EXAM 3 MMG 220

Efforts to reduce phosphate pollution in freshwater lakes have been found to have the unintended consequence of increased coastal/estuary pollution. Why?

(A) High phosphorus loading to lakes leads to the assimilation of large amounts of both nitrogen (N) and phosphorus (P) by algal blooms. N fixation ensures that the algae can take advantage of all available phosphorus. Export of algal tissues from the surface to the lake bottom leads to the burial or denitrification of assimilated N. Under these conditions, little N is exported from the lake to downstream rivers. (B) The results reported by Finlay et al. demonstrate that as phosphorus inputs decline, algal growth and nitrogen assimilation also decline, and the biological pumping of nitrogen into lake sediments slows down. Without high rates of assimilating and sinking together with burial or denitrification, more nitrogen remains in the surface waters of these clearer lakes. This unused nitrogen is then exported downstream. Which then reaches the marine environment causing an extreme bloom

Chemical transformations within the nitrogen and sulfur cycles are carried out primarily by bacteria and archaea. What limited roles do plants and fungi have in these two cycles?

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Cyanobacteria carry out photosynthesis, carbon fixation and nitrogen fixation. However, all of these processes do not co-exist in the same cell. How and why do cyanobacteria segregate these processes.

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The gases primarily responsible for global warming are H2O and CO2. Despite their relatively low concentrations in the atmosphere, CH4 and N2) also have a significant impact. Why?

CH4 and N2O are better at trapping radiation and heat thus contributing to the warmin

Carbon fixation is obviously essential to biotic carbon cycle. How do the microbial microcompartments termed carboxysomes contribute to carbon fixation?

Calvin Cycle is the most prevalent microbial carbon-fixation pathway. In cyanobacteria, some nitrifying bacteria and thiobacilli (sulfur-oxidizing chemolithotrophs) Calvin cycle is associated with inclusions termed carboxysomes. Carboxysomes are microcompartments that function to sequester enzymes involved in CO2 fixation (RuBisCo, carbonic anhydrase). These compartments concentrate carbon dioxide to overcome the inefficiency of RuBisCO - the predominant enzyme in carbon fixation and the rate limiting enzyme in the Calvin cycle. These organelles are found in all cyanobacteria and many chemotrophic bacteria that fix carbon dioxide.

The Pacific Biosystems SMRT sequencing platform is able to perform real time sequencing on single molecules. How have zero-mode waveguides and unblocked fluorescently-tagged dNTPs allowed for single molecule sequencing?

DNA sequencing is performed on SMRT chips, each containing thousands of zero- mode waveguides (ZMWs). Utilizing the latest geometries available in semiconductor manufacturing, a ZMW is a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate. Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zeptoliters (10-21 liters). At this volume, the activity of a single molecule can be detected amongst a background of thousands of labeled nucleotides. The ZMW provides a window for watching DNA polymerase as it performs sequencing by synthesis. Within each chamber, a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume. Phospholinked nucleotides, each type labeled with a different colored fluorophore, are then introduced into the reaction solution at high concentrations which promote enzyme speed, accuracy, and processivity. During this time, the engaged fluorophore emits fluorescent light whose color corresponds to the base identity. Then, as part of the natural incorporation cycle, the polymerase cleaves the bond holding the fluorophore in place and the dye diffuses out of the detection volume. Following incorporation, the signal immediately returns to baseline and the process repeats. Due to the small size of the ZMW, even at these high, biologically relevant concentrations, the detection volume is occupied by nucleotides only a small fraction of the time. In addition, visits to the detection volume are fast, lasting only a few microseconds, due to the very small distance that diffusion has to carry the nucleotides. The result is a very low background.

Dimethyl sulfide is a volatile degradation product of the microalgal osmolyte DMSP. What is the potential impact of this gas on warming in the Arctic ocean?

Following the release of (DMSP) from the phytoplankton that produce it, the compound is subjected to bacterial degradation by two general pathways. The first of these, the demethylation pathway, leads to the production of (MMPA), whereas the cleavage pathway yields dimethyl sulphide (DMS) and either acrylate or 3-hydroxypropionate (3HP). The emitted DMS can then be further transformed by DMS-consuming bacteria or released into the atmosphere, where it can be converted to dimethyl sulphoxide (DMSO) or sulphate aerosols. These can act as cloud condensation nuclei (CCN), leading to an increase in (the amount of sunlight reflected back into space). A major step in the global sulphur cycle is the return of these compounds to the Earth's surface, via rain or snow, thus transferring the element from sea to land. DMS is also important as a chemoattractant for zooplankton, seabirds and marine mammals

What types of questions might be addressed by combining stable isotope probing (SIP) with metaproteomic analysis?

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What is the purpose of each of the three basic types of nitrate reduction (denitrification)? What are the end products of each type?

1. Assimilatory Reduction: nitrate (NO3-) reduction to ammonium and incorporation into biomass 2. Nitrate Respiration: : mixture of gaseous end products (including N2, N2O) 2NO3- + 5H2 +2H-> N2 + 6 H2O 3. Dissimilatory Nitrate Reduction to Ammonium (DNRA) NO3- + 4H2 + 2H -> NH4 + 3H2O The substrates for nitrous oxide (N2O) production, ammonium (NH4+) and nitrate (NO3−), enter soils in various forms. Atmospheric dinitrogen (N2) can be deposited in the soil following fixation by soil microorganisms and is subsequently converted to NH4+; alternatively, reactive forms (mainly NO3 and NH3) can be deposited in precipitation or as dry deposition. Sources of N2O, including fixed N2, can also be released from organic residues from plants and animals, animal waste and nitrogen fertilizers. The major source of anthropogenic substrate is agricultural application of nitrogen fertilizers and manure. In soil, a considerable amount of NH4+ is used by plants and microorganisms, and the remaining portion is transformed into NO3− by NH3-oxidizing bacteria and archaea through nitrification. Most NO3− is converted into N2 via various nitrogen oxides (including N2O) by denitrification processes (carried out by denitrifying bacteria), and these then escape in the atmosphere. Some nitrate is leached into the groundwater, and some is used by plants.

In contrast to pyrosequencing which uses untagged and unblocked dNTPs, Illumina sequencing by-synthesis uses fluorescently-tagged, 3'-blocked dNTPs. How are these dNTPs used in Illumina sequencing?

1. Randomly fragment genomic DNA and ligate adapters to both ends. 2. Bind single-stranded fragments to the inside surface of the flow cell channels. 3. Add unlabeled nucleotides and enzyme to initiate solid-phase bridge amplification. 4. The enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate. 5. Denaturation leaves single-stranded templates anchored to the substrate. 6. Several million dense clusters of single-stranded DNA are generated in each channel of the flow cell. 7. First chemistry cycle: to initiate the first sequencing cycle, add all four labeled reversible terminators, primers and DNA polymerase to the flow cell. 8. After laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. Record the identity of the first base for each cluster. 9. Second chemistry cycle: to initiate the next sequencing cycle, add all four labeled reversible terminators and polymerase to the flow cell. 10. After laser excitation, collect the image data as before. Record the identity of the second base for each cluster. 11. Repeat cycles of sequencing to determine the sequence of bases in a given fragment a single base at a time. 12. Align data, compare to a reference, and identify sequence differences. Each fluorescence signal originates from a clonally amplified template cluster.

Anammox bacteria are able to generate energy by using ammonia as an electron donor and nitrite as an electron acceptor. How does the anammoxosome enable these bacteria to generate ATP?

A model for anammox biochemistry, which is dependent on the unique type of compartmentalization in these planctomycetes. The catabolic anammox reactions coupled over the anammoxosome membrane result in electron transport via cytochrome c and consequent translocation of protons from the pirellulosome to the anammoxosome. The resulting proton gradient generates a proton-motive force that drives subsequent ATP synthesis via the membrane-embedded ATPase. bc1, cytochrome bc1 complex; Hao, hydrazine-hydroxylamine oxidoreductase; Hh, hydrazine hydrolase; Nir, nitrite reductase; Q, coenzyme Q. Experimental evidence for the anammoxosome compartment of anammox bacteria being a dedicated energy generator Use ammonia to generate energy

The conversion of short chain fatty acids (such as propionate and butyrate) to acetate is an endothermic reaction (+delta G) under standard conditions. How are acetogens able to produce energy from these reactions?

Acetogens oxidize low molecular weight fatty acids (ethanol, butyrate, propionate, fermentation end products) to H2, CO2 and acetate. These reactions are thermodynamically unfavorable under standard conditions (+delta G). These reactions are thermodynamically possible only if methanogens remove H2 as it is produced (resulting in a partial pressure of H2 of less than 1/1000 atm). Acetogens key trait: produce acetic acid via Wood-Ljungdahl pathway relies on acetyl-CoA synthase for CO2 fixation AND in terminal electron-accepting energy conservation.

Anaerobic oxidation of ammonia (Anammox) is carried out by a subset of bacteria that reside within the phylum Planctomycetes. What are some of the rather bizarre characteristics of the organisms within this phylum?

Anaerobic Ammonium Oxidation A unique autotrophic metabolism that includes the ability to oxidize NH4+ to N2, using NO2- as an electron acceptor, with the accompanying reduction of CO2 Anammoxosome: consists of a single bilayer membrane across which a PMF is generated (may be functional equivalent of mitochondrion as both organelles generate a non-photosynthetic PMF). This is the organelle-like structure in which the energy-generating process involving the combination of ammonia with nitrite takes place. The anammoxosome membrane, which consists of the ladderane lipid bilayer, and the anammox reaction pathway. Intermediates in the cycle are hydrazine (N2H4) and hydroxylamine (NH2OH), which are highly toxic. The dense, impermeable membrane may serve to contain them. lack peptidoglycan cell wall actually made of glycoproteins lipids->Ladderane has both ester and ether linkages functions to generate ATP Anammox Planctomycetes: major contributors to the global nitrogen cycle. Estimated that 50% of the nitrogen molecules in the atmosphere have been generated by these organisms. Planctomycetes: Bacterial phylum that possesses distinct features: a) Intracellular compartmentalization b) Lack of peptidoglycan c) Membrane-bound nucleoid composed of an envelope with two membranes (genus Gemmata), analogous to eukaryotic nucleus

Methanotrophs are the "gatekeepers" of methane reservoirs that are sequestered in ocean sediments. Yet in many (if not most) cases they can't do the job alone. How do sulfate reducing bacteria function in the metabolism of methane?

Anaerobic methanotrophic archaea (ANME) oxidizes the methane in a sequence opposite to that of methane formation in methanogenesis. An unknown intermediate is exchanged between the ANME and sulphate reducing bacteria (SRB). The sulphate reducers act in a relationship that ultimately allows the carbon in methane to be oxidized to CO2 and the electrons generated by that reaction reduce sulfate (SO42-) to hydrogen sulfide (H2S) AOM: Anaerobic Oxidation of Methane; 'gatekeepers' of methane reservoirs sequestered in ocean sediments AOM carried out by uncultured archaeal (ANME)-bacterial (SRB) syntrophic communities ANME are responsible for the removal of up to 25% of total global, biologically produced methane Anaerobic methanotrophic archaea (ANMEs) oxidize methane (CH4) and convert it to CO2 and water, in cooperation with sulphate-reducing bacteria (SRB), which convert sulfate (SO42−) to hydrogen sulfide (H2S).

What is emulsion PCR? What is bridge PCR? What is the common objective of both of these techniques? (in addition to simple template amplification)

Emulsion PCR: The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Each bead carries a unique single-stranded DNA library fragment. The bead-bound library is emulsified with amplification reagents in a water-in-oil mixture resulting in microreactors containing just one bead with one unique sample-library fragment. One Fragment = One Bead. Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results in a copy number of several million per bead. Subsequently, the emulsion PCR is broken while the amplified fragments remain bound to their specific beads. The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing Bridge PCR: DNA is fragmented and then ligated with adaptors; DNA is attached to the surface; unlabeled nucleotides and enzymes were added to initiate solid phase bridge amplification; double stranded bridges are built on the surface; denaturation leaves single stranded DNA attached to the surface; several million dense clusters of double stranded DNA are generated in each of the flow cell

The overall impact of rising temperatures on the net production of greenhouse gases from terrestrial environments is exceedingly complex. One potential result of climate change may be an alteration of local precipitation. If drought conditions were to lower the water table in a wetland or peatland environment, what would you predict to be the most likely impact on greenhouse gas emissions?

Key biological processes in the carbon cycle of permafrost environments: Permafrost thawing at the transition zone introduces previously unavailable organic matter into the expanded active layer of soil. Enzymatic hydrolysis decomposes complex organic matter into soluble substrates for microbial fermentation, producing a mixture of organic acids, alcohols and microbial biomass. Methanogenic archaea convert acetate, methylated compounds or H2 and CO2 into CH4 that can be released to the atmosphere through ebullition, diffusion or aerenchyma. Methanotrophs oxidize some of this CH4, converting it to CO2.

While hydrogen, oxygen, nitrogen, carbon, phosphorous and sulfur comprise ~96% of cell mass, several transition state metals are also essential to life. What types of functions do metals have within cells? Why are transition state metals thought to represent a link between contemporary life forms and the earliest events in the evolution of life on Earth?

Metals are required as cofactors in approximately 2/3 of all enzymes; microbially mediated processes that maintain the biosphere require the types of metallic micronutrients; without inclusion of the metals within the enzymatic structures, the catalysts would be ineffective. May represent a link between inorganic components of the geosphere and evolution of life to contemporary biosphere function Strong links between inorganic components of the geosphere and the evolution of life [from the prebiotic earth to the iron sulfur world to the RNA and DNA worlds] through to contemporary biosphere function. The presence in microorganisms of element-specific, genetically encoded transport mechanisms further confirms the biological and evolutionary significance of the targeted elements. Well characterized nutrient uptake transport systems in prokaryotes include those for K+ Mg2+ Fe3+ Mn2+ Zn2+ Na+ PO43- & SO42- Eliminate toxicity by transforming the element or by mobilizing the element away from the cell; detoxification mechanisms are manifest as efflux pumps, reduction and/or methylation.

What are the principal steps in the recycling of complex organic matter (such as proteins, polysaccharides, an lipids) into inorganic components (such as C02) within an anaerobic environment such as soil and sediments? Why do some anaerobic environments generate CH4 as well as CO2?

Proteins, polysaccharides, and lipids are broken down into simpler compounds: amino acids, fatty acids, and sugars by enzymes in a process called hydrolysis. The products of hydrolysis are further broken down by acidogenic bacteria into ammonia, carbon dioxide and hydrogen sulfide, in a process called acidogenesis. The molecules created through the acetogenesis phase are further digested by acetogens to produce largely acetic acid as well as carbon dioxide and hydrogen. Methanogens utilize the intermediate products of the preceding stages and convert them into methane, carbon dioxide and water.

Purple sulfur and green bacteria occupy habitats in which they have access to both sunlight and H2S. Why?

Purple Sulfur Bacteria: Phototrophy Coupled to H2S, S0 and H2 Oxidation (primarily anaerobic photolithotrophs) Cyclic photophosphorylation Green Sulfur Bacteria: Phototrophy Coupled to H2S, S0 and H2 Oxidation (primarily anaerobic photolithotrophs)

What is assimilatory sulfur reduction?

Sulfate is fully oxidized, thus it serves as the terminal electron acceptor in anaerobic respiration, termed dissimilatory sulfate reduction. Alternatively, plants and microbes can donate the S atom to organic compounds during assimilatory sulfate reduction. H2S is fully reduced, so it can serve as an electron donor during chemo- and photolithotrophy. Because elemental sulfur and thiosulfate are neither fully oxidized nor reduced, they can serve as either electron donors or acceptors. The sulfur cycle is similar to the nitrogen cycle in that, depending on the oxidation state of the sulfur species, it can serve as an electron acceptor, electron donor, or both. Assimilatory Reduction: plants and microbes can donate the S atom to organic compounds

Why are H2S oxidizing bacteria essential for the maintenance of manatee habitat?

Sulfide-driven coevolution: tripartite mutualistic interactions among seagrasses, lucinid bivalves and sulfide oxidizing bacteria in their gills generate a higher fitness of all species involved under sulfidic conditions.

Microbes comprise >90% of the ocean biomass, and thus lytic viruses (phage) are likely to have a significant impact on carbon cycling. What are the likely fates of the particulate organic matter (POM), dissolved organic matter (DOM) and recalcitrant dissolved organic matter (RDOM) that are released from microbial cells by viral lysis? What is the relevance of the stoichiometry of carbon, nitrogen, and phosphorous in each of these carbon pools (POM, DOM, RDOM) to carbon cycling in the ocean?

The biological pump is a process whereby CO2 in the upper ocean is fixed by primary producers and transported to the deep ocean as sinking biogenic particles POM or DOM. The microbial loop is a pathway in the aquatic food web whereby DOM is taken up by bacteria and archaea, which are consumed by protists, which are in turn consumed by metazoans. The viral shunt reflects virus-mediated lysis of microorganisms, which returns the POM to the DOM pool. The proposed microbial carbon pump is a conceptual framework for understanding the role of microbial processes in the production of RDOM. RDOM can persist in the ocean for millennia and is therefore a reservoir for carbon storage in the ocean. Three major pathways have been identified in the microbial carbon pump: direct exudation of microbial cells during production and proliferation (path 1); viral lysis of microbial cells to release microbial cell wall and cell surface macromolecules (path 2); and POM degradation (path 3). The viral shunt moves material from heterotrophs and photoautotrophs into particulate organic matter (POM) and dissolved organic matter (DOM). In this process there is a stoichiometric effect, such that the chemical composition of the POM and DOM pools are not necessarily the same as the composition of the organisms from which the material was derived. Highly labile materials, such as amino acids and nucleic acids, tend to be recycled in the photic zone, whereas more recalcitrant carbon-rich material, such as that found in cell walls, is probably exported to deeper waters. Thus, the material that is exported to deeper waters by the viral shunt is probably more carbon rich than the material from which it was derived. This would increase the efficiency of the biological pump. The numbers in parentheses are the estimated ratios of carbon:nitrogen:phosphorous (in atoms).

Oxford Nanopore has developed a single molecule sequencing technique that does not require DNA synthesis. What is the basic principle that allows this platform to read DNA sequences?

Trace of the ionic current amplitude through an alpha-hemolysin pore. Nanopore is inserted into a membrane created by a synthetic polymer, it has very high electronic resistance. Single molecules that enter the nanopore causes disruptions in the current, measuring the disruptions the molecule can be identified. In strand sequencing an intact DNA strand is sequenced as it passes through the nanopore. An enzyme unzips the DNA and feeds it through the nanopore. A characteristic disruption of the current is indicated by the presence of a particular combination of bases. So specific that it can be used to determine the order of DNA bases on that strand. (DNA sequence information is obtained by changes in the ionic current running along the DNA through the nanopore, which occur as the DNA is ratcheted through the pore by the polymerase.) Hairpin structure allows it to read both side of the strand, the sense and anti-sense strands.

Fermenter-methanogen syntrophy

a type of H2-coupled syntrophy, in which H2 is produced by fermenters and consumed by methanogens. Fermenters produce H2, when intracellular substrates for fermentation are exhausted; electrons are then transferred from reduced coenzymes to H+ to yield H2. However, this reaction is endergonic and tends to be reversed under natural conditions, leading to the accumulation of reduced coenzymes and resulting growth inhibition. Methanogens benefit from fermenter-produced H2 as an energy source, resulting in the alleviation of the H2-inhibition on the fermenters. Organics are converted to methane by the syntrophy.

methanogenic environment

an anoxic environment in which organic matter is degraded and protons and CO2 act as the main electron accpetors. This is an energetically limited environment in which alternative electron acceptors are absent and conditions are close to thermodynamic equilibrium.

In principle, why does proteomic (and in particular, metaproteomic) analysis rely on previously established genomic or metagenomic databases?

mass spec gives you a max of 9 amino acids, this isnt enough proteomic peptide, if you translate the entire genome and then you can find the 9 amino acids in a certain protein

The Ion Torrent sequencing machine is basically a pH meter. How does one sequence DNA with a pH meter?

semi-conductor chip, the wells capture chemical information from DNA sequencing and translate it into digital information. DNA is cut up into fragments and then is copied using an emulsion bead producing millions of fragments. Then each bead goes into a well and is flooded with one of the four DNA nucleotides. When ever a nucleotide is incorporated into a single strand of DNA a hydrogen ion is released. Ion torrent sequences DNA by reading the chemical change derived directly on the chip. The hydrogen ion directly alters the pH. The nucleotide added is changed every 15 seconds and the pH is monitored thus the sequence can be determined. The Ion Personal Genome Machine™ (PGM™) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded and no base will be called. If it is a match, a hydrogen ion is released and the pH is adjusted.

How has single cell sequencing aided in the development of therapeutic drugs from microbial secondary metabolites?

single cell sequencing allows you to sequence genomes to find genes able to make the therapeutic drugs then you can tranfer it to another organism and have it make the product you want

syntropy

syntrophy: A nutritional situation in which two or more organisms combine their metabolic capabilities to catabolize a substrate that cannot be catabolized by either one of them alone. The Gibbs free energy changes involved in syntrophic metabolism are very low, close to the minimum free energy change needed to sustain microbial growth

Thiomargarita nambiensis is currently the record holder for prokaryotic cell size (by volume). How does it overcome the apparent diffusion limitations on cell size? In the case of this sulfur oxidizing bacterium, what is the advantage of being so large?

the organism is shown suspended in sediment with high concentrations of sulfide. Under such conditions, sulfides are stored in sulfur inclusions and can be metabolized for energy. the sediment resuspensions expose the organism to nitrates which are also stored in cell vacuoles