© 2015 Pearson Education, Inc. CH4 - PRACTICE QUESTIONS DNA replication always proceeds in only one direction because the ________ of the incoming nucleotide is attached to the free ________ of the growing DNA strand. A) 5'-phosphate / 3'-hydroxyl B) 3'-phosphate / 5'-hydroxyl C) 5'-deoxyribose / 3'-base D) 3'-base / 5'-deoxyribose

© 2015 Pearson Education, Inc. CH4 - PRACTICE QUESTIONS DNA replication always proceeds in only one direction because the ________ of the incoming nucleotide is attached to the free ________ of the growing DNA strand. A) 5'-phosphate / 3'-hydroxyl B) 3'-phosphate / 5'-hydroxyl C) 5'-deoxyribose / 3'-base D) 3'-base / 5'-deoxyribose

© 2015 Pearson Education, Inc. CH4 - PRACTICE QUESTIONS Which of the following is NOT correct regarding DNA and RNA synthesis? A) The overall direction of chain growth is from the 5' to 3' end. B) Both processes require an RNA primer to begin. C) The template strand is antiparallel to the newly synthesized strand. D) DNA is the template for both DNA and RNA synthesis.

© 2015 Pearson Education, Inc. CH4 - PRACTICE QUESTIONS Which of the following is NOT correct regarding DNA and RNA synthesis? A) The overall direction of chain growth is from the 5' to 3' end. B) Both processes require an RNA primer to begin. C) The template strand is antiparallel to the newly synthesized strand. D) DNA is the template for both DNA and RNA synthesis.

PowerPoint ® Lecture Presentations prepared by John Zamora Middle Tennessee State University C H A P T E R © 2015 Pearson Education, Inc. Microbial Growth and Growth Control 5

© 2015 Pearson Education, Inc. 5.1 Binary Fission Growth: increase in the number of cells Generation time: time required for microbial cells to double in number During cell division, each daughter cell receives a chromosome and cell “stuff” to exist as an independent cell Abbott Pantry Door

© 2015 Pearson Education, Inc. Figure 5.1 Cell elongation Septum formation Completion of septum; formation of walls; cell separation Septum One generation Binary fission: cell division following enlargement of a cell to twice its minimum size

© 2015 Pearson Education, Inc. Figure 5.2 The divisome (cell division apparatus) FtsZ: forms ring around center of cell; related to tubulin ZipA: anchor that connects FtsZ ring to cytoplasmic membrane FtsA: helps connect FtsZ ring to membrane and also recruits other divisome proteins 5.2 Fts Proteins and Cell Division FtsZ PC

© 2015 Pearson Education, Inc. 5.2 Fts Proteins and Cell Division DNA replicates before the FtsZ ring forms Location of FtsZ ring is facilitated by Min proteins MinC, MinD, MinE FtsK protein mediates separation of chromosomes to daughter cells Figure 5.3

© 2015 Pearson Education, Inc. 5.3 MreB and Cell Morphology Prokaryotes contain a cell cytoskeleton that is dynamic and multifaceted MreB: major shape-determining factor in prokaryotes related to actin Forms simple cytoskeleton in Bacteria and probably Archaea Forms spiral-shaped bands around the inside of the cell, underneath the cytoplasmic membrane (Figure 5.5a and b) Not found in coccus-shaped bacteria

© 2015 Pearson Education, Inc. 5.3 MreB and Cell Morphology MreB: Prokaryotic cell cytoskeleton Localizes peptidoglycan synthesis and other cell wall components along the cell during growth Figure 5.5 MreB

© 2015 Pearson Education, Inc. 5.3 MreB and Cell Morphology Crescentin: shape-determining protein produced by vibrio-shaped cells of Caulobacter crescentus Crescentin protein organizes into filaments ~10 nm wide that localize on the concave face of the curved cells Figure 5.5c related to intermediate filaments

© 2015 Pearson Education, Inc. 5.4 Peptidoglycan Biosynthesis Production of new cell wall material is a major feature of cell division In cocci, cell walls grow in opposite directions outward from the FtsZ ring In rod-shaped cells, growth occurs at several points along length of the cell

© 2015 Pearson Education, Inc. Figure 5.6 Beginning at the FtsZ ring, small openings in the wall are created by autolysins New cell wall material is added across the openings Wall band: junction between new and old peptidoglycan 5.4 Peptidoglycan Biosynthesis

© 2015 Pearson Education, Inc. 5.4 Peptidoglycan Biosynthesis Bactoprenol: carrier molecule that plays major role in insertion of peptidoglycan precursors Bonds to NAG/NAM/pentapeptide precursor Figure 5.7 C 55 Figure 5.8a

© 2015 Pearson Education, Inc. Figure 5.8b Transpeptidation: final step in cell wall synthesis 5.4 Peptidoglycan Biosynthesis

© 2015 Pearson Education, Inc. II. Population Growth 5.5Quantitative Aspects of Microbial Growth 5.6The Growth Cycle 5.7 Continuous Culture

© 2015 Pearson Education, Inc. Figure 5.9 Logarithmic plot Arithmetic plot 5.5 Quantitative Aspects of Microbial Growth

© 2015 Pearson Education, Inc. 5.5 Quantitative Aspects of Microbial Growth Generation time (g) of the exponentially growing population is g = t/n t is the duration of exponential growth n is the number of generations during the period of exponential growth

© 2015 Pearson Education, Inc. 5.6 The Growth Cycle Typical growth curve for population of cells grown in a closed system is characterized by four phases Figure 5.11 Viable count Turbidity (optical density) StationaryExponentialLagDeath Growth phases

© 2015 Pearson Education, Inc. 5.6 The Growth Cycle Lag phase Interval between inoculation of a culture and beginning of growth Exponential phase Cells in this phase are typically in the healthiest state Stationary phase Growth rate of population is zero Either an essential nutrient is used up, or waste product of the organism accumulates in the medium Death phase If incubation continues after cells reach stationary phase, the cells will eventually die

© 2015 Pearson Education, Inc. 5.7 Continuous Culture Continuous culture: an open-system microbial culture of fixed volume Chemostat: most common type of continuous culture device Both growth rate and population density of culture can be controlled independently and simultaneously Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out Concentration of a limiting nutrient

© 2015 Pearson Education, Inc. Figure Continuous Culture The growth rate is controlled by dilution rate The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient

© 2015 Pearson Education, Inc. Figure 5.13 In a batch culture, nutrient concentration affects both growth rate and yield 5.7 Continuous Culture

© 2015 Pearson Education, Inc. 5.7 Continuous Culture Chemostat cultures are sensitive to the dilution rate and limiting nutrient concentration At too high a dilution rate, the organism is washed out At too low a dilution rate, the cells may die from starvation Increasing concentration of a limiting nutrient results in greater biomass but same growth rate

© 2015 Pearson Education, Inc. Figure Continuous Culture

© 2015 Pearson Education, Inc. III. Measuring Microbial Growth 5.8 Microscopic Counts 5.9 Viable Counts 5.10 Spectrophotometry

© 2015 Pearson Education, Inc. 5.8 Microscopic Counts Microbial cells are enumerated by microscopic observations (Figure 5.15) Figure 5.15 Ridges that support coverslip Coverslip Sample added here. Care must be taken not to allow overflow; space between coverslip and slide is 0.02 mm ( mm). Whole grid has 25 large squares, a total area of 1 mm 2 and a total volume of 0.02 mm Microscopic observation; all cells are counted in large square (16 small squares): 12 cells. (In practice, several large squares are counted and the numbers averaged.) To calculate number per milliliter of sample: 12 cells X 25 large squares X 50 X 10 3 Number/mm 2 (3 X 10 2 ) Number/mm 3 (1.5 X 10 4 ) Number/cm3 (ml) (1.5 × 10 7 )

© 2015 Pearson Education, Inc. 5.8 Microscopic Counts Limitations of microscopic counts Cannot distinguish between live and dead cells without special stains Small cells can be overlooked Precision is difficult to achieve Phase-contrast microscope required if a stain is not used Cell suspensions of low density (<10 6 cells/ml) hard to count Motile cells need to immobilized Debris in sample can be mistaken for cells

© 2015 Pearson Education, Inc. 5.9 Viable Counts Viable cell counts (plate counts): measurement of living, reproducing population Figure 5.16 Spread-plate method Pour-plate method Sample is pipetted into sterile plate Sample is spread evenly over surface of agar using sterile glass spreader Sterile medium is added and mixed well with inoculum Solidification and incubation Incubation Typical spread-plate results Typical pour-plate results Surface colonies Surface colonies Subsurface colonies Sample is pipetted onto surface of agar plate (0.1 ml or less)

© 2015 Pearson Education, Inc. 5.9 Viable Counts To obtain the appropriate colony number, the sample to be counted should always be diluted Figure 5.17

© 2015 Pearson Education, Inc. 5.9 Viable Counts The great plate anomaly: direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates Why is this? Microscopic methods count dead cells, whereas viable methods do not Different organisms may have vastly different requirements for growth

© 2015 Pearson Education, Inc Spectrophotometry Turbidity measurements are indirect, rapid, and useful methods of measuring microbial growth Most often turbidity is measured with a spectrophotometer, and measurement is referred to as optical density (OD) Figure 5.18a

© 2015 Pearson Education, Inc. Figure Temperature Classes of Microorganisms

© 2015 Pearson Education, Inc. Figure 5.20 Polyextremophile Deinococcus radiodurans 5.11 Temperature Classes of Microorganisms

© 2015 Pearson Education, Inc Microbial Life in the Cold Extremophiles Organisms that grow under very hot or very cold conditions Psychrophiles Organisms with cold temperature optima Inhabit permanently cold environments Psychrotolerant Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC More widely distributed in nature than psychrophiles

© 2015 Pearson Education, Inc. Figure Microbial Life in the Cold Core of frozen sea water from Antarctica Polaromonas sp. Continuously frozen lake 0◦C water column

© 2015 Pearson Education, Inc Microbial Life in the Cold Production of enzymes that function optimally in the cold; features that may provide more flexibility More α-helices than β-sheets More polar and less hydrophobic amino acids Decreased interactions between protein domains Transport processes function optimally at low temperatures Modified cytoplasmic membranes (high unsaturated fatty acid content)

© 2015 Pearson Education, Inc Microbial Life in the Cold

© 2015 Pearson Education, Inc Microbial Life in the Cold Shewanella frigidimarina Salmonella spp.∆Salmonella spp.

© 2015 Pearson Education, Inc Microbial Life at High Temperatures Above ~65ºC, only prokaryotic life forms exist Thermophiles: organisms with growth temperature optima between 45ºC and 80ºC Hyperthermophiles: organisms with optima greater than 80ºC Inhabit boiling hot springs and seafloor hydrothermal vents Figure 5.23

© 2015 Pearson Education, Inc Microbial Life at High Temperatures Enzyme and proteins function optimally at high temperatures; features that provide thermal stability: Critical amino acid substitutions in a few locations provide more heat-tolerant folds An increased number of ionic bonds between basic and acidic amino acids resists unfolding Production of di-inositol phosphate, diglycerol phosphate Modifications in cytoplasmic membranes Bacteria have lipids rich in saturated fatty acids Archaea have lipid monolayer rather than bilayer

© 2015 Pearson Education, Inc Effects of pH on Microbial Growth The pH of an environment greatly affects microbial growth Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)

© 2015 Pearson Education, Inc. Figure Effects of pH on Microbial Growth

© 2015 Pearson Education, Inc Effects of pH on Microbial Growth Acidophiles: organisms that grow best at low pH (<6) Some are obligate acidophiles; membranes are destroyed at neutral pH Stability of cytoplasmic membrane is critical Alkaliphiles: organisms that grow best at high pH (>9) Some have sodium motive force rather than proton motive force

© 2015 Pearson Education, Inc Effects of pH on Microbial Growth The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic Internal pH has been found to be as low as 4.6 and as high as 9.5 in extreme acidophiles and alkaliphiles, respectively

© 2015 Pearson Education, Inc Osmolarity and Microbial Growth Typically, the cytoplasm has a higher solute concentration than the surrounding environment; thus, the tendency is for water to move into the cell (positive water balance) When a cell is in an environment with a higher external solute concentration, water will flow out unless the cell has a mechanism to prevent this

© 2015 Pearson Education, Inc. Figure Osmolarity and Microbial Growth

© 2015 Pearson Education, Inc Osmolarity and Microbial Growth Mechanisms for combating low water activity in surrounding environment involve increasing the internal solute concentration by Pumping inorganic ions from environment into cell Synthesizing or concentrating organic solutes Compatible solutes: compounds used by cell to counteract low water activity in surrounding environment

© 2015 Pearson Education, Inc Oxygen and Microbial Growth a.Aerobes: require oxygen to live b.Anaerobes: do not require oxygen and may even be killed by exposure c.Facultative organisms: can live with or without oxygen d.Microaerophiles: can use oxygen only when it is present at levels reduced from that in air Figure 5.27 a.Aerobes: require oxygen to live b.Anaerobes: do not require oxygen and may even be killed by exposure c.Facultative organisms: can live with or without oxygen d.Microaerophiles: can use oxygen only when it is present at levels reduced from that in air e.Aerotolerant anaerobes: can tolerate oxygen and grow in its presence

© 2015 Pearson Education, Inc. Figure Oxygen and Microbial Growth Gas packs Glove Box/Anaerobic Chamber Anaerobes require specialized anoxic environments

© 2015 Pearson Education, Inc Oxygen and Microbial Growth Conversion of toxic forms of oxygen Figure 5.30 Figure 5.31 Catalase assay H2O2H2O2

© 2015 Pearson Education, Inc. Sterilization The killing or removal of all viable organisms within a growth medium Inhibition Effectively limiting microbial growth Decontamination The treatment of an object to make it safe to handle Disinfection Directly targets the removal of all pathogens, not necessarily all microorganisms 5.17 General Principles and Growth Control by Heat

© 2015 Pearson Education, Inc General Principles and Growth Control by Heat Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids Does not kill all organisms, so it is different from sterilization The autoclave is a sealed device that uses steam under pressure Allows temperature of water to get above 100ºC It's not the pressure, but the high temperature, that kills the microbes

© 2015 Pearson Education, Inc. Figure General Principles and Growth Control by Heat

© 2015 Pearson Education, Inc. Figure General Principles and Growth Control by Heat

© 2015 Pearson Education, Inc Other Physical Control Methods: Radiation and Filtration Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth UV UV has sufficient energy to cause modifications and breaks in DNA Ionizing radiation Electromagnetic radiation that produces ions and other reactive molecules

© 2015 Pearson Education, Inc Other Physical Control Methods: Radiation and Filtration Filtration allows liquid or gas to pass through, but pores of filter are too small for organisms Depth filters: HEPA filters (Figure 5.36a) Membrane filters: Function more like a sieve (Figure 5.36b and c) Figure 5.36

© 2015 Pearson Education, Inc Chemical Control of Microbial Growth Antimicrobial agents are classified according to their effect on growth and recovery Figure 5.39 bacteriostaticbacteriocidal bacteriolytic

© 2015 Pearson Education, Inc Chemical Control of Microbial Growth Minimum inhibitory concentration (MIC) is the smallest amount of an agent needed to inhibit growth of a microorganism Varies with the organism used, inoculum size, temperature, pH, etc. Figure 5.40

© 2015 Pearson Education, Inc. Figure Chemical Control of Microbial Growth Disc diffusion assay uses solid media Antimicrobial agent added to filter paper disc MIC is reached at some distance Zone of inhibition Area of no growth around disc

© 2015 Pearson Education, Inc Chemical Control of Microbial Growth These antimicrobial agents can be divided into two categories Products used to control microorganisms in commercial and industrial applications Examples: chemicals in foods, air conditioning cooling towers, textile and paper products, fuel tanks Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces Sterilants, disinfectants, sanitizers, and antiseptics