Download 1. overall goals a. general knowledge of microbiology b. in

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I.
Introduction and History
A. Introduction to the course (syllabus)
1. overall goals
a. general knowledge of microbiology
b. integration/explanation of fundamental biological processes
c. knowledge applicable to everyday life
d. improved critical thinking, problem solving, scientific literacy
2. general background
a. ubiquitous, wherever life = microbes
(1) 1E8/g soil, 200 - 500 lb/acre fertile soil
(2) majority of biomass
(3) only microbe-free environment is inside healthy tissues
b. layman associates bacteria (germs) with disease
(1) all over skin surfaces (1013 body cells vs 1014 microbes)
(2) all food not sterilized or freshly cooked
(3) drinks, soil, dust, air, etc
c. majority are harmless, many beneficial, pathogens in the minority, except where
sickness is prevalent
d. life as we know it could not exist without microbes
(1) evolution
(2) photosynthesis, oxygen production
(3) elemental cycles (C, N, O, S, metals)
(4) nutrition
(5) waste degradation
3. why study microbiology
a. basic biological science
(1) accessible research tools for investigating life processes
(2) deepest understanding of living processes due to studies with microbes
b. role in the biosphere
(1) important in food chains
(2) mineral cycling
c. applied biological science
(1) impact on medicine, agriculture, industry
(2) many diseases (especially communicable)
(a) disease major cause of death at turn of century, better sanitation and health practices
have lowered risk tremendously
(b) microbial disease still a major cause of death in many developing countries
(3) soil fertility and animal production
(a) N2 fixation
(b) rumen microbiology
(c) nutrient cycling
(4) food microbiology
(a) preservation (antimicrobial)
(b) fermented foodstuffs (dairy, grain, vegetables)
(c) chemical production (fructose from corn syrup, amino acids for aspartame, citric acid
for soft drinks)
(5) biotechnology (especially pharmaceuticals, stock chemicals, genetic engineering)
(6) sanitation (food treatment, landfills, sewage treatment)
(a) wastes carried away with flowing water (pipelines) over 4000 years ago
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(b) today, 15,000,000 children die each year from poor sanitation and lack of
immunization and simple medical treatment
B. Overview of Microbiology and Cell Biology
1. Classes of microorganisms
a. bacteria
(1) prokaryotes = cells that lack a true, membrane-enclosed nucleus, having genetic material
in a nucleoid instead
(2) Archaea = archaebacteria
(3) Bacteria = eubacteria
b. algae
(1) eucaryotic = cells that have a membrane-delimited nucleus
(2) photosynthetic = convert light to energy (via chloroplasts)
(3) lack roots, stems, and leaves, but have chlorophyll, like other plants
(4) microscopic (e.g. Euglena) to macroscopic (e.g. brown kelp)
(5) phycology or algology is the study of algae
a) paralytic shellfish poisoning
b) neurotoxic shellfish poisoning
c. fungi
(1) "eucaryotic, spore-bearing organism that has absorptive nutrition and lacks chlorophyll;
that reproduces asexually, sexually, or by both methods; and that normally has
filamentous hyphae surrounded by cell walls, which usually contain chitin" (Prescott)
(2) important as decomposers
(3) molds, yeasts, mushrooms (toadstools, truffles)
d. protozoa
(1) eucaryotic, animal-like, usually motile, unicellular
(2) important in food chains; many are animal parasites; some have become useful in
molecular biology
e. viruses
(1) simple, acellular entities consisting of DNA or RNA enclosed in a protein coat
(2) obligate intracellular parasites
(3) specific viruses infect bacterial, plant, animal cells
2. Microorganisms as Cells
a. the cell is the fundamental unit of living matter
(1) membrane = barrier
(2) nucleus or nucleoid
(3) cytoplasm = contains machinery for cell growth and function
b. cell components = proteins, nucleic acids, lipids, polysaccharides
3. Cells are nonrandom entities that transform energy to maintain order
a. energy generation is an important component of metabolism (catabolism)
b. chemical reactions catalyzed by enzymes encoded by DNA
C. History of early microbiology (Chapter 1)
1. Francesco Redi demonstrated that maggots do not arise from spontaneous generation
a. meat was left uncovered or covered with gauze or paper
b. maggots developed in uncovered meat
c. maggots did not develop in meat covered with paper
d. flies laid eggs on gauze where they developed into maggots, meat was not infested
e. important toward development of scientific method (reproducible, controls)
2. microorganisms first seen and described by Antony van Leeuwenhoek (1676)
a. simple, single lens microscopes (50 - 300x magnification)
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3.
4.
5.
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b. huge curiosity, observed anything he could
(1) animalcules (intestinal organisms, including Giardia)
(2) bacteria from overnight pepper infusions
(3) seeds and plant embryos
(4) small invertebrates
(5) spermatozoa
(6) red blood cells
(7) essentially all main unicellular organisms we know today
(a) yeasts
(b) protozoa
(c) algae
(d) bacteria
c. "I have had several gentlewomen in my house, who were keen on seeing the little eels in
vinegar; but some of them were so disgusted at the spectacle, that they vowed they'd never
use vinegar again. But what if one should tell such people in the future that there are more
animals living in the scum on the teeth in a man's mouth, than there are in a whole
kingdom?"
d. superior observation skills but did not allow others to copy his techniques and verify his
results
e. compound microscopes, at that time, provided higher magnification, but quality of dual
lenses was lower than Leeuwenhoek's single lens
f. renewed argument for spontaneous generation
(1) animimalcules formed spontaneously
(2) arose from "seeds" or "germs" of animalcules always present in air
(3) difficult to prove a negative
Louis Pasteur
a. filtered air through guncotton
(1) dissolved cotton in alcohol/ether and found trapped objects resembling plant spores,
protozoan cysts, and other microbial cells
(2) placing the cotton in sterile medium resulted in growth
b. sterilized nutrient broth in open flasks with swan necks
(1) no growth occurred
(2) if necks were broken (shortened), growth occurred immediately
c. By 1861 Pasteur had almost completely resolved the controversy and demonstrated how to
keep solutions sterile
Pasteur's studies led him to discover the existence of life forms that can live only in the absence
of free oxygen
a. microscopic observation of butyric acid bacteria showed loss of motility at margin of
flattened drop
b. cells in center (away from air) remained motile
c. introduced the terms aerobic and anaerobic
d. "fermentation is life without air"
Sterilization is killing all microorganisms in or on objects
a. sterile = devoid of life
b. sterilization procedures were essential to development of microbiology as a science
c. basis of the canning preservation of many foods
Aseptic technique = handling of objects so as to prevent contamination
Pasteur termed the microbial spoilage of wine and beer "diseases"
a. considered that microorganisms could act as agents of infectious disease
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(1) already known that fungi could cause disease in wheat and rye
(2) a fungus was responsible for the great Potato Blight of Ireland
b. introduction of anesthesia (1840) allowed longer, more complicated surgeries
(1) surgical sepsis increased
(2) bacteria in air (demonstrated by Pasteur) considered as possible cause
8. Ignaz Semmelweiss improved hospital sanitary practices
a. observed mortality rates lower with midwives than doctors
(1) strep infection = child bed fever or puerperal sepsis
(2) got fired
b. friend died of P.S. from cut during autopsy of P.S. victim
c. hypothesized an invisible agent responsible for disease
d. sanitized hospitals by required handwashing and changing lab coats (fired again)
e. died in insane asylum of P.S.
9. John Lister sterilized instruments with heat and used phenol on dressings and sometimes on
wounds
(1) phenol kills bacteria
(2) less wounds became infected
(3) indirect evidence for role of bacteria in infection
10. Direct evidence for role of bacteria in disease was by Robert Koch, working with anthrax
(1) injected a series of 20 healthy mice with anthrax bacilli
(2) inoculated broth with spleen from infected mouse
(3) isolated anthrax bacilli spores
(4) injection of spores into mice resulted in anthrax
11. Koch's Postulates
a. The organism should be present in every case of the disease, but absent in healthy
individuals
b. The suspected microorganism must be isolated and grown in pure culture
c. The disease must result when the isolated microorganism is inoculated into a healthy host
d. The same microorganism must be isolated again from the diseased host
12. Vaccination developed by Pasteur
a. against cholera and rabies
b. named in honor of Edward Jenner (vacca is Latin for cow)
(1) Jenner saw that milkmaids who had cowpox were immune from smallpox
(2) started intentional inoculation with cowpox to prevent smallpox
13. The germ theory of disease (from Pasteur’s and Koch’s work)describes the demonstration that
microbes can be the agents of disease
a. general belief was that epidemics were penalties of God
b. greatest impetus for development of microbiology
c. contagious diseases spread through populations by contagions
d. after discovery of microbes, contagions = microorganisms
14. Koch developed spread plate techniques on solid nutrient surfaces
a. started with potato slices
b. added gelatin to nutrient fluid (e.g. blood serum)
(1) digested by many bacteria
(2) melted above 28o C
c. agar suggested by Fanny Angelina Eilshemius the wife of Walter Hesse, one of Koch's
assistants
(1) not attacked by most bacteria
(2) did not melt below 100o C
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II.
15. biochemistry born when Buchner discovered that sugar added to disrupted yeast (physical
grinding) was fermented
a. key to chemical analysis of energy-yielding metabolic processes
b. similarities later observed between glycolysis by muscle and alcohol fermentation by yeast
c. vitamins required by animals identical to growth factors required by some bacteria and yeast
D. Microbes and human welfare
1. microbes are critical for some of the steps in nature’s mineral cycles
2. natural abilities of microorganisms are exploited to treat solid wastes and recycle water
3. bacteria and fungi are used to clean up environmental pollutants (bioremediation)
4. experiments conducted this century with microbes helped establish the science of genetics and
molecular biology
a. mechanisms of genetic transfer
b. establishment of DNA as hereditary material
c. biotechnology (recombinant genetics)
d. gene therapy
E. Detrimental effects
1. corrosion/spoilage
2. disease
F. Microbes and human disease
1. normal flora are the microorganisms that co-exist on us
a. perform functions that help us
b. protects us from invasion by pathogenic bacteria
2. microbes (bacteria, protozoa, fungi, viruses) can cause disease
3. emerging diseases are those that have come to our notice within the last two decades
a. AIDS (HIV)
b. mad cow disease = Creutzfeldt-Jakob in humans (spongiform encephalopathy)
c. Ebola
d. hantavirus
Chemistry
A. The structure of atoms and how they form bonds
1. components of the atom
a. electrons
b. protons
c. neutrons
2. atomic number = number of protons
3. atomic mass or weight = number of protons plus number of neutrons
4. isotope = elements whose atoms have different numbers of neutrons (some may be radioactive)
5. chemical bonds are the result of interactions involving electrons
6. ions are atoms that have gained or lost an electron
a. no longer a balance between positive and negative charges
b. negative ions are attracted to positive ions and form an ionic bond
c. negative ions = anions, positive ions = cations
d. ionic bonds can dissociate in water
7. covalent bonds form when two atoms share electrons
a. orbit both nuclei
b. very strong bond
8. hydrogen bonds are an attraction between hydrogen covalently bound to one molecule with
oxygen or nitrogen bound to another molecule
a. hydrogen is weakly positive
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b. oxygen or nitrogen is weakly positive
c. overall bond is weak
B. Chemical reactions
1. reactions progress from more energy to less energy (energy given off)
2. activation energy is the energy required for a reaction to get started (Figure 2.5)
a. a catalyst lowers the activation energy and speeds up the reaction
b. enzymes are biological catalysts made of protein
c. catalysts are not permanently altered by the reaction
C. Organic Compounds
1. Four important classes of macromolecules
a. carbohydrates
b. lipids
c. nucleic acids
d. proteins
2. carbohydrates contain carbon, hydrogen, and oxygen
a. polymers (polysaccharides) made up of sugar monomers (saccharides)
b. the same monomers can make up different polymers, depending on the types of bonds
(1) cellulose is made of glucose with $-1,4 linkage
(2) starch is made of glucose with "-1,4 linkage
c. sugars are components of many key cell structures
(1) nucleic acids (ribose or deoxyribose)
(2) lipopolysaccharide
3. lipids contain carbon, hydrogen, and oxygen
a. a typical lipid has a backbone of glycerol linked to fatty acids (Figure 2.10)
(1) glycerol part is usually hydrophilic (water-loving)
(2) fatty acid region is hydrophobic (water-hating)
b. important components of membranes (Figure 2.11)
(1) form bilayers
(2) glycerol (with phosphate attached) on the outsides
(3) fatty acid chains on the inside
4. nucleic acids contain carbon, hydrogen, oxygen, nitrogen, phosphorus
a. composed of nucleotides (Figure 2.17)
(1) nitrogen base (adenine, guanine, cytosine, thymine, uracil)
(2) sugar (ribose or deoxyribose)
(3) phosphate
b. DNA occurs as a two-strand helix, held together by hydrogen bonds between the bases
(1) backbone is made of phosphate (negative charge) and deoxyribose
(2) center contains bases in complementary pair between strands
(a) adenine - thymine (2 hydrogen bonds)
(b) guanine - cytosine (3 hydrogen bonds)
(3) RNA contains ribose instead of deoxyribose
(a) occurs as a single strand
(b) uracil replaces thymine
(c) can form secondary structures (hydrogen bonds between complementary bases)
5. proteins contain carbon, hydrogen, oxygen, nitrogen, and may contain sulfur
a. made up of chain of amino acids connected by peptide bonds (Figure 2.15)
(1) chain of amino acids = polypeptide
(2) functional polypeptide = protein
b. proteins can have several levels of structure (conformation) (Figure 2.16)
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III.
c. primary structure = sequence of amino acids (peptide bond is a strong covalent bond)
d. secondary structure occurs through hydrogen bonding between amino acids
(1) two primary structures
(a) "-helix
(b) $-sheet or pleated sheet
(2) easily denatured
e. tertiary structure is additional folding of secondary structure
(1) may involve disulfide bridges (between cysteines), hydrogen bonding, other interactions
(2) more difficult to denature than secondary structure
f. quarternary structure is when the mature protein is made up of subunits
(1) not all proteins have subunits
(2) subunits held together by hydrogen bonds
g. denaturation is when bonds are broken, altering the conformation, and affecting the activity
of the protein
(1) harsh denaturation by heat is almost always irreversible
(2) gentle denaturation (urea) can often be reversed, with activity restored
6. isomer = chemical compounds with the same formula but different structures
7. stereoisomer = isomers that exist as mirror images
a. L- and D- forms
b. enzymes are so specific they can only react with a specific stereoisomer
(1) L-amino acids are used by biochemical systems
(2) D-sugars are used by biochemical systems
Nutrition and Growth
A. General nutrition
1. general requirements
a. nutrients = substances required for microbial growth
(1) biosynthesis
(2) energy production
b. analysis of cells shows that 95% of dry weight is made up of carbon, oxygen, hydrogen,
nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron
(1) termed the macroelements or macronutrients
(2) COHNSP are components of carbohydrates, lipids, proteins, and nucleic acids
(3) cations have several roles, many of them enzyme related
c. micronutrients = microelements = trace elements
(1) manganese, zinc, cobalt, molybdenum, nickel, copper
(2) small amounts required (contaminants in water, glassware, media components often
enough for growth)
(3) usually part of enzymes and cofactors
2. growth factors
a. organic compounds that are essential cell components that cannot be synthesized by the cell
b. three major classes
(1) amino acids (required for protein synthesis)
(2) purines and pyrimidines (required for nucleic acid synthesis)
(3) vitamins
(a) small organic molecules
(b) all or part of enzyme cofactors
3. media composition (enrichment, pure culture)
a. culture medium = nutrient mixture prepared for the growth of microorganisms
(1) culture = microbes that grow in or on a culture medium
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(2) many different types of media, depending on the organism of interest or the application
(a) no single medium to grow all organisms
(b) some organisms have specific requirements
(c) estimated only 10% of bacteria are culturable
(d) different media used to test anti-microbials, water qualities, vitamin concentrations,
etc.
(e) enrichment media
b. defined medium = synthetic medium = a medium in which all components are known
(1) allows accurate assessment of nutrient utilization
(2) can limit contamination
(3) "clean" medium often useful in physiology/ecology studies
c. complex medium = medium that contains some ingredients of unknown chemical
composition
(1) extracts from meat, yeasts, plants, soil, water, rumen, etc.
(2) digests of proteins
(3) very rich
(4) good for growth of a wide variety of bacteria
d. solid medium
(1) useful for isolation, enumeration
(2) agar (gelling agent made from seaweed derivative) most common agent
(a) generally non-bacterially degraded
(b) solid below 100o C
(c) suggested for use by Fanny Angelina Hesse
(3) gelatin can be used but is degraded by many bacteria and liquifies above 28o C
(4) gel-rite, a gellan gum, also in use
e. general purpose media support a wide range of organisms
f. selective media favor the growth of particular organisms, often inhibiting the growth of
others
(1) addition of basic fuschin or crystal violet inhibits G+
(2) used to enrich for bacteria with specific traits
g. differential media distinguish between different groups based on identifiable reactions
h. selective and differential media can be combined
(1) MacConkey agar contains bile salts and crystal violet that inhibit G+, and lactose identify
lactose fermentors
(2) Mannitol salts agar contains 7.5% NaCl to select for Staphylococcus and a pH indicator
to differentiate between mannitol fermentors and non-fermentors
B. General growth
1. growth = increase in cell number or cell mass (usually refers to number in case of bacteria)
2. most bacteria reproduce by binary fission
a. cell numbers increase by powers of 2
b. generation time = amount of time required for population to double (reproduce)
c. cell numbers increase exponentially, so numbers are usually plotted on a semi-log scale to
provide a straight line
3. bacterial growth curve = plot of cell growth over time, usually in a batch culture or closed
system
a. lag phase = period of little or no cell division
(1) "gearing up" phase
(2) intense metabolic activity, particularly DNA and enzyme synthesis
b. log or exponential phase = period of most rapid growth, where cells are in continual state of
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cell division
(1) generation time reaches a constant minimum based on genetic potential and medium
limitations
(2) cells most active metabolically
(3) microorganisms particularly sensitive to adverse conditions
(4) single bacterium weighing 9.5 x 10-13 g with doubling time of 20 minutes could,
theoretically, result in 80,000 tons of cell material
c. stationary phase = period of equilibrium between growth and death of cells
(1) depletion of nutrients and accumulation of waste products
(2) changes in pH
d. death phase = period when more cells are dying than dividing
(1) logarithmic decline phase (constant proportion dies with time)
(2) population can diminish or become extinct
4. batch vs. continuous culture
a. batch = closed system (no addition or removal of materials)
b. continuous culture = open system (continual introduction of nutrients and removal of wastes)
(1) bacteria maintained in exponential phase
(2) growth rate dependent on dilution rate (rate of introduction of fresh medium)
(3) cell density dependent on concentration of limiting nutrient
(4) if dilution rate is greater than the growth rate, cells are washed out
5. influence of environmental factors (role of physical parameters on bacterial growth)
a. water activity
(1) describes the availability of water to the organism
(2) aw = ratio of vapor pressure of air over a substance or solution divided by vapor pressure
of water at the same temperature
(3) high water activity = dilute environment
(4) low water activity inhibits bacterial growth
(a) high solute concentration
(b) method of preservation
(5) halotolerant = able to survive with salt, but less than optimal growth
(6) halophiles = require salt for growth
(7) osmophiles = live in environments with low water activity (could be due to non-salt
solutes)
(8) xerophiles = live in dry environments
b. pH
(1) each organism has a pH range where growth is possible, along with an optimum pH
(2) most life occurs around neutrality, but ranges from 2 - 10
(3) acidophiles live at low pH
(4) alkalinophiles live at high pH
(5) neutraphiles live around pH 7
c. temperature
(1) every organism has a minimum temperature, a maximum temperature, and an optimum
temperature
(2) psychrophile opt. # 15o C
(a) oceans (avg temp 5o C)
(b) organisms growing under ice in polar regions (snow alga)
(c) membranes rich in unsaturated fatty acids
(3) mesophile opt about 35o C
(a) best studied group
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(b) corresponds to pathogens
(4) thermophile opt about 60o C
(a) hot springs
(b) water heaters
(c) compost heaps and digestors
(d) membranes rich in saturated fatty acids
(e) changes in enzyme structures
(5) extreme thermophile opt$ 85o C
(a) thermal vents
(b) steam vents
d. oxygen concentration
(1) bacteria vary in their need for and tolerance
(2) obligate anaerobes are killed by oxygen
(3) aerotolerant anaerobes can grow in the presence of oxygen but cannot use it as an
electron acceptor
(4) facultative anaerobes can use oxygen, but can also grow without it
(5) microaerophiles require oxygen, but at lower levels than in air (2 - 10%)
(6) aerobes require oxygen for growth
(7) toxic forms of oxygen
(a) superoxide anion, O2i) occurs during reduction of O2 to H2O
ii) highly reactive
iii) can cause oxidative destruction of lipids and other biochemical components
(b) peroxide, O22i) formed during respiratory processes
ii) often used as a disinfectant
(c) hydroxyl free radical, OH.
i) formed by ionizing radiation
ii) formed by reaction of superoxide anion with peroxide
(8) several enzymes to protect against oxygen toxicity
(a) catalase
i) H2O2 + H2O2 6 2H2O + O2
ii) present in aerobes and facultative anaerobes
(b) peroxidase
i) H2O2 + NADH + H+ 6 2H2O + NAD+
ii) no oxygen produced
(c) superoxide dismutase
IV.
i) O2- + O2- 6 H2O2 + O2
ii) present in aerobes, facultative anaerobes, and aerotolerant anaerobes
e. pressure
(1) most cells are barotolerant
(2) barophilic organisms grow more rapidly at high pressures
(a) isolated from guts of deep-sea invertebrates
(b) hard to study (cultivate)
f. radiation
Metabolism
A. Energetics
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1. overview
a. energy = ability to do work
(1) ultimate source = sun (exception = chemoautotrophs)
(2) photosynthetic organisms convert solar energy into chemical energy
b. chemical energy resides in bonds between atoms, specifically in the electrons
(1) potential energy = bonds remain unchanged
(2) kinetic energy = bond energy freed for cellular work
(a) redox reactions = rearrangement of electron distribution
(b) can be used for synthesis, growth, movement
c. exergonic = reaction releases energy
d. endergonic = reaction requires input of energy
2. coupled reactions and the role of ATP
a. ATP functions as an energy "storehouse"
(1) energy released in exergonic reactions drives phosphorylation of ADP to ATP
(2) ATP can release the energy to drive endergonic reactions
(3) "metabolic money" - can be earned , banked, saved, spent, exchanged
(4) not really "high-energy" bonds, more like "unstable"
(a) not a large amount of energy released
(b) energy released quickly and easily (kerosene)
b. energy extraction in biological systems involves transfer of electrons, termed redox
reactions
(1) oxidation = loss of electrons
(2) reduction = gain of electrons
(3) redox reactions always occur in pairs, with an electron donor and an electron acceptor
(a) conjugate or redox pair
(b) may involve a temporary electron carrier
(4) energy released from redox reactions can be coupled to phosphorylation (addition of an
inorganic phosphate, Pi, to ADP or some other carrier)
(a) usually refer to electron transfer, but generally occurs as part of atom, like hydrogen
(b) hydrogen ions (in addition to electrons) have a role in energetics
(c) dehydrogenation = removal of hydrogen during a redox reactions
c. electron carriers = coenzymes that shuttle electrons between molecules
(1) often transfer both electrons and hydrogens, but can be electrons only
(2) NAD is most common carrier
(a) 2 electrons, 2 protons
(b) actual reduced state = NADH + H+, but often written as NADH or NADH2
(3) electrons are transferred through a series of redox reactions (carriers) = electron transport
chain or respiratory chain
(4) other carriers = FAD, NADP, CoA, cytochromes
d. substrate level phosphorylation = energy released directly from substrate to phosphorylate
ADP
(1) no external electron acceptor
(2) redox reactions occur between portions of the substrate molecule
e. oxidative phosphorylation - ATP formed through a series of redox reactions through a
respiratory pathway
f. photophosphorylation = ATP formed through sunlight-driven reactions
B. thermodynamics
1. )E = EB - EA = Q - W
a. EA = energy of a system at the start
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b. EB = energy at end
c. Q = heat absorbed by the system
d. W = work done by the system
2. 1st law (energy neither created or destroyed) cannot predict if reaction is spontaneous
a. the change in energy of a system depends only on the initial and final state and not on
the path of the transformation
b. explained by 2nd law: process occurs spontaneously only if the sum of the entropies of the
system and its surroundings increases:
3. So, the change in free energy of a reaction depends on changes in internal energy and entropy of
the system
4. a reaction can occur spontaneously only if the )G is negative
a. the )G of products must be less than the )G of reactants
b. )G of a reaction is independent of the path or mechanism of the transformation
c. )G is unrelated to the reaction rate
5. for the reaction A + Bö C + D
a. )G = )Go + RT ln ([C][D]/[A][B])
b. )Go = standard free energy change, at pH 7, use )Go'
c. )Go' = -RT ln ([C][D]/[A][B]) = -RT K'eq
6. the overall free energy change for a chemically coupled series of reactions is equal to the
sum of the free-energy changes of the individual steps
7. a thermodynamically unfavorable reaction can be driven by a thermodynamically
favorable reaction that is coupled to it
C. energy required for work, active transport, synthesis
1. energy from external source
a. chemotrophs use chemical energy
b. phototrophs use light energy
2. ATP is the most common energy carrier in biological systems
3. several other biological compounds have high phosphate group transfer potential (Tab. 13-1)
these are high energy bonds because free energy is released when they are hydrolyzed
D. Enzymes
1. protein catalysts with high specificity for the reaction catalyzed and molecules acted upon
a. catalyst = increases rate of reaction without being permanently altered itself
b. acts on reactants or substrates to form products
2. general mechanisms
a. enzymes effect the rate, but not the energy yield or requirement
b. in a reaction, reactants come together an form a transition-state complex that resembles
both reactants and products
(1) activation energy = energy required to bring reacting molecules together in the correct
way to reach transition state
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(2) transition-state complex decomposes to yield products
(3) enzymes accelerate reactions by lowering the activation energy
c. active site or catalytic site = specific binding site for substrates on enzyme
(1) enzyme may bring substrates together at active site
(a) in effect, concentrates substrates
(b) aligns substrates in correct orientation to form transition-state complex
(2) effectively accelerate reaction 2-3 logs over uncatalyzed reaction rates
(a) allows reactions to occur at lower temperatures
(b) essential to life processes
E. General metabolism
1. catabolism = molecules broken down into simpler molecules with the release of energy
a. some energy trapped and made available for work
b. produces compounds for biosynthesis
c. generates reducing equivalents for energy or biosynthesis
2. anabolism = synthesis of complex molecules from simpler ones with an input of energy
3. amphibolic pathways function catabolically and anabolically
a. glycolysis and TCA cycle
b. most reactions are reversible or other enzymes exist to bypass irreversible catabolic steps
c. two separate enzymes are important for independent regulation
F. Catabolism (energy yielding reactions)
1. Three products
a. energy (ATP)
b. reducing equivalents (NADH, FADH2)
c. precursor molecules (pyruvate, acetyl-CoA, etc.)
2. can be broken down to 3 stages
a. stage 1 = large nutrient molecules are hydrolyzed to simpler molecules
b. stage 2 = products of 1st stage degraded to simpler compounds that are key intermediates in
amphibolic pathways
(1) acetyl CoA, pyruvate, TCA intermediates
(2) ATP, NADH, FADH2
c. stage 3 = molecules completely oxidized to CO2 in TCA cycle
(1) high yield of energy (ATP, NADH, FADH2)
(2) can use oxygen or other electron acceptor
3. substrate level phosphorylation = phosphorylation of ADP coupled to exergonic breakdown of
substrate molecule
4. glycolysis (Emden-Meyerhoff pathway)
a. most common pathway for glucose degradation to pyruvate
(1) stage 2 of catabolism
(2) functions in presence or absence of O2
(3) occur in all major groups of microorganisms
b. can be divided into 2 parts, 6-carbon and 3-carbon
(1) 6-C, glucose phosphorylated twice to fructose-1,6-bisphosphate
(2) no energy yield; 2 ATP consumed
(3) 3-C stage starts with cleavage of F-1,6-P
(4) 2 ATP/3-C compound (= 4/glucose)
(5) also generates 2 NADH which must be oxidized to NAD
c. pentose phosphate or hexose monophosphate pathway = another pathway for sugar
breakdown to pyruvate
(1) can operate at same time as EMP pathway
1113- p.14
(2) produces high levels of NADPH (for synthetic reactions)
d. Entner-Doudoroff pathway = another hexose to pyruvate pathway, substitutes for EMP
pathway
(1) generally in Pseudomonas, Rhizobium, Azotobacter, Agrobacterium, and a few other
gram negative bugs
(2) rare in gram positives; Streptococcus faecalis
5. fermentation = all metabolic processes that release energy from a sugar or other organic
molecule, do not require oxygen or an electron transport system, and use an organic molecule as
the final electron acceptor
a. uses organic compounds (part of substrate) as terminal electron acceptors
(1) substrate level phosphorylation
(2) reducing equivalents recovered in organic endproducts
b. yields small amount of ATP
c. other definitions in use
(1) any process that produces alcoholic beverages or acidic dairy products (general use)
(2) any spoilage of food by microorganisms (general use)
(3) any large-scale microbial process occurring with or without air (industrial definition)
(4) any energy-releasing metabolic process that takes place only under anaerobic conditions
(oversimplification)
d. typical products of pyruvate fermentation
(1) alcohol (beer, wine, whiskey)
(2) solvents (acetone, butanol)
(3) organic acids (lactic, acetic)
(4) dairy products, soy sauce
6. tricarboxylic acid cycle (TCA cycle, citric acid cycle, Kreb’s cycle)
a. stage 3 of catabolism
b. pyruvate degraded to CO2
c. overall, the TCA cycle generates 2 CO2, 3 NADH, 1 FADH2, and 1 GTP (equivalent to 1
ATP) for each acetyl CoA oxidized
d. TCA cycle enzymes widely distributed among microorganisms
(1) even microorganisms that lack complete cycle
(2) one major function is to provide compounds for biosynthesis
7. respiration = ATP-generating process in which molecules are oxidized and the final electron
acceptor is (almost always) an inorganic molecule
a. electron transport chain is an essential part of aerobic respiration
(1) O2 is final electron acceptor aerobically
(2) anaerobically, final electron acceptor is an inorganic molecule other than O2 or, rarely, an
organic molecule
(a) sulfate, nitrate, carbonate, ferric iron
(b) fumarate
b. electron transport chain = sequence of membrane associated electron carrier molecules that
are capable of oxidation and reduction
(1) ETS have two basic functions
(a) accept electrons from a donor and pass them to an acceptor
(b) conserve some of the energy released during electron transport for ATP synthesis
(2) electrons passed through the chain, in direction of higher reducing potential, with
stepwise release of energy
(3) energy released drives the chemiosmotic generation of ATP
(4) located in plasma membrane of prokaryotes, mitochondrial membrane of eucaryotes
1113- p.15
8. anaerobic respiration = respiration in which electron acceptors other than O2 are used
a. typically NO3-, SO42-, CO32-, as well as some organics (e.g., fumarate)
b. less energy generated than with O2, due to lower potentials
9. chemolithotrophy = energy generation from inorganic molecules
a. lithothroph = rock eating
b. typically H 2, H2S, NH3
c. usually involves aerobic respiratory processes
d. possess ETS components and generate a PMF
e. most are autotrophs (use CO2 as carbon source)(vs. heterotrophs, which use organic
compounds as their source of C)
10. phototrophy
a. photosynthesis = conversion of light energy into chemical energy
(1) carbon fixation = synthesis of sugars from CO2
(2) electrons come from hydrogens in water
b. light reactions = light energy converts ADP to ATP (photophosphorylation) and NADP is
reduced to NADPH
c. dark reactions = electrons and energy used to reduce CO2 to sugar
d. light energy is absorbed by chlorophyll molecules
(1) chlorophyll a used by green plants, algae, cyanotobacteria
(a) membranous thylakoids in chloroplasts of plants, algae
(b) thylakoids in cyanobacteria
(2) bacteriochlorophylls used by other bacteria
(3) bacteriorhodopsin = pigment in Halobacterium
e. excited electrons jump from chlorophyll through an electron transport chain
(1) similar to respiration
(2) ADP converted to ATP by chemiosmosis
f. green and purple bacteria are anoxygenic
(1) use H2, H2S, So, reduced organics as electron source
(2) can also grow chemotrophically in dark
11. catabolism of carbohydrates, lipids, and proteins
a. carbohydrates are hydrolyzed and/or converted to glucose and fed into a glycolytic pathway
b. lipids
(1) glycerol moieties enter glycolytic pathway
(2) fatty acids $-oxidized to acetyl CoA, which is fed into TCA cycle or used for biosynthesis
c. proteins are hydrolyzed external to plasma membrane; amino acids enter cell by specific
transport mechanisms
(1) amino acids are deaminated
(2) resulting organic acid converted to pyruvate, acetyl CoA, or a TCA intermediate
G. Anabolism (synthetic reactions)
1. general principles
a. cells use free energy (ATP) to synthesize more complex molecules from smaller, simpler
precursors
b. macromolecules = polymers of smaller units; protein, nucleic acids, lipids, polysaccharides
c. synthesis from simple precursors (monomers) that are common intermediates requires less
genetic information, raw material, and energy
d. many enzymes used for anabolism and catabolism
(1) amphibolic pathways often contain different enzymes at key steps for anabolism and
catabolism
(2) allows independent regulation
1113- p.16
(a) often by endproducts
(b) concentrations of ATP, ADP, AMP, NAD+
e. different cofactors
(1) NADH catabolic (electron transport)
(2) NADPH anabolic (electron donor)
(3) fatty acid catabolism uses acyl CoA
(4) fatty acid anabolism uses acyl carrier protein thioesters
f. many macromolecules can form more complex structures by self assembly
(1) flagella
(2) ribosomes
2. precursor molecules do not contain nitrogen or sulfur
a. sulfur can be obtained from inorganic or organic sources
(1) inorganic sulfur must be reduced to sulfide
(2) cysteine (synthesized from inorganic sulfur) or methionine
b. nitrogen can be used as N2 (a few bacteria), ammonia, or nitrate
(1) nitrate gets reduced to ammonia
(2) ammonia gets incorporated into amino acids
(3) N2 gets "fixed" into ammonia
3. amino acids are synthesized from intermediates from glycolysis, TCA cycle, and pentose
phosphate pathway
a. 5 major pathways for aa synthesis
b. key intermediates:
(1) glucose (to erythrose-4-P): phe, tyr, trp
(2) 3-phosphoglycerate: serine ö cysteine, glycine, histidine (purines)
(3) oxaloacetate: aspartate ö asparagine, threonine, isoleucine, methionine, lysine
(pyrimidines)
(4) α-KG: glutamate, glutamine, proline, arginine
(5) pyruvate, acetylCoA: alanine, lysine, isoleucine, valine, leucine.
4. phosphorus assimilation differs from sulfur and nitrogen
a. phosphorus is neither oxidized or reduced during assimilation
b. inorganic phosphate can be assimilated directly by phosphorylation in catabolic pathways
c. ATP is the major phosphate donor in cells