1. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 Chapter 7 Microbial Growth and Reproduction

2. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 Reproductive Strategies the reproductive strategies of eukaryotic microbes –asexual and sexual, haploid or diploid Bacteria and Archaea –haploid only, asexual - binary fission, budding, filamentous –all must replicate and segregate the genome prior to division

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5. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 Bacterial Cell Cycle cell cycle is sequence of events from formation of new cell through the next cell division –most bacteria divide by binary fission two pathways function during cycle –DNA replication and partition –cytokinesis

6. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 Chromosome Replication and Partitioning - 1 most bacterial chromosomes are circular single origin of replication – site at which replication begins terminus – site at which replication is terminated, located opposite of the origin replisome – group of proteins needed for DNA synthesis DNA replication proceeds in both directions from the origin origins move to opposite ends of the cell

7. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 Figure 7.3

8. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 8 Chromosome Partitioning replisome pushes, or condensation of, daughter chromosomes to opposite ends MreB (murein cluster B) – an actin homolog, plays role in determination of cell shape as spiral inside cell periphery, and chromosome segregation –new origins associate with MreB tracks –if MreB is mutated, chromosomes do not segregate

9. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 9 Figure 7.4

10. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 10 Plasmid Segregation plasmids replicate independently and carry proteins necessary for segregation E. coli R1 plasmid produces three proteins essential for its inheritance –ParM – similar to MreB, actin homolog forms long filaments –ParR (repressor) and ParC (centromere- like) both bind to origins and link to ParM –ParM filaments elongate and separate plasmids to opposite ends of cell

11. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11 Figure 7.5

12. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12 Cytokinesis - Septation septation – formation of cross walls between daughter cells several steps –selection of site for septum formation –assembly of Z ring –linkage of Z ring to plasma membrane (cell wall) –assembly of cell wall synthesizing machinery –constriction of cell and septum formation

13. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 13 Z Ring Formation - Role in Septation protein FtsZ –tubulin homologue, found in most bacteria and archaea –polymerization forms Z ring, filaments of meshwork MinCDE system in E. coli limits the Z ring to the center of the cell –MinC, MinD, MinE oscillate from one side of cell to other –link Z ring to cell membrane –Z ring constricts and cell wall synthesis of septal wall

14. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 14 Figure 7.6

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16. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 16 Table 7.1

17. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 Cellular Growth and Determination of Cell Shape determined by peptidoglycan synthesis in bacteria –penicillin binding proteins (PBPs) – link peptidoglycan strands and catalyze controlled degradation for new growth –autolysins – PBP enzymes that degrade peptidoglycan and site new units added

18. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18 Figure 7.8

19. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19 Cell Wall Growth and Cell Shape Determination cocci divisome - new peptidoglycan forms only at the central septum –FtsZ determines site of cell wall growth –FtsZ may recruit PBPs for synthesis of septum rods are similar but elongate prior to septation –MreB determines cell diameter and elongation as Z ring forms in center

20. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 20 Figure 7.9

21. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 21 Cell Wall Growth and Cell Shape Determination vibrio (comma-shaped bacteria) –FtsZ – forms Z ring –MreB – helical polymerization throughout cell –crescentin – intermediate filament homologue cytoskeletal protein localizes to short, curved side of cell asymmetric cell wall synthesis forms curve

22. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 22 Figure 7.10

23. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 23 Growth increase in cellular constituents that may result in: –increase in cell number –increase in cell size growth refers to population growth rather than growth of individual cells

24. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 The Growth Curve observed when microorganisms are cultivated in batch culture –culture incubated in a closed vessel with a single batch of medium usually plotted as logarithm of cell number versus time has four distinct phases –lag, exponential, stationary, senescence, and death

25. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 25 Figure 7.11

26. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 26 Lag Phase cell synthesizing new components –e.g., to replenish spent materials –e.g., to adapt to new medium or other conditions varies in length –in some cases can be very short or even absent

27. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 27 Exponential Phase also called log phase rate of growth and division is constant and maximal population is most uniform in terms of chemical and physical properties during this phase

28. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 28 Balanced Growth during log phase, cells exhibit balanced growth –cellular constituents manufactured at constant rates relative to each other

29. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 29 Unbalanced Growth rates of synthesis of cell components vary relative to each other occurs under a variety of conditions –change in nutrient levels shift-up (poor medium to rich medium) shift-down (rich medium to poor medium) –change in environmental conditions

30. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 30 Figure 7.12

31. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 31 Stationary Phase closed system population growth eventually ceases, total number of viable cells remains constant –active cells stop reproducing or reproductive rate is balanced by death rate

32. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 32 Possible Reasons for Stationary Phase nutrient limitation limited oxygen availability toxic waste accumulation critical population density reached

33. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 33 Stationary Phase and Starvation Response entry into stationary phase due to starvation and other stressful conditions activates survival strategy –morphological changes e.g., endospore formation –decrease in size, protoplast shrinkage, and nucleoid condensation –RpoS protein assists RNA polymerase in transcribing genes for starvation proteins

34. 34 Starvation responses Bacteria in culture may inter into stationary phase in response to starvation. This may lead to ; –morphological changes; e.g., endospore formation –decrease in size, protoplast shrinkage, and nucleoid condensation –production of starvation proteins that makes the cell more resistant to damage by different ways; Increase peptidoglycan cross-linking and cell wall strength. Increase in the production of DNA binding proteins to protects DNA Chaperon proteins protect against protein denaturation and renature damaged proteins As a result of these measures, starved cells become hard to kill, they become resistant to starvation, resistant to chemicals such as chlorine, damaging temperatures and oxidative stress. This leads to; long-term survival increased their virulence

35. 35 Death Phase It was argued that when cells are in the decline phase, they are dead, however, there is a debate over this statement. There are two alternative hypotheses explaining the phenomenon; –Cells are Viable But Not Culturable (VBNC) Cells alive, but dormant. Cells were genetically programmed to survive. They will not be grown under laboratory conditions but in animal passage they might grow again. –Programmed cell death Fraction of the population genetically programmed to die (commit suicide). Those will leak their nutrients to be consumed by other surviving bacteria that will not lyse and might grow later when cells are passed through an animal.

36. 36 Loss of Viability Figure 6.8In all cases, the survivors will not be able to grow under regular laboratory conditions.

37. 37 Loss of viability A; cells leave stationary phase due to starvation and accumulation of toxic waste and inter decline phase. B; it is believed that a fraction of microbes go into programmed cell death and nutrients released from dead cells are consumed by other living cells. C; some part of the culture inters the state of viable but non- culturable due to starvation. They can only grow through passage through animals. 37

38. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 38 Prolonged Decline in Growth Why it happen? bacterial population continually evolves as they are better able to use released nutrients upon the death of other cells. process marked by successive waves of genetically distinct variants with more capability of survival natural selection occurs

39. 39 Prolonged Decline in Growth Prolonged growth experiments reveal that an exponential decline phase is some times replaced by gradual decline in the number of culturable cells which can last for months or years. The most strong cells will utilize nutrients released from dead cells and tolerate toxic waste. i.e natural selection occurs bacterial population continually evolves process marked by successive waves of genetically distinct variants Figure 6.9

40. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 40 Measurement of Growth Rate and Generation Time Figure 7.16

41. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 41 Table 7.2

42. 42 Figure 7.15 Exponential microbial growth as drawn from data in table 7.2. population is doubling every generation Direct plot Logarithmic

43. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 43 Figure 7.17; generation time determination

44. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 44 Table 7.3

45. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 45 Measurement of Microbial Growth can measure changes in number of cells in a population can measure changes in mass of population

46. 46 Measurement of Cell Numbers Most obvious method is direct cell counts –Counting chambers; Petroff-Hausser counting chamber can be used for counting prokaryotic cells. While hemocytometers can be used for counting both prokaryotic and eukaryotic cells. –Electronic counters; such as Coulter counters are used for direct counting of protists and yeast or cells can be counted by flow cytometer. –Membrane filters; used mainly for counting bacteria from large liquid volumes. Viable cell counts –plating methods –membrane filtration methods

47. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 47 Counting Chambers easy, inexpensive, and quick useful for counting both eukaryotes and prokaryotes cannot distinguish living from dead cells Figure 7.18

48. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 48 Direct Counts on Membrane Filters cells filtered through special membrane that provides dark background for observing cells cells are stained with fluorescent dyes useful for counting bacteria with certain dyes, can distinguish living from dead cells

49. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 49 Flow Cytometry microbial suspension forced through small orifice with a laser light beam movement of microbe through orifice impacts electric current that flows through orifice instances of disruption of current are counted specific antibodies can be used to determine size and internal complexity

50. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 50 Viable Counting Methods spread and pour plate techniques –diluted sample of bacteria is spread over solid agar surface or mixed with agar and poured into Petri plate –after incubation the numbers of organisms are determined by counting the number of colonies multiplied by the dilution factor –results expressed as colony forming units (CFU)

51. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 51 Viable Counting Methods membrane filter technique –bacteria from aquatic samples are trapped on membranes of known pore size –membrane soaked in culture media –colonies grow on membrane –colony count determines number of bacteria in original sample

52. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 52 Figure 7.19

53. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 53 Figure 7.20

54. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 54 if microbe cannot be cultured on plate media dilutions are made and added to suitable media turbidity determined to yield the most probable number (MPN) Viable Counting Methods

55. 55 Measurement of Cell Mass An increase in the cell growth is accompanied by an increase in cell mass. Dry weight is the most direct method of cell mass estimation –time consuming and not very sensitive Measuring the quantity of a particular cell constituent –e.g., protein, DNA, ATP, or chlorophyll –useful if amount of substance in each cell is constant Turbidometric measures (light scattering) of cultures –Amount of scattered light is directly proportional to the biomass of the cells. High absorbance means low transmittance = high microbial growth –quick, easy, and sensitive

56. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 56 Figure 7.21

57. 57 The Continuous Culture of Microorganisms Growing bacteria in a flask is a closed system as no addition of new nutrients and no removal of waste therefore the log phase only stays for short period of time. Growth in an open system means –continual provision of nutrients –continual removal of wastes and toxins This continuity maintains cells in log phase at a constant biomass concentration for extended periods achieved using a continuous culture system (i.e fermenter)

58. 58 The Chemostat Rate of incoming fresh medium = rate of removal of exhausted medium from vessel An essential nutrient is in limiting quantities which determines the final growth rate Figure 6.16

59. 59 Dilution Rate and Microbial Growth dilution rate D, is the rate at which medium flows through vessel relative to vessel size. F is the flow rate, v is the vessel volume. D = f/v

60. 60 Dilution rate reflects the amount of new nutrients added to the media, so at first, when dilution rates are low the microorganisms grow well because of the accumulation of the nutrients but soon the essential nutrient will be depleted. Dilution rate increase means more nutrition so the generation time decreases i.e growth increases and the biomass maintained high at a wide dilution rates. This goes up to a level were the dilution rate is too high, so at this time, nutrient concentration keeps increasing and the generation time keeps decreasing. But the biomass will decrease sharply because this high increase in dilution rates (which will be at some time greater than the maximum growth rate) will wash out the bacteria from the culture vessel and this will sharply decrease the biomass.

61. 61 The Turbidostat This is another type of continues growth vessel but with a photocell that regulates the flow rate of media through the vessel to maintain a predetermined turbidity or cell density. This system differs from the chemostat in the following: –dilution rate varies –no limiting nutrient –turbidostat operates best at high dilution rates while chemostat does not operate well at high dilution rates.

62. 62 Importance of continuous culture methods Industrial 1.Constant supply of cells in exponential phase growing at a known rate. This could be beneficial if the organism is used for vaccine production. 2.Food and industrial microbiology benefits. i.e production of nutrients such as vitamins or certain proteins that is used as drugs …etc. Research 1.Study of microbial growth at very low nutrient concentrations, close to those present in natural environment 2.Study of interactions of microbes under conditions resembling those in aquatic environments

63. 63 The Influence of Environmental Factors on Growth Most organisms grow in fairly moderate environmental conditions however some procaryotes can live in very harsh environments. Example; Extremophiles –grow under harsh conditions that would kill most other organisms. e.g some bacteria can live under 1.5 miles below earth surface, no O 2 and temperature > 60˚ C. –Table 7-4 summarizes the way in which microorganisms are categorized in response to environmental factors.

64. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 64 Table 7.4

65. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 65 Solutes and Water Activity changes in osmotic concentrations in the environment may affect microbial cells –hypotonic solution (lower osmotic concentration) water enters the cell cell swells may burst –hypertonic (higher osmotic concentration) water leaves the cell membrane shrinks from the cell wall (plasmolysis) may occur

66. 66 Solutes and Water Activity…con Water activity (a w ) or water potential –amount of water available to organisms –reduced by interaction with solute molecules (osmotic effect) higher [solute]  lower a w – reduced also by adsorption to surfaces (matrix effect) It is important that bacteria are able to respond to changes in their osmotic concentrations in their environment. Example microbes in hypotonic environment can reduce osmotic concentrations in their cytoplasm by the use of inclusion bodies (collecting the small proteins in large bulk) or some other organisms will stretch the pores in their membranes and allow solutes to leave to prevent water from accumulating inside. Or some protists use contractile vacuoles to expel water outside such as the Paramesium.

67. 67 Water Activity (a w ) water activity of a solution can be expressed quantitatively and is equal to 1/100 of the relative humidity of solution. also equal to ratio of solution’s vapor pressure (Psoln) to that of pure water (Pwater) Aw = Psoln/ Pwater Aw of a solution can be measured by sealing the solution in a chamber and measure its relative humidity (RH) after the system has come to equilibrium. If RH reads 95 then Aw would be 0.95. Aw is inversely related to osmotic pressure.

68. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 68 Microbes Adapt to Changes in Osmotic Concentrations reduce osmotic concentration of cytoplasm in hypotonic solutions –mechanosensitive (MS) channels in plasma membrane allow solutes to leave increase internal solute concentration with compatible solutes to increase their internal osmotic concentration in hypertonic solutions –solutes compatible with metabolism and growth

69. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 69 Extremely Adapted Microbes halophiles –grow optimally in the presence of NaCl or other salts at a concentration above about 0.2M extreme halophiles –require salt concentrations of 2M and 6.2M –extremely high concentrations of potassium –cell wall, proteins, and plasma membrane require high salt to maintain stability and activity

70. 70 Osmotolerant organisms Some microorganisms grow over wide ranges of water activity and keep the osmotic concentrations above that of the habitat by using Compatible solutes to increase their internal osmotic concentration –solutes that are compatible with metabolism and growth i.e they do not interfere with metabolism. Example; –Many microorganisms increase their internal osmotic concentration in hypertonic environment by producing; choline, betaine, proline, glutamic acid and other amino acids and have some elevated levels of potassium ions. Some have proteins and membranes that require high solute concentrations for stability and activity those are called halophiles.

71. 71 Effects of NaCl on microbial growth Halophiles –grow optimally at >0.2 M Extreme halophiles –require >2 M and saturation up to 6.2 M. example; The archaeon Halobcterium can be isolated form the Dead Sea or the salt lake in Utah. Those organisms accumulate enormous amounts of potassium (up to 4-7 M) to remain hypertonic to their environment. In addition, plasma membranes and cell wall have high amounts of NaCl that is necessary for their function. Figure 7-24

72. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 72 pH measure of the relative acidity of a solution negative logarithm of the hydrogen ion concentration Figure 7.25

73. 73 pH Acidophiles –growth optimum between pH 0 and pH 5.5. Most fungi prefer pH 4-6 Neutrophiles –growth optimum between pH 5.5 and pH 7. most bacteria and protists. Alkalophiles –growth optimum between pH 8.5 and pH 11.5 –Drastic changes in pH disrupts the plasma membranes of bacteria and might inhibit the activity of enzymes and transport proteins.

74. 74 pH Microorganisms respond to external pH changes using different mechanisms; –Most acidophiles and alkalophiles maintain an internal pH near neutrality Their plasma membrane is impermeable to protons –Some bacteria synthesize proteins that provide protection e.g., chaperon proteins such as acid-shock proteins or heat shock proteins. These prevent acid denaturation and aid in their refolding. a proton translocating ATPase contribute to protecting the bacteria from acidic media by making more ATP or pumping protons out of the cell

75. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 75 pH most microbes maintain an internal pH near neutrality –the plasma membrane is impermeable to proton –exchange potassium for protons acidic tolerance response –pump protons out of the cell –some synthesize acid and heat shock proteins that protect proteins many microorganisms change the pH of their habitat by producing acidic or basic waste products

76. 76 Effect of temperature The most important factor influencing the effect of temperature is the temperature sensitivity of the enzyme- catalyzed reactions with the speed of the enzyme reactions double for every 10 ° C rise. Above certain temperatures the speed slows and very high temperature become lethal as this temperature will affect both structure and function aspects of the microbe Very low temperatures solidify the bacterial membranes thus affecting their function but not the structure. Organisms therefore have characteristic temperature - dependence with distinct cardinal temperatures.

77. 77 Temperature Organisms exhibit distinct cardinal growth temperatures –minimal –maximal –Optimal These cardinal temperatures are not fixed, i.e based on certain changes in the media they might be changed. Table 6-5 shows some of these Temperatures. Figure 6.20

78. 78 Classes of organisms based on temperature Psychrophiles; grow well at 0 and have optimum at about 15 ° C and maximum at 20 C. The cell membranes of the bacteria living in this environment contains high amount of unsaturated fatty acids that keeps the membrane semi-fluid. Psychrotrophs or facultative psycrophiles; grow at 0-7 ° C with optimum between 20-30 ° C and maximum at about 35 ° C. this type of bacteria and fungi are major food spoilage organisms. Mesophiles; are microorganisms with growth optimum 20-45 ° C. Minimum T is 15-20 ° C and maximum 45 ° C. Most microbes fall in this category and of course almost all human pathogens fall in this category i.e 37 ° C.

79. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 79 Temperature Ranges for Microbial Growth (summary) psychrophiles – 0 o C to 20 o C psychrotrophs – 0 o C to 35 o C mesophiles – 20 o C to 45 o C thermophiles – 55 o C to 85 o C hyperthermophiles – 85 o C to 113 o C

80. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 80 Table 7.5

81. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 81 Figure 7.27

82. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 82 Adaptations of Thermophiles protein structure stabilized by a variety of means –e.g., more H bonds –e.g., more proline –e.g., chaperones histone-like proteins stabilize DNA membrane stabilized by variety of means –e.g., more saturated, more branched and higher molecular weight lipids –e.g., ether linkages (archaeal membranes)

83. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 83 Oxygen Concentration growth in oxygen correlates with microbes energy conserving metabolic processes and the electron transport chain (ETC) and nature of terminal electron acceptor

84. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 84 Oxygen and Bacterial Growth aerobe –grows in presence of atmospheric oxygen (O 2 ) which is 20% O 2 obligate aerobe – requires O 2 anaerobe –grows in the absence of O 2 obligate anaerobe –usually killed in presence of O 2

85. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 85 Oxygen and Bacterial Growth microaerophiles –requires 2–10% O 2 facultative anaerobes –do not require O 2 but grow better in its presence aerotolerant anaerobes –grow with or without O 2

86. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 86 Figure 7.28

87. 87 Oxygen Concentration need oxygen prefer oxygen ignore oxygen oxygen is toxic < 2 – 10% oxygen Figure 6.22

88. 88 Classification of organisms based on O 2 requirements Obligate aerobes; contain SOD and Catalase and can not live without O2. Oxygen serves as the terminal electron acceptor for electron transport chain. It participates in sterol and fatty acid synthesis in multicellular eukaryotes. Facultative anaerobes; contain SOD and catalase. do not require O2 for growth but grow better in its presence. Aerotolerant; contains SOD but not catalase. Ignores the presence of O2 and grow equally well whether its present or not. Strict or obligate anerobe; does not contain SOD or catalase. Can not live in its presence. Thus they obtain their energy through fermentation. Microaerophiles; contain SOD and low levels of catalase. Need 2-10% O2 not regular 20 %.

89. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 89 Basis of Different Oxygen Sensitivities oxygen easily reduced to toxic reactive oxygen species (ROS) –superoxide radical –hydrogen peroxide –hydroxyl radical aerobes produce protective enzymes –superoxide dismutase (SOD) –catalase –peroxidase

90. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 90 Strict Anaerobic Microbes all strict anaerobic microorganisms lack or have very low quantities of –superoxide dismutase –catalase these microbes cannot tolerate O 2 anaerobes must be grown without O 2 –work station with incubator –gaspak anaerobic system

91. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 91 Figure 7.29; anaerobic work station

92. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 92 Figure 7.30

93. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 93 Pressure microbes that live on land and water surface live at 1 atmosphere (atm) some Bacteria and Archaea live in deep sea with very high hydrostatic pressures

94. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 94 Pressure barotolerant –adversely affected by increased pressure, but not as severely as nontolerant organisms barophilic (peizophilic) organisms –require or grow more rapidly in the presence of increased pressure –change membrane fatty acids to adapt to high pressures

95. 95 The Electromagnetic Spectrum Radiation Figure 7;31

96. 96 Radiation Damage Ionizing radiation; is very harmful to microorganisms. It has very short wavelength and high energy. There are two major forms of ionizing radiation; –x rays and gamma rays. Low levels of these radiation will cause mutations  death –Radiation disrupts chemical structure of many molecules, including DNA. It destroys; –Hydrogen bonds and oxidize double bonds. –Destroys ring structures and polymerize some molecules Oxygen enhances these changes by producing hydroxyl fee radicals –damage may be repaired by DNA repair mechanisms however, higher levels are lethal. Therefore, –Ionizing radiation is used for sterilization. –Deinococcus radiodurans extremely resistant to DNA damage

97. 97 Radiation Damage… Ultraviolet (UV) radiation; can kill all types of microorganisms due to its short wavelength (10-400) and high energy with the most lethal UV radiation is 260 nm because this wavelength is the most absorbed by DNA. –mutations  death. –UV light causes formation of thymine dimers in DNA which inhibits DNA replication and function. –DNA damage can be repaired by several repair mechanisms. –Longer UV wavelengths (325-400) are also harmful by inducing the breakdown of tryptophan to toxic products.

98. 98 Radiation damage… Visible light is so important but at high intensities it become harmful by the action of pigments (chlorophyll, flavins, bacteriochlorophyll and cytochromes) which absorb light and get excited and act as photosensitizers. These will then transfer its energy to O 2 generating the singlet oxygen ( 1 O 2 ) It is powerful oxidizing agent and will quickly destroy a cell. This is also employed by phagocytic cells to destroy bacteria after engulfing. Many microorganisms avoid the harmful damage of extensive light by carotenoid pigments. This pigment protect many light-exposed microorganisms from photooxidation by quenching singlet O 2 by converting its energy back to the unexcited ground state.

99. 99 Growth Limitation by Environmental Factors Leibig’s law of the minimum –total biomass of organism determined by nutrient present at lowest concentration. An increase in limiting essential nutrient such as phosphate will increase the population until some other nutrient become limiting. Shelford’s law of tolerance –above or below certain environmental limits, a microorganism will not grow, regardless of the nutrient supply. Such as extreme temperature or pH, O 2 level, pressure or the presence of other inhibitory substances.

100. 100 Responses to low nutrient levels (oligotrophic environments) The organisms become more competitive in nutrient capture and use of available resources Developing morphological changes to increase surface area and ability to absorb nutrients. So, some microbes will change their shape from rod to mini- or ultra micro cells (figure 6-26). Developing mechanisms to sequester certain nutrients such as iron (Fe) making it less available for their competitors

101. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 101 Biofilms most microbes grow attached to surfaces (sessile) rather than free floating (planktonic) these attached microbes are members of complex, slime enclosed communities called a biofilm biofilms are ubiquitous in nature in water can be formed on any conditioned surface

102. 102 Figure 7;33

103. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 103 Figure 7.34; biofilm heterogeneity

104. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 104 Biofilm Formation microbes reversibly attach to conditioned surface and release polysaccharides, proteins, and DNA to form the extracellular polymeric substance (EPS) additional polymers are produced as microbes reproduce and biofilm matures

105. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 105 Biofilms a mature biofilm is a complex, dynamic community of microorganisms heterogeneity is differences in metabolic activity and locations of microbes interactions occur among the attached organisms –exchanges take place metabolically, DNA uptake and communication

106. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 106 Biofilm Microorganisms the EPS and change in attached organisms’ physiology protects microbes from harmful agents –UV light, antibiotics, antimicrobials when formed on medical devices, such as implants, often lead to illness sloughing off of organisms can result in contamination of water phase above the biofilm such as in a drinking water system

107. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 107 Cell to Cell Communication Within the Microbial Populations bacterial cells in biofilms communicate in a density-dependent manner called quorum sensing produce small proteins that increase in concentration as microbes replicate and convert a microbe to a competent state –DNA uptake occurs, bacteriocins are released

108. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 108 Quorum Sensing acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram- negative organisms to regulate biofilm formation and increase virulence factors –diffuses across plasma membrane –once inside the cell it induces expression of target genes that regulate a variety of functions –many microbes produce effect

109. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 109 Figure 7.35; representative cell-cell communication molecules

110. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 110 Quorum Sensing Systems processes regulated by quorum sensing involve host-microbe interactions; this might lead to; –symbiosis – Vibrio fischeri and bioluminescence in squid –Increase pathogenicity and increased virulence factor production – lead to DNA uptake for antibiotic resistance genes

111. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 111 Figure 7.36