Final

Question 1

2. Describe in detail how ATPase works for ATP production and why cells may use their ATPase in reverse.

ATPase and ATP production

Answer 1

ATPase (complex V) is a large membrane enzyme complex that catalyzes conversion of PMF into ATP and has two parts:

1. Multi-subunit headpiece of alpha and gamma called F1 on cytoplasmic side of membrane.
2. Proton-conducting channel called F0 that spans membrane.

• F1/F0 complex catalyzes a reversible reactions between ATP and ADP + Pi
• Proton movement through subunit a of F0 drives rotation of c protein, generating a torque that is transmitted to F1 by gammaE subunit
• Energy transferred to F1 through coupled rotation of gammaE subunits
• Cause conformational change in beta subunits, a form of potential energy tapped to make ATP
• Possible because conformational change to beta subunits allows for sequential binding of ADP + Pi to each subunit
• Conversion to ATP occurs when beta subunits return to original conformation
• The primary function of b2 subunits of F1 is to prevent alpha and beta subunits from rotation with gamma-eipsilon so conformational changes in beta can occur. 3-4 protons consumed by ATPase per ATP produced.

Question 2

Why ATPase in reverse?

Answer 2

• The F1/F0 motor is reversible
• ATP hydrolysis can be used to create a H+ gradient by the reversal of the ion flux
• F1 rotary motors works in the forward motion to hydrolyse ATP and to drive the F0 motor n reverse to create a H+ gradient
• The generation of a H+ gradient can then be used to maintain ionic balance, as well as for active transport to drive substrate accumulation.

Question 3

3. Describe the diversity of pigments and membrane systems used in bacteria to utilise light as an energy source. How do some bacteria adapt to life at low light intensity?

Pigments

Answer 3

There is a wide diversity of pigments and membrane systems used in bacteria to use light energy as an energy source.
Photosynthesis= process that converts light energy to chemical energy.
-Different species have different pigments so that unrelated organisms can co exist in an environment, each using wavelengths of light the other is not using.

Pigments:

Chlorophylls and Bacteriochlorophylls

• Light sensitive pigments
• Chlorophyll a is the main one, absorbs read and blue light and transmits green
• Bacteriochlorophyll a absorbs between 800-925 nm, others absorb in the regions of the visible and IR spectrum
• Found in photosynthetic membranes where the light reactions of photosynthesis are carried out.

Carotenoids

• Hydrophobic pigments embedded in the membrane
• Absorb light in the blue region
• Can transfer energy to the reaction centre but also functions as a photoprotective agent. Carotenoids quench toxic oxygen species produced form photooxidation caused by bright light.

Phycobilisomes

• Very efficient energy transfer form biliproteincomplex to chlorophyll a, allows for growth of cyanobacteria at low intensities
• Phycobilisomes content increases in cells as light intensity decreases
• Accessory pigments allow the organism to capture more of the available light
• Cyanobacteria contain phycobiliproteins, their main light harvesting pigments are red of blue. Absorb light in the range of 550-650 nm.

Question 4

3. Describe the diversity of pigments and membrane systems used in bacteria to utilise light as an energy source. How do some bacteria adapt to life at low light intensity?

Membrane systems

Answer 4

Oxygenic Photosynthesis

• Z scheme
• Contains PSI and PSII
• PSII absorbs at a short wavelength, P680
• PSI absorbs at a longer wavelength, P700
• Starts with PSII, P680 absorbs light and water is split into oxygen and hydrogen with an e- donated to P680.
• e- travels through PSII where is donates it to the protein plastocyaninin and then the e- gets donated to PSI which leads to the reduction of NADP+
• Not a closed circuit, needs an exogenous e- donor
• Transfer of e- from acceptor in PSII to PSI generates a PMF where ATP can be made= non cyclic photophosphorylation
• If sufficient reducing power us present, ATP can also be produced when e- travel from ferredoxin to cytochrome bf complex from which e- transport returns the e- to P700, this flow generates a membrane potential and synthesis of ATP- cyclic photophosphorylation.

Anoxygenic Photosynthesis

• Light excites P870 to P870*
• At the higher state, a cascade happens where the energy gets passed to Bchl then Bph and through a series of e- carriers in the process
• This is coupled with the transfer of protons across the membrane creating the PMF→ drives ATPase making ATP.
• The e- returns the P870 at the end of the chain so to can be used again once light excites the reaction centre.

Location of photosynthetic pigments

• In prokaryotes, chloroplasts are absent. Photosynthetic pigments are integrated into internal membrane system
• In organisms e.g. cyanobacteria, these pigments are found in chloroplast.

Question 5

3. Describe the diversity of pigments and membrane systems used in bacteria to utilise light as an energy source. How do some bacteria adapt to life at low light intensity?

Low light intensities

Answer 5

1. Reaction centres surrounded by more numerous light harvesting or antenna chlorophylls, which funnel light energy to the reaction centre. At low light intensities, this allows the capture and use of photons that would otherwise be insufficient to drive the reaction centre alone
2. Green sulfur bacteria contain chlorosome. Giant antenna system, Chlorosomes absorb low light intensities and transfer the enrgy to bacteriochlorophyll a in the reaction centre in cytoplasmic membrane
3. Phycobilisomes, very efficient transfer from the biliprotein complex to chlorophyll a, allows for growth of cyanobacteria at low light intensities. Phycobilisome content increases in cells as light intensities decrease. Accessory pigments allow the organism to capture more of the available light

Question 6

4. Describe anoxygenic and oxygenic photosynthesis. Highlight the differences in electron transport systems, generation of ATP, and reducing power for fixing CO2.

Anoxygenic Photosynthesis

Answer 6

Anoxygenic photosynthesis is the phototrophic process where light energy is captured and converted to ATP, without the production of oxygen.

• Light converts a weak e- donor, p870 into a very strong e- donor, p870*. It’s excited by the absorption of light. It becomes more willing to give up that e-.
• When at the higher state, a cascade happens where the energy gets passed to Bchl then Bph and through a series of e- carriers, transferring e- in the process.
• This is often coupled with the transfer of protons across the membrane that ultimately gives you energy → Proton motive force.
• The e- return to p870 at the end of the chain so it can be used again once light excites the reaction centre.

ATP generation

• Synthesis of ATP results for the formation of the proton motive force (PMF) generated by proton extrusion during e- transport and the activity of ATPase in coupling the dissipation of the proton motive force to ATP formation
• This method of making ATP is called cyclic photophosphorylation as e- move around a closed circle. No net gain or loss of e-

CO2 fixation

• Autotrophic growth requires reducing power (NADH of NADPH) and ATP so that CO2 can be reduced to the level of cell material
• Reduced substances (e.g. H2S or S2O32-) are oxidised by c type cytochromes and e- end up in the quinone pool
• E0’ of quinone is not negative enough to reduce NAD+ directly so e- from quinone pool must be forced backwards against the thermogradient to reduce NAD+ to NADH, a process called reverse electron flow which is driven by ATP.
• e- are coming from external e- donors.
• Not very efficient as you need to spend more energy to make more energy to reduce CO2.

Question 7

4. Describe anoxygenic and oxygenic photosynthesis. Highlight the differences in electron transport systems, generation of ATP, and reducing power for fixing CO2.

Oxygenic Photosynthesis

Answer 7

Use light to generate both ATP and NADPH with e- for reducing power coming from the splitting of water into oxygen and electrons

• Two systems of light reactions are photosystem I and photosystem II, have spectrally distinct forms of reaction centre chlorophyll a.
• Z scheme
• First water is split into oxygen and hydrogen atoms with an electron donor to p680
• Light energy is absorbed by PSII, exciting p680 making it a good e- donor that reduces the first member of the electron transport chain, Ph.
• e- then travel through PSII where it finally a copper containing protein, plastocyanin donates e- to PSI, which leads to the reduction of NADP+ to NADPH.

ATP synthesis
-Water is split to generate electrons used to reduce p680 back to its resting state. The protons (H+) from water act to create the proton motive force.

Transfer of electrons from acceptor in PSII to reaction centre chlorophyll in photosystem I generates a proton motive force form with ATP can be produced=Non cyclic photophosphorylation
When sufficient reducing power in present ATP can also be produced when electrons travel from ferredoxin to cytochrome of complex form which electron transport returns the electrons to p700, this flow generates a membrane potential and synthesis of ATP= cyclic photophosphorylation

Question 8

6. Describe how the concept of chemolithotrophy emerged from the studies of sulfur bacteria by the great Russian microbiologist Sergei Winogradsky. Discuss how chemolithotrophic aerobic H2-oxiding bacteria use H2 as an energy source and fix CO2.

Describe how the concept of chemolithotrophy emerged from the studies of sulfur bacteria by the great Russian microbiologist Sergi Winogradsky.

Answer 8

Winogradsky studied sulfur bacteria Beggiatoa and showed they were only found in waters rich in H2S, as the H2S dissipated, sulfur bacteria were no longer present. So Winogradsky suggested that their development was dependent on the presence of H2S. when sulfur bacteria Beggiatoa filaments were starved of H2S, they lost their sulfur granules, which were rapidly restored if a small amount of H2S was added. So Winogradsky concluded H2S was being oxidized to elemental sulfur. He showed that when sulfur granules disappeared, sulfate appeared in the medium.
H2S → S0 → SO4-
Because these organisms seemed to require H2S for development in the springs, he postulated that this oxidation was the principal source of energy for these organisms - origin of chemolithotrophy.

Question 9

6. Discuss how chemolithotrophic aerobic H2-oxidizing bacteria use H2 as an energy source and fix CO2.

Answer 9

-Chemolithotropic aerobic H2 oxidising bacteria use H2 as an energy source and fix CO2 by the Calvin cycle.
6H2 + 2O2 + CO2 → (CH2O) + 5H20
-The Calvin cycle is the most widespread mechanism for CO2 fixation into cell material.
-pathway is widely distributed and present in purple bacteria, cyanobacteria, algae, green plants etc.
-When readily usable organic compounds are present, synthesis of Calvin cycle and hydrogenase enzyme is repressed.
-Most grow under microaerobic conditions, as hydrogenases are oxygen sensitive enzymes
-Nickel also required for Chemolithotrophic growth as virtually all hydrogenases contain Ni2+ as a metal co factor
-Some hydrogen bacteria can grow on CO as energy source.

• Required NAD(P)H, ATP and 2 key enzymes: RubisCO and phosphorikinase.
• First step in CO2 reduction is catalysed by RubisCO
• RubisCO catalyzes formation of two molecules of PGA from Ribulose biphosphate
• PGA is then phosphorylated and reduced to a key intermediate of glycolysis, glyceraldehyde 3-phosphate
• Final step is phosphorylation of ribulose 5-phosphate with ATP by Phosphoribulokinase which like RubisCO is unique to the Calvin cycle.
  1. Light independent reactions are initiated; CO2 is fixed from an inorganic to an organic molecule
  2. ATP and NADPH are used to reduce 3-PGA into G3P. ATP and NADPH are converted to ADP and NADPH
  3. RuBP is regenerated, which enables the system to prepare for more CO2 to be fixed.

Question 10

7. Compare and contrast the use of CO2 and H2 as substrates for both acetogenesis and methanogenesis.

Acetogenesis

Answer 10

Acetogenesis and methanogenesis, strictly anaerobic prokaryotes can use Co2 is the e- acceptor in energy metabolism,

• H2 is a major e- donor for both.
• Both result in the generation of an ion gradient either H+ or Na+ which drives ATPase.

Acetogenesis
-Homoacetogens carry out the reaction
4H2 + H+ + 2HCO3- → CH3COO- + 4H2O
-In addition to H2, e- donors include a variety of C1 compounds, sugars, organic and amino acids etc.
-Homoacetogens convert CO2 to acetate by the acetyl coA pathway.
-Homoacetogens can grow at the expense of the reactions of the acetyl coA pathway
-Homoacetogens can grow chemoorganotrophically by fermentation of sugars. They ferment glucose via the glycolytic pathway converting glucose to 2 molecules of pyruvate and 2 molecules of NADH and acetate is then produced.
-They can grow chemoorganotrophically through reduction of CO2 to acetate with H2 as the electron donor.
-ATP synthesis is during the conversion of acetyl coA to acetate and via sodium motive force. An input of ATP is initially needed to make a Na+ motive force and therefore more energy

Question 11

7. Compare and contrast the use of CO2 and H2 as substrates for both acetogenesis and methanogenesis.

Methanogenesis

Answer 11

• Methanogenesis is the production of methane by organisms.
• Production of methane is carried out by anaerobic Archaea called methanogens
• Methanogenesis occurs through a series of reactions involving novel coenzymes
• Those involved in carrying C1 units from initial substrate Co2 to final product CH4 and those that function in redox reactions to supply electrons necessary for reduction of co2 to CH4.

CO2 + 4H2 → CH4 + 2H2O
H2 is the external electron donor for CO2 reduction.

11 substrates have been shown to be converted to methane by pure cultures of methanogens.
Methane is produced by 3 major pathways
1. Reduction of CO2
2. Fermentation of acetate
3. Using methyl substrates, reduced using an external donor.

-Unlike Acetogens, methanogens may need to interact with syntrophs to get H2 or other substrates.

Question 12

8. Methanogens can utilize three main groups of substrates for the production of methane: carbon dioxide, methyl compounds, and acetate. Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.

Methyl Substrates

Answer 12

There are 11 substrates that have been shown to convert to CH4.

• The substrates are divided into 3 classes: CO2 substrates, methyl substrates and acetotophic substrates.
• All reaction are exergonic and can be used to synthesize ATP.

Methyl Substrates
CH3OH + H2 → CH4 + H2O (-131 kJ)
Methanol is reduced to CH4 and H2 is oxidized to H20.

In the absence of H2
4CH3OH → 3CH4 + CO2 + 2H20 (-319 kJ)
e,g, methanol

• Methyl compounds (i.e., methanol) are catabolised by donating methyl groups to a corrinoid protein to form CH3-corrinoid.
• The CH3-corrinoid complex donates methyl group to CoM, yielding CH33-CoM from which methane is formed in the same way as from CO2 reduction.
• If reducing power (such as H2) is not available to drive the terminal step then some methanol must be oxidised to CO2 to yield electrons, this occurs by reversal of steps in methanogenesis.
• Other than methanol, these other substrates can be used: methylamine, dimethylamine, trimethylamine, methylmercaptan and dimethylsulfide. It has an environmental significance in that even in the absence of H2, the substrates can still be used.

Question 13

8. Methanogens can utilize three main groups of substrates for the production of methane: carbon dioxide, methyl compounds, and acetate. Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.

Acetotrophic substrates.

Answer 13

CH3COO- + H20 → CH4 + HCO3- (-31 kJ)
e.g. acetate, pyruvate

• Acetate is first activated by acetyl-CoA, which can interact with carbon monoxide dehydrogenase (CODH) of the acetyl-CoA pathway.
• The methyl group of acetate is transferred to the corrinoid enzyme to yield CH3-corrinoid and then goes through the CoM mediated terminal step of methanogenesis.
• Another acetotophic substrate is pyruvate. Only a few methanogens are acetoclastic; this has an environmental significance in that they produce a lot of methane for such a small diversity of them.
• In an experiment done on measurements of methane formation in sewage sludge, 2/3 of the methane produced was from acetotrophic methanogens.

Question 14

8. Methanogens can utilize three main groups of substrates for the production of methane: carbon dioxide, methyl compounds, and acetate. Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.

CO2 type substrates

Answer 14

CO2 + 4H2 → CH4 + 2H2O (-131 kJ)
e.g. CO2(e- derived from H2, alcohols, pyruvate), formate, CO

• CO2 is reduced to CH4 and H2 oxidized to H20
• The reduction of CO2 to CH4 is generally H2-dependent, but formate, CO, and organic compounds such as alcohols can supply electrons for CO2 reduction.
• First, CO2 is activated by methanofuran-containing enzyme and reduced to formyl.
• The formyl group is then transferred to methanopterin, dehydrated and reduced to methylene, then to methyl.
• The methyl group is transferred to coenzyme M and methyl-CoM is reduced to methane by the methyl reductase system in which F430 and CoB are involved.
• F430 removes CH3 group from CH3-CoM, forming a Ni2+-CH3 complex, which is reduced by electrons from CoB generating CH4 and a disulfide complex of CoM and CoB (CoM-S-S-CoB).
• Free CoM and CoB are regenerated by reduction of this complex with H2

Environmental significance: CO2 is common and abundant in nature any types of methanogens can use CO2-type substrates and so its very easy substrate to take in.

Question 15

9. Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations: (i) homofermentative and heterofermentative lactic acid fermentation, (ii) mixed acid fermentation by enteric bacteria, and (iii) butyric acid fermentation by Clostridium species.

Homofermentative and Heterofermentative

Answer 15

Lactic acid can be produced during fermentation into two pathways: Homofermentative and Heterofermentative.
Homofermentative is the process of producing lactic acid in a single yielding pathway. Homofermentative lactic acid bacteria contain aldolase and produce a molecules of lactate from glucose by the glycolytic pathway.
Glucose → 2 Lactate + 2H+

In comparison to this,
Heterofermantative produces additional products mainly ethanol and CO2. The reason for this is it lack aldolase and cannot easily breakdown fructose biphosphate to triose phosphate. To achieve redox balance it must go through the process below.

Glucose 6-phosphate (oxidation) -> Phosphogluconic acid (Decarboxylated) -> Pentose phosphate (convert to) -> Triose phosphate & Acetyl phosphate. Key enzyme: Phosphoketolase.
Ethanol is reduced from acetyl phosphate. CO2 will be observed.
Glucose → Lactate + ethanol + CO2 + H+ + ATP

To differentiate between the two, one produces CO2 which can be observed.

Question 16

9. Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations: (i) homofermentative and heterofermentative lactic acid fermentation, (ii) mixed acid fermentation by enteric bacteria, and (iii) butyric acid fermentation by Clostridium species.

Mixed Acid Fermentation

Answer 16

-Characteristic of enteric bacteria
-This is the process in which acids are generated from fermentation of sugars through glycolysis. The acids produced in this process are acetic, lactic and succinic acids and the process produces additional substances such as Ethanol, CO2 and H2. It is also able to generate other neutral products e.g. Butanediol.
-This process produces more CO2 than mixed-acid fermenters.
-Therefore, the process does not acidify its environment, this means that the organisms’ are unable to tolerate more acidic environment.
2 pyruvate + NADH → 2 CO2 + Butanediol.

Question 17

9. Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations: (i) homofermentative and heterofermentative lactic acid fermentation, (ii) mixed acid fermentation by enteric bacteria, and (iii) butyric acid fermentation by Clostridium species.

Clostridial Sugar Fermentation sugars.

Answer 17

-This process produces butyric acid by fermenting sugars
-Butanol and acetone are by products of this process.
-The early stage of the fermentation, butyrate and acetate are produced but as the pH drops it affects the synthesis of acid that will result in accumulation of acetone and butanol.
-Acid production will only continue if the media is buffered to keep it neutral.
-In relation to this idea, lowering the pH will trigger de-repression of genes for solvent production.
Glucose → butyrate + 2CO2 + 2H2 (3 ATP/glucose)
2 Glucose → acetone + butanol + 5 CO2 + 4H2 (2ATP/glucose)

Question 18

10. Fermentations are characterised by the generation of ATP via substrate-level phosphorylation; however, there are a number of fermentations which lack substrate-level phosphorylation. Describe using examples how the small amount of energy released is used to generate ATP?

Propionigenium modestum

Answer 18

Fermentation of the certain compounds does not yield sufficient energy to synthesize ATP. Other processes are able to produce ATP by catabolising compounds that can be linked to ion pumps that established a proton or sodium motive force. Following examples are able to yield small amount of ATP by certain processes.

1. Propionigenium modestum. This process catabolizes succinate under strictly anoxic conditions and is able to yield ATP by establishing a sodium motive force.
   - Succinate is oxidized to propionate, which gets pumped across
   - The energy associated with this is transferred to a sodium extruding decarboxylase which pumps Na across the membrane.
   - This generates a sodium motive force → ATP

Question 19

10. Fermentations are characterised by the generation of ATP via substrate-level phosphorylation; however, there are a number of fermentations which lack substrate-level phosphorylation. Describe using examples how the small amount of energy released is used to generate ATP?

Oxalobacter formigenes

Answer 19

2. Oxalobacter formigenes. This process catabolizes oxalate that produces formate. -Formate is excreted from the walls
   - Export of formate form the cell establishes a PMF which forms ATP
   - Have oxalate and its oxidized to form formate a weak acid. 1 hydrogen is consumed.
   - As you pump formate out and pump oxalate in. Outside is slightly more positive as formate contains one less H. Inside is less positive. The positive change then pushes protons through ATPase.

The small amount of energy released is couple to pumping of ions across the cytoplasmic membrane. Minimum energy requirement to pump a single ion is estimated to be -12 kJ.
Reactions that release less energy should not be able to drive ion pumps and not be energy conserving reactions

Question 20

11. Syntrophy is the cooperation of two or more organisms to degrade a substrate that neither can degrade alone and typically involves interspecies H2 transfer. Describe how the energetics of syntrophy work when transfer is coupled to methanogenesis, and also describe the oxidation of the fatty acid butyrate to acetate plus H2 by the syntroph Syntrophomonas.

Answer 20

Syntrophy is the process whereby 2 or more microbes cooperate to degreade a substance that neither can degrade alone.
-Syntrophic reactions are important for the anoxic portion of the carbon cycle.
-Most reactions are based on interspecies hydrogen transfer
-H2 production by one partner is linked to H2 consumption by the other
-There are many methanogens that interact with syntrophs to get H2 for methanogenesis.
-One syntrophic reaction is ethanol fermentation
(2 CH3CH2OH + 2 H2O → 4 H2 + 2 CH3COO- +2 H+)
-Methanogenesis is 4 H2 + CO2 → CH4 + 2 H2O
-The coupled reaction is
2 CH3CH2OH + CO2 → CH4 + 2 CH3COO- + 2 H+
-H2 is produced from ethanol fermentation and syntrophically transferred to the methanogen for methanogenesis to produce CH4.
-This helps with energy conservation in that methanogens save energy by getting H2 syntrophically and not from an indirect source.
-Energy conservation is based on substrate level phosphorylation and oxidation phosphorylation.
-Syntrophs still however pose a significant challenge to understanding of minimal requirements for energy conservation in bacteria.
-Fermenting ethanol in not energetically favourable. Its an energy consuming pathway. But its waste product can be used in methanogenesis
-When coupled, now have a net energy gain

Question 21

11. Syntrophy is the cooperation of two or more organisms to degrade a substrate that neither can degrade alone and typically involves interspecies H2 transfer. Describe how the energetics of syntrophy work when transfer is coupled to methanogenesis, and also describe the oxidation of the fatty acid butyrate to acetate plus H2 by the syntroph Syntrophomonas.

Oxidation of FA

Answer 21

Oxidation of fatty acid butyrate to acetate plus H2 by the syntroph Syntrophomonas is endothermic

• Process starts with acetyl-CoA coming from one of the final steps in the process to butyrate and binding to butyrate.
• 4 more reactions occur making intermediates and the reaction then makes 2 molecules of acetyl-S-CoA where one goes to make acetate and the other goes to make acetyl-P then to acetate and ATP.
• Production of H2 is driven by reverse electron flow during the process.
• H2 consumption affects the energetics of the reaction carries out by the H2-producer, allowing the reaction to be exothermic.

Question 22

12. Methanotrophs oxidise methane to carbon dioxide. Describe the pathway for methane oxidation and the two alternative pathways for assimilation of carbon into cell material in these bacteria. The first step in the oxidation of methane by methane- oxidising bacteria involves a unique enzyme which can be present in two different forms, describe how the two forms of this unique enzyme differ. 


Answer 22

Methane → Methanol → Formaldehyde → Formate → Carbon dioxide

• The pathway for methane oxidation in methanotrophs starts off with methane being oxidised to methanol (CH3OH) using the enzyme, methane monooxygenase.
• It is then further oxidized to formaldehyde a central intermediate in the pathway
• Roughly ½ of the formaldehyde is further oxidized to CO2 to generate energy. The other ½ is assimilated into cell carbon via either the RuMP pathway or the Serine pathway

Methane monooxygenase can be found in two forms: pMMO (periplasmic) and sMMO (soluble).

pMMO is membrane associated,

• Has a narrow substrate range
• High degree of identity with ammonia monooxygenase (Amo)
• Found in all methanotrophs.

sMMO is cytoplasmic

• Wide substrate range, has
• No identity with aMO, is
• Only present in some methanotrophs,
• Only expressed in low copper, it is a non-heme iron monooxygenase, oxidises methane to methanol and also co-oxidizes other organics such as alkanes.

Question 23

12. Methanotrophs oxidise methane to carbon dioxide. Describe the pathway for methane oxidation and the two alternative pathways for assimilation of carbon into cell material in these bacteria. The first step in the oxidation of methane by methane- oxidising bacteria involves a unique enzyme which can be present in two different forms, describe how the two forms of this unique enzyme differ. 


Serine Pathway

Answer 23

Serine Pathway
-Acetyl coA is synthesize form one molecule of formaldehyde and one molecule of CO2
-Required introduction of reducing power and energy in the from of two molecules each of NADH and ATP from each acetyl coA synthesized.
-Serine pathway employs a number of enzymes of citric acid cycle and one unique enzyme, serine transhydroxymethylase. Eventually acetyl coA is made and can be used in biosynthesis.
Type II methanotrophs utilize this pathway; they contain membranes arranged around periphery of the cell and are alpha-Proteobacteria.
Formaldehyde + CO2 + 2NADH + 2ATP → Acetly-S-CoA + 2H2O

Question 24

12. Methanotrophs oxidise methane to carbon dioxide. Describe the pathway for methane oxidation and the two alternative pathways for assimilation of carbon into cell material in these bacteria. The first step in the oxidation of methane by methane- oxidising bacteria involves a unique enzyme which can be present in two different forms, describe how the two forms of this unique enzyme differ. 


RuMP Pathway

Answer 24

RuMP Pathway
-More efficient than serine pathway as carbon atoms for cell material are derived more directly from formaldehyde
-Glyceraldehyde-3-phosophate (G3P) is synthesized from formaldehyde and only one molecule of ATP per G3P
-No reducing power (NADH/NADPH) required as formaldehyde is at same oxidation level as cell material
-Due to lower energy requirements of RuMP pathway, the cell yield of type I methanotroph is higher than for type II
-Enzyme hexulosephosphate synthase and hexuloase-6-P isomerase are unique.
-Type I methanotrophs utilize this pathway; they contain bundles of membranes and are gamma-Proteobacteria.
3 Formaldehyde + ATP → G3P

Question 25

1a. Bacteria play key roles in the biogeochemical cycling of nitrogen and sulfur. Describe in detail the individual redox reactions, their associated biochemical pathways, and microorganisms involved in either the nitrogen or sulfur cycle.

Answer 25

5 key steps: Nitrification, Denitrification, N2 Fixation, Ammonification, Anommox.

Question 26

Nitrogen fixation

Answer 26

- Energy intensive process that produces ammonia (NH3) - Inhibited by NH3, carried out under anaerobic or oxygen limited conditions. - In soil, mainly by bacteria associated with nodules on the roots of legumes (25 % of total nitrogen fixed) - In aquatic and marine environments, predominantly cyanobacteria (35% of total nitrogen fixed). - All nitrogen fixing archaea are methanogens - Catalyzed by nitrogenase, a two-part enzyme complex: Dinitrogenase contains two copies of FeMo-co, dinitrogenase reductase contains iron. - The enzyme is inhibited by oxygen. The structure contains both Fe and S. They are all highly reactive so if exposed to oxygen, it will destroy them. Nitrogenase is protected from O2 in aerobic nitrogen fixers. -The nitrogen triple bond makes nitrogen extremely inert. Nitrogen fixation is a very energy intensive process. 8 e- are consumed but only 6 e- are transferred t o nitrogen atoms. -Electrons are supplied by dinitrogenase reductase to dinitrogenase one at a time. Two ATPs are hydrolyzed to supply one electron. Pyruvate donates electrons to flavodoxin. Flavodoxin reduces dinitrogenase reductase. Electrons are transferred to dinitrogenase one at a time. 2 ATP are consumed per electron. (N2 → HN=NH → H2N-NH2 → NH3) -Diazotrophs: microorganism that fix N2. Nitrogenase is highly conserved. Free living anaerobes. Aerobic diazotrophs employ strategies to keep O2 away from Nitrogenase.

Question 27

Anammox

Answer 27

- Anammox stands for anaerobic ammonium oxidation. (NO2- + NH4+ → N2 +2H2O). -NO2- acts as the e- acceptor and is the product of aerobic ammonia oxidation (nitrification). - An example of a microbe that uses this process is: Brocadia anammoxidans. An “organelle” within the cell called anammoxosome contains a strong reductane: N2H4. It lacks the Calvin cycle enzymes and instead uses the acetyl-CoA pathway to fix CO2.

Question 28

Ammonification and ammonium assimilation

Answer 28

Ammonification uses organic-N and produces NH4+. Many microbes can do this. Ammonium incorporated into cellular components (e.g. amino acids, purines, pyrimidines). Nitrogen assimilation is the formation of organic nitrogen compounds (e.g. amino acids) form inorganic nitrogen compounds present in the environment. C:N if C is 20 times more, ammonium assimilation is occurring. If C is less, ammonification is occurring.

Question 29

Nitrification

Answer 29

NH4+ → NO2- → NO3- (oxidative reactions coupled with oxygen) -Carried out in 2 steps by two kind of nitrifiers: ammonium oxidizers and nitrite oxidizers. -Net energy producing reactions carried out by Chemolithotrophic (and mostly autotrophic) bacteria and archaea. -Some heterotrophic fungi and bacteria can also perform nitrification, although no energy is produced. Nitrosifying bacteria 1. NH3 + O2 + 2e- + 2H+ → NH2OH + H2O Carried out by ammonia monooxygenase (AMO). Produces hydroxylamine (NH2OH). Requires two electrons. 2. NH2OH + H2O + 1/3 O2 → NO2- + 2H2O + H+ Carried out by hydroxylamine oxidoreductase (HAO), Produces nitrate (NO2). Generates 4 electrons. Sum: NH3 + 1 ½ O2 → NO2- + H2O Only 2 electrons are actually generated. Nitrifying bacteria NO2- + ½ O2 → NO3- -carried out by nitrite oxidoreductase. -Very short ETC -Generates a PMF through cytochrome aa3 which ultimately drives ATP synthesis. -CO2 is fixed using the Calvin cycle. -All nitrifying bacteria are Proteobacteria except Nitrospira which forms its own phylum. -Wide spread in soil and water, particularly environments rich in ammonia (from ammonification)

Question 30

Denitrification

Answer 30

NO3- → N2 - Nitrite used as terminal electron acceptor. - occurs under microaerophilic or anaerobic conditions - Primary type of dissimilatory nitrate reduction in soil. - Not inhibited by NH4+. Reduction reactions. ``` nitrate (NO3-) --> nitrite (NO2-) - nitrate reductase. nitrite --> nitric oxide (NO) -nitrite reductase. nitric oxide --> nitrous oxide (N2O) -nitric oxide reductase. Nitrous oxide--> dinitrogen (N2) nitrous oxide reductase. ``` - The 4 enzymes used are sensitive to oxygen and are inter-regulated. Nitrate is required for the full expression of the enzymes. N2O is the main product under high O2 conc or low NO3- conc. - This process is mostly carried out by respiratory heterotrophs.

Question 31

2a. Discuss the metabolic and phylogenetic diversity of nitrification. What are the roles of different groups of microorganisms involved, and what are the key enzymes involved? Explain why many nitrite oxidizers are chemoorganotrophs from the perspective of energetics.

Answer 31

NH4- → NO2- → NO3- -Nitrification is the oxidation of ammonia. Oxidative reaction coupled with oxygen. - Carried out in two steps by two kinds of nitrifiers: ammonium oxidizers and nitrate oxidizers. - Net energy producing reactions carried out by Chemolithotrophic and mostly autotrophic bacteria and archaea - Some heterotrophic fungi and bacteria can also perform nitrification although no energy is produced. - Nitrification occurs at the membrane. Nitrosifying bacteria Nitrifying bacteria

Question 32

Nitrosifying bacteria Nitrifying bacteria

Answer 32

Nitrosifying bacteria 1. NH3 + O2 + 2e- + 2H+ → NH2OH + H2O Carried out by ammonia monooxygenase (AMO). Produces hydroxylamine (NH2OH). Requires two electrons. 2. NH2OH + H2O + 1/3 O2 → NO2- + 2H2O + H+ Carried out by hydroxylamine oxidoreductase (HAO), Produces nitrate (NO2). Generates 4 electrons. Sum: NH3 + 1 ½ O2 → NO2- + H2O Only 2 electrons are actually generated. Nitrifying bacteria NO2- + ½ O2 → NO3- -carried out by nitrite oxidoreductase. -Very short ETC -Generates a PMF through cytochrome aa3 which ultimately drives ATP synthesis. -CO2 is fixed using the Calvin cycle. -All nitrifying bacteria are Proteobacteria except Nitrospira which forms its own phylum. -Wide spread in soil and water, particularly environments rich in ammonia (from ammonification)

Question 33

2a. Explain why many nitrite oxidizers are chemoorganotrophs from the perspective of energetics.

Answer 33

- more favourable energetically to obtain the energy from the oxidation of reduced compounds. - They don’t produce enough energy for themselves due to the small transport chain. -They also use the Calvin cycle to fix CO2. -This puts severe energy constraints on them. - They have low growth yield due to this. - All nitrifying bacteria are Proteobacteria except for Nitrospira.

Question 34

4a. Discuss the biochemistry of sulfur oxidation, the unusual ecology of sulfur oxidizing bacteria, and at least one example of strategies used by sulfur oxidizers in non-acidic environments to obtain inorganic nutrients.

Answer 34

- Oxidation of hydrogen sulfide (H2S), elemental sulfur (S0) or thiosulfate (S2O32-) to ultimately sulfate (SO42-l - Sulfide (HS-) is oxidized to sulfite (SO32-) through the transfer of 6 e- -If starting from elemental sulfur (S0) first reduce it to sulfide before oxidizing it to sulfite. S2O32- is first split into S0 and SO32- -Sulfite is eventually oxidized to sulfate (SO42-). Sulfite oxidase is the most common pathway. A few chemolithotrophs use the APS reductase in reverse. -The Sox system oxidizes sulfide directly to sulfate -Chemolithotrophic sulfur oxidation (aerobic) generally tolerates and often requires low pH. -Photoautotrophic sulfur oxidation (anaerobic and Anoxygenic). Limited to green and purple sulfur bacteria. CO2 + H2S → S0 + fixed carbon.

Question 35

4a. The unusual ecology of sulfur | oxidizing bacteria

Answer 35

The unusual ecology of sulfur oxidizing bacteria is that sulfide reacts with oxygen rapidly but not instantaneous. Therefor these organisms have evolved to somehow reconcile the simultaneous need for sulfide and oxygen. Sulfur oxidizers live in environments where they need both oxygen and H2S. This is unusual as these two things normally react spontaneously. The fact that these organisms live where they don't react together is unusual.

Question 36

4a. Discuss the biochemistry of sulfur oxidation, the unusual ecology of sulfur oxidizing bacteria, and at least one example of strategies used by sulfur oxidizers in non-acidic environments to obtain inorganic nutrients. Examples Thiothrix Desulfobaulbaceae

Answer 36

- use filaments to hold position in fast flowing water rich in O2 and H2S. - Finds a place where you have mixing occurring. Where water flows, your going to have oxygen, disturbing the sediment also releases H2S. Uses filaments to hold onto stable places. Can then use the oxygen. Desulfobaulbaceae -Filamentous multicellular bacteria (aka cable bacteria) -Centimeter long structures containing thousands of Bacteria inside an outer membrane, -Electrons obtained from sulfide oxidation are transported via ‘bacterial micro cable’ to the surface for oxygen reduction. -Half reactions occur more than a centimeter apart. -Also capable of using nitrate and nitrate as electron acceptors. They carry sulfur reduction at the bottom. Can dump electrons onto oxygen. Just need to dump electrons for the reaction to continue.

Question 37

5a. Describe the conditions that lead to the self-propagating oxidation of pyrite, how the process contributes to the formation of acid mine drainage, and the ecological significance of acid mine drainage.

Answer 37

-Pyrite (FES2) is one of the most common forms of iron. Present in bituminous coals and metal ores. A combination of chemical and bacterial oxidation of FeS2 leads to acidification 1. FeS2 is oxidized to O2, generating HS, which is then oxidized to SO42- 2. Biological oxidation of Fe2+ to Fe3+ 3. Fe3+ spontaneously reacts with FeS2 and produces more SO42- In nature in terms of mining, it is very rare to contain a pure metal or a very specific metal. One of the most common substance in mining in terms of metal is Pyrite (FeS2). Elemental metal iron spontaneously reacts with sulphur that results to pyrite. This is one of the most common form of iron. Digging deeper into a mine or metal mines, you will find a combination of chemical and bacteria oxidation of pyrite can lead to acidification of the environment.

Question 38

5a. Describe the conditions that lead to the self-propagating oxidation of pyrite, how the process contributes to the formation of acid mine drainage, and the ecological significance of acid mine drainage. Propagation cycle

Answer 38

Pyrite (FeS2) oxidized to Ferrous oxide (FeO) then ferrous oxidise can be further oxidised by a bacteria or spontaneously to Ferrous ion. - At this point, the environment will be acidified because of the sulphuric acid (2SO42- + H+). - The ferrous ion is stable and this spontaneously react to ferrous oxide. - The initiation of this process is oxygen dependent. However, the actual propagation cycle is NOT oxygen dependent because the process has Ferrous oxide reacting as the reductant. - Cycle becomes self propagating. Don't need an electron acceptor at this point.

Question 39

5a. Describe the conditions that lead to the self-propagating oxidation of pyrite, how the process contributes to the formation of acid mine drainage, and the ecological significance of acid mine drainage. Acid mine drainage

Answer 39

Acid mine drainage: FeS2 + 14 Fe3+ + 8H2O → 15 Fe2+ + 2SO42- + 16 H+ -self propagating process The problem with this production is when it rains or when ground water seeping through. This reactions gets intiated. Water plays an important role in this process as once it slips through in coal-mining operation, the self-propagating cycle gets initiated. - Once the process is initiated, it does not require further addition of oxygen and water. -The process results in acidifying the environment (pH <1). - This pH 1 (acidified environment) dissolves heavy metals. - Leaking takes place of the heavy metals this results affects the surrounding the fresh water system where the mine is operating. - Ferroplasma is an Archaeon that acts as an indicator when there is an acid mine drainage problem as it grows from pH O in 50 degrees. A major problem in all surface coal mining operations - pH <1 - dissolves heavy metals - degrades water quality in rivers and lakes

Question 40

6a. Describe the physiology of microbial nanowires as currently understood and the metabolic advantages possessed by microorganisms that have microbial nanowires.

Answer 40

Electrically conductive appendages produced by certain Fe3+ reducing bacteria, e.g. Genobacter and Shewanella sp - Useful in environments depleted of electron acceptors. - Transfer electrons to insoluble materials e.g. ferric oxides. - In Shewanella: extensions of outer membrane that conduct through cytoplasm - In Geobacter: modified pili - Electrons shuttling by nanowires can occur over large distances. Electron transfer across biofilm layers. Sheaths are used to translocate the electrons. - Produced by Shewanella under oxygen-deprived conditions. Conductivity similar to semiconductors. - Use nanowire to obtain electrons from electron donors. - Only want to do this if you don’t have access to good terminal electron acceptors - This organism act as the terminal electron acceptor for other organisms - Get electrons from other organisms. As electrons go through the e- transport chain this makes a PMF and so ATP. - Once you make a circuit where electrons can flow, can tap into this. This makes a battery. - Can steal these electrons and use for fuel cells. These organisms form the basis of microbial fuel cells. Generate electrical currents by collecting electrons from microbial oxidation of toxic or waste material (e.g. landfills) via microbial nanowires.

Question 41

7a. What is the defining characteristic of halophiles? Describe physiological adaptations in halophiles specifically associated with this characteristic. What are the characteristics of osmophiles and xerophiles, and what are the key features of compatible solutes?

Answer 41

Halophiles have a requirement for NaCl in order for them to grow. They require 1-12 % NaCL and extreme halophiles require >9 % and grow at up to 32 % NaCl. This cannot be replaced by other salts e.g. K+, Mg2+ - Halotolerant microorganisms grow best in the absence of solutes - Most extreme halophiles are Archaea. Nearly all are obligate aerobes. Halobacterium salinarum, an archaeon, -requires Na+ and K+. Na+ is required on the outside of its membrane to balance the negatively charged proteins that are next to the membrane inside the cell. - It stabilises the cell wall containing glycoproteins rich with negatively charged amino acids. - Lower conc. of Na+ causes the negatively charged proteins to repel each other. -Proteins in the cytoplasm are acidic so it requires K+ for stability and activity. - This has the effect of balancing osmotic pressure in terms of countering Na+ on the outside of the cell. Bacteriorhodopsin is a mechanism found in many halophilic archaea that facilitates phototrophic generation of ATP under anoxic conditions (not photosynthesis). -It absorbs light energy and uses it to pump a proton across the cytoplasmic membrane. -They are found as red and orange carotenoids, a similar structure to the rhodopsin found in the human eye. In H. salinarum, the mechanism is linked to Na+ and H+ pumping, using it to maintain the balance between Na+ and K+. An Na+ - H+ antiport system achieves this.

Question 42

7a. What is the defining characteristic of halophiles? Describe physiological adaptations in halophiles specifically associated with this characteristic. What are the characteristics of osmophiles and xerophiles?

Answer 42

Osmophiles -Not the same as halophiles. Both groups are adapted to high osmolarity in the environment . Osmophiles do not require high osmolarity They are adapted to high solute concentrations. Glycerol is the primary compatible solute. Cause spoilage in high sugar food items. Generally not pathogenic Xenophiles -Organisms adapted to very low moisture content (and possibly high osmolarity)

Question 43

7a. What is the defining characteristic of halophiles? Describe physiological adaptations in halophiles specifically associated with this characteristic. What are the characteristics of osmophiles and xerophiles, Key features of compatible solutes?

Answer 43

Compatible solutes are needed to maintain a positive water (slight positive osmotic pressure) so that they have access to water. ---Must have compatible solutes to offset the high salt concentration outside of the cell membrane. - These solutes must be acquired or synthesised in large amounts to have the effect and not interfere with cellular processes. - Examples in halophiles include glycine, betadine and glycerol.

Question 44

8a. Describe in detail at least four mechanisms commonly adopted by thermophiles to overcome the challenges of surviving at high temperatures.

Answer 44

Prokaryotes are more thermal tolerant that eukaryotes. Archaea are more thermal tolerant than Bacteria. There is a clear upper limit temperature for photosynthesis. 1. Heat stable enzymes that function better at high temperatures 2. Stabilization of proteins 3. DNA stability 4. Ribosomal RNA tRNA stability 5. Cytoplasmic membrane stability

Question 45

8a. Describe in detail at least four mechanisms commonly adopted by thermophiles to overcome the challenges of surviving at high temperatures. 1. Heat stable enzymes that function better at high temperatures 2. Stabilization of proteins 3. DNA stability

Answer 45

Heat stable enzymes that function better at high temperatures - Increased ionic bonds between basic and acidic amino acids - Greater hydrophobicity in the interiors. - Increased disulfide bonds between cysteine residues Stabilization of proteins - Chaperonins (thermosome in Archaea) help refold partially denatured proteins - Intracellular solutes (e.g. diglycerol phosphate and mannosylglycerate) help stabilize proteins - Increases the temperature at which organisms can survive but not necessarily grow. DNA stability - Increased intracellular solutes reduce depurination and depypyrimidization - Reverse DNA gyrase introduced positive supercoils into the DNA of hyperthermophiles - Histones in Euryarchaeota compact DNA into nucleosome like structures and prevent DNA strains from separating

Question 46

8a. Describe in detail at least four mechanisms commonly adopted by thermophiles to overcome the challenges of surviving at high temperatures. 4. Ribosomal RNA tRNA stability 5. Cytoplasmic membrane stability

Answer 46

Ribosomal RNA tRNA stability - Higher proportion of GC nucleotides in the rRNA and tRNA of hyperthermophiles - G-C pairs are mores stable that A-U pairs, allowing rRNA to maintain secondary structures Cytoplasmic membrane stability - More long chained (e.g. higher melting point) and saturated (higher hydrophobicity) fatty acids in lipid bilayer membranes - Lipid monolayer membranes in practically all hyperthemophilic archaea

Question 47

9a. Describe in detail three environmental factors that influence known upper temperature limits for microbial life in various habitats.

Answer 47

Hyperthermophiles are defined as having their optimal growth >85 degrees. - Archaea can grow at 122 C. Many of the species grow optimally above 100 C. - Chemolithotrophic or chemoorganotrophic Water boils at 100 C. Requires nucleation points. These environments have been hot so long (thousands of years) that their nucleation points are gone; if you were to drop a pebble which has lots of nucleation points, the water would boil up high. -High pressure is another environmental factor that influences the upper temperature limit. -In high pressure environments such as subsurface aquifers (>250 C) and deep sea hydrothermal vents (up to 400 C). -Another environmental factor is the interaction between temperature and pH. Majority of hyperthermophiles at very high temperatures at a near neutral pH. -If you have an acidic environment, e.g. pH 2, 80 C is the temp limit: heat and acidity is a lethal combination for microorganisms. -If you look at acidic hot pools above 80oC, they are mostly likely sterile as we are not aware of any microorganism that can survive high T and low pH. Macromolecules have been detected in hydrothermal vent water at 150 C. This tells us that if these types of macromolecules can be found, then maybe life is possible.

Question 48

9a. Describe in detail three environmental factors that influence known upper temperature limits for microbial life in various habitats, Three examples of why life is unlikely to be present at supercritical (e.g., >250°C) temperatures.

Answer 48

However, at superheated hydrothermal vent water (>250oC), life is unlikely to be present because they contain no DNA, RNA or protein which are molecules essential for life. These are unable to exist at such high temperatures as they denature, therefore life cannot exist. Biochemical issues at super critical temperatures - ATP is degraded almost instantly at 150 C - Amidation (spontaneous cleaving of peptide bonds) occurs above 130 C. Proteins will completely break. - Critical chemical reactions maybe unfavourable (or become spontaneous) at higher temperatures.

Question 49

10a. Explain the difficulties encountered by acidophiles and list at least two examples of how these difficulties are overcome. What evidence suggests that the intracellular pH of acidophiles is close to pH 7.0?

Answer 49

Acidophiles live in environments below pH 7. There is a high number of H+ outside the cell. Natural proton motive force: large pH gradient (up to 5 orders of magnitude) across the cytoplasmic membrane. -Microbes must maintain intracellular pH homeostasis. Protons also leak through cytoplasmic membrane. Internal pH must remain near neutrality. pH 5-9 - DNA is acid labile - RNA is alkaline labile - RNA will breakdown in alkaline environments while DNA will break down in acidic environments. - Intracellular enzymes have a pH optima near 7 and denature at extracellular pH levels. Physiological adaptations to deal with extreme pH.

Question 50

10a. Explain the difficulties encountered by acidophiles and list at least two examples of how these difficulties are overcome. What evidence suggests that the intracellular pH of acidophiles is close to pH 7.0? Adaptations of acidophily: Examples of how these difficulties are overcome

Answer 50

Cytoplasmic membrane requires a high [H+] to maintain stability. The massive concentration of protons on the outside keeps the membrane together. ``` Active solutions -Proton efflux systems (requires energy) -Using electrons generated by oxidative phosphorylation and oxygen to convert H+ to H2O. -Reverse membrane potential Pumps protons out but require energy. ``` Passive solutions -Reduce membrane permeability (e,g, tetraether lipids) -Coating the cell wall with positively charged proteins -Cytoplasmic buffering molecules Repulsion membrane potential-the repulsion between the positively charged membrane keeps the protons away at a distance making it less likely they come in the cell. Secondary adaptations -abundance of secondary transporters that use PMF to transport nutrients -repairing acidity-induced damages to DNA and protein. Use a lot of secondary transporters that use the PMF to transport nutrients. Most organisms make a PMF and then use it, they don't waste it. Acidophiles have an influx of protons so they might as well use it. They couple the transport of nutrients with the PMF and they just deal with the influx of protons. There is no ‘universal’ adaptation to acidophily.

Question 51

11a. Explain the difficulties encountered by alkaliphiles and list at least two examples of how these difficulties are overcome. What evidence suggests that the intracellular pH of alkaliphiles is close to pH 7.0?

Answer 51

1. They have a very high concentration of OH- around the cells. - OH- spontaneously combines with H+ to form H2O. Makes it hard for the organisms to form a PMF for the production of energy (ATP) - Natural reverse proton gradient. To overcome this, they use a sodium motive force to generate ATP and drive transporters. Alkaliphile habitats are often rich in Na+. -Some alkaliphiles can use a PMF to make ATP but the mechanism is unclear. Likely involves keeping proteins extremely close to the outer cytoplasmic membrane and way from OH-. Other uses for a sodium gradient - Na+ symports for nutrient transport - Drive flagella using the Na+ gradient - how to maintain a Na+ gradient Active acidification - Na+/H+ antiporters found in all alkaliphiles. - If you have an antiporter, pumps in protons with the pumping out of sodium so you can maintain a sodium gradient while acidifying the cytoplasm (RNA is acid labile) Passive acidification - Cell wall rich in polymers made of organic acids (e.g. glutamic acid and gluconis acid), which prevents the entry of OH-) - Higher hexosamine and amino acid content in the peptidoglycan. - If you have the negatively charged membrane, this repulses the negatively charged OH- ions making it less likely for them to come inside.

Question 52

11a. Explain the difficulties encountered by alkaliphiles and list at least two examples of how these difficulties are overcome. What evidence suggests that the intracellular pH of alkaliphiles is close to pH 7.0?

Answer 52

Internal pH must remain near neutral. - DNA is acid labile - RNA is alkaline labile - RNA will breakdown in alkaline environments while DNA will break down in acidic environments. - Intracellular enzymes have a pH optima near 7 and denature at extracellular pH levels.

Question 53

12a. What were the conditions in Earth’s oceans four billion years ago? How would the conditions have shaped the dominant microbial metabolism at the time? List one example of those metabolic pathways that we still see in microorganisms today.

Answer 53

Cellular life started about 4 billion years ago. The early oceans were: - For the most part, Earth was anoxic - high temperatures - limited organic carbon as there was no photosynthesis. - Rich in H2S (from volcanoes) (and FeS), CO2 and H2. These conditions set boundaries on what microbial life had to be like. Bacteria and archaea diverged before photosynthesis. Anoxygenic photosynthesis. Cyanobacteria then came, combined with chlorophyll to produce oxygen. This killed a lot of the microbial life. Then came the formation of the ozone layer. Without it, life could not leave the sea as then would be bombarded with UV. So important for life to evolve on land.

Question 54

12a. What were the conditions in Earth’s oceans four billion years ago? How would the conditions have shaped the dominant microbial metabolism at the time?

Answer 54

Around 4 billion years ago, microbes would have likely used H2 as electron donor (I,e, fuel). - It formed spontaneously from H2S and H+ (acid). Have multiple sources of hydrogen and primitive hydrogenase which oxidises hydrogen and passes electrons along to elemental sulfur, this drives substrate level phosphorylation of ATP - Hydrogen based metabolism requires the fewest proteins so lower barriers to cross when acquiring energy - Likely reduced S0 (elemental sulfur) as fewer enzymes are needed than sulfate reduction. This drives oxidative phosphorylation of ATP at primitive ATPase (using primitive PMF). Microbial metabolism had to be autotrophic as there’s no substrate for them to burn apart from hydrogen, and no other carbon source; they produced acetate and methanol. -There also most likely no biological nitrogen fixation; it a complicated pathway, if there is a low demand for nitrogen then things like lightning that can fix nitrogen is sufficient.

Question 55

12a. What were the conditions in Earth’s oceans four billion years ago? List one example of those metabolic pathways that we still see in microorganisms today.

Answer 55

-One example of the metabolic pathways we still see today in microorganisms is H2 metabolism. There are many microorganisms that use H2 as an energy source such as chemolithotropic aerobic H2-oxidising bacteria e.g. Beggiatoa. There are also microorganisms that produce H2 such as purple photosynthetic bacteria.

Question 56

1.b Describe the three stages of biofilm development and at least four benefits that biofilms provide for the inhabitants. . 


Answer 56

- Biofilms are assemblages of bacterial cells attached to a surface and enclosed in an adhesive matrix secreted by the cells. - A matrix is a mixture of polysaccharides, proteins and nucleic acids. - They typically contain porous layers. 3 Stages of Biofilm development: Attachment -Adhesion of a few motile cells to a suitable solid surface -Starts with random collision of cells with a surface, resulting in attachment. -Attachment achieved through appendages (e.g. pili, flagella) or cell surface proteins. -Attachment triggers the expression of biofilm specific genes. →c-di-GMP signals switch to biofilm growth →Production of extracellular polysaccharides for matrix formation →Loss of motility Colonization - Intercellular communication, growth and polysaccharide formation - Intercellular communication also aid in biofilm formation, via concentrations of signaling molecules Development - More growth and polysaccharide - Environmental cues (e.g. oxygen depletion) may trigger active dispersal. (E.g. secretion of proteases that cleaves surface adhesion proteins).

Question 57

1b. Four benefits that biofilms provide for the inhabitants.

Answer 57

1. Self defense against phagocytosis (e.g. protozoa or macrophages), physical forces and antibiotics. - Inner layers of biofilm cells have more time to initiate stress response 2Allows cells to remain in favourable niches - Nutrient rich surfaces - Flowing areas where nutrients are replenished - Nutrient depletion creates zones of altered activity. These anoxic environments may favour certain microbes. Creates new niches. 3. Allowing cells to remain in close proximity to one another - Intercellular communications - Exchange of nutrient and genetic material 4. Maybe the default mode of growth in nature - Doesn't require much energy for the organisms, quick way to protect themselves to some extent.

Question 58

1b. Give one example of quorum sensing involvement in the development or functioning of biofilms

Answer 58

Quorum sensing= Mechanism to assess the presence and density of other microbial cells, usually of the same species. Aliivibrio fischeri - A marine bioluminescent bacterium - Luciferase expression (e.g. lux operon) is activated by LuxR - LuxR is induced by a sufficient level of N-3-oxohexanoul homoserine lactone (an AHL) - The luxI gene encodes an enzyme that makes the A.fischeri AKL - Leads to the discovery of quorum sensing. - These organisms can coordinate when they become bioluminescent. - Other organisms start making AHL, it diffuses into a cell. This activates laxR, which activates the expression of luciferases. AHL is also produces.

Question 59

2b. Why are freshwater bodies in temperature climates often stratified, and why is such stratification less common in colder climates?

Answer 59

- Most freshwater bodies are stratified in temperate climates. Stratification is influenced by temperature. - The surface is close to the atmospheric conditions. - As you go deeper, the penetration of sunlight decreases and so temperatures drop. This is not in a linear fashion though. - Over time the colder water stays at the bottom since its denser relative to warmer water. - Separated by temperature and density. As you go further and further down, the temperature drops. There are three layers: -Epilimnion (surface layer) -Thermocline (in the middle)= steep change in temperature -Hypolimnion (deeper layer). The layers are separate so transfer of material between layers normally facilitated by diffusion rather than mixing (to move between boundaries). - Epilimnion is fairly well mixed since the wind blows on the surface of the lake, well oxygenated. - But when it hits the thermocline there is a transition. - Not all the oxygen can diffused across the thermocline. - Results in a deep drop in the oxygen concentration. There is even less oxygen in the hypolimnion. So the bottom waters can experience extended anoxia. Cold temperatures - Stratification is not always maintained. - The surface before it ices, cools very fast, making it way denser than the water in a thermocline. - The cold oxygenated water sinks displacing the hypolimnion. This causes an inversion of the strata. Temperate environment - The water is not well mixed and the water is very stratified, the bottom water is often anoxic and fairly rich in nutrients. - This results in water that are not clear but cloudy. In e.g. southland, you see lakes that are clear and can see to the bottom. These lakes being in a colder area are well mixed throughout the year. They are nutrient deprived. The entire water may stay oxygenated. A pristine lake is not as productive as a habitat. - H2S increases slowly with increasing depth within the hypolimnion. - Across the strata, as oxygen decreases, temperature decreases, but concentration of H2S increases and is present in the hypolimion layer.

Question 60

2b. Describe how concentrations of O2 and H2S, as well as temperature, change across the strata. How does this stratification phenomenon affect the distribution of microorganisms? 


Answer 60

- In freshwater microbial ecology they contain both oxygenic phototrophs (e.g. algae and cyanobacteria) and oxygen consuming heterotrophs). - Primary producers largely determine the activity and diversity of heterotrophs. - Heterotrophs are relying on the primary producers to generate the carbon they consume. - So the amount and timing of the carbon produced determines the heterotrophs. - Paradoxically, primary production boom can lead to anoxia in bottom water due to respiration of excessive organic material. - Lots of algal and cyanobacteria mass being generated, they will eventually die. - When they die, it’s often because the nutrient input has been used up. - This means that boom in primary production is not being sustained. - The dead primary produced will then be respired by the heterotrophs. - Have a sudden crash in photogenetic phototrophs and a sudden boom in heterotrophs. - Heterotrophs required oxygen as they respire the primary production biomass. - These heterotrophic bacteria will use up all the oxygen, which leads to anoxia. - This kills all the metazoans that can live in oxic conditions. -The freshwater is dominated by Proteobacteria, Actinobacteria, Bacteroidetes and Cyanobacteria.

Question 61

3b. Describe pelagic waters’ characteristics as a microbial habitat. How do those characteristics affect the physiology of pelagic microorganisms?

Answer 61

Pelagic waters typically contain very little key inorganic nutrients for phototrophic organisms. E.g. N, O, Fe. - Mostly are well oxygenated. - Microbial cell levels an order of magnitude lower than freshwaters, but overall productivity still higher. - Most organisms in ocean waters are very small. This is an adaptive feature of an oligotrophic lifestyle. - They have a hard time obtaining nutrients due to low concentrations in the environment. - Want to minimize the surface area to volume. A smaller volume means organisms require less energy for cellular maintenance. But since they can rely on nutrients to diffuse across the membrane they requires more effective nutrient transport system. - These organisms have nutrient transport systems to obtain the limiting nutrients in the environment. - These transporters aren’t necessarily found in freshwater organisms.

Question 62

3b. How do coastal waters differ from pelagic waters, and why do those differences lead to oxygen depletion? 


Answer 62

Ocean is divided into pelagic and coastal waters. -Coastal waters are more nutrient rich and variable. -They contain variable salinity and have and input of terrestrial nutrients. -Have a much higher primary productivity that sometimes leads to anoxia. -Regions of oxygen-depleted waters are between depths of 100 and 1000 m. • Over wide expanses of the open ocean and coastal ocean • Associated with nutrient rich regions of high surface productivity and limited mixing • Warming of surface waters increases stratification and reduces oxygen transfer. -Cell abundance decreases with depth. Surface water contains 10-1000 times more cells than water at 1000m. Bacteria predominate above 1000 m.

Question 63

4b. Compare the geologic origins of and environmental conditions at Cold Seeps and Hydrothermal Vents and describe how their differences and similarities shape ecosystems found at these habitats. 
 Cold seeps Differences and similarities.

Answer 63

- Cold seeps occur mostly along continental margins where gases (CH4, H2S) seep through the sediments and provide energy to sustain large endemic biomass. - Metazoans e.g. tubeworms, soft coral, crabs mussels rely on symbiotic chemoautotrophic bacteria to harvest energy and nutrients from the environment. - This is because they have no way of getting nutrients on their own. Some feed on other species but the ultimate energy source come from chemoautotrophy. - The ecosystem is driven by chemoautotrophy. The differences and similarities between them shape the ecosystems found at these habitats. - They determine what type of organisms are found, what type of relationships there are between the organism and what type of environment it is, especially in terms of temperature. - Organisms living in or around hydrothermal vents are adapted to the high temperatures whereas the organism in and around cold seeps are adapted to much lower temperature. - There are different nutrients available as well which in turn determines the organisms and symbiotic relationships.

Question 64

4b. Compare the geologic origins of and environmental conditions at Cold Seeps and Hydrothermal Vents and describe how their differences and similarities shape ecosystems found at these habitats. 
 Hydrothermal vents

Answer 64

-Deep-sea hydrothermal vents are volcanic springs occurring at or near mid ocean ridges. -Depths from <1000 to >4000 m. -Seawater enters the Earths crust through openings in the sea floor (rifts) and becomes hydrothermal fluid (very hot) rich in reduced inorganic compounds (H2S, CO, H2 and metal ions). -When water comes out, it come out in 2 forms; hot vents, or out a series of channels. -The warm diffuse vents are 5-50 C while the hot vents (black smokers) are 270 to >400 C. -In deep-sea hydrothermal vents, they contain rich and diverse metazoan populations that rely on chemoautotrophic bacteria to fix carbon and harvest energy. -Some reside on the inside walls of chimneys. Some vents microbes are endosymbiotic but many are free living. Free-living hydrothermal vent microbes are dominated by chemolithotrophs especially Epsilonproteobacteria which oxidizes sulphide and sulfur using O2 or nitrate as TEA. Most are presumed to be thermophilic or hyperthermophilic and lives in the gradients between hot vents and surrounding water. A lot are free-living but some are both symbiotic and free-living.

Question 65

5b. What is Rhizobia and what benefits does it provide to/gain from its host?

Answer 65

Rhizobia - Collection of microbes that is found near the roots of legumes which is composed of nitrogen fixing alpha and beta proteobacteria - Inhabit root nodules and provide nitrogen nutrients to the host - Can also be free-living - Highly specific symbiosis - They are acquired horizontally. The plants initially grow with the microbes but then they acquired them from the environment. The plant has to be free living at some point of its life. - They provide huge benefits to the host. Benefits to Rhizobia - Leghemoglobin provides protection from O2. Microbes gain protection from oxygen. Nitrogenase is oxygen sensitive. Leghemoglobin moves oxygen away from it. - Only produced by the nodule when it’s inoculated with the correct rhizobial species.

Question 66

5b. Describe the biochemical processes that occur in root nodules and the sources of nutrients involved. 


Answer 66

- Rhibozia gets carbon substrates from the plant. - Plant makes electron donors through photosynthesis, (e.g. succinate, malate and fumarate). These organic acids then feed into the TCA cycle. - TCA cycle generates a PMF and eventually ATP. - ATP is used to facilitate nitrogen fixation, also gaining reductive power from pyruvate. - Nitrogen fixation gives you ammonia - N2 → NH3 (ammonia). NH3 is assimilated by glutamine synthetase in the plant cytoplasm. - Glutamine synthase turns ammonia into glutamine in the plant cytoplasm. This can be transported around as an organic nitrogen compound.

Question 67

6b. What is the goal of wastewater treatment? Describe the main components of a modern wastewater treatment system and three potential environmental issues created by untreated wastewater. 


Answer 67

- Wastewater includes sewage, gray water (non consumable water) and industrial wastewater. - The primary goal of wastewater treatment is to reduce nutrient and toxic material loads. This is measured in terms of reduction in BOD. Typical domestic wastewater is about 200 BOD units and industrial wastewater can reach 1500 BOD units. The goal is less than 5 BOD units. Wastewater goes first through primary treatment and then secondary treatment. Primary treatment consists of screening then sedimentation. Secondary treatment consists of anaerobic digestion and aerobic oxidation. The wastewater is then disinfected. Primary treatment - Physical separation steps to remove solid and particular materials - Outflow can still have very high BOD Secondary treatment-Anaerobic -Anaerobic degradative and fermentative reactions in sludge digesters • Breakdown of suspended macromolecules (polysaccharidases, proteases and lipases) • Soluble nutrients are removed through fermentation. First generate fatty acids, H2 and CO2. Fatty acids are then fermented by syntrophs to produce acetate. Products are consumed by archaeal methanogens to produce CH4. -Used for wastewater with very high BOD Secondary treatment- Aerobic -Aerobic degradation of organic materials in wastewater with low BOD (e.g. household wastewater) -Activated sludge • Continuous aeration. Slime forming bacteria grows and forms aggregated masses (i.e. flocs). • Flocs are settled and removed. A small amount is retained as inoculant • BOD reduction (95%) mostly through aggregation • Addition of oxygen is an energy intensive process. -Tricking filter methods • Spread wastewater on crushed rocks (about 2m thick) • Biofilms develop on rock surfaces and help oxidise organic material. Disinfection with UV, chlorination or ozone -Treated effluent to discharge.

Question 68

6b. Three potential environmental issues created by untreated wastewater. 


Answer 68

1. Pharmaceuticals - Most medicine taken is not metabolized so ends up in the water. - Synthetic estrogen creates hermaphrodite fish - Wastewater treatment aren’t necessarily designed to deal with estrogen - Oxazepam changes fish feeding behavior. 2. Cosmetics - Face scrubs micro beads (0.004-1.25 mm) absorb and concentrate chemical pollutants. - Fish often mistake these beads for food and so they ingest them thus increasing chemical pollutants in the environment and may kill the fish 3. Algal Blooms - Directly attributable to nitrogen and phosphorus nutrient pollution. - Nitrogen: animal waster and excessive fertilizer use - Phosphorus: household cleaning products - Directly linked to climate change. Increased storm frequency and intensity. Elevated temperature promotes algal growth. Most commonly Cyanobacteria create anoxia and decimate aquatic life. - caused by high nutrient content in water. - Many algae produce natural toxins. - Results in shellfish poisoning - Many toxins are concentrated through filter feeding. 4. Introduction of inorganic nutrients. - Kill organisms and disrupt the ecological niche. - Untreated wastewater would also basically kill many organisms and habitats wherever it is discharge. - Not only does it affect the environment, but also humans; diseases and deaths

Question 69

7b. Describe enrichment as a technique in microbial ecology and the three requirements for a successful enrichment attempt.

Answer 69

Enrichment is a culture dependent method. - Design a medium and a set of incubation conditions that are selective for the desired organism and counter selective for undesired ones. - Replicate (as much as possible) the conditions of the organism’s ecological niche - Must start with the right and viable inoculum (e.g. containing the organism of interest) - Hundreds of enrichment strategies exist. - Need to have some knowledge of the ecology and physiology of the culture you are trying to grow. - Cultures must be duplicated as closely as possible to the resources and conditions of the organism’s niche. It must also be replicated as much as possible. - Must start off with the right and viable inoculum (containing the organism of interest), thus the making of an enrichment culture may begin with collecting a sample from the appropriate habitat to serve as the inoculum. - Enrichment cultures are established by placing the inoculum into selective media and incubating under specific conditions. Conditions are set to be selective for the desired organism and counter-selective for the undesired ones. e.g. when growing cyanobacteria you want a medium without a lot of carbon, otherwise you will get a lot of other organisms growing instead. - Need to take the ecology of the microbe into account. When taking a sample, need to take it in a certain way e.g. taking a soil sample, letting it dry will kill some of the microbes present.

Question 70

7b. List and explain three significant shortcomings for enrichment as a technique for studying microbial ecology. 


Answer 70

Negatives - Impossible to determine true negatives - Positive outcomes do not imply ecological significance. Sometimes the dominant microbe grown is not necessarily the most important microbe in the ecology, could just be a fast grow culture or may not replicated the exact conditions form where is was taken from. - The organism of interest maybe in the sample but the resources and conditions of the laboratory culture maybe insufficient for growth. This may result in positive outcomes, which may not be a true reflection of the microbes in that environment. - The most dominant microbe growth may not be the true microbe that dominants in the environment. -Isolation of the desired organisms form an enrichment culture says nothing about the ecological importance or abundance of the organism in its habitat. A positive enrichment proves only that the organism was present in the sample/inoculum.

Question 71

8b. Describe the most-probable-number (MPN) technique and its use in microbiology. List three types of isolation procedure—which one of these is MPN most common used with?

Answer 71

MPN is based on the assumption that the last tube with growth in the dilution series has 10 of fewer cells. It is used to estimate the concentration of viable microbes in a sample by means of replicate liquid broth in ten fold dilutions. Carry out 10 fold dilutions; each tube concentration is decreased by 10-fold dilution. You keep doing dilutions until you assume you have only one colony. Isolation procedure is when a pure culture is yield (containing only one species) and begins with an enriched culture. Three types of isolation procedures are: 1. Streaking agar: doesn’t work for all microbes 2. Agar shake: useful for anaerobic microorganisms. 3. Liquid dilutions: a serial 10-fold dilution until no growth is observed. Liquid dilution is the procedure that MPN is most commonly used with.

Question 72

8b. List three ways in which the purity of a microbial isolate can be verified and why these attributes individually cannot verify the purity of a potential isolate.

Answer 72

Verifying purity 1. Cell morphology 2. Colony characteristics 3. Growth in other media (esp. media that favours potential contaminants) 4. Molecular genetic techniques (as supplemental information) These attributes individually cannot verify the purity of a potential isolate because microbes can be similar in one attribute but diff with another, therefore all ways of verification are needed. E.g. one microorganism may have similar morphology to another but their colony characteristic may be different, if only morphology was used for verification. I would be possible that the pure culture may not by the culture you wanted.

Question 73

10b. What are phylogenetic markers? List four examples of protein-coding genes that can be used as phylogenetic markers. 


Answer 73

Phylogenetic marker= is a fragment (locus) of either coding or non-coding DNA which is used in phylogenetic reconstructions, i.e. which is known to have no or predictable variation with a given species and which sequences are available for most or all species of a genus. The two ribosomal genes are organized in an operon. These RNA molecules function as RNA molecules and so are well conserved at the nucleotide level. Protein coding genes are well conserved at the nucleotide level due to degeneracy and wobbly codons. If you don't have good conservation at the nucleotide level, wont be able to design good PCR primers. Examples of protein coding genes: Can take a gene and use it to tell what metabolic process is present. May not be a true representation of all the microbes using that process as PCR is very sensitive. narG - Metabolic process: Denitrification - Encoded enzyme: Nitrate reductase. nifH - Metabolic process: Nitrogen fixation - Encoded enzyme: Nitrogenase amoA - Metabolic process: Nitrification - Encoded enzyme: Ammonia monooxygenase apsA - Metabolic process: Sulfate reduction - Encoded enzyme: Adenosine phosphosulfate reductase.

Question 74

10b. Describe the differences between rRNA genes and the ITS region in terms of their use as phylogenetic markers.

Answer 74

16S and 23S ribosomal RNA is for bacteria, - They are joined together with the internal transcribed spacer (ITS). It basically links up the two genes. - The entire operon is transcribed as one thing and then the ITS gets chopped out. ITS does not serve any function. - Since it is not used, there is no evolutionary pressure to conserve the sequence. So the sequence and its length can be variable because there is no function and no pressure to keep it stable. - ITS can vary between strands of the same species. E.g., E. coli there is lots of strands, some pathogenic, some harmless. Can use ITS to distinguish between them which will have identical 16S ribosomal RNA. - Use 16 S to get a high level over view. - To get fine resolution, use ITS. - Since ITS is not functional, its not conserve in any way. So can’t use it to predict what organism it is. ITS (internal transcribed spacer) - Non-functional, variable length - Useful phylogenetic marker for subspecies level resolution of bacterial diversity.

Question 75

11b. Describe how DNA is sequenced using the Sanger technique. How are the four nucleotides (A, T, C, and G) distinguished, and what specific attribute of the technique allows only one nucleotide to be read at a time?

Answer 75

Sanger Sequencing - Fluorescently labeled dye terminators - Analyzed using capillary electrophoresis and bases are read by fluorescence detector automatically - One sequence at a time. - DNA is first separated into two strands. The strand that is to be sequenced is copies used chemically altered bases. - These altered bases cause the copying process to stop when a particular letter is added into the growing chain. - This process is carried out for all 4 bases and then the fragments are put together like a jigsaw to reveal the sequence of the original DNA. - Regular dNTPs are mixed with fluorescently terminal labeled dye terminators. - For a DNA strand to keep growing need to have a 3’OH group. - With the dye terminators, we attach a dye to that position instead so you can’t add another nucleotide instead. - The ddNTP is specific to one nucleotide and will stop elongation when that nucleotide is incorporated in the growing chain, thus forming different sized fragments of DNA. - Each base has a different colour. As this travels through a capillary in a DNA sequencer this is read by a laser and fluorescent detector This method limits you to sequencing one sequence at a time. - Wouldn’t work if you have two sequences as you will have a point where there are 2 peaks from two bases. Won’t be able to tell what base you have. - This can only be done one nucleotide at a time because if more than one nucleotide was used, then more than one ddNTP would be used, thus wrong fragments that doesn’t match the sequence of the original DNA. Next generation sequencing - High throughput (3 to 3000 million reads per run) - Read length approaching or exceeding Sanger sequencing (as long as 30 Kb per read) - Simple sample preparation (<24 hours by a single technician) - Low cost (as low as $5 per GB) - Requires small amounts of DNA (<1ug)

Question 76

11b. Contrast these with one of the next-generation sequencing platforms (i.e., Illumina, Ion Torrent, or PacBio). 
 Ion Torrent Sequencing

Answer 76

Ion Torrent sequencing technology - Semiconductor-based, non-optical detection of nucleotide incorporation - Detect chemical signals associated with nucleotide incorporation - Low cost instrument and reagents. - Ion chips have many wells covering those pixels. The wells captures chemical information from DNA sequence signals and translate it into digital information. - The sequencing process starts when DNA is cut up into millions of fragments each fragment then attaches to its own bead and it copied until it covers the bead. - This automated process covers the different beads with the different sized fragments. These beads flow across the chip each depositing in a well. - The wells are flooded with one of the 4 DNA nucleotides. - Whenever a nucleotide is incorporated into the single stranded DNA, a hydrogen ion is released. - This is how the ion torrent sequencing sequences DNA by reading this chemical change directly on the chip. - The hydrogen ion changes the pH of the solution in the cell. - An ion sensitive layer beneath the well measures that change in pH and converts it to voltage. This voltage change is recorded indicating which nucleotide is incorporated. Each well works as the smallest pH meters. - If a nucleotide is washed over and its not complementary to the strand, its not incorporated, no change in pH or voltage. - If there are two identical bases next to each other, two nucleotides are incorporated and the voltage doubles, recording two bases added. - This process happens simultaneously in million of wells. Doesn’t matter if you have chips with a million wells or a billion, the sequencing process occurs in a few hours.

Question 77

12b. Describe in detail two ways in which analyses of stable isotopes can be used to study microbial ecology. 
 Stable isotope probing

Answer 77

Not all isotopes are radioactive. E.g. 95% of carbon in nature is 12C, 5% is 13C and very little is 14C. Only 14C is radioactive. Isotopes are metabolized different by microorganisms. - Enzymes typically prefer lighter isotopes - Therefore, biologically fixed carbon is depleted in 13C (compared with inorganic carbon) and shows isotopic fractionation. Stable Isotope Probing - Add substrates containing less common isotopes (i.e. 13C, 15N, 18O) and identify organisms that have incorporated these isotopes into cellular material. - Use substrate to specify the pathway of interest. - Typically used to reveal the diversity behind specific metabolic transformations in the environment - Coupled with molecular genetic analyses. - Particularly useful for studying a specific pathway in a metabolically diverse community (e.g. methane consumption in forest soils). - Stable isotopes are typically heavier. If it’s the only thing available, organisms will use it. - If you add stable isotopes in the form on carbon substrates (e.g. CO2, ammonia), even though biological systems don't like it, if it’s the only thing available, they will use it. - SIP reveals microbial diversity by yielding isotope-labelled DNA that can be used to analyse specific genes or the entire genome of the organism(s) that consumed that labelled substrate. - SIP uses substrates to specify the pathways of interest. When coupled with molecular genetic analysis SIP is particularly useful for studying a specific pathway in a metabolically diverse community (e.g. methane consumption in forest soils). Example - We have an environmental sample, we take out all the air/substrate and replace it with 13C labeled glucose. Only the cells involve for example in methyltrophy. - If you only want to find the methylotrophs, you feed then 13C labeled methane. Only the methylotrophs will take up the 13C methane. You incubate it for a bit. You then extract the final DNA sample and put into a fast centrifuge. - Spinning for a few days, will eventually be able to separate the DNA bands on the basis of stabilized content. 12C DNA gets separated from the 13C DNA due to different weights. - The only things that can be present in the 13C DNA fraction are the things that can use the substrate added. This is called stabilize isotope probing. By looking at the fraction, you know which organisms used the substrates added, and the other fraction contains things that are dead or are not involved in the pathway you are looking at. - Can then remove and analyze (PCR 16S rRNA or metabolic genes or do genomics)

Question 78

12b. Describe in detail two ways in which analyses of stable isotopes can be used to study microbial ecology. 
 Isotopic Fractionation

Answer 78

- The two elements most useful for stable isotope studies in microbial ecology are carbon and sulfur. Carbon exists as 12C, 95% abundance, and 13C, 5% abundance. - Sulfur has four stable isotopes, the dominant isotope is 32S, some sulfur is found as 34S and very small amounts of 33S and 36S. - The relative intensities of these isotopes change when C or S is metabolised by microorganisms because they typically favour lighter isotopes. - The isotopic composition of a material can reveal its past biological or geological origins. - Methane produced by methanogenic archaea is isotopically extremely light, indicating that methanogens discriminate strongly against 13CO2 when they are reducing CO2 to CH4. - By contrast, carbon in isotopically heavier marine carbonates are clearly of geological origins. - Therefore, biologically fixed carbon is depleted in 13C (compared with inorganic carbon) showing isotopic fractionation.