Exam 2

John Martin The Iron Hypothesis

"Give me a half a tanker of iron and I will give you another ice age"

Keller et al. (2013) Study Design

#1: 4 days of vancomycin colon wash and fecal donation #2: 4 days of vancomycin, no wash or donation #3: 4 days of vancomycin, colon wash, but no donation

Temp and growth rate

(from low to high) -psychrophiles -psychrotrophs -mesophiles -thermophiles -hyperthermophiles

Chlamydomonas nivalis (alga)

(in ice-looks red) Carotenoid pigment protects chloroplast Absorbs heat Liquifies snow

Multicellular algae come in different shapes

- Spirogyra forms long multicellular filaments. -Volvox forms colonies of cells. -Ulva forms large sheets of cells in two layers. -Cymopolia forms calcified stalks of cells.

Oxidation-Reduction Reactions

-(i.e. "redox" reactions) -•Energy generated by moving electrons (e-) from stronger e- donors to stronger e- acceptors -•Redox couple =NAD+ and NADH

Temperature limit

--80% of Earth experiences temperatures below 5°C --Temperature limit for life ~ -20°C

Protists in freshwater systems

--Alveolates •Ciliates -Cilia used for: ---Propulsion ---Food acquisition --Dinoflagellates ---Phototrophs and predators ---Secondary/tertiary endosymbionts

Biosynthesis and pathogenesis

--Legionella pneumophila --seen in lungs

Green Bacteria Phototrophy

--Light absorption by bacteriochlorophyll excites 2 electrons to higher energy state. --Subsequent e- movement to NAD+ results in NADH and proton gradient for ATP.

Dark reactions: Fixation of Carbon Dioxide

--Light-independent (aka "dark") reactions - Calvin cycle. --Running cycle requires lots of energy input - both ATP and NADPH. --How do phototrophs generate these energy sources?

Phototrophic Microbial Mats

--Microbial mat, Elkhorn Slough, CA --Cyanobacteria & diatoms --Purple bacteria --Green bacteria --Sulfate reducers --Exopolysaccharides give the layers a mat-like consistency.

Shelled Amebas

--Radiolarians -Shells (tests) made of silica --Foraminiferans -Shells made of CaCO3 -Laid down in helical succession -Shells make up reefs, beach sand, sedimentary rock, units for geological analysis

Purple Bacteria Phototrophy

--Wavelength picked up by this bacteriochorophyll very long = lower energy --Can pump protons via ETS, making ATP --Electrons from donor (Bchl) get returned to Bchl - cyclic phototrophy --NADH generated by using ATP to pump electrons against energetic gradient --ATP diversion to reduce NAD+ results in slow growth relative to green bacteria.

Water as a Microbial Habitat

--What are most of the microbes? •Important chemical/physical factors -dissolved oxygen content -temperature -pH -light penetration

CO2 fixation: Reverse TCA

--acetyl phosphate is a global signal of the nutritional environment --excreted until needed for further catabolism --Requires less energy to run than Calvin Cycle

1. Carbon source: Different trophic groups

--autotroph (CO2): require nutrients from other source --heterotroph (organic): usually do not require nutrients from other source

Partitioning the Light Spectrum

--chlorophyll a: Cyanobacteria --bacteriochlorophyll: purple and green bacteria

E. coli catabolizes lactose

--lactose metabolism in E.coli

Barophile

--optimal growth: 500-600 atm --growth range: down to 1 atm --depth found: 5-6km

extreme barophile

--optimal growth: 700-800atm --growth range: only above 700 atm -depth found: down to 11 km

Do SS Also Help Aphids?

-Acyrthosiphon pisum--pea aphid --aphidius ervi--parasitic wasp --ss effects on Parasitoid Larvae more eggs in mean number found in H. defensa

Quorum Sensing (QS)

-Along with external sources, signals controlling gene regulation can be generated by bacteria. -Autoinducer made by cell diffuses freely through membrane -low concentration of autoinducer; no gene expression -high concentration of autoinducer inside bacteria: gene expression activated

Primary Endosymbiotic Algae

-Chlorophyta (green algae) -Rhodophyta (red algae) -Cell wall is made of cellulose and glycoproteins -Contractile vacuole removes excess water -Pyrenoid for CO2 fixation -Starch bodies for energy

Red Algae (Rhodophyta)

-Deep waters -Unicellular, filamentous, multicellular forms -Agar and agarose -Human food sources: Porphyra -Coralline algae: base of coral reefs

Secondary endosymbiotic algae

-Diatoms -Unicellular -Shell = frustule -"Diatomaceous earth" -Coccolithophores -Shell = CaCO3 -Brown algae (kelps) -Provide habitat for food web

fungal

-Ectomycorrhizal fungi (ECM) -Arbuscular mycorrhizal fungi (AMF)

Chemotrophy makes ATP and stores energy in Ion Gradients: ETS

-Electron transport systems: involve the movement of electrons that results in pumping of protons (H+) across cell membrane. -Flow of H+ back in to the cell has many functions, including ATP synthesis, flagellar rotation, drug pumps, and sugar uptake.

Two-component complex

-Fe protein -FeMo protein

Keller et al. (2013) Study Results

-Fecal transplant much more effective than vancomycin. -Apparent mechanism: restores microbial diversity. -Trial stopped because considered unethical to keep some patients on only antibiotics!

Lichens dominate in extreme environments

-Hold moisture -Protect seeds -Prevent erosion -Input N

Protection Mechanism III: biochemical

-Leghemoglobin: -Globin portion provided by the plant, heme portion by the bacteria -Similar to hemoglobin in animals, but has a higher oxygen affinity.

Methicillin-resistant Staphylococcus aureus (MRSA) Resistance

-Methicillin absent -Methicillin present and binding -methicillin destroyed

c) Endophytes grow within plants

-Neotyphodium coenophialum -Dichanthelium lanuginosum with Curvularia protuberata with CThTV

Energy Acquisition Summary

-Organotrophy: organic compounds donate electrons ----fermentation: catabolism (electron acceptor:organic)(systems for energy acquisition-glycolysis and other catabolism) ----organic respiration (catabolism with inorganic or small organic electron acceptor)(electron acceptor: O2, and NO3-, SO42-, Fe3+, prother) (systems for energy acquisition: glycolysis and other catabolism TCA cycle, electron transport system) -Lithotrophy: inorganic compounds donate electrons ----lithotrophy or chemoautotrophy (electron acceptor: O2 or NO3-) (systems for energy acquisition: electron transport system) ----methanogenesis (electron acceptor: CO2)(systems for energy acquisition: methanogenesis) -Phototrophy: light absorption provides electrons ----photoautotrophy: light absorption drives CO2 fixation (systems for energy acquisition: Photosystem I or II) ----photoheterotrophy: catabolism with light absorption (examples of energy-yielding reactions: photolysis of H2S, HS-, or small organic molecules, light-driven H+ pump or Na+ pump) (systems for energy acquisition: photosystem I or II, Bacteriorhodopsin or proteorhodopsin)

Controls over biosynthesis

-Regulation --Catabolite repression -Secondary products --Antibiotics -Genome degeneration --Parasites

Controls over biosynthesis

-Regulation -Catabolite repression -Secondary products -Antibiotics -Genome degeneration -Parasites -Spatial arrangement -Carboxysomes

Direct Interaction with Regulatory Proteins

-Repressors: --scenario 1: repressors bind the DNA and represses target gene; inducer causes repressor to release --scenario 2: respressor-corepressor complex binds DNA and represses target gene -Activatrs: --activator binds ligand; complex binds the regulatory sequence and activates target gene; removing ligand stops transcription -then target gene is inactivated

The Transcription Complex

-Sigma factor (σ): protein that helps RNA polymerase recognize and bind to promoter. -RNA polymerase

Example: Temperature Response

-Sigma factor H needed to turn on heat-shock genes in E. coli. -Controls on Sigma H: At lower temperatures, Sigma H that is produced is targeted for degradation. -At higher temperatures greater production to due higher translation.

Coral mutualisms

-Symbiodinium spp (Zooxanthellae) -healthy coral: polyps and zoo-xanthellae -bleached coral -dead coral covered in algae --Corals have fungi, too

Lichens and humans

-Usnea filipendula ("beard lichen") -Letharia vulpina ("wolf lichen") -Evernia prunastri ("oakmoss lichen")

Freshwater microbial communities

-Usually oligotrophic -Oxygenic phototrophs are in the epilimnion -When receives large concentrations of nutrients, can become eutrophic. -Causes algal blooms: --Phosphates --Nitrates --Organic pollutants

pH

-acidophile: growth optimum between pH 0 and 5.5 -neutrophile: growth optimum between pH 5.5 and 8.0 -alkalophile: growth optimum between pH 8.0 and 11.5

Aphid-Buchnera Symbiosis

-aphid -Buchnera endosymbiont: Bacteriocyte

Ex: QS in Vibrio fisheri

-autoinducer -acyl homoserine lactone (AHL)

Polyhydroxyalkanoates used for medical devices

-bacterial polyester: poly-4-hydroxybutyrate

pressure

-barophile: growth more rapid at high hydrostatic pressures

Viruses have high count numbers in the ocean

-biomass: 90% bacteria and archaea, 5% protists, 5% viruses -abundance: 94% viruses, 5% bacteria and archaea, 1% protists

why haven't all bacteria died as well?

-bottom dissolved oxygen contours

Lakes are highly stratified

-cyanobacteria at the top -O2 at the top -chloroflexi and sulfur bacteria -H2S -sulfur bacteria and sulfate-reducing organisms -oligotrophic lake (diluted nutrients) -eutrophic lake (rich nutrients) ---have a higher amount of sulfur bacteria and sulfate-reducing organisms

Gradients in Microbial Mats

-cyanobacteria, diatoms -purple bacteria -green bacteria -sulfate reducers

Microbial Mat Light Absorption

-cyanos -greens -purples (in decreasing wavelengths)

Fermentation completes reduction of pyruvate by NADH

-fermentation pathways

NADH dehydrogenase pumps 4 H+ across the membrane

-four protons are pumped across transmembrane alpha helices

Geobacter: Anode reduction

-fuel: latate, acetate->bacterial biofilm (inside) -oxidation (inside)->CO2 outside -reduction->H2O out

Heliobactor pylori and Ulcers

-gastric pit forms -urease activity -HCl goes in

Bacteria and archaea use three main routes to catabolize glucose

-gycolysis (EMP) -Entner-Doudoroff (ED) -Pentose Phosphate pathway (PPP)

other physiological responses to pH stress

-in strongly acidic (pH2) environment, amino acis decarboxylases drain proteins from the cell -under acidic conditions (PH 5) cells use the transport K+ or other cations to maintain pH homeostasis. how is unclear -under alkaline stress (pH9) Na+/H+ antiport systems scavenge protons from the environment -E.coli cytoplasm is negatively charged relative to the exterior

Ecosystem effects: Plant communities

-increase plant biomass and nutrition -increase plant diversity

Hydrolyzing gut bacteria: part of the human genome?

-maltose sensor and regulator: trenscriptional activation of the sus operon -marine bacteria that catabolize porphyran -human bacterial genes from gut and marine bacteria

Effects of Mycorrhizas on Plants

-mycorrhizal -non mycorrhizal

oxygen concentration

-obligate aerobe: completely dependent on atmospheric =2 for growth -faculatative anaerobe: does not require O2 and dies in its presence -microaerophile: requires O2 levels between 2-10% for growth and is damaged by atmospheric O2 levels (20%)

Adaptation to oxygen concentration

-obligate aerobe: need O2 -facultative anaerobe: prefer O2 -aerotolerant anaerobe: ignore O2 -strict anaerobe: O2 is toxic -Microaerophile: <2-10% O2

Overview of aquatic systems

-oceans: 96.1% -polar ice and groundwater: 3.7% -surface freshwaters 8soil water, lakes, streams, rivers, etc.): 0.2%

Fermentation transfers electrons back to organic molecules through enzymes

-outside cell membrane (periplasm) -protons pumped per electron pair -pyruvate turns into lactate---produces NAD+ from NADH, H+

How do microbes get energy to build biomass?

-precursor carbon->ATP->macromolecules (proteins, nucelic acids, lipids) -precursor carbon->monomers->macromolecules (proteins, nucelic acids, lipids) -reducing electrons, then goes into monomers, then macromolecules (proteins, nucleic acids, lipids)

temperature

-psychrophile: grows at 0ºC and has an optimum growth temperature and 15ºC or lower -psychrotroph: can grow at 0-ºC, has an optimum between 20 and 30ºC and a max of about 35ºC -mesophile: has growth optimum between 20-45ºC -thermophile: can grow 55ºC or higher, optimum often between 55 and 65ºC -hyperthermophile: has an optimum between 85 and about 113ºC

1. Energy (ATP) source: movement of electrons

-rearrangement of external molecules-phototroph OR chemotroph

Aphid Secondary Symbionts (SS)

-secondary bacteria in aphids, section of aphid labelled with specific probes in Buchnera and the secondary bacterium Hamiltonella defensa

Microbes acquire N through assimilation

-secreted products: antibiotics, toxins, quorum signals, biofilm matrix -autotrophy: CO2 fixation, calvin cycle, reverse TCA cycle -heterotrophy: catabolism of sugars, amino acids, fatty acids

The Embden-Meyerhof-Parnas Pathway

-substrate energy changes during the Embden.Meyerhof-Parnas pathway (glycolysis)

Microbial Syntrophy cleans up oil!

-sulfate-reducing bacterial partner -Syntrophus: --Benzoate + 7H2O → 3 acetate + HCO3- + 3H+ + 3H2 --DG0' = +70, does not proceed spontaneously -Desulfovibrio: --4H2 + SO4- + H+ → HS- + 4H2O --DG0' = -152, proceeds spontaneously -Living together, DG for Syntrophus metabolism is negative!

1. Formation of glucose 6-phosphate

-the large negative change in G'º for ATP hydrolysis drives the reaction, coupled reactions drive endergonic processes

Fungi are the primary decomposers in ecosystems

-they circle and grow -co2 out enzymes out -uptake

Variation

-variation less within than between -diversity varies by habitat

Nitrogen is reduced in four cycles

1. Fe protein tranfers 2e- to FeMo center 2. FeMo protein binds 2H+, which is reduced to H2 gas 3. N2 takes the place of H2 at the binding site 4. Successive pairs of H+ and electrons reduce N2->HN=NH->H2N-NH2->2NH3

fate of carbon from the pelagic zone to the bottom of the ocean

1. air and sea exchange CO2 2. Phytoplankton take up CO2 to grow 3. Zooplankton eat phytoplankton and respire CO2 4. Fragments of decaying phytoplankton and fecal pellets from zooplankton both contain carbon 5. separately or in aggregations (called "marine snow"), these carbpon-containing particles sink 6. Only 5 to 50% of the total carbon from blooms reaches 100m about 2 to 25% sinks between 100 and 500m 7. Microbes decompose particles further, zooplankton eat some of this material 8. CO2 from organic matter respiration recirculates back to surface waters 9. zooplankton migrate up at night to feed and back to the depths during the day 10. perhaps only 1 to 15% of the original carbon in surface water sinks below 500m

Trophic groups: Soil food web

1. decomposers 2. symbionts 3. pathogens

CO2 fixation: the Calvin Cycle

1.Bacteria and chloroplasts 2.Not in archaea/cytoplasm of eukaryotes 3.Evolved after divergence of 3 domains of life

Copepods carry Cholera

1.Copepod carries bacteria on surface 2.Bacteria eat chitin of egg cases 3.Humans disperse new copepods through diarrhea -travels through water sources

Aquatic microbes 2: lecture summary

1.Microorganisms are systemic throughout aquatic ecosystems (water column, aquatic animals) - revealed through metagenomics 2.Photosynthetic and non-photosynthetic microorganisms provide the base of the aquatic food chain 3.Extremophiles with different adaptations to temperature, oxygen, and pressure dominate the lower water column 4.Aquatic microbes are sensitive to global changes, including climate warming and pollution

marine microbes: Lecture Summary

1.Microorganisms are systemic throughout aquatic ecosystems (water column, aquatic animals) - revealed through metagenomics 2.Photosynthetic and non-photosynthetic microorganisms provide the base of the aquatic food chain 3.Extremophiles with different adaptations to temperature, oxygen, and pressure dominate the lower water column 4.Marine microbes are sensitive to global change

Terrestial microbes: Lecture Summary

1.Microorganisms are systemic throughout ecosystems (soil, plants, animals) 2.Microorganisms are major players in decay, symbiosis/mutalism, and parasitism/pathogenesis 3.Microbes can accelerate carbon and nutrient flows through ecosystems

Fate of catabolism products

1.Respiration: electrons removed are ultimately transferred to an inorganic electron acceptor such as oxygen

Fate of catabolism products

1.Respiration: electrons removed are ultimately transferred to an inorganic electron acceptor such as oxygen 2.Fermentation: all the electrons from organic substrates are put back onto the organic products

Topic 2.10 - Terrestrial microbes

1.Where are microbes found in ecosystems? 2.Which ecological processes are microbes responsible for in ecosystems? 3.What are some different relationships between microbial activity and nutrient flow in terrestrial ecosystems? 4.What are some relationships between microbial activity and ecosystem development? --Microbes are found throughout terrestrial systems

Microbes in aquatic systems

1.Which major ecological processes do microbes drive in aquatic systems? 2.What kind of environmental stresses to microbes deal with in aquatic environments? 3.What are some ways that microbes deal with these environmental stresses?

Microbiota Comparison Among Animals

16S rRNA gene sequences of intestinal microbiota Similar lineages across animals - evolutionary implication?

Describe two ways in which the unique membrane lipids of archaea protect them from environmental stress: one for pH and one for temperature.

Acidophililic archaea often have high amounts of tetraether lipids. Altered cell membrane structures decrease proton permeability. In addition, archaeal membranes are more stabilized to high temperatures by a variety of means. The terpenoid lipids of archaea are more saturated (i.e. have regular methyl groups) and are more linear than bacterial membranes. In addition, the tetraether lipids of archaea will be higher molecular weight compared to those of bacteria (i.e. they create a phospholipid monolayer in the cell).

Which of the following processes is inhibited in the presence of oxygen?

Activity of the Fe-S protein in nitrogenase

How do the structures in primary endosymbiotic algae (i.e. true algae) facilitate its growth in aquatic environments? How is the cell wall of these algae similar or different from the cell wall of plants?

Algae contain vacuoles, which maintain osmolarity of the cell. A pyrenoid concentrates bicarbonate (HCO3-) and converts it to CO2 for carbon fixation via dark reactions in the cell. The pyrenoid is surrounded by starch bodies for energy storage. The starch is broken down, followed by glycolysis and respiration in the mitochondria. Algal cell walls are made of sugar molecules, as they are in bacteria, archaea, and other eukaryotes, The Chlamydomonas cell is encased in a cell wall composed predominantly of glycoprotein. Other green algae have cellulose cell walls similar to those of plants.

Protists in freshwater systems:

Alveolates

Compare and contrast the different types of protists in freshwater systems. Which could be used to track a lake's environmental conditions?

Amebas (or amoebas) are free-living predators that engulf prey by phagocytosis. Free-living amebas can harbor bacterial pathogens such as Legionella pneumophila, which contaminates water supplies and air ducts. Amebas move by extension of lobe-shaped pseudopods. Foraminiferans are a type of ameba that make needle-like pseudopods that radiate in all directions. Their shells are made of calcium carbonate (CaCO3), which are found in reefs, beach sand, and sedimentary rock. Because these shells capture the isotopic signature of oxygen, they can be used as units for geological analysis. The oxygen captured in CaCO3 has an isotopic signature of the water where the ameba lives. If the water is warm, the lighter oxygen isotopes (16O) will evaporate slightly more than the heavier oxygen isotopes (18O). Thus, the water will have a heavier isotopic signature of oxygen (higher 18O/16O) as water temperatures rise. If a foram lives during a time when the Earth's temperature is warm, it will have a heavier oxygen isotope ratio in its shell than a foram living during a time when the Earth's temperature is cool. Alveolates are known for their complex outer covering, or cortex. The cortex contains networks of vesicles called cortical alveoli that store calcium ion and in some species grow protective plates. In contrast to Amebas, alveolates move by cilia and flagella. Alveolates equipped with paired flagella or cilia are known as flagellates and ciliates, respectively. Flagella (singular, flagellum) and cilia (singular, cilium) are essentially equivalent organelles composed of microtubules and enveloped by the cell membrane. They are covered by an extension of the plasma membrane. Ciliates contain short microtubule projections, driven by ATP. Cilia are shorter than flagella and more numerous, and usually cover a broad surface. Cilia beat in coordinated waves that maximize motility (cell propulsion) and food acquisition.

Describe the ameba motility strategy.

Amoeba move through the extention and turnover of pseudopods. The extension of these lobe-shaped pseudopods has been studied closely for its relevance to human white blood cells. The mechanism of pseudopod motility remains poorly understood, but it is known to involve a sol-gel transition between cortical cytoplasm (just beneath the cell surface) and the cytoplasm of the deeper interior. The tip of the pseudopod contains a gel of polymerized actin beneath the cell membrane. From the center of the ameba, liquid cytoplasm (sol) containing actin subunits streams forward along microtubular "tracks," powered by ATP hydrolysis. The actin subunits stream into the pseudopod, where they polymerize, forming a gel. Then, the gel region grows, pushing the membrane forward. As the gel is pushed backward, it resolubilizes to continue the cycle.

Symbiosis Outcome Affected By Environment

An example of a symbiotic interaction that can have a range of outcomes is present with hydras and algae. Hydras are small freshwater Cnidarians that live in ponds and slowly moving rivers. They feed on small animal rotifers present in the water column. Some hydra are green because they contain algae from the genus Chlorella inside their bodies. The algae photosynthesize and they release substantial amounts of photosynthetically fixed carbon to the animal cells in the form of maltose (a sugar). Green hydra can easily be cultivated in the laboratory, and they can also be "bleached" of their algae by incubation at very high light intensity. Both hydra containing algal cells (symbiotic hydra) and those deprived of their algae (aposymbiotic hydra) reproduce rapidly by asexual budding. So large numbers of genetically identical symbiotic and aposymbiotic hydra can be generated for experiments. --performance of green hydra under different conditions of illumination and feeding -hydra: starved in light (survival) -symbiotic aposymbiotic >10 weeks, <2 weeks, the hydra benefit -interaction: + effect on alga, + effect on hydra

How do environmental conditions determine the type of symbiosis that can occur between two organisms? Use the Hydra-Chlorella (algae) system as an example.

An example of a symbiotic interaction that can have a range of outcomes is present with hydras and algae. Hydras are small freshwater Cnidarians that live in ponds and slowly moving rivers. They feed on small animal rotifers present in the water column. Some hydra are green because they contain algae from the genus Chlorella inside their bodies. The algae photosynthesize and they release substantial amounts of photosynthetically fixed carbon to the animal cells in the form of maltose (a sugar). Green hydra can easily be cultivated in the laboratory, and they can also be "bleached" of their algae by incubation at very high light intensity. Both hydra containing algal cells (symbiotic hydra) and those deprived of their algae (aposymbiotic hydra) reproduce rapidly by asexual budding, so large numbers of genetically identical symbiotic and aposymbiotic hydra can be generated for experiments. To investigate whether green hydra benefit from their symbiosis with Chlorella, the performance (that is survival and growth) of symbiotic and aposymbiotic hydra can be compared under different environmental conditions. When hydra are maintained in the light without food, the non or aposymbiotic hydra die within a few weeks. Green hydra benefit from the symbiosis, provided they are illuminated. Well-fed aposymbiotic and symbiotic hydra grow at approximately the same rate when light is present. The algae release sugars, but these sugars play a minor role in their diet. Fed cultures of the hydra can also be maintained indefinitely in the dark. In this case, the algal population drops by about 60% but the animals remain pale-green. In the darkness, green hydra grow slower than non-green hydra, probably because nutrients derived from the food are diverted to maintain Chlorella, which in the absence of light, contribute nothing to the hydra. This example makes it clear that the environment plays a major role in determining the outcome of symbioses. In this case, the hydra benefit when food is scarce, but Chlorella are of no discernible value to fed animals in the light and they are detrimental, reducing the growth of fed hydra, in the dark.

Describe two ways that repressor mechanisms operate in antibiotic resistance; one way in which an inducer ligand (antibiotic) is transmitted directly into the cell, and one way in which an inducer is sensed on the cell surface and a signal is sent into the cell.

An inducer ligand (antibiotic) is transmitted directly into the cell: tetracycline efflux pumps. In E.coli, when tetracycline is absent from the cytoplasm, a repressor protein called tetR binds to an operator that blocks transcription of the tetB gene. tetB makes the protein (TetB) that is the efflux pump that removes tetracycline from the cell. When tetracycline comes into the cell, it acts as an inducer - it then binds to TetR. That binding causes the release from the operator DNA, thereby allowing high expression of the TetB pump protein. An inducer is sensed on the cell surface and a signal is sent into the cell: methicillin-resistant Staphylococcus aureus (MRSA). The BlaR1 protein of methicillin-resistant Staphylococcus aureus (MRSA) is a membrane protein involved in sensing of the presence of β-lactam antibiotics and transduction of the information to the cytoplasm. The process of antibiotic recognition on the cell surface leads to activation of the zinc protease (BlaR2) in the cytoplasmic domain. The protease activity degrades BlaI, a repressor protein, resulting in derepression of the antibiotic resistance genes. These genes encode BlaZ, which is a lactamase enzyme that binds to B-lactam antibiotics, rendering them inactive.

In the aphid-Buchnera symbiosis, what is the secondary symbiont to the wasp?

Antagonistic competitor

Protection Mechanism II: spatial

Aquatic cyanobacteria develop special cells called heterocysts to fix N2 --nitrogen fixation requires specialized structures (there's a heterocyst) -light vs fluorescence microscopy -A cyanobacteria, Anabaena, with fluorescent vegetative cells and 'dark' heterocyst cells.

Thermophiles

are adaptive to high temperatures

How can microbial processing of N in the environment lead to infant deaths?

Bacteria are efficient at producing nitrate in the environment through the oxidation of ammonium. If too much nitrate is produced by these bacteria (as in areas where soil is over-fertilized with urea or ammonium fertilizer), it can lead to nitrate-contaminated groundwater. Methemoglobinema is a blood disorder in which hemoglobin is inactivated. This happens when babies are exposed to high nitrate groundwater. The infant stomach is not yet acidic enough to prevent growth of the denitrifying bacteria, which reduce nitrate to nitrite. So, denitrifying bacteria grow in the infant stomach and produce a lot of nitrite. Nitrite produced by the bacteria oxidizes iron in the hemoglobin, eliminating its capacity to carry oxygen. Failure to carry oxygen leads to a bluish appearance in babies. Affected gray skin color and may become irritable or lethargic, depending on the severity of their condition. The condition can progress rapidly to cause coma and death if it is not recognized and treated appropriately.

What are some challenges of using mutations only to adapt to changing environmental conditions? How does gene regulation overcome these challenges?

Bacteria have two basic strategies for adaptation: Mutations can produce desirable new traits, as is the case with mutations that make bacteria resistant to the antibiotics we take to kill them. But there are three main issues with mutation: 1) mutations occur randomly. Because of this, they can be lethal if they make a vital protein inactive. The random nature of mutations also means that a lot of bacteria must die so that enough mutations can be tested to find the ones that are successful. 2) some desirable traits may actually require multiple mutations, which can take considerable time. Bacteria faced with a new and hostile environment may not have a lot of time to adapt. 3) imagine the environment changes again back to its original condition. Mutations are only reversible by additional rounds of mutations, which brings us back to the original two problems. So basically mutations are not an easy on/off switch. A bacterium living in the real world must be able to alter multiple activities within minutes, then alter them back if conditions change once more. So in addition to mutation, bacteria also need a rapid, reversible strategy for change. This short-term strategy involves controlling which proteins are produced under a given set of conditions (i.e. gene regulation).

Repressor Ex. 1: Tetracycline Resistance

Bacterium that is resistant to tetracycline

Nutritional symbiosis:

Buchnera sp. in aphid gut

DG varies based on concentration ratios

DG = DG0 + 2.3RTlog([C][D]/[A][B]) •The log of fraction is negative! •Ex: DG0 = +20, won't proceed. •Ex: DG = +20 + (-30) = -10, will proceed.

Antibiotic resistance in both MRSA and tetracycline-resistant bacteria involves regulating the expression of genes that remove antibiotics from the cell. Both types of gene regulation involve:

De-repressing transcription when antibiotics bind to repressors as inducers

Amenalism: microbial antagonism

Competition 1.Food 2.Space --Exploitative: reduce resource to low concentrations --Interference ---Antagonism at a distance ---Hyphal interference

Describe the role of decomposer microbes in the Earth system.

Decomposer microbes recycle plant nutrients and create soil organic matter. Soil organic matter is important because it: • retains nutrients • maintains soil structure • holds water for plant use Soil creates the substrate that holds plant roots, making it possible for land plants to establish. In addition, decomposers release large amounts of CO2 to the atmosphere through oxidative respiration, which is one of the largest biogenic fluxes of greenhouse gases through the biosphere.

How are microbes involved in coral bleaching under global warming?

Coral bleaching happens when the symbiosis between the coral animal and it's photosynthetic microbial symbiont is disrupted. Symbiodinium is an endosymbiotic (symbiotic inside the host) dinoflagellate (algal protist). They enter the host cell through phagocytosis. Symbiodinium is a genus of diverse endosymbiotic algae with genus members commonly referred to as zooxanthellae. Coral are born azooxanthellate, symbiont free, and are infected with the symbiont horizontally while in the polyp stage through either feeding or phagocytosis by gastrodermal cells. Symbiodinium provide photosynthetically fixed sugars to coral while coral provides inorganic nutrients, a high light environment, and protection from the threat of aquatic herbivores With global warming (+1-2oC), corals lose their symbionts (60-90%) or they lose the symbiont's photosynthesis pigments (50-80%). There is evidence of irreversible damage to photosystem II in heat-stressed symbiotic dinoflagellates within corals during a natural bleaching event. The dysfunction of PSII correlates with the loss of the PSII reaction-center protein. It is hypothesized that host corals expel the dysfunctional symbionts because they no longer produce fixed C. When this happens, the coral turns white because the calcium skeleton is showing through.

Example: Respiration in Lakes

Coupling organic e- donor with different e- acceptors results in microbial succession.

Which group(s) of organisms are most likely to fix CO2 via the Calvin cycle?

Cyanobacteria

Describe "historical exposure" (also known as historical contingency) and how it applies to the human microbiome.

Differences in historical exposures (e.g. microbes available to colonize) could lead to differences in the species composition of microbial communities in different human habitats (i.e. locations on or inside the body). For example, when a microbial community from the tongue is transplanted onto the forearm, it tends to remain similar in composition to the original transplant (tongue) community (in terms of phylogenetic diversity and species composition). This suggests that if a microbial species can colonize the forearm, it is likely to remain there as a member of the community.

autoinducers languages

Different Autoinducers Create Specific "Languages"

The Electron Tower

E0' reaction (reduction potential) = E0'acceptor - E0'donor •Goal: pair stronger e- donor & stronger e- acceptor. Glucose (donor) + 6O2 (acceptor) à 6CO2 + 6H2O +820 - (-430) = 1250 mV •DG0' = -nFE0' -n = # electrons transferred, F = Faraday constant (96.5) •+E value = -G value •-482.5 = -4 * 96.5 * 1.25 V

Oxidative (aerobic) respiration transfers electrons to oxygen

E0' reaction = E0'acceptor - E0'donor NADH + H+ + 1/2O2 à NAD+ + H2O E0' reaction = 820 mV - (-320 mV) = 1,140 mV 1,140 mV = -220 kJ/mol (DG)

What are the similarities and differences between obligate pathogens and free-living saprotrophs in the ways they control the rate and type of biosynthesis?

Each species makes an evolutionary "choice" whether to maintain the expense of a particular assimilatory or biosynthetic pathway, or to lose the pathway and become dependent on other species in the environment. Parasitic microbes that grow only inside a host cell show extensive loss of biosynthetic genes. Since energy is required to run this biosynthetic machinery, as well as maintain and replicate the genome, parasites that can rely on their hosts for biosynthesis and energy have lost many abilities to generate energy themselves (or conserve it in molecules like ATP and NADH). This is referred to as genome degeneration. Free-living bacteria, by contrast, often divert carbon resources away from growth to produce secondary products, which are not essential nutrients, but can enhance nutrient uptake in low-nutrient environments or inhibit competitors. The free-living bacteria that run the Calvin cycle also control rates of biosynthesis by concentrating CO2 near Rubisco using carboxysomes. However, both pathogens (such as Legionella pneumophila) and free-living bacteria (such as certain E. coli strains) regulate certain biosynthesis pathways in similar ways. For example, because fatty acid synthesis is expensive and needs to be balanced with growth of the cell, it is regulated by starvation in both species. Fatty acid synthesis can be regulated by other mechanisms as well: 1) Acetyl-CoA carboxylase regulates its own transcription (similar to catabolite repression). Transcription of an operon encoding two subunits of acetyl-CoA carboxylase (AccB, AccC) is repressed by one of its subunits, protein AccB. As AccB increases in concentration, it binds the promoter of the accBC operon, repressing further transcription. 2) In E. coli, low temperature favors unsaturated fatty acids because they are less rigid and maintain membrane flexibility. Cold will initiate the dehydratase enzyme that desaturates the fatty acid bond.

Describe some differences between ectomycorrhizal fungi and arbuscular mycorrhizal fungi.

Ectomycorrhizae colonize the rhizoplane, the surface of plant rootlets—the most distal part of plant roots. The fungal mycelia never penetrate the root cells. They form a thick mantle surrounding the root and growing between the root cells, then extend long mycelia away from the root to absorb nutrients. Ectomycorrhizal fungi then give plants a portion of these nutrients in exchange for carbon (i.e. sugars from photosynthesis). Arbuscular mycorrhizal fungi also colonize the rhizoplane of plant roots, but unlike ectomycorrhizal fungi, they penetrate the root cells and make structures inside the plant cell for nutrient exchange and storage. Arbuscules are sites of nutrient exchange (obtain sugars as hexoses) and vesicles are storage organs. Like ectomycorrhizal fungi, arbuscular mycorrhizal fungi trade nutrients for carbon with the plant. Unlike ectomycorrhizal fungi (which colonize trees and shrubs), arbuscular mycorrhizal fungi colonize many of our crop plants. Arbuscular mycorrhizal fungi are also ancestors of other fungi - they form their own monophyletic group. Ectomycorrhizal fungi diverged from saprotrophic fungi at least 50 times, so retain the capacity to break down soil organic matter (unlike arbuscular mycorrhizal fungi).

How do endolithic (living inside rock) microbes make a living in bedrock beneath soil? What kind of trophy do they engage in?

Endoliths (microbes that live inside of rock) live off of decay of uranium. Uranium-238 decay generates hydrogen radicals that combine to form H2 gas. H2 gas + CO2 provides an e- donor and a carbon source for methanogens and other endolithic lithotrophs. Because they use CO2 as their carbon source (to make methane), they are considered chemolithoautotrophs.

What is the difference between chemotrophy and phototrophy? How is it related to lithotrophy and organotrophy?

Energy is obtained from chemical reactions triggered by the absorption of light (phototrophy, what photosynthetic organisms use) or from redox reactions (chemotrophs) that transfer e- from high energy compounds to make products of lower energy. Chemotrophs fall into two classes that use different sources of electron donors: lithotrophs and organotrophs. Phototrophs fall into these two classes as well - they can obtain electrons from oxidation of inorganic compounds (like H2S, in the case of green photosynthetic bacteria), or from the oxidation of organic compounds.

Polyketide antibiotics are broad-spectrum antibiotics

Erythromycin •Produced by actinomycetes •Secondary metabolites •Synthesized by a modular enzyme Erythromycin •Produced by actinomycetes •Secondary metabolites •Synthesized by a modular enzyme -Behie et al. (2017) Molecules to Ecosystems: Actinomycete Natural Products In situ. Front. Microbiol. Erythromycin •Produced by actinomycetes •Secondary metabolites •Synthesized by a modular enzyme Nonribosomal peptides •Vancomycin •Stendomycin •Modular enzymes

Why is there so much microbial photosynthesis taking place on Earth?

Every year photosynthesis converts 10% of atmospheric CO2 to biomass. Most of the photosynthesis on Earth comes from microbes (particularly in marine systems, which account for > 50% of photosynthesis on Earth). This is because there is a lot of light energy on the ocean surface to drive phototrophy. Photosynthesis involves coupling phototrophy (known as a light reaction) to fixation of CO2 (known as a dark reaction). Fixation of CO2 occurs via the Calvin cycle, which requires a lot of both energy and reducing power to occur. Autotrophs use phototrophy, because it can produce large amounts of both ATP and NADH. The fact that phototrophy is very efficient at providing both types of molecules explains why so many photosynthetic microbes use the two (phototrophy and autotrophy) in combination.

What types of microbial products are synthesized by modular enzymes? How can these products both threaten and assist humans?

Fatty acids are synthesized by the modular enzyme complex "fatty acid synthase complex". Legionella synthesizes polyester granules. It is a human pathogen, the leading cause of legionellosis (chronic lung disease). This polyester helps the bacteria live in water sources like air conditioners. Intracellular energy reserves, such as poly-3-hydroxybutyrate (PHB), may also promote environmental persistence. However, related condensation pathways yield materials such as polyesters and polyketide antibiotics, such as vancomycin and erythromycin. Products in a class called polyhydroxyalkanoates (a form of polyester) are made naturally by Legionella pneumophila and soil bacteria such as Ralstonia eutropha. Ralstonia eutropha bacteria form storage granules of a polyhydroxyalkanoate, poly-3-hydroxybutyrate. By overexpressing a modified form of the polyester in E. coli, we can produce large amounts of the product. TephaFLEX surgical sutures, which are absorbed by the body, are manufactured from poly-4-hydroxybutyrate, synthesized by engineered Escherichia coli.

How has infection by Francisella tularensis been combated through manipulation of quorum sensing?

Francisella tularensis is a bacterium known to cause a disease called rabbit fever. This disease is typically confined to other mammals like rabbits that serve as reservoirs, where the disease is actually contracted by humans through biting insect vectors such as ticks and mosquitoes. Pneumonic tularemia includes skin ulcers (where the bacteria enter the body) followed by swelling of the lymph nodes. Part of the virulence of this pathogen is the production of pedestals on the cells they are infecting. These pedestals are produced only when bacterial densities are high - a classic example of quorum sensing. To try to inhibit the virulence of Francisella, a group of scientists identified a molecule called LED 209, which appeared to be very similar to the autoinducer of Francisella, yet not toxic to animals. This AI mimic inhibited binding of the real AI molecule and therefore limited the expression of the virulence genes. The researchers took things a step further and looked at how applying this molecule in a mouse model affected mouse survival when challenged with the pathogen.

Microbes are energetically powerful!

Glucose + 6O2 -->6CO2 + 6H2O

Halophiles require high salt concentrations

Halophilic archaea require 2-4M NaCl Can grow at an aw of 0.75 Low intracellular [NaCl] Ion pumps secrete sodium, replace with potassium

Lichen Micromorphology

Haustoria

Describe the types of microorganisms you might expect to find near a hydrothermal vent at the bottom of the ocean, in descriptive terms (i.e. halotolerant, strict anaerobe, etc.). Be sure to include what you might imagine it's optimal temperature, oxygen concentrations, pH, and water/salt environment to be.

Microbes present in hydrothermal vents have to deal with extreme heat, as well as salinity, low oxygen availability, and high pressure. This means that they are likely to be thermophilic, halotolerant anaerobes and extreme barophiles. They are likely strict anaerobes, because there is probably little oxygen available at the bottom of the ocean. In terms of pH, perhaps they are neutrophiles, because the pH at the bottom of the ocean may not be too extreme.

Describe how microbes build large biomolecules from either (1) the products of catabolism (i.e. in heterotrophy) or (2) the fixation of CO2 (i.e. in autotrophy). What is the role of ATP and reducing equivalents like NADH/NADPH?

Heterotrophy breaks down organic molecules. Pathways of breakdown include hydrolytic and oxidative reactions that produce small monomers that can enter the glycolysis pathway, then the TCA cycle or fermentation pathways. Many of the intermediates from glucose catabolism and the tricarboxylic acid (TCA) cycle can be used as substrates for biosynthesis reactions in the cell. In addition, microbes reverse these pathways to build larger molecules (carbon skeletons) from the smaller molecular products of catabolism (sugars, amino acids). For example, products of heterotrophy can enter reverse glycolysis (gluconeogenesis). Autotrophy fixes CO2 into sugar. This happens through cycles like the Calvin cycle and reverse TCA cycles. In the Calvin cycle, there are three main phases: carboxylation of ribulose 1,5-bisphosphate to a 6-C molecule and splitting into 3-phosphoglycerate (PGA): 6C → 2[3C]; reduction of PGA to glyceraldehyde 3-phosphate (G3P); and regeneration of ribulose 1,5-bisphosphate through the consumption of additional ATP. One of every six G3P is converted to glucose. The other five molecules enter a series of reactions that regenerate three molecules of ribulose 1,5-bisphosphate. Reverse TCA, on the other hand, involves fixation of CO2 by several intermediates, including succinyl-CoA, 2-oxoglutarate, and acetyl-CoA. Reduction (addition of 2H+ + 2e-) is performed by NADPH or NADH and by reduced ferredoxin (FDH2). In addition, ATP is consumed when citrate is converted to acetyl-CoA, and the acetyl group is then used to create pyruvate. Pyruvate is then used to re-generate oxaloacetate, consuming a 3rd ATP molecule. Biosynthesis usually reduces the substrate by hydrogenation and by removing oxygen. Cell components such as lipids and amino acids are more reduced than substrates such as CO2 and acetate, so their biosynthesis requires a reducing agent such as NADPH. All biosynthesis requires energy from ATP and electrons from reducing cofactors such as NADPH.

Why is the coral endosymbioant, zooxanthellae, sensitive to global warming?

High temperatures damage the reaction center of Photosystem II in zooxanthellae

Treponema pallidum: genomic analysis of metabolism

If Treponema doesn't use TCA, how does it respire and generate NADH?

Why do photosynthetic microbial mats assemble in the way they do (in terms of green bacteria, purple bacteria, cyanobacteria, and sulfate reducers)? Be sure to explain why the species assemble in the order they do (from top to bottom) and why the layers of different species may be more or less thick compared to others.

If you look closely at the mat, you will see a series of colored layers atop a black layer. The green layer at the top of these mats is occupied by cyanobacteria and diatomaceous algae. The next two layers of are also photosynthetic but neither one produces oxygen. The final layer, which is black, is where the sulfate reducers are present. So why are they arranged in this order? One reason is the amount of light energy that each group is able to utilize. Cyanobacteria take out the more high energy light in the 600 and 700 nm wavelengths. Next in the mat are the purple bacteria and take up light in the far end of the visible spectrum above 800 nanometers. The light that is left for the Green bacteria is in the middle of those two ends. Its chlorophyll absorbs at around 700-800 nm. However, another reason for the stratification is the availability of electron donors. You can see gradients in sulfur compounds, for example. In this case, there is decreasing sulfate with depth, do its abundance in seawater and consumption by sulfate reducers. H2S is generated by sulfate-reducing bacteria, which are chemotrophs, and so reside underneath the green sulfur bacteria. Green sulfur bacteria are below purple bacteria because they need a supply of H2S as electron donors. This is also good, but sulfides are toxic to most organisms and their movement out of the sediments could be harmful to other organisms. All of these gradients are set up by microbes and the layers optimally arranged for each of the groups involved. If you look closely at the environmental conditions in these mats they are definitely characterized by lots of gradients. Light only penetrates to a certain depth, but it is present in all the layers where photosynthesis is occurring. Oxygen is produced by the cyanobacteria and diatoms, but diffuses up into the water and air column or it is consumed by other bacteria and does not penetrate into the lower layers. This is good for the purple and green bacteria, which only photosynthesize when oxygen is not present.

Thought question

In E. coli, genes coding for membrane transport proteins are regulated by σH, as well as housekeeping σD, extracytoplasmic stress σE, flagellar σF, nitrogen limitation related σN, and starvation-induced σS. If each sigma factor recognizes a different promoter, how does the cell manage to transcribe these membrane transport protein genes that respond to multiple stresses, each involving a different sigma factor?

How is glucose involved in catabolite repression of lactose degradation?

In E. coli, the sugar lactose induces transcription of genes that encode beta-galactosidase (lacZ) and lactose permease (lacY). But in the presence of glucose, a preferred carbon source, lac transcription is halted. Halting lac transcription enables preferential catabolism of glucose. Substrates are selected through gene regulation. lacZ and lacY are part of an operon in E. coli called the lac operon. The operon is inducible by lactose to the maximal levels when cAMP (cyclic adenosine monophosphate) and CAP (catabolite activator protein, an activator protein) form a complex. (a) Under conditions of high glucose, a glucose breakdown product inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP. (b) Under conditions of low glucose, there is no breakdown product, and therefore adenylate cyclase is active and cAMP is formed. (c) When cAMP is present, it acts as an allosteric effector (and in inducer), complexing with CAP. (d) The cAMP-CAP complex acts as an activator of lac operon transcription by binding to a region within the lac promoter.

Compare and contrast photosynthesis conducted by green photosynthetic bacteria (photosystem I), purple bacteria (photosystem II), and cyanobacteria ("Z pathway").

In all cases, there is light absorption by a pigment that gets excited by a photon of light. This separates electrons from a molecule coupled to an ETS that is homologous to those of respiratory ETS. In photosystem I, absorbed energy allows the transfer of e- from P840 to ferredoxin through a quinone. The e- then gets transferred to Ferredoxin-NAD+ reductase, which reduces NADP+ to NADPH. Bacteriophyll P840 absorbs light over a variety of wavelengths. PSI receives electrons associated with H from H2S (hydrogen sulfide) HS- (hydrogen bisulfide) or H2, or even from Fe2+. Purple bacteria capture light not used by other phototrophs. The peak wavelength absorbed by bacteriochlorophyll P870 lies so far into the infrared (800-1,100 nm) that the photon energy is insufficient to reduce NAD(P) to NAD(P)H. In purple bacteria, Bchl P870 donates an energized electron to a quinone (Q). Two of these donated electrons complete the conversion of quinone to quinol (QH2). Electrons flow from QH2 to cytochrome bc, then are coupled to pumping of protons across the membrane in cytochrome bc. The proton potential drives synthesis of ATP. The cytochrome bc complex transfers the electrons back to P870, where they can be re-excited by photon energy (this is refered to a cyclic phototrophy). Since the reduction potential is too small to reduce NADP+ to NADPH, photosystem II requires reverse electron flow. In reverse electron flow, an electron donor reduces an ETS with an unfavorable reduction potential, requiring input of energy. Purple bacteria obtain this energy by spending ATP to increase the proton potential The "Z" pathway of photolysis found in cyanobacteria and chloroplasts combines key features of both PS I and PS II to produce oxygen. In the PSII reaction center, the photoexcitation of chlorophyll P680 yields enough energy to split H2O. Both chlorophylls absorb photons of shorter wavelengths (higher energy) than the P840 and P870. Energy absorption boosts the electrons into a higher energy state. This causes the chlorophyll molecule to strip an electron from water, generating molecular oxygen. When this process happens twice, the molecular oxygens can combine to make O2. The electrons from PS II do not cycle back to the PS II reaction center, as they do in purple bacteria. Instead, they are transferred to PS I by a protein called plastocyanin. The energy of the electron transferred by plastocyanin is augmented through absorption of a second photon by the chlorophyll of PS I. A second photon excites P700, transferring e- to ferredoxin and then to NADPH. Subsequent electron flow through ferredoxin can now generate NADH or NADPH. The proton gradient drives ATP synthesis. The energy (ATP) yields are high enough to generate NADPH and fix CO2 into biomass. The components of photosystems I and II (PS I and PS II) run anaerobically, producing sulfur or oxidized organic by-products, but not O2. The Z pathway ultimately generates O2.

Describe the ways in which marine dead zones threaten human society.

In marine dead zones, many electrons are being passed through the ETS to generate energy. This is because excess nutrients and carbon from human activities will stimulate the growth (and thereby oxidative respiration) of aerobic microbes. As a result, many molecules of oxygen are consumed, and the water begins to become anoxic. This causes die off of many marine animals, including fish. In this process, ammonium from runoff can be converted to nitrate (ammonium oxidation). As oxygen levels decline, nitrous oxide also builds up in marine dead zones, because the nitrates from ammonium oxidation are used as an electron acceptor. There are many denitrifying bacteria in this zone, reducing nitrate to nitrite and N2O. Bacterial denitrification and ammonia oxidation in polluted ocean water may contribute to global warming, because N2O captures about 200 times more heat than CO2.

Can you write an energy story for the overall process of glycolysis via the EMP pathway? For energy terms, just worry about describing things in terms of whether they are energy producing (exergonic) or energy consuming (endergonic). Make sure your story starts with glucose and ends with pyruvate.

In the EMP pathway, a glucose molecule undergoes a stepwise breakdown to two pyruvate molecules. The first part is an energy consuming (endergonic) process, where glucose is activated through two substrate phosphorylations by ATP, meaning that it uses up ATP initially. The direction of flow is determined by these key irreversible reactions that consume ATP. These steps drive the pathway by spending energy. Glucose is "activated" by phosphorylations that ultimately convert it into fructose-1,6 bisphosphate. However, the breakdown of glucose to two molecules of pyruvate is ultimately an energy producing (exergonic) process, coupled to net production of two ATP and two NADH. Furthermore, in the cytoplasm, as intermediate products form they are quickly consumed by the next step, so the pathway flows in one direction.

Characterize the types of symbioses involved in the ant-wasp-aphid-buchnera-secondary symbiont system. Which pairs of species interactions do you think represent symbioses? Which pairs of interactions do you think are too weak or distant to be considered symbioses?

In the early to mid- 1990s, Angela Douglas discovered that pea plant aphids could not live without a certain species of bacteria in their gut. The pea plant aphid obtains most of its essential amino acids not from it's food source (phloem), but from an endosymbiotic bacterium (Buchnera) that synthesizes the amino acids directly. Aphids typically feed on phloem, which is the sugary water that plants produce as a result of photosynthesis. They exude a small drop of liquid from the end of their back of their abdomen. Amino acids constitute only about 1% of phloem, so you have to suck a lot of sap to get what you need. Aphids don't defend themselves very well because they are stuck sucking sap most of the time. Ants actually serve as bodyguards and harvest the drops for sugar themselves. To get a more completely balanced diet, however, aphids associate with a specialized endosymbiotic bacteria called Buchnera. These bacteria are stored in specialized cell structures called bacteriocytes in their guts. In the bacteriocytes, aphids supply sugars and other compounds to the bacteria, which in turn produce amino acids to supplement the nitrogen-poor plant sap diet of the aphid. Buchnera makes up about 90% of the microbial cells inside of aphids, but there are other microbes present. The role of these secondary symbionts was not particularly well understood until relatively recently. Wasps parasitize the aphids by laying their eggs inside of them and when the larvae hatch they start to eat the aphid and kill it. The presence of other the secondary symbiont bacteria can significantly reduce aphid parasitism by the wasp, primarily by reducing the health of wasp larvae inside the aphid. Direct symbiosis between organisms in this system could include: 1) Ant-aphid synergism 2) Aphid-Buchnera mutualism 3) Aphid-Secondary symbiont mutualism 4) Aphid-Wasp parasitism Indirect symbiosis between organisms in this system could include: 1) Ant-Wasp amenalism 2) Ant-Buchnera synergism 3) Ant-Secondary symbiont mutualism 4) Buchnera-Wasp amenalism 5) Secondary symbiont-Wasp amenalism

Describe the different types of microbial competition and the role of microbial metabolites in mediating those interactions.

Microbial competition can be: 1) Exploitative, whereby one species draws down a common resource to a level below which other species can survive 2) Interference, whereby one species uses a common resource to synthesize secondary metabolites (i.e. molecules not essential for the growth of the organism) that kill competitors. This can happen at a distance (if metabolites diffuse through the resource to kill the competitor) or through cellular interference (when the cells of the competitors come into contact).

Describe how stomach ulcers can develop in some people (but not others) due to the activity of native microorganisms

In the stomach, there are bacteria that can live there, although they typically colonize areas that have a higher pH than 2 that coats the stomach and protects the stomach lining from the acid. These areas are created by the mucin layer. In the mucin layer, the pH is actually right about 7 and species of Lactobacillus and Heliobacter as well as a yeast in the genus Torulopsis live there. One of these microbes, Heliobacter pylori, is now known to be the cause of most gastric ulcers. To get to the mucin layer, this bacterium has flagella that propel it through the stomach's acidic liquid. For the bacteria to get to the mucin layer can take a while, even with flagella. To protect it from stomach acid, Heliobacter pylori secretes an enzyme called urease, which cleaves urea into ammonia and CO2. The CO2 diffuses away, the ammonia stays close to the bacterium. It forms a cloud around the bacterium that neutralizes stomach acid in its immediate vicinity. After reaching the mucin layer, the bacteria burrow into the mucin. Here, H. pylori exhibits another interesting adaptation to its human host. The lipopolysaccharide of H. pylori does not elicit the type of intense inflammatory response elicited by the LPS of E. coli or other Gram-negative bacteria. This isn't well understood, but it has to do with a change in the LPS that causes it to mimic an oligosaccharide called the Lewis antigen. The Lewis antigen is found on human cells and helps our immune system recognize self versus non-self cells. This mimicry is the reason why most people who are colonized with H. pylori do not develop ulcers. For people who do respond to the bacteria, the immune response is not successful at eliminating the bacteria in the mucin layer because the host killer cells can't penetrate it successfully. The ulcer is created by the damage to the gastric mucosa caused by our own immune system. Then HCL and pepsin attach to the stomach wall. Some strains produce a protein toxin called VacA that leads to vacuolation and apoptosis of its host gastric cells. The role of this toxin in ulcer formation is still controversial, but it may make the inflammatory response to the bacteria more severe. Why some people respond and others don't is a question we don't currently know the answer too. Two possible answers are 1) people differ genetically in their response to H. pylori LPS and 2) some strains cause greater inflammatory response than others.

What is the iron hypothesis and why has it not been supported?

John Martin put forth the iron hypothesis, stating "Give me a half tanker of iron and I'll give you another ice age". His studies indicated a scarcity of iron micronutrients in the open ocean (i.e. pelagic zone) that was limiting phytoplankton growth and overall productivity in these "desolate" regions. Martin hypothesized that increasing phytoplankton photosynthesis could reduce the amount of greenhouse gas in the atmosphere - slowing or even reversing global warming - by sequestering large amounts of CO2 in the sea. It turns out, however, that C sequestration has not increased with increased photosynthesis due to iron fertilization. It is not clear why this happens, but it may be because so little of the C gets trapped on the ocean floor.

TCA (Kreb's) cycle

Kreb's cycle: -acetyl phosphate is a global signal of the nutritional environment -excreted until needed for further catabolism •Generates NADH, CO2, ATP •NADH donates e-s to the ETS •Gram-negative bacteria •Gram-positive bacteria •Most eukaryotes

Lactic Acid and Cavities

Lactic acid bacteria •Aerotolerant anaerobes •Glucose à pyruvate à lactate through fermentation •High sugar intake = lactate secretion = fast tooth decay

Describe lichen morphology, specifying the location of the fungus and the photosynthetic symbiont. What are some of the neat uses of lichens by humans?

Lichen are composed of a fungus (mycobiont) and a photobiont (algae, cyanobacteria, or both). The alga or bacterium provides photosynthetic nutrition, while the fungus provides minerals and protection (from UV light damage). The mycobiont makes up the body of the lichen. The photobiont cells are typically present towards the upper surface where light levels are highest. The mycobiont is present throughout the lichen, and anchors the lichen to solid surfaces. New tissues produced by a lichen that has been turned upside will orient in the regular position. This suggests the fungus can sense light and readjust to enable the photobiont to receive adequate illumination. Cortex of a lichen is a dense, protective skin of fungal tissue. The fungus actually surrounds each of the photobionts with hyphae. It uses specialized structures called haustoria to penetrate the photobiont cell. Through the haustoria, fungi extract the sugars produced by photosynthesis and the nitrogen from n-fixation. The third layer is the medulla, which is largely made up of fungal cells. Another layer, the lower cortex, is where the lichen is attached to the substrate by hair-like strands. The entire structure of any lichen is known as a lichen body, or thallus. In the Russian Far East, Usnea filipendula ("beard lichen") was used as a powder to treat wounds. When it was tested for antibacterial activity, the results were quite positive. "Wolf lichen" (Letharia vulpina) was the most widely used dye lichen for native peoples in North America. "Oakmoss lichen" is an important ingredient in fine perfumes. It is harvested commercially in large quantities in south-central Europe.

What types of molecules serve as C sources for catabolism and what are their similarities/differences?

Major C sources for catabolism in microbes include polysaccharides, lipids, proteins, and aromatics. Some of these substrates are used more rapidly (e.g. polysaccharides) because they require less activation energy or fewer types of enzymes to break down. In addition, different C sources have different breakdown products. Polysaccharides are hydrolyzed ultimately to products like glucose. By contrast, lipids are hydrolyzed to glycerol and fatty acids. Proteins are hydrolyzed to amino acids, which are further broken down in one of two ways: decarboxylation (removal of CO2) to produce an amine, or deamination (removal of NH3) to produce a carboxylic acid. Amine products include putrecine and cadaverine, which cause noxious odors. Fungi and soil bacteria catabolize lignin to oxidized benzene derivatives such as benzoate and vanillin. Many bacteria also metabolize benzene derivatives, and even polycyclic aromatic molecules, either partly or all the way to CO2. For all of these molecules, some fraction of their products ultimately enters central metabolic pathways. Glucose from polysaccharides and glycerol from lipids commonly enter central catabolic pathways such as glycolysis. Other molecules, like carboxylic acids from protein, fatty acids from lipids, and benzene derivatives can enter the TCA cycle when a terminal electron acceptor is available; alternatively, they enter fermentation.

What are the major trophic groups of terrestrial microbes and where in terrestrial ecosystems are they found?

Major groups of microbes in terrestrial ecosystems include decomposer microbes, root symbionts like mycorrhizal fungi (both ectomycorrhizal and arbuscular mycorrhizal) and N fixers, endophytes of plant tissues, and both plant and animal pathogens. Microbes are found throughout terrestrial ecosystems, but certain parts of terrestrial ecosystems have more microbes than others. Decomposers are found in dead plant tissue like wood, soil, and even bedrock. Root associated microbes are found mostly in the rhizosphere and the rhizoplane, and in fact this is where most soil microbes are found as well (because there is so much plant carbon available through the root system). Endophytes can be found throughout plants, as can plant pathogens.

Energetics: Lecture Summary

Microbes can break the basic rules of thermodynamics using syntrophy Energy sources (e- donors) and sinks (e- acceptors) for microbes can be organic or inorganic compounds Microbes store energy in membrane potentials and high-energy bonds (NADH, ATP)

Obligate mutualisms: Lichens

Mycobiont forms majority of lichen body (= thallus). Photobiont located at the top of the thallus. Photobiont can move in response to orientation!

a) Mycorrhizal fungi

Mycorrhiza: mutualistic association between a fungus and a plant root where carbon flows primarily from plant to fungus, and inorganic resources move from fungus to plant

Denitrification in Marine Dead Zones

N and C loads usually happen together = O2 used in ETS during catabolism = NO3 used in ETS

How does oxygen impact N assimilation by microbes? How do microbes protect against the detrimental impact of oxygen on N assimilation?

N-fixation - like the fixation of carbon - is a very energy demanding process. Therefore, it requires oxidative respiration (O2 as the terminal electron acceptor in the ETS) to generate enough ATP to run the N fixation process. Aside from the high energy cost, a major issue regarding nitrogen fixation is that the enzyme responsible for n-fixation is highly sensitive to oxygen. O2 sensitivity occurs because of the large reducing power needed to make NH4+ (O2 wants the e-s!). Also, In the presence of oxygen the Fe protein, which is the first in the two-component system, is damaged in a way that cannot be reversed. To protect against the detrimental effect of O2 on nitrogenase activity, N-fixers can: (1) separate phototrophy from N fixation temporally (during the day, the bacteria grow photosynthetically and generate ATP and reducing power. At night, photosynthesis ceases and nitrogenase becomes active) (2) separate phototrophy from N fixation spatially (one example is free-living cyanobacteria with specialized cells called heterocysts. In the vegetative cells, photosynthesis goes on as normal and no nitrogenase is produced. In the heterocyst cells, which are thick-walled that oxygen can't easily diffuse in, nitrogenase is produced and can be active.) (3) bacteriods (which are bacterial cells from the Rhizobia genus living in the roots of legumes) let plant leghemoglobins bind to O2 and transport it directly to the bacteria's ETS (rather than letting the O2 float around free in the cell). (4) The transcription of genes coding for nitrogenase is turned off in the presence of O2 (when oxygen levels are high, NifA is blocked from activating transcription of nitrogenase genes by binding NifL) Ammonium oxidation (i.e. nitrification) requires oxygen to be present (so that ammonium can be oxidized!), but denitrification is a strictly anaerobic process.

Microbes cycle N uniquely in other ways

N2 reduction->nitrogen fixers->NH4+->Nitrifiers->NO3->Denitrifiers->anaerobic reduction-->(go back to start)

How can we use the genome of a microbe to infer its metabolism?

Now that so much metabolism is understood in model organisms, much of the metabolic capacity of other organisms can be deduced by comparison of their genomes. As an example, the TCA cycle, in whole or in part, is found in all microbial species except for degenerately evolved pathogens that are dependent on host metabolism, such as Treponema pallidum, the cause of syphilis. The genome encodes key enzymes of glycolysis, as well as those of fermentation to lactate or acetate. However, enzymes of the TCA cycle are completely absent, as are the proteins of electron transport. Thus, T. pallidum can generate ATP only by substrate-level phosphorylation, such as the ATP formation steps of glycolysis. In addition, human gut bacteria (strains of Bacteroides plebeius), have obtained genes from marine bacteria enabling digestion of marine seaweed polysaccharides such as porphyran. Analyzing from individuals with a long dietary history of eating Porphyra seaweed (nori, used to wrap sushi), research has shown that their intestinal bacteria include a strain of Bacteroides plebeius whose genes encode enzymes and Sus-like proteins for digestion of the sulfonated polysaccharides unique to the seaweed. The genes show homology with similar genes from marine bacteria associated with the seaweed (Zobellia and Microscilla). In effect, the human-associated bacteria appear to have "learned" to digest the seaweed by picking up the necessary genes through horizontal transfer. Humans who have never consumed the seaweed lack bacteria with these particular genes. However, in individuals who consume a lot of seaweed, the genomes of their gut bacteria are functionally part of their human metagenome.

Habitats: Oral Microbiota

•A complex community that develops over time. •~500 taxa present. •Contains both aerobic and anaerobic members. •Many bacteria don't attach directly to teeth, instead they form biofilms (i.e. plaque).

Describe why increasing depth in lakes leads to succession of microbes that have different modes of respiration. How are electron receptors other than oxygen involved?

Oxygen is the favored electron acceptor because it provides the greatest energy gain, when paired with any particular electron donor. But in many lakes, especially ones where there is not a lot of mixing, oxygen becomes less and less available at depth. Many bacteria have no problem with this and simply switch their electron acceptors to use molecules other than oxygen (like NO2-). Others specialize on the zone that best favor their growth. The lower in the lake, the less of a difference in E zero there is between reactants (electron acceptors) and their products, so there is less energy gained with these alternate electron acceptors.

Photosynthetic microorganisms can live near deep sea hydrothermal vents, in the absence of light energy. How do these organisms make a living near the hydrothermal vent? Be sure to include sources of energy, electrons, and carbon for these organisms.

Photosynthetic H2S oxidizers (green sulfur bacteria, like Chlorobium species) fix carbon from CO2 at the bottom of the ocean. These microbes use oxidation of sulfur (from H2S) to get electrons and they obtain energy not from sunlight, but from heat that is emitted from the reaction zone of hydrothermal vents. Chlorobium species use Photosystem I to get energy, which has a bacteriochlorophyll that absorbs energy at 840 nm. Heat energy from hydrothermal vents is in this range, so can be used by Chlorobium to excite electrons in its Photosystem I. Chlorobium can then run Photosystem I to generate energy for the cell.

Algae

Phototrophs or Heterotrophs? •Single-celled plants and simple multicellular plants •Primary producers: phytoplankton •Evolved by engulfing phototrophs --Primary endosymbionts -Green algae -Red algae --Secondary endosymbionts -Cryptophyte algae -Kelp -Diatoms

Which types of human habitats harbor the most bacterial phylogenetic diversity? Which habitats harbor the most bacterial taxon diversity? What does the difference between phylogenetic diversity and taxon diversity suggest about these different habitats?

Phylogenetic diversity tends to be greatest on outside habitats (i.e. skin) rather than inside habitats (i.e. external auditory canal). For example, the oral cavity has a lot of bacterial phylogenetic diversity, but not nearly as much as the skin. By contrast, the human gut harbors a large amount of bacterial diversity, particularly in the colon (i.e. large intestine), at the species/taxon level. The colon actually has higher diversity than skin at the species/taxon level (i.e. more different taxa colonizing the colon than the skin). This suggests that the colon has been very selective in the types of microbes that it gets colonized by, and that those lineages colonizing the colon have radiated into many different taxa after colonization. Phylogenetic diversity is the total branch length between taxa over the entire bacterial phylogenetic tree that is found within the skin community). Higher phylogenetic diversity on skin indicates that a greater diversity of bacterial lineages (perhaps at the order or family level) can colonize the skin, perhaps because it is in direct contact with.

Your relatively undiverse tongue microbiome is shaped primarily by environmental factors like temperature and osmotic stress. If you transplanted microbes from your forearm onto your tongue and then re-sampled your tongue 8 hrs later, what would you expect to find?

Phylogenetic diversity would have dropped to tongue levels

Describe the tri-partite symbiosis between tropical panic grasses, fungal endophytes, and viruses. Which microbial partner is more beneficial to the plant - the fungus or the virus?

Plant vessels are colonized by endophytic microbes. Tall fescue is a grass grazed by cattle in the southeastern US. It is well-known for hosting foliar and stem endophytes. The fungus produces alkaloids that deter insect predators, pathogens, and root-feeding nematodes. Other kinds of endophytes protect plants from heat, salt, and drought. A plant-fungal symbiosis between grass and a fungus allows both organisms to grow at high soil temperatures (65oC) in Yellowstone National Park. When root zones are heated up to 65°C, non-symbiotic plants either become shriveled and chlorotic or simply die, whereas symbiotic plants tolerate and survive the heat regime. When grown separately, neither the fungus nor the plant alone is able to grow at temperatures above 38°C. However, the plant can only grow at high temperatures when it is infected with both the fungus (Curvularia protuberata) and it's viral symbiont (Curvularia thermal tolerance virus; CThTV). When plant roots are maintained at 65°C for 10 hours under greenhouse conditions, plants that are nonsymbiotic (NS) and symbiotic die. The wild-type plant inoculated with a virus-infected isolate of the endophytic fungus C. protuberata (Wt) and a hygromycin-resistant isolate of C. protuberata newly infected with the virus both persist under these conditions. However, when a plant is inoculated with a virus-free hygromycin-resistant isolate (VF) of C. protuberata, it is not able to withstand the heat.

N-fixation exclusive to microbes

Plant-assimilated NH4+ drives AA production and provides reducing power This is catalyzed by nitrogenase, a 2-component enzyme complex

Why do the purple bacteria occupy a thinner layer of the mat that than the green and the cyanobacteria?

Purple bacteria use ATP diversion to reduce NAD+ indirectly

What is a reduction potential (E), and how is it related to ΔG?

Reduction potential (E) is the tendency of a molecule to accept electrons, measured in volts (V) or millivolts (mV). A reduction potential exists between the oxidized form of a molecule (electron acceptor) and its reduced form (electron donor). The better the molecule is at accepting electrons (rather than donating), the higher its reduction potential, relative to it's reduced form. A positive value of E has a negative ΔG, as described by this equation: ΔG°′ = −nFE°′ This means that for molecules that are good electron acceptors (i.e. have high reduction potential), when they accept electrons, it is a highly favored reaction (i.e. has a large negative delta ΔG).

How do Rhizobia bacteria colonize root cells to conduct N fixation?

Rhizobia are attracted to the legume by chemotaxis toward plant flavonoids. Nod factors (protein molecules composed of chitin with lipid attachments) establish species-specificity for the plant to detect the Rhizobium species. The Nod factor induces an epidermal root hair to curl around the bacterium and take it up into the infection thread, a tube of plant cell wall material. The plant nucleus directs then tube growth inside the plant cell. The thread eventually penetrates the plant cortical cells, where the bacteria lose their cell walls and become nitrogen-fixing bacteroids. The bacteriods remain sequestered within a sac of plant-derived membrane called a symbiosome. The membrane of the symbiosome contains transporters that exchange nutrients between bacteriods and plant cell.

What is a secondary endosymbiont and how are they related to algae?

Secondary endosymbionts are organisms that engulfed a primary symbiont (perhaps through phagocytosis), which had also previously engulfed another organism. All algae possess chloroplasts (which evolved by engulfing cyanobacteria). These primary endosymbiotic algae, or "true algae," are products of a single ancestral endosymbiosis that also gave rise to land plants. Secondary endosymbiotic algae arose from protists that engulfed a primary endosymbiont. Cryptophyte algae are an example of secondary endosymbiotic algae that contain chloroplast, the vestigial primary-host cytoplasm, and the vestigial nucleus or nucleomorph from the engulfed primary endosymbiont.

Sorcerer II Expedition 2003 - 2007

•6 million new genes discovered •Every 200 miles, 85% of sequences are unique •Marine species are endemic •25 million species identified Things that determine microbial communities: -Environment: Weather, Sunlight, Nutrients -history: dispersal, evolution Photosynthetic genes are highly expressed in the ocean

What is a sigma factor? How does elevated temperature induce transcription of heat shock proteins in bacteria via a sigma factor?

Sigma factor (σ) is a protein that helps RNA polymerase recognize and bind to a promoter region of DNA. Regulation of the heat-shock sigma factor sigma H (also called RpoH) in E. coli illustrates how the synthesis of a sigma factor can be controlled at the level of translation and degradation. At 30°C, mRNA from the sigma H gene (rpoH) adopts a secondary structure that buries the ribosome binding site, so rpoH mRNA is poorly translated. Inappropriate expression of heat-shock genes at 30°C is prevented by the DnaK-DnaJ-GrpE chaperone system, which interacts with sigma H and shuttles it to various proteases for digestion. To get the process of transcription going, the RNA polymerase needs to find the right part of the DNA helix to start with. Excessive heat, above 42°C for E. coli, causes proteins to denature and membrane structure to deteriorate. All cells subjected to heat above their comfort zone (optimal growth range) will express a set of proteins called heat-shock proteins. These proteins include chaperones that refold damaged proteins, and a variety of other proteins that affect DNA and membrane integrity. The transcription of many E. coli heat-shock genes requires sigma H. So, one of the first consequences of growth at elevated temperature is an increase in the amount of sigma H present in the cell. A sudden rise in temperature melts the secondary structure of the mRNA from rpoH, exposing the ribosome-binding site, allowing translation to occur more readily. Thus, heat shock increases sigma H synthesis, which in turn increases transcription of the heat-shock genes whose products include chaperones and proteases. At 42°C, misfolded cytoplasmic proteins siphon off the chaperone trio and release sigma H to direct transcription of the heat-shock genes. The sigma factor attaches to the RNA polymerase and it is actually what recognizes the promoter sequence which is located upstream of the gene of interest. Once it finds its match, the RNA polymerase then opens up the helix and start transcribing the mRNA to make a protein. As soon as this starts, the sigma factor is released and can go off and find another RNA polymerase to help in finding the right promoter region.

How does the UV-resistant alga Chlamydomonas nivalis contribute to the melting of glaciers?

Snow is a surprisingly harsh environment with conditions such as low temperature, low nutrient, and high irradiation of UV. Chlamydomonas nivalis is a green alga that owes its red color to a red carotenoid pigment. This pigment protects the chloroplast from intense ultraviolet radiation by absorbing it. It also absorbs heat, which provides the alga with liquid water as the snow melts around it. As a result, Chlamydomonas nivalis photosynthetic activity deepens the sun cups, and accelerates the melting rate of glaciers and snowbanks.

Catabolism

Substrates are hydrolyzed to products that enter central pathways of catabolism

Define symbiosis. What types of interactions could be considered symbioses?

Symbiosis is the intimate association of two unrelated species. The term symbiosis was first widely used in the mid 1800s by a scientist named de Bary to describe the association between fungi and algae in lichens. De Bary explicitly treated parasitic interactions as symbioses, but he excluded those that were only of short duration. According to de Bary, both the Schistosoma and Aphid-buchnera examples would be considered symbioses but the insect-plant interaction would not. Different people have different definitions of symbioses. Many, for example, particularly parasitologists, don't consider parasitic interactions to be symbioses. Others argue that any type of interaction with a significant outcome, no matter how short, should be considered a symbiosis. This would make the insect-plant association a symbiosis. One of the more commonly held beliefs about symbioses is that they are associations in which all organisms benefit.

Do microbes use the same enzymes or different ones?

TCA cycle and Acetyl-CoA cycle

Which groups of organisms typically use the Calvin cycle? Which groups use reverse TCA? How are these two mechanisms of CO2 fixation related to different types of phototrophy conducted by these different groups of organisms?

The Calvin cycle is performed by several categories of organisms: bacteria and chloroplasts. It is not found in archaea or in the cytoplasm of eukaryotes. Therefore, it appears to have evolved after the divergence of the three domains of life. It requires a large amount of energy (9 ATP x 2 cycles to generate 2xG-3-P to make one 6-carbon glucose molecule) and reducing power (NADH) to run, so is typically coupled to oxygenic photosynthesis (i.e. "Z" pathway). In some anaerobic bacteria and archaea, the entire TCA cycle runs in reverse. This is believed to be the most ancient form of CO2 fixation - the original cycle of biomass generation in the ancestors of all three living domains. In the reverse TCA cycle, the acetyl group enters biosynthesis, recycling the coenzyme A. The Reverse TCA cycle requires less energy to run (3 ATP as opposed to 9 ATP in the Calvin Cycle), so it is usually used by organisms that don't use the Z pathway for photosynthesis. For example, reverse TCA is used by Chlorobium (green sulfur bacteria), which conducts anoxygenic photosynthesis by photolyzing H2S (electron donor) using only photosystem I.

Describe the coupled redox reactions that lead to fixation of N2 into ammonia.

The coupled redox reactions that lead to N fixation are driven by the enzyme nitrogenase. Electrons flow through from e- donors (like NADH) to ferredoxin, which binds ATP, and transfers e- to the Fe-S cluster (Fd is oxidized and FeS is reduced - requiring 8 ATP). This reduces the FeMO protein (FeS is oxidized and FeMo is reduced). Finally, the electrons are passed to N2, which is reduced to NH3 (then - usually - protonated to NH4+). Nitrogen fixation requires four reduction cycles through nitrogenase: 1. Fe-S protein acquires 2e- from an electron transport protein such as ferredoxin, and then transfers them to the FeMo center. 2. The FeMo center binds 2H+, which is reduced to H2 gas. 3. N2 can now bind to the active site by displacing the H2. 4. Successive pairs of H+ and e- reduce N2 → HN = NH → H2N - NH2 → 2NH3

Describe stratification of environmental factors in lakes, and how this changes with eutrophication. Mention how eutrophication changes the location and abundance of different types of microorganisms living in lake systems.

The depth profile of an oligotrophic lake includes the oxygenated zone (epilimnion) reaches to the thermocline, below which only anaerobes grow. Large amounts of nutrients can be deposited from runoff from agricultural fertilizer or septic systems These nutrients include phosphates, nitrates, and organic pollutants. Because phosphorus is commonly a limiting nutrient (nutrient in shortest supply) for algae, addition of phosphates from detergents and fertilizers can lead to an algal bloom. Nitrogen from sewage effluents and agricultural fertilizer runoff can lead to algal blooms as well, by relieving nitrogen limitation of growth (sometimes, algae and cyanobacteria are limited by the availability of nitrogen). Organic pollutants from sewage effluents overfeed heterotrophic bacteria, depleting the epilimnion of oxygen. In a eutrophic lake, the oxygenated epilimnion is much shallower, and microbial concentrations are tenfold higher than in an oligotrophic lake. This oxygen loss causes the anoxic hypolimnion to reach nearly up to the surface of the lake. In general throughout lakes, as depth increases, minerals become increasingly reduced. Anaerobic forms of metabolism predominate, with microbes using various alternative electron acceptors.

Describe the active processes (i.e. involving activation and repression mechanisms) and passive processes (i.e. not involving activation and repression mechanisms) of quorum sensing in Vibrio fisheri.

The phenomenon of quorum sensing was first discovered in the bacteria that are able to produce light like fireflies (Vibrio fisheri). This system involves active control of an enzyme called luciferase, which is responsible for light emission, by the passive diffusion of autoinducers into (and out of) the bacterial cell. LuxI is the protein that makes the autoinducer. For Vibrio fisheri, it makes an autoinducer that is an acyl homoserine lactone (AHL). Those passively diffuse out into the environment. When enough V. fisheri cells are in the same place, the density of AHLs goes way up. They then passively diffuse back into the cells and bind to the second protein luxR, which is an activator protein. This is where the active part starts: LuxR helps the RNA polymerase complex bind to beginning of the lux operon, and all the lux genes get transcribed at a high level (inducer + activator protein). lux B and luxA genes encode luciferase. Luciferase generates a redox reaction in the cell that eventually leads to light formation.

Describe the different steps involved in the electron transport system of E. coli. How do electrons and protons move between flavin, ferredoxin, quinone, and cytochrome oxidase?

The electron transport system, ETS (or electron transport chain), stores energy from electron transfer as ion gradients across the membrane of the cell or an organelle. Note that NADH forms by receiving two electrons plus 2H+ from an organic product of catabolism (designated RH2). The first thing that happens with NADH is that it gets oxidized. NADH donates electrons to NADH dehydrogenase (NADH:quinone oxidoreductase, NDH-1). Those all get transferred to a flavoprotein. The flavin part of the protein (flavin mononucleotide - FMN) is an electron and proton acceptor. The flavoprotein transfers two electrons to the next protein in the system, one at a time. This protein that has an iron-sulfur cluster is more commonly known as a ferredoxin. To prepare to receive more electrons, the iron-sulfur protein transfers the electrons it currently holds to a quinone. The quinone can accept 2 electrons and 2 protons (on board), so it picks up more protons from the cytoplasmic side of the membrane. Once again the quinone interacts with a protein that only handles 2 electrons - cytochrome. So the protons get dumped outside the cell. To get more electrons, cytochrome interacts with an enzyme called cytochrome oxidase. This enzyme takes the electrons, which oxidizes cytochrome, and transfers them to the terminal electron acceptor, which in this case is oxygen. This whole process involves the pumping of protons out of the cell, which sets of a gradient of more outside than inside. The cell then uses that gradient to create ATP as the protons are allowed to diffuse through the ATP synthase pathway. *Note that the structure of each cofactor must allow transition of an electron between closely spaced energy levels to avoid "spending it all in one place." If all energy were spent in one transition, most of it would be lost as heat instead of being converted to several small processes, such as pumping H+ across the membrane.

What are the characteristics of the most highly expressed genes in the ocean? How abundant are they in microbial genomes (across all microbes) and what sorts of metabolic processes are they involved in?

The most highly expressed DNA sequences in the open ocean are microbial genes that happen to be most rare in microbial genomes. They include elements of photosynthesis (light harvesting proteins and Rubisco) and genes for DNA repair (likely due to UV damage).

Where are microorganisms found in the ocean ecosystem? Describe the different zones of the ocean, their abiotic characteristics (e.g. temperatures, pressures, salt content, pH), and which types of organisms live there.

The ocean is salty throughout. However, the water column is not uniform throughout. Diffusion rate of oxygen in water is about 10,000 times less than in air. So, the oxygenated parts of the ocean are usually the top parts (neuston zone and euphotic zone). Photosynthetic microbes (i.e. producers or phytoplankton) that fix half the world's carbon inhabit these zonesThe neuston is the air-water interface (10 microns thick). It contains the highest concentration of microbes. By contrast, the euphotic zone contains the upper 200-300 meters of the open ocean. In this zone, we find phytoplankton, as well as zooplankton (i.e. microbial predators of phytoplankton), as well as decomposers. The coastal shelf can decrease the size of the euphotic zone to a couple of meters. The aphotic zone has no light penetrating, so does not support the growth of phototrophs (only heterotrophs). The benthos zone is where the water column meets the ocean floor, and harbors microorganisms that live near hydrothermal vents, for example. pH is mostly determined by the dissolution of CO2 in the water, such that pH is low near the top of the ocean (due to CO2 from air dissolving at the surface, creating carbonic acid). In the benthos, hydrothermal vents can emit pH ~10 fluids, increasing the pH of the surrounding water significantly. In the benthos, psychrophiles and barophiles grow, although slowly, because little organic matter is hitting the ocean floor. By contrast, thermophiles grow in black smoker thermal vents. Reduced minerals support lithotrophic bacteria, including mutualists of giant clams and worms. H2S oxidizers fix carbon from the carbonates, generating organic metabolites that feed the animal hosts. The tube worm Riftia is colored bright red by a pigment that carries H2S and O2 through its circulatory fluid. It has evolved such dependence on its microbes that it has lost its own digestive tract.

Describe how dental plaques develop, including the different types of bacteria that colonize (and when) and the different types of metabolism they exhibit that contributes to gum and tooth decay.

The oral microbiome is a diverse community (~ 500 taxa) that contains both aerobic and anaerobic members. Bacteria often develop biofilms called plaques in the gingival crevice. If plaque growth in the gingival crevice is not inhibited, the microbial population can shift from a mix of Gram positive and Gram negative species to one being dominated by Gram negatives. After that, spirochete bacteria begin to appear. These new populations releases proteases that destroy gum tissue. The resulting inflammatory response gives rise to bleeding gums and eventually receding gums and tooth loss. Another set of bacteria that are common in the human mouth are lactic acid bacteria (Gram positives). These bacteria are what are referred to as aerotolerant anaerobes, meaning they don't use oxygen but can grow in its presence. These guys are important to us because of their metabolism. They breakdown sugars like glucose into pyruvate, then reduce pyruvate to lactate through fermentation. The lactate that is leftover is no good for the bacteria, so they excrete it into their surroundings. These bacteria do not generate much ATP, so they make up for this by processing more glucose molecules per unit time than if they used pyruvate in aerobic cellular respiration. The lactic acid bacteria grow so rapidly and produce so much lactic acid, which inhibits the growth of other bacteria, that they soon come to dominant the enrichment culture. The problem for us with lactic acid bacteria involves the amount of sugar in our diet. When our sugar intake is low, these bacteria do not produce enough lactic acid in a short enough time period to damage the enamel on our teeth. A high sugar diet, however, allows them to produce high concentrations of lactic acid, which undermines the integrity of the tooth enamel and results in a cavity.

At a family barbeque, you tell your family that you are taking microbiology this semester. Your aunt proceeds to gag and say how gross microbes are. How would you describe to your aunt three examples of human-associated microbes actually improving or maintaining human health?

There are many ways in which human-associated microbes can benefit the body. For example, in the large intestine, prokaryotes break down complex polysaccharides that cannot be digested with human intestinal enzymes. Examples would include plant polysaccharides like cellulose and pectin. Also entering the colon are human-produced polysaccharides like mucins, which are released when small intestinal cells slough off. The main end products of polysaccharide fermentation are compounds like acetate, propionate, and butyrate. These compounds can be absorbed by the colon mucosa and used as sources of carbon and energy. The amount of energy the human body derives from the fermentation by bacteria has been estimated to about 15-20% of the total energy obtained from our diets. So, the colon can really be considered a second organ of digestion, but unlike the stomach, it consists mainly of prokaryotic cells. People taking certain antibiotics that are particularly effective against colonic bacteria can experience dramatic decreases in the numbers of normally predominant anaerobes. Under such conditions, Clostridium difficile, which is normally present at low numbers, can overgrow other bacteria and produce toxins that cause severe damage to the lining of the colon. Death can result in the matter of a few days. This example highlights the fact that keeping our native microbiota intact is a big way we fight off pathogens, particularly because our native gut microbial communities are very diverse. Because of this, the community consumes nutrients that might otherwise support the growth of pathogenic species. Other protective roles of human-associated microbes are also related to deterring the growth of disease-causing microbes. For example, Lactobacillus sp., which normally inhabits the female reproductive tract, defends the host against colonization of pathogens such as C. albicans. Evidence suggests that Lactobacillus reduces the adhesion of C. albicans to epithelial cells either by (a) outcompeting fungal cells for adhesion sites, such as cellular receptors to which Lactobacillus has higher affinity, or (b) by secreting biosurfactants such as surlactin that physically decrease fungal binding. Additionally, when L. acidophilus breaks down food in the intestine, (c) several substances are formed (such as lactic acid and hydrogen peroxide) that create an unfriendly environment for "bad" bacteria.

Describe phylogenetic diversity of human-associated microbes, including what the phylogenetic tree of human-associated bacteria is shaped like. Where on the tree is the most diversity found (i.e. phylum level? class level? Species or strain level?)?

There are more than 50 bacterial phyla on earth, but human-associated communities are dominated by only 4 of them. Most of the diversity is concentrated at the strain level and not at deeper phylogenetic divisions. This suggests that the human body has been somewhat selective about which microbes it allows it gets colonized by, but those microbes that have colonized the human body have radiated into a number of different niches. More phylogenetic diversity is found between people than within the same person on any given day. In addition, more diversity is found between habitats, people, and months of sampling than within habitats, people (i.e. sampling multiple spots on the same person), or within the same month.

What are two different fates of the products of catabolism? What is the role of these next steps in microbial growth?

Two fates of catabolism products include: 1. Respiration: complete breakdown of organic molecules with electron transfer to a terminal electron acceptor such as O2. The benefit for the cell is that it can store energy as a proton potential, which can be used to generate ATP for the cell. Respiration yields far more energy from catabolism than does fermentation. For humans, respiration is synonymous with breathing; but in the absence of O2, many microbes use alternative electron acceptors, such as nitrate or sulfate. The use of a terminal electron acceptor other than O2 is called anaerobic respiration. Thus, microbes—unlike humans and other animals—have the capacity for anaerobic respiration. 2. Fermentation: partial breakdown of organic food without net electron transfer to an inorganic terminal electron acceptor. Microbes compensate for the low efficiency of fermentation by consuming large quantities of substrate and excreting large quantities of fermentation products. An advantage of fermentation is that the rapid accumulation of acids or ethanol can inhibit growth of competitors. Fermentation products are then excreted from the cell. The large quantities of substrate consumed in fermentation generate large amounts of waste products to be excreted.

How is Geobacter able to generate energy for fuel cells?

We are now harnessing bacterial electron transfer to power our own electrical devices These bacteria use the extension of electron-conducting "nanowires" made of protein. The nanowires help bacteria form a biofilm that connects them to each other and connects their cytochromes to the metal electron acceptor. In a fuel cell, the bacteria form a biofilm on the anode (electron-attracting electrode). As the bacteria oxidize organic fuel, they cause charge separation between electrons and hydrogen ions. The electrons then pass within a current to the cathode. The hydrogen ions migrate through a polymer membrane, whereas the electrons enter the anode leading to an electrical wire. The remaining carbon and oxygen atoms of the fuel are released as CO2. To complete the circuit, the electrons from the wire current ultimately react with oxygen and hydrogen ions to form water, as in aerobic respiration.

Protozoa in freshwater systems

What kind of habitat favors pseudopod motility? •Amebas: •Use phagocytosis •Cause disease (Legionella) •Form pseudopods

You find an unusual colony of E. coli floating on the surface of your Coke (pH 3). Describe three mechanisms by which those bacterial cells may withstand the low pH environment of soda by regulating their internal cellular pH.

When cells are placed in pH conditions below their optimum, protons can enter the cell and lower internal pH to lethal levels. Therefore, many microbes have ways to regulate their internal pH levels. a) Proton extrusion mechanisms: E. coli changes the pH of the medium using amino acid decarboxylases (removal of CO2 from amino acid, formation of amines). It consumes organic acids when growing at low pH, but produces these acids when growing in alkaline conditions. For example, E. coli has an antiporter protein on it's cell surface that brings glutamate into the cell in exchange for the decarboxylation product gamma-aminobutyric acid (GABA), which is transported out of the cell. The combined action of the decarboxylase and antiporter removes H+ from the cell. Microbes can also prevent the unwanted influx of protons in other ways: b) Acidophiles will transport other cations (K+ or Na+) into the cell to make it more basic. cs) Acidophiles often have high amounts of tetraether lipids. Altered cell membrane structures decrease proton permeability. d) Alkaliphiles use antiport systems to scavenge protons. They will recruit protons to cell in exchange for expelling Na+.

Where can most microbes live?

Xerophilic fungi: Most tolerant organisms to low water activity (<0.85)

b) N-fixing bacteria

are branching out like a tree 1. Flavonoids from plant bind Nod protein of Rhizobium, attracting bacteria to plant 2. Rhizobium cell binds to root hair 3. Root hair is induced to curl around Rhizobium 4. Plant cell envelope opens tube inward as infection thread 5. Rhizobium bacteria travel down infection thread, plant nucleus travels ahead 6. Rhizobium bacteria reach cortical cells 7. Bacteria enter cortical cells and differentiate to bacteroids ---N-fixing bacteria are integral to historical and modern agriculture

Regulation of the Calvin Cycle

by carboxysomes

The process by which Geobacter metallireducens (which can be used to create fuel cells) removes insoluble Uranium6+ through oxidation of acetate in polluted water is a form of:

chemoorganotrophy

Ice nucleation by psychrophiles

if you touch water it'll turn into ice immediately

Colon-Associated Diseases

infant botulism,. pseudomembraneous colitis

Microbial diversity in humans

is low

Swiss cheese production involves two fermentation stages

lactobacillus helveticus and streptococcus salvarius

Most microbes are in the environment

most taxa are poorly described

Barotolerant

optimal growth: 1atm growth range: up to 400atm depth found: 4km

solute and water activity

osmotolerant: able to grow over wide range of water activity or osmotic concenytration halophile:requires high levels of sodium chloride, usually above 0.2M to grow

Where do AMF and EMF live?

soil nutrients: -high: nonmycorrhizal plants -arbuscular mycorrhizal plants -low: Ectomycorrhizal plants

Substrates for biosynthesis are generated from catabolic reactions microbes use the same enzymes or different ones?

substrates for biosynthesis

A new species of microbe is discovered near a hydrothermal vent on the ocean floor by DNA sequencing. It can regulate many intracellular factors in order to survive in the vent. Which of these factors can the microbe NOT regulate?

temperature

glacier ice

there's high microbial diversity in glacier ice

Colonic Fermentation

theres a process between human enzymes and bacterial enzymes -bacterial enzymes: acetate propionate butyrate, and undigested material

Energetically favorable reactions help cells build biomass

ΔG0 reaction = ΔGf0 products- ΔGf0 reactants Glucose + 6O2 à 6CO2 + 6H2O ΔG0 reaction = [(6 × ΔGf0CO2) + (6 × ΔGf0H2O)] - [(ΔGf0Glucose) + 6 × (ΔGf0O2)] ΔG0 reaction = [(6 × -394.4 kJ/mol) + (6 × -237.1 kJ/mol)] - [(-910.4 kJ/mol) + 6 × (0 kJ/mol)] = -2,879 kJ/mol Oxidation of organic molecules is often an energy-releasing process. Why does oxidation of glucose generate so much more free energy than oxidation of ethanol? hint: think about the molecular composition of glucose vs. ethanol and how many oxygens are already there

Linking Microbiota and Obesity

•12 obese patients fed 1 of 2 diets for 1 year. •Compared gut microbiota to lean patients. •Strong evidence weight may be controlled by microbes. •Suggests potential new type of treatment.

Strategies for Adaptation to Immediate Changes to Environment

•Adaptation strategy differs depending on timeline - seconds to generations. •Long-term strategy = mutation, which can be beneficial but also risky. -Issues with mutation: •mutations occur randomly. •Some traits require multiple mutations •Not exactly reversible •Short-term strategy = gene regulation, which is rapid and reversible.

Eukaryotes in aquatic systems

•Algae •Protists (Algae, Protozoa)

Ways to Inhibit QS

•Approach #1: Modify the signal -Analog substitution in AI production pathway that stops synthesis -Modify AI molecule so it does not bind with receptor •Ex: Erwinia response: lactonolysis via pH change •Approach #2: Modify the receptor -Destroy receptor, so signal can't be "heard" -Use an AI mimic to competitively inhibit receptor

Microorganisms in the Soil Environment

•Archaea - on surfaces of soil particles •Prokaryotes on surfaces within smaller soil pores •Filamentous fungi - bridge open areas between soil particles or aggregates --polysaccharide secretion by plants binds clay particles ---OXYGENATED; water drained -organic (O) horizon: organic matter undergoing decomposition -aerated (A) horizon: decomposed organic particles and minerals -eluviated (E) horizon: insoluble particles leached by rainwater, gray or grayish brown -B horizon: clay and minerals ---WATER TABLE--- ---ANOXIC; water saturated -water-saturated horizon: organic matter, clay and minerals leached from above -mineral particles broken from bedrock below -bedrock

Cyanobacteria Phototrophy

•Combines key features of PSI and PSII. -the cycle for a single electron is shown -four electrons (4e-) cycle per O2 photolyzed -two electrons (2e-) cycle per NADP+->NADPH -Feredoxin-NAD+ reductase

Fatty acid biosynthesis

•Condensation of CO2 + acetyl-CoA •Rest needs fatty acid synthase complex •Regulated by -transcription repressor (AccB of acetyl-CoA carboxylase) -Starvation -Temperature (dehydratase) •Need fatty acid synthase complex -Condensation of CO2 + acetyl-CoA •Regulated by -transcription repressor (AccB of acetyl-CoA carboxylase) -Starvation -Temperature (dehydratase) •Similar pathways used for: -Polyesters -Polyketide antibiotics

Controlling Gene Expression

•Constitutive expression: genes expressed at the same level under all conditions. •Regulated expression: genes transcribed under only certain conditions. •How can bacteria manipulate expression levels? -One common way: by promoting (with sigma factors and activators) or inhibiting (with repressors) RNA polymerase activity.

Dominant Structuring Factor: Habitat

•Costello et al. (2009) Science 326, 1694. •8 healthy subjects, 5 male, 3 female. •Sampled on two consecutive days in June and Sept. •Data from up to 18 different locations. •Pyrosequencing of 16S rRNA = 106 seqs.

DG0 vs. DG

•DG0 calculated under 'ideal' conditions. •DG takes into account the actual concentrations of reactants and products. •For reaction A + B à C + D: DG = DG0 + 2.3 * RT * log([C][D]/[A][B])

Barophiles

•Deep ocean contains barophiles that can tolerate atmospheric pressures up to 1,100 atm •Extreme barophiles are often psychrophiles -due to overlap between depth and low T? -due to common strategies for overcoming reduced fluidity and enzyme activity?

Probiotics

•Definition: a preparation of living, defined organisms that alters community associated with a host and confers a health benefit. •Increasing evidence probiotics can be helpful in preventing disease. -Lactobacillus GG reduces incidence of diarrhea and speeds recovery. •Typically have transient effects - don't establish as residents. -May play a particularly important role following antibiotic treatment by altering community susceptibility to secondary infections.

Sigma Factor Variation & Abundance

•Different sigma factors recognize different promoters. -By using one sigma factor for multiple genes, bacteria can coordinate function. •Sigma factor abundance directly affects RNA polymerase activity: making more of a sigma factor will lead to more transcription of associated gene(s). •Typically environmental sensing systems control sigma factor production.

Habitat: Human Colon

•Different type of environment than mouth, stomach and the small intestine. •Bacterial concentrations in the colon are extremely high (1012/g, >5000 taxa). •Some methane-producing archaea are present at low levels •Colon is designed to be a holding tank for microbes, like rumen in cattle.

sea microbes

•Discovery of Synechococcus (1979) and Prochlorococcus (1988) •Prochlorococcus most abundant phototroph in sea (probably on Earth!)

Bacteria: Quick-Change Artists?

•Example 1: A bacterium living in pot of dressing at a salad bar, suddenly transported into a pool of stomach acid. •Example 2: A bacterium living inside an insect host, suddenly injected into a human arm.

Ex: Francisella Pathogenicity

•F. tularensis - cause of "rabbit fever". •Can infect humans and cause lethal effects - pneumonic tularemia. -quorum sensing inhibitor

Light reactions: getting energy from light!

•First types of photosynthesis did not evolve oxygen as a product. -Came much later with cyanobacteria •Cyanobacteria (and green plants) use the two-part "Z" pathway: photosystem I + photosystem II •Other bacteria can take the two-part "Z" pathway and run each part independently -Green bacteria = photosystem I alone -Purple bacteria = photosystem II alone

Habitat: Human Stomach

•For many years, doctors thought the stomach was free of microbes. •However, some bacteria do colonize the stomach, particularly areas where pH is higher. •Stomach is coated with a mucin layer that protects stomach lining. •pH in the mucin layer ~ 7, colonization by bacteria and fungi (~200 taxa).

Fecal Bacteriotherapy?

•For patients with chronic colonic illness, re-establishing a healthy microbiota possible with fecal transplant (FT). -Application for patients with chronic C. difficile infection. •Study 1: 24 of 26 patients completely cured of any C. difficile infection following FT (J. Clin. Gastroenterology study, 2012) •Study 2: FT vs. vancomycin treatment (Keller et al. 2013).

Regulation & Environmental Sensing

•Gene regulation is done to respond to environmental changes - how are those changes sensed? 1.Some changes can transmit directly into the cell

Regulation & Environmental Sensing

•Gene regulation is done to respond to environmental changes - how are those changes sensed? 1.Some changes can transmit directly into the cell - tetracycline resistance! 2.Others are sensed on the cell surface and a signal is sent into the cell

gene regulation lecture summary

•Gene regulation provides a rapid and reversible strategy for dealing with changes to environmental conditions. •Regulatory proteins can be used to both increase (sigma factors, activators) and decrease (repressors) transcription levels. •Signals affecting regulation can interact directly with transcription machinery or indirectly through phosphorylation relays. •Bacteria can use QS to census the biotic environment and control group-related activities, which may also be used to fight disease.

Where Are We?

•Goal: capture energy to build order in cell. •So far, energy source has been electron exchange to ETS from organic or inorganic compounds (small energy transitions). •Light can also provide energy to move electrons = phototrophy (large energy transition) •Microbes often couple phototrophy with autotrophy (Photosynthesis) -involves both phototrophy (light reactions) & biosynthesis (dark reactions).

Genetic Regulation

•How do bacteria quickly respond to changes in environmental conditions? •What mechanisms do bacteria use to sense environmental changes? •Can bacteria "talk" to each other and, if so, why is that important? •How can we use bacterial "talking" as a new way to fight disease?

Topic 2.5 - Chemotrophy

•How do microbes use thermodynamics to move electrons between molecules? •Why can microbes use lots of other compounds beside oxygen to breath? •How do microbes store energy created from electron movement in cells?

Topic 2.9 - Nitrogen Cycling

•How does nitrogen get into the system? •How do different N-fixing organisms protect nitrogenase from oxygen damage and how is N-fixation regulated? •How does fixed nitrogen get recycled in the environment, and what are the products of nitrification and denitrification?

Topic 2.3 - Human-Associated Microbes

•How does our microbiota influence our health and metabolism? -Oral microbiome -Stomach microbiome -Intestinal microbiome

The Human Microbiome

•Humans normally carry at least 10 times more prokaryotic cells than eukaryotic cells (and 100x more prokaryotic genes). •Microbial populations develop just after birth and remain there throughout entire lifespan. •Not just contaminants; our indigenous microbiome provides many protective and nutritional roles. -Microbes colonize wherever our body meets the environment

Carnivorous fungi!

•Hyphal ring •Adhesive hyphae •Nematoxic compounds

Definitions of Symbiosis

•In the mid 1800s, the term was coined by Anton De Bary - literally "living together". •Often defined by interaction outcome: originally included the full range of interactions. •In 20th century, definition changed to focus on mutually beneficial interactions. •Other parts of a definition: timing - transient vs. life-long; necessity - facultative vs. obligate; specificity - narrow vs. broad; location - inside (endo) vs. outside (ecto).

Which of these are symbioses?

•Interaction 1: Schistosoma is a parasitic trematode worm. The adults live in the blood vessels of mammals and birds. It infects ~20 million humans, causing diarrhea and malaise. •Interaction 2: Many flowering plants are pollinated by insects. The insect transfers pollen as it visits plants to feed on nectar or pollen. •Interaction 3: Aphids are insects which feed on phloem sap of plants. They require bacteria in the genus Buchnera, which are contained within special aphid cells in the body cavity. If the bacteria are eliminated by antibiotics, the aphids fail to grow and die without producing offspring.

Catabolism: Lecture summary

•Lipids, polysaccharides, proteins, and aromatics serve as C sources for organotrophs •Different types of catabolic mechanisms offer different amounts of energy-rich products •Genome analysis can be used to infer the metabolism of organisms

Aquatic systems

•Marine & brackish systems -Ocean -Coastal regions -Estuaries -Mangrove swamps -Salt marshes --Freshwater systems -Streams, rivers -Lakes, ponds -Swamps, bogs

Lithotrophy: Inorganic e- Donors

•Microbes can use inorganic compounds as e- donors (all animals can't!). •Example: Iron oxidation. •Ex: Fe2+ = e- donor & O2 = e- acceptor. •E0' = 820 - 770 = 110 mV. •Generally lower energy yields than organotrophy, but still sufficient for microbial growth.

Organotrophy: With (aerobic) & Without (anaerobic) O2

•Microbes can use other e- acceptors when oxygen is absent (animals can't!). •Ex: Glucose = e- donor & Nitrite (NO2) = e- acceptor. •= 440 - (-430) = 870 mV. •Alternative e- acceptors = Lower (but still relatively high) energy yield.

Marine viruses

•Most numerous biological entities in ocean (> 10 million particles per ml) •Contain more carbon than 75 million blue whales •~50% of prokaryotes are lysed each day

Biosynthesis: Lecture summary

•Multiple pathways are used to fix CO2, and likely evolved under different conditions •Biosynthesis spends energy, so microbes have evolved controls over production •Precursors to biosynthesis are generated from catabolic reactions •Fatty acids are synthesized via enzyme complexes (not ribosomes), as are other primary and secondary biopolymers used by humans

Types of Symbiosis

•Mutualism -Species specific, organisms can't grow apart •Synergism -Both species benefit, but can be separated •Commensalism -One species benefits, while the other species experiences no effect •Amenalism/Antagonism -One species benefits by harming another: competition •Parasitism -One species benefits by harming another, species-specific, obligate

N2 is fixed by microbes

•Needed by all organisms for basic cellular processes: ~80% of air is gaseous nitrogen. -But atmospheric form, N2, is very stable. •To be assimilated, N2 gas must be converted to ammonia (NH3/NH4+). N2 + 8H+ + 8e- + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi •NH4+ is then assimilated by microbes and plants to form amino acids (e.g. glutamate)

Nitrification and Agriculture

•Nitrification is a significant agricultural issue. •Nitrates are readily leached from soil into groundwater. --Nitrate contaminated water can cause methemo-globinema ("blue baby syndrome") due to growth of nitrite-producing bacteria in infant stomach.

Nitrogen cycling: lecture summary

•Nitrogen fixation by microbes plays an essential role in making N bioavailable. •Microbes have evolved ways to protect nitrogenase from O2 and regulate fixation. •N-cycle reactions are coupled, and result in a number of intermediate products •N-fixation, nitrification and denitrification form the basic ecosystem N-triangle

Nitrogenase and Oxygen

•Nitrogenase is highly sensitive to oxygen. •O2 from photosynthesis needed to generate ATP for fixation -N-fixation is often coupled with photosynthesis •Most N-fixers live in well oxygenated environments and respire aerobically. -A range of different strategies have evolved to protect nitrogenase.

Protection Mechanism IV: genetic

•Nitrogenase production has multiple environmental checks. •When [NH4] is low, nitrogenase operon get turned on in a two-step process. •When [NH4] is high, nif genes do not get transcribed. •When [O2] is high, NifA gets complexed and nitrogenase operon is turned off.

Effects of Indigenous Microbes

•Normally play a protective role: native colonization deters the growth of disease-causing microbes - how? •Occupation of adhesion sites •Production of antagonistic substances •Lower pH helps prevent STD microbes. • •Nutrient utilization - intestinal microbiota nutrient consumption prevents C. difficile colonization.

human microbe summary

•Opportunistic microbes can create disease (oral cavity) •Native microbes can contribute to chronic conditions (e.g. gastric ulcers) •Our gut microbiome also plays a major role in our nutrition and prevention of disease.

Geobacter: Uranium reduction

•Oxidizes acetate (e- donor) and reduces Uranium (e- acceptor) •Reduces U6+ to U4+ CH3CO2- + 4 U6+ + 4 H2O → 2HCO3- + 4 U4+ + 9 H+ -Produces insoluble UO22+ •Remediation in Colorado River

Psychrophiles and thermophiles

•Psychrophiles -slow growth -heavy metal transport -metal resistance proteins •Thermophiles -Biofilms -Lithotrophs -Symbionts •Thioautotrophic symbionts -Redox of inorganic compounds -H2S (electrons) -Reduce carbonates •Can have photosynthetic bacteria! -Chlorobium (green sulfur bacterium) -How do they get energy?

Protection Mechanism I: temporal

•Single-celled cyanobacteria found in open ocean (e.g. Synechococcus and Prochlorococcus). •Separation of phototrophy and nitrogen fixation in time (temporal). -During the day, oxygenic phototrophy generates ATP and reducing power. -Nitrogen fixation is then carried out at night.

Denitrification

•Strictly anaerobic process, N species used as alternate electron acceptors. •Nitrate (NO3-) è Nitrite (NO2-) è Nitric Oxide (NO) è Nitrous Oxide (N2O) è Dinitrogen (N2).

Microbial community ecology: lecture summary

•Symbiosis represents a range of interactions, from parasitic to mutualistic. •Outcome of symbioses can depend greatly on the environmental context in which they occur. •Pair-wise interactions between hosts can have important direct yet diverse effects (Buchnera - nutrition, SS - parasite protection). •Symbiotic interactions can also involve three- or more strong interactors.

Enviromental microbiome summary

•Temperature, pH, osmolarity, oxygen, and pressure are major selective forces on microbes •Extremophiles have physical and biochemical adaptations to accommodate environmental stresses

human microbiome summary

•The human microbiota is integral and diverse part of who we are. •It appears to be largely structured by the habitats on and inside the body.

Habitat: Small Intestine

•The pH is much closer to neutral (~8). •Problem for microbes: the rapid flow of contents through intestines. -Rapid flow helps human cells compete for nutrients. •Even places with highest concentrations still relatively low (105/g).

How do we know if chemical reactions can support life?

•To determine whether a reaction will go forward: •DG0 reaction = sum DG0 products - sum DG0 reactants. •Assumptions = pH 7, 25°C, and [1 M]. •If DG0 is negative, the reaction will proceed spontaneously, If DG0 is positive, the reaction won't proceed spontaneously.

Topic 2.8 - Biosynthesis

•What are the major primary and secondary pathways of biosynthesis (anabolism) in different microbial groups? •What are the major products of anabolism in microorganisms? •Which primary and secondary products of microbial metabolism have been most helpful to human society?

Linking Pattern and Process

•What generates these diversity patterns? •Hypothesis 1: Differences in current environmental factors (e.g. nutrients available, local chemistry). •Hypothesis 2: Differences in historical exposures (e.g. microbes available to colonize). •Need an experiment to separate these factors

Topic 2.13 - Microbial Symbioses

•What types of interactions are considered symbioses? •What kinds of effects do symbionts have on their hosts? •How do environmental factors affect symbiotic outcomes? •What happens in cases where more than one symbiont is present?

Topic 2.7 - Catabolism

•What types of molecules serve as C sources for catabolism? •Which energy-rich products are generated in the main pathways of organic molecule catabolism in microbes? •How can we use information about catabolic activities of microbes to solve environmental problems?

Quorum Quenching

•With no autoinducer, the QS system is not activated and no transcription occurs. •If host can block QS communication, that can stop pathogenicity of bacteria. -Interference with QS called "quenching". •Because the pathogen is still alive, there is less selective pressure for resistance.

Glucose induces catabolite repression

•cAMP-CAP complex activates lac operon transcription •cAMP: cyclic adenosine monophosphate •CAP: catabolite activator protein •Lactose breakdown -->inhibits enzyme adenylate cyclase •Less lactose breakdown -->adenylate cyclase active

Adaptations of thermophiles >55°C

•protein structure stabilized by a variety of means -e.g., more H bonds -e.g., more proline, less glycine -e.g., chaperones •histone-like proteins stabilize DNA •DNA gyrase tightly coils DNA •membrane stabilized by variety of means -more saturated, more linear, higher molecular weight lipids

1. Decomposers

•recycle plant nutrients •create soil organic matter -retains nutrients -maintains soil structure -holds water for plant use

Root-associated microbes

•rhizosphere -volume of soil around the root influenced by materials released from root •rhizoplane -surface of root •plant roots receive between 30% to 60% of the net photosynthesized carbon -of this, ~40% to 90% enters the soil as a wide variety of materials which create unique environments for soil microbes

Solutes and Water Activity: major stressor

•water activity (aw) -amount of water available to organisms -reduced by interaction with solute molecules (osmotic effect) higher [solute] Þ lower aw -Measured as vapor pressure over solution relative to pure water