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Microbiology Y2 > Hitchcock L4-9 > Flashcards

Hitchcock L4-9 Flashcards

1
Q

What is the process of glycolysis simply?

A

Breaks glucose (6C) into two glyceraldehyde 3-phosphate (3C) requiring 2ATP and then two pyruvates (3C) producing 4ATP. The phase from glucose to G3P is called investment phase which goes into pay out phase to form pyruvate.
Does not need oxygen and occurs in the cytoplasm.

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2
Q

What is the yield from glycolysis?

A

2ATP by substrate level phosphorylation and 2NADH.

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3
Q

What is the process of the link reaction simply?

A

Pyruvate (3C) is oxidised to acetyl CoA (2C) using pyruvate dehydrogenase in mitochondrial matrix. Reaction is catalysed by three enzyme pyruvate dehydrogenase complex which releases CO2.

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4
Q

What is the yield of the link reaction?

A

One NADH per pyruvate so 2NADH per glucose.

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5
Q

What is the yield of the citric acid cycle?

A

Per acetyl CoA it is 1ATP, 3NADH and FADH2. So yield is doubled due to two acetyl CoA from one glucose.

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6
Q

What is the process of oxidative phosphorylation?

A

Electrons enter the chain from NADH at complex 1 or succinate (complex 2) and reduce quinones. Quinols are oxidised by complex 3 reducing soluble cytochrome C in inner membrane space. Reduced cytochrome C acts as electron donor to complex 4 where oxygen is reduced to water. Proton motive force is used by ATP (complex 5) to generate ATP.

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7
Q

What is the ratio of H+ / ATP in oxidative phosphorylation and what are the consequences?

A

10 H+ are translocated across the membrane per NADH oxidised. 6 H+ are translocated across the membrane per FADH2 oxidised. Costs 2.7 H+ / ATP. Therefore can produce 3.7 ATP per NADH and 2.2 ATP per FADH2.* However, the cost of actively importing NADH, pyruvate, ADP and Pi increases the H+/ATP ratio to >4.
Taking this into account the ATP yield per mol of glucose is ~30 (including glycolysis and Krebs cycle)

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8
Q

What modes of metabolism does E.coli have?

A

All three - aerobic, anaerobic and fermentation, it is a facultative anaerobe.

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9
Q

How does E.coli perform glycolysis and Krebs?

A

Performs glycolysis and Krebs the same way as in mitochondria but with processes occurring in cytoplasm.

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10
Q

What are the alternative complexes in E.coli?

A

Nuo and Ndh are alternative NADH complexes. SDH is the succinate dehydrogenase. There are no alternative complex 3 or cytochrome C. Two alternative terminal quinol oxidases called Cyo and Cyd.

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11
Q

What is the process of aerobic respiration in E.coli?

A

Electrons are donated from NADH or succinate (FADH2) via dehydrogenase enzymes and reduce quinones to quinols.
E. coli lacks complex III and a cytochrome c oxidase (complex IV) - instead it has two different respiratory terminal oxidases, Cyo and Cyd, which directly oxidise the quinols produced by the NADH dehydrogenase and succinate dehydrogenase.

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12
Q

What is Cyo?

A

Alternative terminal quinol oxidase. Cytochrome bo3 heme-copper oxidase consisting of heme b, heme o3, Cu centre and four subunits of CyoABCD. It releases two H+ from quinol oxidation to the p-side. Also pumps 2H+ from n-side to p-side having a higher H+/e- ratio than Cyd. It does have a lower affinity for oxygen working under oxidative conditions.

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13
Q

What is Cyd?

A

Alternative terminal quinol oxidase. Cytochrome bd oxidase consisting of 3 hemes (two heme b and one heme d) and four subunits of CydABHX. It releases two H+ from quinol oxidation to the p-side. Does not pump protons so lower H+/e- ratio than Cyo but has a higher affinity for oxygen so works under toxic conditions. More resistant to sulphide, hydrogen peroxide and nitric oxide.

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14
Q

What is the H+/e- while using Nuo and Cyo?

A

8 H+ are translocated per NADH oxidised with a H+/e- ratio of 4. Costs 3.33 H+/ATP synthesised so 10 protons per 3 ATP in E.coli. Can produce 2.4 ATP per NADH oxidised via Nuo and Cyo.

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15
Q

What is the H+/e- while using Ndh and Cyo?

A

4 H+ are translocated per NADH with H+/e- ratio of 2. Costs 3.33 H+/ATP synthesis so 10 protons per ATP in E.coli. Can produce 1.2 ATP per NADH oxidised via Ndh and Cyo.

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16
Q

Why does E.coli use Ndh instead of Nuo despite doubling the H+/e- ratio so double the ATP?

A

Differing H+/e- ratios allow bacteria to optimize the efficiency of energy generation under varying conditions (e.g., donor and acceptor concentrations). Ndh has a higher turnover number – favours increased metabolic flux/growth rate over maximal energy efficiency of the respiratory chain. Nuo is utilised under micro aerobic (low oxygen) conditions with the Cyd oxidase or with alternative electron acceptors in the absence of oxygen.

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17
Q

What is anaerobic respiration?

A

Use of a membrane embedded electron transport chain to generate a PMF but with a terminal acceptor other than oxygen.

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18
Q

What are the alternative terminal acceptors in anaerobic respiration?

A

Nitrate, nitrite, fumarate, trimethylamine N-oxide and dimethyl sulfoxide.

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19
Q

What type of quinone does E.coli use?

A

Ubiquinone under aerobic conditions and menaquinone under anaerobic conditions as menaquinone is slightly better electron donor and slightly worse electron acceptor.

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20
Q

What does E.coli use the Krebs cycle for?

A

Uses it to make substrates for biosynthesis. It is amphibolic meaning it does both catabolism and anabolism. It forms oxidative and reductive branches meaning it is no longer cyclic. Oxidative branch forms alpha ketoglutarate and reductive branch forms succinate.

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21
Q

What happens to the substrates under anaerobic conditions in E.coli?

A

The pyruvate dehydrogenase complex is inhibited and pyruvate is converted to formate and acetyl CoA by pyruvate formate lyase. Acetyl CoA is converted to acetate by phosphor transacetylase and acetate kinase generating ATP. Formate can act as the electron donor to anaerobic ETC via formate dehydrogenase enzyme.

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22
Q

How does fumarate act as a terminal electron acceptor?

A

Fumarate reductase is a multi subunit enzyme containing a flavin cofactor and three FeS clusters. No redox loop is present so protons liberated upon quinol oxidation are released to the cytoplasm. Nuo coupled to Frd = 4H+ / 2e- = 2H+ / e- with 1.2 ATP per NADH oxidised.

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23
Q

What is the best alternative terminal electron acceptor in E.coli?

A

Nitrate as it has the highest midpoint potential. Nitrate reductase and Formate dehydrogenase are structurally very similar except Fdn is periplasmic facing whereas Nar is cytoplasmic facing.

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24
Q

What is fermentation?

A

Fermentations use endogenous organic molecules as electron acceptors in the absence of oxygen and a respiratory electron transport chain.

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25
Q

How is ATP produced?

A

ATP production is limited to substrate level phosphorylation.

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26
Q

What acts as the terminal electron acceptor in fermentation?

A

Pyruvate or a derivative.

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27
Q

What is the importance of NADH in fermentation?

A

NADH generated must be re-oxidised to regenerate NAD+ restoring redox balance and keep glycolysis/ATP production going. Re-oxidation of NADH also forms fermentation products which are more reduced than organic starting substrate.

28
Q

What happens to the organic starting substrate in fermentation?

A

Most of it is routed to fermentation products excreted as waste so little biomass is produced.

29
Q

What happens to pyruvate in fermentation?

A

It remains in the cytoplasm and is reduced to lactate using NADH as the electron donor regenerating NAD+ and restores redox balance allowing glycolysis to continue to make ATP by substrate level phosphorylation.

30
Q

What carries out homolactic fermentation?

A

Lactic acid bacteria.

31
Q

What occurs in heterolactic fermentation?

A

Catabolises sugars by the pentose phosphoketolase pathway. Generates pentose (5C) sugar that is cleaved into G3P (3C) and acetyl P (2C). G3P is metabolised to pyruvate and then lactate. Acetyl P is converted to ethanol via acetyl CoA and acetyl aldehyde intermediates.

32
Q

What occurs in alcohol fermentation?

A

Glucose is converted to ethanol and CO2. Pyruvate is decarboxylated to acetaldehyde which is reduced to ethanol by alcohol dehydrogenase using NADH as electron donor, also regenerating NAD+. Carried out by yeast and some bacteria.

33
Q

What occurs in mixed acid fermentation?

A

Carried out by gram negative enterobacteriacae such as E.coli. Pyruvate is converted to variable mixture of end products including ethanol, formate, acetate, H2 + CO2 and lactate + succinate.
Proportions of end products vary depending on growth conditions to balance ATP production and redox balance.

34
Q

What occurs in acetone butanol ethanol fermentation?

A

Carried out by gram positive clostridium species. Produces solvents in ratio 3:6:1 acetone: butanol: ethanol. Interest to produce butanol as renewable biofuel. Generates extra ATP and re-assimilate acetic and butyric acid products generating acetone and re-generating acetyl CoA and butyryl CoA which can convert to ethanol and butanol to reform NAD+.

35
Q

What occurs in malolactic fermentation?

A

Secondary fermentation in wine production performed by lactic acid bacteria. Takes up malic acid and decarboxylates it to form lactic acid which is excreted. Antiport of malate and lactic acid by MleP generates a membrane potential and decarboxylation of malate by MleA consumes a proton in cytoplasm contributing to pH gradient. ATP synthesis is chemiosmotic and no redox balancing required.

36
Q

What is growth?

A

Co-ordinated synthesis of macromolecules. Macromolecular synthesis leads to cell division and an increase in cell numbers. Growth rate is the change in cell number/ cell mass per unit of time.

37
Q

How do you measure microbial growth?

A
  1. Cell dry weight - doesn’t give indication of if cells are alive or dead.
  2. Cell number - either total count or viable count.
  3. Optical density - Usually about 600nm and requires standard curve to link to direct measure of cell number.
  4. Specific cell component such as protein, chlorophyll etc.
38
Q

What is batch culture growth?

A

Culture of a fixed volume in a vessel - closed system. Cells divide by binary fission leading to exponential growth. Time between division is generation time or doubling time.

39
Q

Why is unrestrained growth not possible in batch culture growth?

A
  1. Essential nutrient is depleted as no new media is added.
  2. Metabolism leads to accumulation of end products causing auto inhibition.
40
Q

How do you calculate the number of cells that an exponential culture has at the end of growth?

A

N = N0 X 2n
Where N = Final cell number
N0 = Initial cell number
n = Number of cell generations during exponential growth

41
Q

How do you calculate the number of generations in exponential culture?

A

n = log2 (N/N0) and g = t/n
Where g = generation time
t = length of exponential growth
n = number of generations

42
Q

What is the specific growth rate constant?

A

The rate at which the population is growing at any instant.

43
Q

What are the limitations of batch culture growth?

A
  • Growth curve is a laboratory artefact
  • Properties of cells vary continuously throughout the growth curve
  • Cells grow at maximum rate so physiology cannot be studied at submaximal growth rates under nutrient limited conditions.
44
Q

What is continuous culture growth?

A

Fresh medium is continuously (chemostats) or periodically (turbidostats) added to culture and an equal volume of spent culture is siphoned away.

45
Q

What are the consequences of continuous culture growth?

A
  1. Substrates/ nutrients are added for growth
  2. Auto inhibited products are diluted
  3. Bacterial populations can be maintained in exponential phase at constant cell density
  4. Growth rate and cell density can be controlled independently.
  5. Allows reproducible cultivation of microorganisms at submaximal growth rates at different growth limitations so culture conditions remain constant
46
Q

What are key features of chemostat culture?

A

Fresh medium is supplied at a constant rate and spent culture is removed at the same constant rate so volume remains constant. Influent medium contains a limiting concentration of one essential nutrient (others in excess). Growth rate of culture adjusts to supply of limiting substrate until dynamic equilibrium is reached.
The amount of cells removed from the vessel by dilution is equal to the increase in cell numbers due to growth supported by the input of the limiting nutrient.

47
Q

What happens when you vary the dilution rate in continuous culture growth?

A

By varying the dilution rate (i.e. the rate at which the limiting nutrient is added to the culture) the growth rate can be varied whilst keeping cell density the same.
Growth rate > dilution rate = Cell no. increases
Growth rate < dilution rate = Cell no. decreases
Growth rate = dilution rate = Cell no. constant.

48
Q

What occurs at low dilution rate in chemostats?

A

Bacterial concentrations is decreased due to requirement of maintenance energy. This is the use of growth limiting substrate for essential cellular functions other than growth. The percentage of total consumed substrate used for cell maintenance compared to that used for growth is more significant, so assumes maintenance of energy independent of growth rate.

49
Q

What occurs at high dilution rate in chemostats?

A

When increased above maximum specific growth rate cells will quickly washout of the chemostat. Cells cannot grow any faster even with the limiting nutrient no longer being limiting and bacterial concentration cannot be maintained. Bacterial concentration decreases and limiting nutrient increases to concentration in the input media.

50
Q

What is a turbidostat culture?

A

Continuous culture with a growth dependent feedback system, in which the dilution rate is controlled by monitoring cell density (TURBIDITY).

51
Q

How is turbidity detected?

A

Using spectrophotometer. Any increase above desired value and pumping rate is adjusted to add fresh medium into vessel. Simple overflow keeps culture volume constant. No growth-limiting substrate and so cells grow at their maximum rate at a constant population density under constant conditions.

52
Q

What is industrial microbiology?

A

Large scale production of commercial products by microorganisms. Typically refers to using microorganisms to produce a compound they already make – engineer organisms and optimise growth conditions to enhance the process.

53
Q

What are examples of microbial products?

A
  1. Antibiotics
  2. Enzymes
  3. Food additives
  4. Chemicals
  5. Terpenes
  6. Alcoholic drinks
54
Q

What are useful properties of microbial products?

A
  • Produce substance of interest
  • Grow rapidly to produce large scale product in short period of time
  • Grow in simple and inexpensive media
  • Metabolic flexibility and adaptability
  • Do not produce toxins or toxic by products
  • Amenable to genetic engineering and are genetically stable
  • Can be stocked or stored
  • Secreted the product into media
55
Q

What are the three methods for using fermentations in industry?

A

Batch. Fed-batch. Continuous.
In batch fermentations, all of the nutrients required for the fermentation are provided in the initial culture medium. Once these nutrients have been consumed, growth of the organism ceases and the fermentation is ended.
Continuous fermentations are performed by continually supplying fresh medium to the culture with the subsequent removal of the same amount of culture, resulting in a steady state being reached in the fermenter. In fed-batch fermentations, nutrients are provided in the batch culture medium. Once consumed, a feed is initiated to provide the culture with additional nutrients and thus allow for further growth of the culture

56
Q

How does fed batch fermentation occur in industry?

A
  1. Initial growth phase in a small fermenter inoculated with freeze dried spores.
  2. Scaled up through two further growth stages in successive larger fermenters to provide a large enough inoculum for production phase.
  3. Fermentation production phase is a fed batch culture with high oxygen levels maintained and C+N feeding.
  4. Carefully monitored to keep fermentation in optimal penicillin production mode during production phase which lasts for 120-200 hours.
  5. Penicillin is excreted into medium and is recovered at the end of fermentation.
57
Q

How do you improve product yield in microbials?

A
  • Mutation and selection
  • Metabolic engineering / synthetic biology
  • Nutritional/physiological approaches
  • Optimising fermentation conditions
  • More than one / all of these
58
Q

What is bioprospecting?

A

The search for organisms, enzymes or natural products with potential commercial applications.

59
Q

What is metagenomics?

A

The study of genes/genetic material from environmental samples.

60
Q

What is gene mining?

A

The process of identifying and isolating genes from environmental samples without having to culture to the organism.

61
Q

What is metabolic engineering?

A

The deliberate redesign of cellular biochemical pathways to enhance production of a product or to produce a novel product.

62
Q

What does metabolic engineering involve?

A
  1. Modifying metabolic pathway to redirect existing metabolism to specific products occurring at branchpoints.
  2. Enhancing precursor and energy/cofactor supply to the pathway by engineering central metabolism.
  3. Engineering transport systems such as channels through membranes
  4. Increasing cellular tolerance to product or substrate
  5. Consideration of regulatory effects such as product feedback inhibition
  6. Decoupling of growth and product formation.
63
Q

What were the four ways in which lysine yield was increased in C.glutamicum?

A
  1. Elimination of allosteric feedback inhibition using anti-metabolites = Concerted allosteric feedback inhibition on LysC (aspartate kinase) by Lys and Thr. The anti-metabolite (substrate analogues) aminoethyl-L-cysteine allosterically inhibits LysC – used to select feedback-resistant LysC mutants.
  2. Promoter engineering to improve metabolic flux at Asp semi aldehyde branch = DapA (dihydrodipicolinate synthase) is the first specific lysine biosynthesis enzyme at the branch point at aspartate semi-aldehyde. Point mutations in dapA promoter result in increased expression – DapA activity increased.
  3. Increasing cofactor supply by over production of transhydrogenase = Transhydrogenase (PntAB) reaction:
    NADH + NADP+ <-> NADPH + NAD+
    Cellular NADH pool greater than cellular NADPH pool - overexpression of pntAB increases NADPH levels.
  4. Increasing lysine secretion by over producing the lysine exporter LysC = Lysine is toxic to the cell at high concentration - C. glutamicum has a lysine exporter (LysE). Lysine is positively charged at neutral pH (pKa of ε-amino group = 10.28) - active export requires either symport with two hydroxyl ions or antiport with two protons. Overproduction of LysE can increase lysine excretion but this can be harmful to the cell membrane – have to use controlled over-expression of lysE gene to achieve the right balance.
64
Q

What is the basic structure of Vitamin B12?

A

It is 1 of 8 B vitamins with a corrin ring containing a central cobalt ion held by upper + lower ligands.

65
Q

How do humans get vitamin B12?

A

Through the diet from animal products. Deficiency is common with vegies and vegans.

66
Q

How was vitamin B12 expressed in E.coli?

A

Optimised expression of genes. Enhanced uptake and chelation of cobalt. Increased metabolic flux to the uroporphyrinogen III starting substrate. Downregulated the competing heme and siroheme biosynthesis pathways. Optimised the fermentation process.

Microbiology Y2 (7 decks)

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