. 3 Introduction The adage “To eat well is to live well” is as true for microbes as it is for humans. 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.

4. 4 We are Carbon-based life forms on Earth All living organisms require: Proteins, which are the building blocks from which the structures of living organisms are constructed (example: enzymes). Nucleic acids, which carry genetic information. Carbohydrates, which store energy in a form that can be used by living cells. Fats, which also store energy, but in a more concentrated form, and which may be stored for extended periods in the bodies of animals.

5. 5 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 - Mg 2+, Ca 2+, Fe 2+, K + Micronutrients - Trace elements necessary for enzyme function - Co, Cu, Mn, Zn Elements of Life

6. 6 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. Microbial Nutrition

7. 7 Table 4-1 Growth factors and natural habitats of organisms associated with disease.

8. 8 Table 4-2 Composition of commonly used media.

9. 9 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. - Body temperature of the armadillo is low enough to favor the growth of the leprosy-causing bacterium Mycobacterium leprae. Figure 1.1

10. 10 How Microbes Obtain Carbon? All of Earth’s life-forms are based on carbon, which they acquire in different ways. - Autotrophs fix CO 2 and assemble into organic molecules (mainly sugars). - Heterotrophs use preformed organic molecules.

11. 11 How Microbes Obtain Energy? In addition to carbon, 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.

12. 12 In short, microbes are classified based on their carbon and energy acquisition as follows: - Autotrophs - Photoautotrophs - Chemoautotrophs (or lithotrophs) - Heterotrophs - Photoheterotrophs - Chemoheterotrophs (or organotrophs) Microbial Nutrition

13. 13 Figure 4.2

14. 14 The Nitrogen Cycle N 2 makes up 79% of Earth’s atmosphere but is unavailable for use by most organisms. Nitrogen fixers possess nitrogenase, which converts N 2 to ammonium ions (NH 4 + ). Nitrifiers oxidize ammonia to nitrate (NO 3 – ). Denitrifiers convert nitrate to N 2.

15. 15 Figure 4.4 The Nitrogen Cycle

16. 16 Nitrogen-fixing bacteria may be free-living in soil or water, or they may form symbiotic associations with plants. - Rhizobium and legumes The Nitrogen Cycle Figure 4.5

17. 17 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

18. 18 Uptake of Nutrients Some nutrients enter by passive diffusion Most nutrients enter by: facilitated diffusion active transport group translocation (form of active transport0

19. 19 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

20. 20 Passive Diffusion and Facilitated Diffusion No energy is spent !

21. 21 Figure 5.3 rate of facilitated diffusion increases more rapidly and at a lower concentration diffusion rate reaches a plateau when carrier becomes saturated

22. 22 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.

23. 23 Figure 4-7 Coupled transport. Co-transport of glucose and Na + in to the cells Sodium-Calcium exchanger

24. 24 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

25. 25 Figure 4-8 ABC transporters.

26. 26 Siderophores Siderophores are specialized molecules secreted to bind ferric ion (Fe 3+ ) and transport it into the cell. - The iron is released into the cytoplasm and reduced to the more useful ferrous (Fe 2+ ) form. Note: Neisseria gonorrhoeae employs receptors on its surface that bind human iron complexes and wrest the iron from them.

27. 27 Iron Uptake ferric iron is very insoluble so uptake is difficult microorganisms use siderophores to aid uptake siderophore complexes with ferric ion complex is then transported into cell Figure 5.8

28. 28 Figure 4-9 Siderophores and iron transport.

29. 29 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.

30. 30 Figure 4-10 Group translocation: the phosphotransferase system (PTS) of E. coli.

31. 31 Culturing Bacteria Microbes in nature exist in complex, multispecies communities, but for detailed studies pure cultures are needed. 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)

32. 32 Figure 4-11 Separation and growth of microbes on an agar surface.

33. 33 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.

34. 34 Figure 4-14 Tenfold dilutions, plating, and viable counts.

35. 35 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 (to grow fastidious organisms). Selective media favor the growth of one organism over another. (bile salts inhibit G+ bacteria) Differential media exploit differences between two species that grow equally well.

36. 36 What is the difference between enriched media and enrichment media?

37. 37 MacConkey medium, both selective and differential. Selects Gram negative organisms Lactose fermenting bacteria such as E.coli, Enterobacter and Klebsiella will produce acid. Non-Lactose fermenting bacteria such as Salmonella, Pseudomonas, Proteus and Shigella cannot utilize lactose

38. 38 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 or spread plate method. Microorganisms can be counted indirectly via biochemical assays of cell mass, protein content, or metabolic rate. - Also by measuring optical density

39. 39 Counting Bacteria Microorganisms can be counted directly by placing dilutions on a special microscope slide, called a Petroff-Hausser counting chamber.

40. 40 Live-dead stain. Fluorescent Dyes: Propidium Iodide – stains dead cells Syto-9 – stains both dead and live cells

41. 41 Figure 4-18 Fluorescence-activated cell sorter.

42. 42 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.

43. 43 The Growth Curve Observed when microorganisms are cultivated in batch culture culture incubated in a closed vessel with a single batch of medium Usually has four distinct phases

44. 44 lag phase no increase log phase maximal rate of division and population growth stationary phase population growth ceases death phase decline in population size Figure 6.6

45. 45 The Mathematics of Growth Generation (doubling) time time required for the population to double in size varies depending on species of microorganism and environmental conditions range is from 10 minutes to several days for some microorganisms

46. 46 Exponential Growth Simple binary fission is not the only kind of division that generates an exponential curve. - e.g.: Plasmodium falciparum invades an RBC, releasing about 20 progeny per generation. Figure 4.20

47. 47 Figure 4-21 Bacterial growth curves.

48. 48

49. 49 The Mathematics of Growth Generation time is the time it takes for a population to double. For cells undergoing binary fission, N t = N o x 2 n where N t is the final cell number N o is the original cell number n is the number of generations

50. 50 Calculation of number of cells, generation times, and growth rates N o = initial population number N t = population at time t n = number of generations at time t g = generation time

51. 51 If in 8 h an exponentially growing cell population increases from 5 × 10 6 cells/ml to 5 × 10 8 cells/ml, calculate g and n.

52. 52 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.

53. 53 Figure 4-23 Relationships between dilution rate, cell mass, and generation time.

54. 54 Figure 4-22 Chemostats and continuous culture.

55. 55 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

56. 56 Figure 4-24 Biofilms.

57. 57 Figure 4-25 Biofilm development.

58. 58 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.

59. 59 Figure 4.26

60. 60 Cyanobacterial Heterocysts Anabaena differentiates into specialized cells called heterocysts. - Allow it to fix nitrogen anaerobically while maintaining oxygenic photosynthesis Figure 4.27

61. 61 Fruiting Bodies Myxococcus xanthus uses gliding motility. - Starvation triggers the aggregation of 100,000 cells, which form a fruiting body. Figure 4.28

62. 62 Streptomyces bacteria form mycelia and sporangia analogous to those of fungi. Eukaryotic-like Structures As nutrients decline, aerial hyphae divide into arthrospores that are resistant to drying. Figure 4.29

63. 63 Figure 4.30

64. 64 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. - Coupled transport - ABC transporters - Group translocation

65. 65 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