Microbiology

Specific regulatory mechanisms

-Control activity of one of a few enzymes -Control synthesis of one or a few proteins Bacteria must often respond to complex environmental changes, or undergo sweeping changes in their physiology; accomplished by global regulatory mechanisms: Bacteria usually live in environments with mixed carbon sources Catabolite repression --> allows the bacterium to use the most efficient carbon source first (often the one most easily fed into the glycolytic pathway; glucose) Diauxic growth curve --> or diphasic growth is any cell growth characterized by cellular growth in two phases; ex. glucose and lactose Lag in growth occurs when glucose is gone, and cells must make new enzymes to use lactose

Writing balanced redox reactions

-Identify electron donor and electron acceptor -Write balanced half reaction (electrons are permitted in half-reactions) -All atoms and charges should be balanced in half-reactions -Electron donor and electron acceptor are reactants -electrons must cancel out

Requirements for metabolism

1) Energy source --> light radiation or chemical 2) Electron donor --> organic or inorganic 3) Electron acceptor --> oxygen, inorganic or organic compounds 4) Nutrition source(s) --> carbon, nitrogen, phosphorous etc When the carbon source and electron donor are the same, this substrate must be partitioned for different uses in the cell

Culture media

1) Rich-complex medium --> not chemically defund, contains complex organic molecules. A wide variety of nutrients is supplied by peptone (pepsin digest of muscle), year extract, blood serum, etc. 2) Defined or minimal medium --> composed of precise amounts of known chemicals. May or may not contain organic compounds, depending on the organism. Cells usually need water, C, N, P, S, K, Mg, Ca, Fe, and traces of Se, Mn, Co, Zn, and Cu (marine microbes may also require NaCl)

Chemotroph

An organism that uses chemicals as both electron donor and energy source

Chemolithotroph

An organism that uses inorganic chemicals as energy source/electron donor Get their carbon and electrons from two different sources, both chemical These organisms are typically autotrophs, meaning that they obtain C for macromolecules from CO2. This is also known as carbon fixation Chemolithotrophs have different proteins to accept electrons from inorganic donors than chemoorganotrophs ---------------------- organism that obtains energy (and electrons) from the oxidation of inorganic compounds some chemolithotrophs are mixotrophs: carbon sources is an organic compound ATP synthesis is by oxidative phosphorylation, powered by the proton motive forse Reducing power is obtained either directly from the inorganic compound or from reverse election transport Cells need NADH and FADH2 for biosynthetic reduction reactions; generated by glycolysis and TCA cycle

Photoheterotroph

An organism that uses light as an energy source, but still must obtain electrons from chemical sources get their energy from light, their electrons from water or another compound, and their carbon from either organic compounds or CO2

Autotroph

An organisms that synthesizes all of its cellular components from CO2 as the sole carbon source

Methanogenesis

Anaerobic respiration system is performed by Archaea (not bac) that are strict anaerobes is the only metabolic option for organisms that perform it-obligate methanogens is a form of anaerobic respiration in which the electron donor is usually H2, and the carbon in CO2, methanol, or acetate is the terminal electron acceptor and is reduced fully to methane, a waste product requires unique one-carbon carriers and electron-carrying cofactors that are not used in other metabolic pathways generates a sodium or proton motive force at one of the final steps in methane generation, which makes it a respiratory process Different carbon compounds can be used for methongensis but the last step is conserved, where a myth group on Co-M (coenzyme M) is reduced to methane released

ORFs

BLAST is an algorithm that compares your input sequence to all other sequences in a database (like Refseq at NCBI) BLAST your predicted protein sequence vs. all other proteins to find homologs Existence of homologs makes it more likely that the ORF really encodes a protein, and homologs may have been studied experimentally in other organisms Look for protein domains (Pfam, CDD) and smaller elements like active sites (PROSITE, MOTIF) within your sequence; domains and motifs can give clues about functions of a protein Predict the structure of a protein, based on known structures of homologs (SWISS-MODEL) Predict the cellular location of a protein (PSORT)

Microbial adaptations to extreme environments: high temperatures

Bacteria do not regulate their internal temp; some bacteria have evolved to live in extremely high or low temps Thermophiles have optimal growth temperatures >45 C Hyperthermophiles have optimal growth temperatures >80 C Typically, proteins in the cell unravel and denature at extremely high temperatures Solution: Individual proteins are more stable because they cantina fewer glycine residues or have increased ionic bonding bw basic/acidic amino acids and their hydrophobic cores. Cells synthesize chaperones that refold denatured proteins. Cells synthesize solutes (di-inositol phosphate, digycerol phosphate) that help stabilize proteins Usually, membranes are too fluid and cannot maintain a barrier against the environment Solution: Cells synthesize phospholipids with saturated fatty acids, either constantly or in response to prolonged heat shock.

Carbon concentrating mechanisms

Carboxysomes of cyanobacteria --> 100 nm diameter compartments surrounded by polyhedral protein shell interior contain a highly concentrate array of RiBisCO and carbonic anhydrase (CA) The icosahedral shell is made of shell proteins that form hexameters (on the faces) and the penners (at the vertices) Protein shell is thought to tightly control what chemical pass into and out of the compartment. Gene cluster can be transplanted to E. coli and it will synthesize carboxysomes Thought that CO2 can't diffuse out of carboxysomes, and O2 doesn't diffuse in very well, favoring the carboxylation reaction.

Oxidative phosphorylation

The production of ATP by an F1F0 ATPase at the expense of PMF

Iron-sulfur clusters

are covalently bound to electron transport proteins via cysteine residues Fe atoms cycle between the 2+ and 3+ oxidation states as an electron is accepted or donated. Fe/S clusters are electron-only carriers The reduction potential of an Fe/S cluster varies with the number of Fe and S and how the cluster is attached to the protein

3 classes of light-harvesting pigments

1) Chlorophylls --> bacteriochlorophyll; ch. a; ch. b; ch. c 2) Phycobilins --> phycocyanobilin; phycoerythrobilin, Phycoerythrin (red accessory pigment), phycocyanin (light blue accessory pigment) 3) Carotenoids --> beta carotene; zeaxanthin chlorophylls and phycobilins are both synthesized bu the tetrapyrrole pathway isoprenoid molecules that function in both light-harvesting and photoprotection ALWAYS found in all types of phototrophs

Counting bacteria

1) Direct microscopic count --> requires a chamber Depth of liquid in counting chamber is 0.02 mm, so the volume under the 25-square grid is 0.02 mm^3 = 0.02 ul = 2x10^-5 ml Number of cells in the 25 square grid = number in 2x10^-5 ml, so number per 1ml can be calculated 2) Viable count or colony count Counts number of cells in a sample capable of growing up into a colony on solid medium (not using streak method) 3) Turbidity - indirect measurement of bacterial numbers As bacteria grow, they convert nutrients into cell mass, cultures appear turbid or opaque because light passing through the sample is scattered by bacteria The turbidity of a culture (its optical density) is proportional to the number of cells present OD proportional to cell number, but the exact relationship is different fro each species due to cell size and shape --> must generate a standard curve that relates OD to cell number counted by an independent method, such as a direct microscopic count.

Two major question in the 1800s

1) Does spontaneous generation occur? -Spontaneous generation: the supposed production of living organisms from nonliving matter, as inferred from the apparent appearance of life in some supposedly sterile environments. Experiments were conducted to determine is microbes could spontaneously generate. NO Broth sterilized and capped --> broth remains sterile Broth sterilized and left uncapped --> broth becomes contaminated Louis Pasteur swan neck flask experiment (1864) -Non-sterile liquid poured into flask -Neck of flask drawn out in flame -Liquid sterilized by heating -Liquid remained sterile Dust and microorganisms were trapped in the bend. When the flask was tipped microorganisms were introduced to the liquid and began to grow Take away: Showed that microbes come only from existing microbes, not from nonliving (sterile) matter; microbes are NOT that different from plants and animals 2) What causes diseases that appear to spread from a particular environment or from person to person? -Germ theory -Before 1800s diseases were though to be caused by noxious air associated with poor sanitation and foul odors With the work done by microbiologist --> assumed that many hospital deaths were caused by the spread of disease from patient to patient, or doctor to patient Koch studied anthrax, caused by the bacterium Bacillus --> linking specific microbes to specific diseases -Showed that a bacterium could still cause disease in a mouse even after growth outside animal host

Principles of photosynthetic electron transport

1) light energy is used to excite a special chlorophyll in the reaction center. This lowers the reduction potential of the chlorophyll, so that it can reduce an intermediate electron acceptor. 2) Electrons flow downhill to carriers with increasing positive E0' (just like any other electron transport chain). 3) electron transport is coupled to proton translocation across membrane to generate proton motive force 4) When the first stable electron acceptor has E0' greater than that of NAD+, reverse electron flow must occur at the expense of energy to generate reducing equivalents (NADH).

Bacterial genome properties

1) sizes range from ~150 kb to 13 Mb -obligate insect symbionts or parasitic bacteria (Mycoplasmas) have smallest genomes -overtime, they have lost many genes and rely on their hosts for many metabolic functions -smallest free-living organisms have genomes of ~1.3 Mb (Pelagibacter) -largest genome belongs to a Myxobacterium with a complex life cycle 2) coding density is remarkably consistent, about 1 ORF per kb 3) Even with so many genomes sequenced, ~30% of the ORFs in any new genome are annotated as "hypothetical," meaning that we can't present their function or any recognizable protein domains 4) Genes are organized in operons -two or more ORFs are often encoded on same mRNA proteins encoded in an operon are almost always part of the same pathway or structure 5) Some genomes contain genomic islands-large regions (20-200 kb) that are unlike the rest of the genome and confer special functions -different codon bias -often absent in closely related strains/species -often flanked by direct repeat sequences (a sign of recombination) -islands encode genes for special processes, such as virulences or metabolic pathways, that aid adaptation to different environments -likely acquired by horizontal gene transfer

Key concepts of fermentation

1. No exogenous electron acceptor involved 2. No electron transport chains involved 3. ATP is generated by substrate-level phosphorylation 4. Energetically inefficient, so organisms that can respire will often choose to respire rather than ferment if conditions are favorable for both

Koch's Postulates

1. The suspected pathogen must be present in all cases of the disease and absent from health animals --> using microscopy staining 2. The suspected pathogen must be grown in pure culture --> pure laboratory cultures 3. Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal --> using experimental animals 4. The suspected pathogen must be reisolated and shown to be the same as the original --> Lab reisolation and culture

Microorganisms

A diverse group of organisms that exist as free-living single cells or cell clusters A single microbial cell can grow, generate energy, reproduce and evolve independently of other cells. (Class will focus on the bacteria and will introduce Archaea) 1) small free-living eukaryotes including algae, fungi, and protists 2) Bacteria 3) Archaea Archaea are very different at the molecular level and have a mixture of bacterial and eukaryotic properties

Consumptions and generation of ATP in glycolysis

A low amount of ATP is generated because the substrate (glucose) is not fully oxidized to CO2 in the absence of an exogenous electron acceptor NAD+ is regenerated during formation of fermentation products, rather than having NADH donate electrons to an electron transport chain. NAD+/NADH is a limiting reagent in the cell, so NAD+ must be regenerated by making fermentation products

Accessory transcription factors

Accessory transcription factors (aside from RNAP + sigma) promote or repress transcription of specific genes They contain a DNA-binding domain and regulatory domain(s) that determine when the protein is active Often act as dimers and bind to direct or inverted repeats on DNA A transcriptional activator turn on tx gene, while a repressor blocks tx

Aerobe vs. Anaerobe

Aerobe --> a microorganism able to use O2 as its electron acceptor in respiration Anaerobe --> A microorganism that cannot use O2 in respiration

How microbes utilize organic nutriments

All cells need C and N, but they can use different chemical forms of these nutrients. Heterotrophs: like us; require organic C source ex. glucose, fumarate, acetate Autotrophs: can get all of the C they need to build cell structures from CO2 in the air. These organic,s perform carbon fixation Animals, plants, and most microbes use NH4+ or NO3- as their nitrogen source. Only nitrogen-fixing bacteria can convert atmospheric N2 to NH4+ and assimilate it into cellular structures (or share it with other organisms).

How ETC help generate ATP

An ETC is a series of electron donor/acceptor molecules that ends in a terminal (exogenous) electron acceptor Protein complexes in an ETC span the cytoplasmic membrane; Some electron transfers in the chain generate enough energy to pump protons across the cytoplasmic membrane from inside to outside the cell The cytoplasmic membrane becomes charged, similar to a battery. When protons are allowed to pass back to the cytoplasm, down their concentration/charge gradient, they release energy and can do work, such as powering ATP synthesis. Each electron carrier in an ETC has a standard propensity to accept/donate electrons just like small molecules (reduction potential E0') The electron transport chain contains proteins with electron carriers of particular E0' Electrons from NADH and FADH2 are passed to a series of cytoplasmic membrane proteins that make up an electron transport chain. Electrons are passed from LOWER to HIGHER E0' in a series of reactions that release energy The energy is conserved as (converted into) another useful source of energy, a proton gradient across the cytoplasmic membrane

Proton motive force (PMF)

An energized state of cytoplasmic membrane resulting from the separation of charge and the elements of water (H+ outside and OH- inside) across the membrane

Heterotroph

An organism requiring organic compounds as a carbon source

Chemoorganotroph

An organisms that uses organic chemicals as energy source (electron donor); get their carbon and electrons from the same source (like us) Some carbons in the substrate are used for synthesis of macromolecules, while other carbons are oxidized and excreted as waste products (CO2 or fermentation products) Animals and many bacteria are aerobic chemoorganotrophs, but many bacteria can use organic carbon sources in combination with alternate (non-O2) electron acceptors obtain electrons from carbon sources using NADH as the intermediate carrier. Their ETC starts with complex I, which receives electrons from NADH.

Ferric iron reduction

Anaerobic respiration system Fe3+ is abundant in nature, but its usually in the solid phase as part of minerals like Fe3O4 (magnetite) or Fe3S4 (griegite) Most of the processes we think about use reactant in the liquid phase, so this presents as extra hurdle for bacteria that reduce ferric iron Bacteria use three distinct mechanisms to access mineralized Fe3+ 1) Produce and excrete chelators which bind and solubilize Fe3+ -observed for iron-reducing bacterium Geothrix fermenting, but the chemical structure of the chelator has not been determined -they could resemble siderophores, which are related by bacteria and function in the acquisition of iron for use in cellular structures (protein active sites, etc) 2) use electron shuttling compounds that are reduced by the cell, then diffuse away and reduce Fe3+ at a distance -quinone-like molecules derived from degradation of plant tissue or produced by the cells can carry electrons from terminal reductases to external Fe3+ -ex. mutations in genes for meaquinone synthesis impair the ability of Shewanella oneidensis to reduce ferric iron; electron shuttling compounds have been described in several species 3) Touch the mineral directly using nanowires -3A) Type IV pills are required for ferric iron reduction by Geobacter sulfurrenducens -Electrons are thought to be conducted along these pili via overlapping pi orbitals of aromatic residues in the PilA protein -The electrons then reach a cytochrome, OmcS, which transfers them to external Fe(III) -one type of nanowire = pili with attached cytochromes -3B) Shewanella oneidensis makes a different type of nanowire consisting of outer membrane extensions -Atomic force microscopy Shewanella oneidensis grown on the surface of a flow chamber with Fe(III) as the sole electron acceptor - Shewanella nanowires are made of connected outer membrane vesicles, and they are much wider in diameter than pili Pili are not required for Shewanella nanowires, which are composed of outer membrane extensions Membrane nanowires are covered with outer membrane cytochromes MtrC and OmcA Electrons are thought to hop directly from cytochrome to cytochrome on the membrane surface, and ultimately onto Fe(III) in the environment

Nitrate reduction

Anaerobic respiration system Two parts --> nitrate reduction and dentrifiation Often performed by facultative aerobes/anaerobes Nitrate reduction: Nitrate (NO3-) --> nitrate reductase reduces NO3 to Nitrite (NO2-) Dentrification is the conversion of nitrite into gaseous compounds that are lost from the soil and water ; gases, released to the atmosphere (denitrification) Nitrite (NO2-) --> Nitric oxide (NO) using nitric oxide reductase --> nitrous oxide (N2O) using nitric oxide reductase --> dinitrogen (N2) using nitrous oxide reductase E. coli can use O2 or NO3- as as electron acceptor; some ETC components are shared by both pathways; nitrate reductase complex is produced instead of cytochrome oxidase E. coli perfoms nitrate reduction, but not full dentrification Fewer protons are transferred out of the cell during oxygen reduction because nitrate reductase is not a proton pump. Oxygen use is more energetically favorable. When the organism has a choice between O2 and NO3-, it will regulate gene expression so as to use O2 first; In E. coli, the nitrate reductase gene is regulated as follows: For nitrate reduction to occur, oxygen must be absent AND nitrate must be present. This ensures that E.coli use the most energetically favorable electron acceptor first

Bacterial adaptation to osmolarity

Bacterial membranes have low permeability to most dissolved molecules (solutes). If a bacterium encounters an environment with a radically different solute concentration than its cytoplasm, internal or external WATER is under pressure to cross the membrane until equilibrium is reached In a hypotonic medium, water tends to ENTER the cell to make it more dilute. The press would ultimately cause cell lysis. Solution: bacteria have rigid cell walls that can withstand internal pressure and prevent osmotic lysis. Bacteria have mechanosensitive channels that are activated by high internal pressures. These can leak specific solutes out of the cell to reduce internal osmolarity In hypertonic medium water tends to exit the cell; loss of water beyond a critical point stops enzyme function and microbial growth. Solution: Bacteria synthesize or import compatible solutes, small molecules or ions that do not distribute cell metabolism but increase the internal osmolarity. These molecules help the cell to retain water. Compatible solutes include proline, glutamic acid, glycerol, and K+

Methanogensis from carbon dioxide

CO2 + 4H2 --> CH4 + 2 H2O deltaG0' = -131 kJ/mol methane Archaea make ~0.5 mol ATP/mol CH4 Occurs where fermenters breaking down complex carbohydrates generate large amount of H2 and CO2 as waste products This process is the final stage in biodegradation of organic matter in anoxic habitats like landfills, animal digestive tracts, and sewage treatment systems Methanogens often live in SYNTROPHIC relationship with fermentative bacteria producing H2 Syntophy= two different organisms corporate to perform an overall metabolic pathway that is not possible (or energetically favorable) for either organism alone Methanogens often live in syntrophy with bacteria that produce H2 as fermentation waste product deltaG0' is calculated assuming 1M reactants and products, but this can be altered if a product like Hs is removed to extremely low levels Methanogens and fermenters are often physically closely associated to facilitate interspecies hydrogen transfer

Overview of metabolism

Catabolism --> energy generation, substrates become products; breakdown of complex molecules in living organisms to form simpler ones, together with the release of energy Anabolism --> energy consumption for biosynthesis, monomers become macromolecules and other cellular constituents; the synthesis of complex molecules in living organisms from simpler ones together with the storage of energy

Respiration

Catabolism in which electrons are extracted from an election donor and passed down an electron transport chain to an electron acceptor, generating a proton motive force. ATP is produced by oxidative phosphorylation In respiration, ATP is produced by oxidative phosphorylation at the expense of the proton motive force Pyruvate is completely oxidized to CO2 using the TCA cycle, rather than being converted to fermentation products For every glucose molecule, this cycle produces 8 NADH, 2 FADH2, and 2 ATP (GTP) by substate-level phosphorylation The electrons on NADH or FADH2 must go somewhere to regenerate NAD+ and FAD (and 38 ATP per glucose) The cycle also generates intermediates of many biosynthetic pathways (aKG --> Glucoses --> Gln --> oxaloacetate --> aspartate --> nucleotide bases) THE POINT--> respiration generates much more energy per substrate molecule than fermentation because glucose is fully oxidized to CO2. The extracted electrons are fed into an electron transport chain to regenerate NAD+, and a H+ gradient is generated to conserve energy Electrons from NADH and FADH2 are donated to an electron transport chain, generating a proton gradient that drives ATP production; If the electrons and protons from NADH/FADH2 were given directly to a terminal acceptor such as O2, a huge amt of energy would be released in an uncontrolled way, largely as unusable heat (like an explosion). Instead, bacteria perform several smaller electron transfers in sequence; this provides several places where the energy of a reaction can be converted into a proton gradient across the IM, which is another form of stored, usable energy e- carriers in respiration: NAD+/NADH carried hydrogen atoms (2 protons plus 2 electrons); it can donate electrons to an ETC or to a variety of biosynthetic reactions in the cytoplasm For almost all eukaryotes, the electron acceptor is oxygen; but many bacteria can use alternative electron acceptors at the ends of their election transport chains; this allows them to grow using respiration in the absence of oxygen

Transcriptomics

Comprehensive transcriptional analysis Measure the expression of Every gene at once, under different environmental conditions or in different mutant backgrounds. Techniques for this are constantly evolving, with RNA-seq being the current favorite

Cytochrome proteins

Contain covalently round heme cofactors. Hemes are porphyrin rings also called tetrapyrroles The Fe coordinated at the center cycles between 2+ and 3+ states as an electron is accepted or donated Hemes are electron-only carriers Different hemes/cytochromes have different reduction protenials, depending on the protein structure

Relationship bw potential and free energy

Conversion of potential difference to free energy: deltaG' = -nF(deltaE0') Where n is the number of electrons transferred in the reaction (given on the electron tower) and F is the faraday constant (96.5 kJ/Vmol e-)

Genome sequencing

Describing the contents of a genome sequence is annotation 1)Find open reading frames (ORFs) An ORF is a series of bases starting with ATG and extending in frame to a stop codon ORFs that produce proteins tend to be 200 bp or longer, but some actual, short proteins are missed by this cutoff This is relatively easy because bacterial genes don't have introns 2) Find ribosome binding sites (RBS) up stream of ORFs An ORF with recognizable RBS is more likely to be expressed and produce a functional protein than one without an RBS 3) Determine codon bias of each ORF compared to others in the genome Some amino acids are encoded by more than one codon; Each organism has certain codons preferred over others An ORF with different codon bias than the rest of the genome is less likely to produce a protein 4) Predict tRNAs and rRNAs-highly conserved across organisms

Electron acceptor/donor

E acceptor: The substance in a reaction that accepts electrons from some other substance, becoming reduced in the process E donor: The substance in a reaction that donates electrons to another substance, becoming oxidized in the process

The electron tower

Electron transfer bw compounds can be viewed as a vertical tower representing the range of redox potentials with the most negative redox couple at the top and the most positive redox couples at the bottom. The reduced substance in the redox pair at the top has the greatest tendency to donate electrons and the oxidized substance at the bottom has the greatest tendency to accept electrons. As electrons from the donor at the top fall down the tower they can be "caught" by acceptors at various level. the difference in reduction potential between the donor and the acceptor is expressed as deltaE0' deltaE0' = (E0' of reduction couple) - (E0' of oxidation couple) If delta E0' is positive, it means that the run is favorable in the direction written The farther the electron fall (or the greater distance bw donor and acceptor on the tower), the greater the energy that is released and available to the microorganism

Properties of ETC

Electrons are passed from lower reduction potential to higher reduction potential Complexes in an electron transport chain span the inner/cytoplasmic membrane The energy released from favorable electron transfers is stored as a proton gradient across the cytoplasmic membrane ETCs are modular. When a bacterium is capable of more than one type of respiration, individual complexes can be substituted, depending on the electron donors and acceptors available in the environment. At the end of an electron transport chain, electrons are passed onto an exogenous acceptor. The reduced acceptor is excreted as a waste product. In this case, the acceptor is O2, and the waste product is H2O. However, some organisms use terminal electron acceptors other than oxygen. They perform anaerobic respiration, and they use different proteins than complex IV at the end of the ETC. Electrons are passed between large, membrane-spanning complexes by quinone, molecules that resides in the cytoplasmic membrane. When a quinone receives electrons from an upstream carrier (like Fe/S), it also needs protons to become fully reduced. Therefore it accepts protons from the cytoplasm. When a quinone donates electrons to a downstream carrier (like a cytochrome), the protons are released into the periplasm and help to generate PMF. Electrons can also be shuttled between large complexes by mobile proteins such as cytochrome c

electron flow in oxidation of sulfur compounds

Electrons from sulfide ad sulfite enter at two different point. PMF is generated as electrons are passed form the quinone pool to cut bc1 and during the reduction of O2 by cytaa3. Sulfide donates electrons at a lower reduction potential that sulfite and yields more PMF than those form sulfite. Electron from sulfide, sulfur, and sulfite are at a higher reduction potential than NAD+/NADH couple, so reverse electron flow is needed to generate NADH. This process USES energy of the PMF

Enrichment cultures

Enrichment culture is the use of certain growth media to favor the growth of a particular microorganism over others, enriching a sample for the microorganism of interest. Martinus Beijerinck Start with an environmental sample (lake water, soil, etc.) Put a small inoculum into selective culture medium -only has certain nutrients -has oxygen/no oxygen -grown at a specific temp -grown in light/dark When organisms grow, transfer a small amount of fresh selective medium and repeat Pure cultures are eventually obtained by streaking on solid media Beijernick and Winogradsky used enrichment cultures to isolate bacteria with novel metabolic properties -Autotrophs acquire their carbon from CO2 in the air -Nitrogen-fixing bacteria convert N2 in the air to NH3 which can be used by themselves and by plants and animals

Cell bio of neutral iron oxidation

Fe2+ only yield one electron per molecule, and Fe2+ is not a great electron donor, even at neutral pH, so bacteria that use this metabolism still oxidize a lot of iron The oxidized iron is insoluble, forming crystals and other physical accumulations How does a bacterium that lives this way avoid being encased in iron mineral? ex. Mariprofundus ferrooxidans with stalks on side of the cell. Stalks are made of exopolysaccharides. As Fe2+ is oxidized, presumably near the source of the EPS, the Fe3+ becomes associated with the EPS and is pushed away from the cell; stalk contain oxidized iron in a polysaccharide matrix

Beginning of microbiology

Field began in the late 1600s with the ability to SEE microorganisms Robert Hooke --> built a compound microscope, could see features that you couldn't see with the naked eye Anton van Leeuwenhoek --> made a simpler microscope with much greater magnification and resolution. (blood cells, sperm cells, algae, nematodes). Made the first recorded observation of bacteria in 1684

Growth phases of bacteria

Graph shows viable count, does not show OD (turbidity). A closed system where bacterial growth depletes nutrients and alters the environment. Lag phase--> Occurs when a stationary phase. culture is diluted into fresh medium or when cells are transferred from rich to minimal medium. Bacteria must sense new environment and synthesize many proteins needed for rapid growth, which were not produced during starvation, or specific proteins needed to produce the nutrients not already present in the culture medium. Stationary phase --> conditions in the beach cultures or local environment change so that microbial growth is limited; i.e., cells run out of an essential nutrient, cell accumulate a toxic waste product. No net increase or decrease in cell number (plateau); that is, they are either NOT dividing, or division = deaths. Not a single physiological state.

Mass spectrometry-based proteomics

Harvest cells grown under different conditions and extract all proteins. Digest w trypsin and identify all peptides by mass spectrometry. (Genome sequence enables you to predict all tryptic peptides, since this enzyme cleaves after K or R, but not before P. magnetic field separates particles based on mass/charges ratio compare list of masses to all the peptide masses that you can predict to its genome This comparison is used to compile a list of proteins expressed under each condition, so that they can be compared to each other, and perhaps also transcriptomes acquired under the same conditions What proteins are present under these conditions? How does protein profile change with time, growth phase, or in mutants?

Energy yielded from redox reactions

How do bacteria store the energy they get from redox reactions? 1) Proton gradient across cytoplasmic membrane 2) High-energy compounds that are used to power unfavorable chemical reactions (ATP, GTP, PEP) In catabolism, electrons are extracted from organic and inorganic molecules and transferred to electron carriers such as NAD+, NADP+, and FAD The reduced forms of these carriers or to other chemical (biosynthetic) reactions in cells Just as ATP is a common currency for runs requiring energy, NAD+ and FAD are common currency for oxidation and reduction. They are used by many different enzymes to oxidize or reduce many different substrates.

CO2 fixation

How is atmospheric CO2 converted into carbohydrates? accomplished by the "dark reactions" of photosynthesis, where the ATP and reducing power generated by phototrophs are used to reduce CO2. Several carbon fixation pathways have evolved i.e., Calvin cycle, TCA cycle. reductive acetyl-CoA, 3-hydroxypropionate 99% of CO2 fixation is via Calvin cycle Bacteria and Archaea account for > ⅓ of earth's CO2 fixation

Assimilative vs. dissimulative reduction

In assimilative reduction, compounds are reduced for the purpose of building cellular macromolecules, and the cell only reduces the amount needed for growth ex. NO3- reduced to NH2 in amino acids, nucleotides SO42- reduced to SH groups in amino acids, cofactors In dismilative reduction, compounds are reduced for the purpose of energy conservation, large amounts are reduced, and the cell excretes the reduced product into the environment as waste. ex. NO3- reduced to NO2-, N2O or N2 SO42- reduced to HS-

Glucose + lactose biphasic growth

In the absence of glucose, transcription of genes for alternative sugar utilization is activated by the catabolite activator protein CAP CAP --> an activator, and cAMP is its co-activator genes that are regulated by catabolite repression contain a binding site for CAP. When CAP binds cAMP, it can bind DNA and activate transcription by RNA polymerase decrease in glucose = increase in cAMP When glucose is present, cAMP is low. CAP is off the DNA and Lac is on, so transcription is repressed When glucose is absent, cAMP is high. CAP is on the DNA and in the presence of the inducer lactose, the Lacl repressor is off, so transcription is activated

Redox couples

Many compounds can act as both electron donors or acceptor depending on the environment and other substances they react with. In the 2H+/H2 redox couple, electrons could be donated to H+ by a reduced compound with a more negative reduction potential, or H2 could donate electrons to an oxidized compound with a more positive reduction potential. When constructing a complete redox reaction: The reduced substance of a redox couple whose reduction potential is more negative donates electrons to the oxidized substance whose potential is more positive.

Catabolite repression

Many operons that have specific repressors or activators are also regulated by catabolite repression ex. lac, mal, and ara operons Specific (ex. lac repressor) and global (CAP) regulatory mechanisms are used in combination ex. Lac repressors cannot bind in the presence of lactose and cAMP-CAP only binds in the absence of glucose

Genomics

Many shot sequence reads from a new organism are assembled into a complete, connected genome "Illumina" sequencing: generates numerous short sequence reads (~100 bp) with very high accuracy Assembly is performed by computer algorithms, based on overlaps between the short sequence reads, The output is long connect sequences called contigs PacBio generates longer reads (10-50 kb) that help to connect contigs to each other. This step is called gap closure. PacBio sequencing used to be too inaccurate to use for whole-genome sequencing alone. But accuracy has been improved, and some people might do the whole thing with PacBio now (although doing a combo with Illumina)

Fermentation process

Non-respiratory catabolism in which one organic compound serves as acceptor, and ATP is produces by substrate level phosphorylation In fermentation, ATP is produced by substrate-level phosphorylation. A phosphate group is added to a substrate, making a high-energy bond. This phosphate group is transferred to ADP to generate ATP Most of the carbon substrate is excreted as a partially reduced end product of energy metabolism and only a small amount of the carbon in the substrate is used in biosynthesis; since each catabolic reaction in fermentation only yields a small amount of ATP, the cell must use almost all of the carbon substrate for energy production, rather than for building biomass

Reverse genetics

Now that we can sequence a whole genome, we can apply reverse genetics, something that couldn't be done before e.g. Identify all predicted proteins of a certain type in a genome and knock out the gene for each one individually. Analyze phenotypes to gain insight into the function of each gene

Bacteria and oxygen

Oxygen can be beneficial or harmful, so different bacteria have evolved different relationships with oxygen. -Obligate/facultative anerobe -Obligate/facultative aerobe -Micro-aerophiles; needs oxygen to survive but at low oxygen levels -Aerotolerant; anaerobic organism capable of surviving or growing despite the presence of oxygen. Oxygen can be converted to reactive oxygen species (ROS); can react with nucleic acids, proteins and lipids an cause cell damage Bacteria produce there's enzymes to detoxify reactive oxygen species O2 + FADH2 --> O2- radical; can forms during respiration superoxide dismutase breaks down O2- radical + H+ to hydrogen peroxide. Hydrogen peroxide can transform into a hydroxyl radical, an ROS. Catalase and peroxidase transforms hydrogen peroxide into water and oxygen, or water and NAD+ respectively

Photoautotroph

Performs two sets of reactions: 1) "Light reactions," in which light energy is conserved as chemical energy (ATP) and reducing power (NADH or Fd). 2) "Dark reactions," in which chemical energy and reducing power are used to reduce CO2 to organic compounds (CO2 fixation)

Two types of phototrophy

Photoheterotrophy; heterotrophic organisms that make use of light energy as their energy source. They also cannot use carbon dioxide as their sole carbon source. Rhodopsins contain retinal, similar to the light sensing molecules in our eyes. Used by photoheterotrophs only. NO photoautotrophs use this. Photoautotrophy; organisms that can make their own energy using light and carbon dioxide via the process of photosynthesis. Photosyntehtic reaction centers (RCs) contain (bacterio) chlorophyll. All photoautotrophs and some photoheterotrophs use RCs.

Reactions of photosynthesis

Photosynthesis is a biological oxidation-reduction reaction in which CO2 is the electron acceptor: CO2 + 2H2A --> (hv) (CH2O) + 2A + H2O In oxygenic photosynthesis, water is the electron donor. Plants, algae, and cyanobacteria perform oxygenic photosynthesis. CO2 + 2H2O --> (hv) (CH2O) +O2 +H2O; deltaG0' = 2840 kJ/mol of glucose In an anoxygenic photosynthesis, H2S or another reduced chemical is the electron donor. Bacteria including purple bacteria, green sulfur bacteria, and heliobacteria perform anoxygenic photosynthesis. CO2 +2H2S --> (hv) (CH2O) + 2S +H2O In both cases, light energy is used to create a proton gradient for ATP synthesis The difference lies in the source of electrons, or reducing power, H2O (oxygenic) vs. H2S or S0; either system is defined by whether or not the process produces oxygen. chlorophyll a (oxygenic) and bacteriochlorophyll a (anoxygenic) are porphyrins, similar to heme. Chlorophyll pigments are associated with photosynthetic membranes via the phytol group, a hydrophobic isoprenoid.

Various e- donors

Principle is that same as for other redox rxns. Better e- donors have LOWER E0' and deltaG0 = -nFdeltaE0' various election donors can be coupled to oxygen as electron acceptor (ferrous iron couple behaves differently at different pH values) If an electron donor has HIGH e0' than NAD+/NADH then the organism will have to perform reverse electron flow to generate NADH for cellular reduction reactions (this process requires energy)

Microbial adaptations to extreme environments: low temperatures

Psychrophiles have optimal growth temperatures < 15C Typically, proteins do not exhibit much thermal motion, so they perform reactions very slowly. Solution: Individual proteins are more flexible than their counterparts in mesophilic or thermophilic bacteria. Usually, membrane fluidity decreases, which inhibits the function of critical proteins that reside in the membrane. Solution: Membrane phospholipids have more unsaturated fatty acids and more short-chain fatty acids, this property can be changes by altering the fatty acid content of newly synthesized phospholipids. Additionally, ice crystals can form in the cytoplasm and puncture the cell wall and extremely low temps Solution: cells produce cryoprotectants (glycerol, sugars) at high concentrations that prevent ice crystal formation (antifreeze!)

RNAP core enzyme

RNAP core enzyme contains subunits sigma subunit or "sigma factor: attaches to the core enzyme and helps RNAP recognizer and bind to the promoter region of a gene tx initiation: A better match of promoter sequence with the consensus sequence for a sigma factor makes a strong promoter, which yields more binding by the sigma factor and more tx of gene Worse match with consensus makes a weak promoter. Extra proteins (transcriptional activators) are often needed to turn on tx at weak promoters The cell regulates global gene expression patterns by controlling which sigma factors are present and active

Sulfur as e-donor

Reduced sulfur compounds or S0 can function as inorganic e- donors H2S (hydrogen sulfide) --> S0 (elemental sulfur) --> ½ S2O32- (thiosulfate) --> SO42- (sulfate) Protons are a by-product of most oxidations of sulfur compounds, so sulfur oxidizers usually acidify their medium and are acid-tolerant or acidophilic. Microbial sulfur oxidation to sulfuric acid can deteriorate concrete structures.

Repressor pathway mutations

Repressor-corepressor: 1) point mutation in operator DNA sequence, results in repressor unable to bind 2) Repressor knockout 3) Point mutation in repressor protein so that it can't bind to the corepressor Repressor-inducer: 1) Repressor knockout 2) Point mutation in operator DNA sequence so that the repressor can't bind 3) Point mutation in repressor protein so that it no longer binds to the inducer Activator-co-activator: 1) point mutation in DNA activator binding site 2) Activator knockout 3) Point mutation in activator protein so that it no longer binds to the co-activator

Repressors

Repressors bind to specific DNA sequences and block transcription of downstream genes 1) End products of biosynthetic pathways can act as corepressors corepressor + repressor --> transcription Arginine is a corepressor that binds to the arginine repressor protein Repressor + corepressor prevent RNAP from transcribing the operon When product is abundant, synthetic enzymes are not transcribed A different way to control repressor activity: Specific enzyme substrates can act as inducers of transcription by blocking the action of repressor proteins inducer + repressor = transcription ex. lac repressor stops transcriptions of lac operon unless lactose is present Lactose is an inducer that binds and inactivates the repressor, allowing transcription of genes for lactose utilization

Fermentation vs. Respiration

Respiration is not confined to aerobes. Oxygen is only one of many possible compounds that can be used as a terminal electron acceptor at the end of an electron transport chain. Different bacteria can respire nitrate, sulfate, sulfur, Fe(III), U(VI), and any number of other inorganic compounds Fermentation occurs in the absence of an electron acceptor that can be used for respiration fermentative bacteria partially oxidize a substrate to a suitable intermediate, which is then reduced as an electron acceptor with the electrons generated in the initial oxidative process

How do we get pure cultures of a single type of organism?

Robert Koch First scientist to grow microbes of a solidified nutrient medium (fist gelatin, than agar --> agar remain solid at high temperatures; found by Koch wife, Angelina Fanny Hesse) Streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria.

RuBisCO

RuBisCO performs two competing reaction Instead of carboxylating RBP, RuBisCO can use O2 as competing substrate to produce 1 PGA and 1 phosphoglycolate (which is toxic) Typically, the carboxylation ran is 15-50x faster than oxygenation, however atmospheric (O2) is >500x greater than that of CO2, AND as temp increases, the aqueous solubility of CO2 is decreased more than O2 CO2 is the natural substrate of RuBisCO, but HCO3- is the dominant species of dissolved inorganic carbon in most aqueous environments The typical dissolved CO2 concentration is ~10um The Km of RuBisCO for CO2 is ~150 um in cyanobacteria Co2 readily passes through lipid membranes Photosynthetic organisms use carbon concentrating mechanisms to achieve high enough CO2 for RuBisCO to function efficiently.

Predict metabolic pathways

Search genome (BLAST) for proteins with known enzymatic activities and connect them in pathways Predict where an organism can survive, what compounds it may use for energy, or what it may provide in a microbial community Streptomycin biosynthesis pathway from Streptomyces griseus genome NOT = transcriptome NOT = proteome NOT = functional proteome

RNA-seq data analysis

Short cDNA sequences are mapped onto this segment of the genome Numbers in c = nucleotide position in the genome cDNA sequences match the non-coding DNA strand in the genome Higher peaks = more reads = more transcription of a particular gene 6-way translation map showing predicted genes

Activators

Some genes needs activators to be transcribes, in addition to RNAP + sigma Like represses, positive regulators recognize specific DNA sequences in the promoters Activators can be turned on by binding to small molecule co-activators co-activator + activator --> transcription ex. maltose operon activation: no transcription without maltose activator proteins and maltose (co-activator) Activators can increase the affinity of RNAP (RNA polymerase) for the promotor by providing extra binding contacts for RNAP itself

RNA-seq workflow

Starting RNA sample --> DNase treatment --> rRNA/tRNA depletion --> First strand reverse transcription --> whole transcriptome single stranded cDNA (directional) Short sequence reads are mapped onto the genome of the bacterium to determine which genes are being transcribed

Reduction potentials

Substances vary in their tendency to become oxidized or reduced. This is denoted as the reduction potential (E0') and is expressed in volts or millivolts. Convention: reduction potentials are expressed for half reactions written as reductions. This "oxidized form" + e- --> "reduced form" e.g. 2H+ +2e- --> H2 By convention, reduction potentials are given for neutrality in biology bc the cytoplasm of most cells is about pH 7 However, since protons can be reactants or products of redox reactions, the prevailing pH will affect how readily the reaction occurs.

Substrate-level vs. oxidative phosphorylation

Substrate-level: phosphate is transferred from another substrate to ADP Oxidative: free phosphate is added to ADP In oxidative phosphorylation, the proton gradient generated by electron transport is used to power ATP synthesis F1 consists of five different polypeptides a-e. F1 is the catalytic complex that interconverts ADP + Pi and ATP Fo is integrated in the membrane and consists of three polypeptides a-c. As protons enter the cell through Foa, the dissipation of the proton motive force drives ATP synthesis. Measured value is ~3H+ per ATP produced. Rotation by the rotor causes conformational changes in F1a and F1b that allow them to bind ADP + Pi, catalyze ATP formation, and release ATP How does this work during fermentation when no terminal electron acceptor is present and electron transport chains aren't functioning? ATP generated by substrate-level phosphorylation is used to run the F1F0 ATPase backward, so that ATP is hydrolyzed to create a proton gradient. The proton gradient is then used to power membrane transport reactions.

iron oxidizing bacteria (FeOB)

The Fe3+/Fe2+ couple has different E0' at different pH values At pH 7, E0' = +0.2, but in the presence of atmospheric oxygen, Fe2+ spontaneously oxidizes to Fe3+ (an abiotic reaction) At pH 2, E0' = +0.76, but Fe2+ is more stable and will not spontaneously oxidize; oxygen is the only viable electron acceptor with E0' greater than iron; The reactions form insoluble ferric hydroxide Fe(OH)3 and other complex iron salts Because the reduction potential of iron and oxygen are so close at pH2 , this process does not yield much energy per electron, and very large amounts of Fe2+ must be oxidized to create a proton gradient and support life. This creates abundant yellow0orange ferric hydroxide as a waste product. At pH 7, iron oxidation can be coupled to either nitrate or oxygen reduction (theoretically). However, the oxygen level in the environment must be low enough that biological iron oxidation is faster than the spontaneous abiotic reaction ------------------- FeOB are chemolithotrophs that use ferrous iron (Fe2+) as their energy source, coupled to the reduction od oxygen or nitrate At acid pH, oxygen is the only viable electron acceptor; at neutral pH, Fe2+ spontaneously oxidizes to Fe3+ in the presence of sufficient oxygen. Therefore, FeOB living at neutral pH also live in anoxic or microoxic environments FeOB are of two types; both are autotrophs, but they have different habitats: 1) acid mine drainage systems, where oxygen is the electron acceptor (pH <3) 2) neutral, anoxic or microaerophilic habitats (pH ~7) where Fe2+ oxidation is coupled to nitrate or oxygen reduction

anoxygenic photosynthesis in purple bacterium

The chlorophyll P870 absorbs light, which converts it to a singlet excited state. This decreases its reduction potential so that it donates an electron to the next acceptor. In cyclic electron flow, the electron is passed through the depicted series of acceptors until it comes BACK to the original pigment that released it. Protons are pumped out by the Q pool to create PMF. When electrons are drown off of the pathway to generate reducing equivalents (NADH or Fd), an electron has to enter the pathway from another source (an electron donor such as H2S) to re-reduce the original P870 pigment ------------------- Comparison: in purple bacteria, reverse electron flow generates NADH in green sulfur bacteria and heliobacteria, the first stable electron acceptor protein, containing an Fe?S cluster, has H0' below that of Fd. Therefore, it can directly reduce Fd, which is used as the electron donor in CO2 fixation.

Electron flow in aerobic hydrogen oxidation

The membrane-bound hydrogenates passes electrons to the quinone pool. Electrons are then passed via cytochromes to oxygen. The PMF generated contributes to ATP synthesis. Some organisms also make a cytoplasmic hydrogenase, which reduces NAD+ to NADH NADH made this way can be used directly in biosynthetic reduction reactions. When a H2 molecule is used for this purpose, its electrons do NOT help to make PMF

The Pasteur effect

The net ATP yield of glucose --> alcohol fermentation is 2 mol ATP/ mol glucose metabolized, which is significantly lower than the ATP yield resulting from aerobic respiration In order to maintain a suitable ATP concentration in the cell (~2 mM), the rate of glucose consumption is significantly increased when yeast are shifted from aerobic metabolism to fermentation. In contrast, a change from anaerobic to aerobic metabolism is accompanied by a reduction in the rate of glucose consumption and an inhibition of alcohol production. This phenomenon is known as the Pasteur effect.

Photosynthetic membranes

The photosynthetic apparatus us usually found in specialized membrane systems In bacteria, the photosynthetic apparatus is located in the cytoplasmic membrane itself (heliobacteria) or in a variety of different internal membrane systems: 1) Thylakoid membranes (cyanobacteria); lines on the outer edges near membrane; attached peripherally to the thylakoid membrane, phycobilisomes absorb light energy and transfer it to the PSII reaction center. 2) chlorosomes (green sulfur bacteria); larger clear areas near the cytoplasmic membrane; contain a tightly packed crystalline array of bacteriochlorophylls inside a protein-lipid monolayer. Excitation energy is transferred from the Bch through intermediate protein/pigments to reaction centers (light or dark brown).

Fermentation

The process of converting carbohydrates to alcohol or organic acids using microorganisms—yeasts or bacteria—under anaerobic conditions. During Pastuer's time, alcohol was thought to be a simple chemical breakdown of sugars, but alcohol and vinegar are actually the products of microbial metabolism (fermentation). Pasteur found that a byproduct of the beet fermentation, amyl alcohol was present in only one optical isomer (each of two or more forms of a compound which have the same structure but are mirror images of each other) 1) Different microbes were present when a fermentation went right vs. wrong 2) Complex organic compounds present that were not predicted by simple chemical breakdown of sugar (Showed that many common industrial processes rely on microbial metabolic activity.)

Two types of photosynthetic reaction centers (RC)

There are two types of membrane-bound, chlorophyll-based photosynthetic reaction centers Type 1 = photosystem (PSI) The first stable electron acceptor is an Fe/S cluster in a protein, so these are called Fe/S-type RCs Type II= photosystem II (PSII) The first stable electron acceptor is a quinone molecule, so these are called Q-type RCs PSI and PSII can be used individually to generate PMF or reducing power Oxygenic photosynthesis uses BOTH PSI and PSII located in the same membrane, and it evolved in cyanobacteria. oxygenic photosynthesis uses a combination of a Q-type R and Fe/S-type RC

How cells respond to environmental conditions

To respond appropriately and efficiently to environmental conditions, cells control 1) gene expression (which portions are made) and 2) enzyme activity (which proteins are on/off) Out of all possible proteins encoded in the genome, a smaller fraction is currently being synthesize and an even smaller fraction are currently active

Calvin cycle

Used radio labeled 14CO2 (radioisotopes) and "lollipop" device to follow incorporation of radiojlabel into specific compounds by the alga Chlorella At time zero, 14CO2 was injected into the lollipop-shaped growth changer containing Chlorella Samples were removed from the chamber at the time intervals after the addition of 14CO2 via the stopcock at the bottom. Chlorella in the samples were killed instantly, and their contents were chemically analyzed to find where the 14C had gone; detection of 14C labeled compounds by thin layer chromatography and autoradiography Small molecules from killed Chlorella were separated in two dimension by TLC and the plates were exposed to film; after 30 sec labeled compounds begin to appear in sugar phosphates and diphosphate and amino acids. ---------------------- 1. Carboxylation using RuBisCo and PRK 2. Reduction 3. Regeneration requires NADPH and ATP

Exponential growth

When bac pop has sufficient nutrients and is not otherwise limited by its environment, it can undergo exponential growth. The # of cells in the pop and the total cell mass INCREASE by a factor of 2 during each generation time. N= N0 x 2^n ---------- N0= initial cell # N= final cell number n= number of generations Generation time or doubling time --> g= t/n ------------ where t= time elapsed during exponential growth

Quinone molecules

are mobile, hydrophobic electron carriers that diffuse in the plane of the cytoplasmic membrane can interact with different donor and acceptor complexes. In their reduced state, each molecule carries 2e- and 2H+ Wen a quinone accepts an electron ONLY from an upstream donor, it also takes up a proton from the cytoplasm. When the quinone passes the electron to a downstream acceptor, the proton can be released into the periplasm, helping to generate the proton motive force. R group is a hydrophobic isoprenoid polymer that kelps the quinone in the membrane

Hydrogen as e- donor

inorganic e- donor 2H+/H2 --> E0'= -.42 V Hydrogen was abundant in early earth history, now a common product of microbial fermentation. Some H2 oxidizers are aerobes, using oxygen as the terminal election acceptor while other anaerobes, coupling H2 oxidation to the reduction of nitrate, sulfite, ferric iron, or other acceptors; aerobic H2 oxidizers can get their carbon from CO2 via carbon fixation. can ALSO grow as chemoorganotrophs (using glucose or other reduced carbon sources as electron donor and carbon sources). When organic carbon sources are present, the bacteria use these preferentially. Sugars repress genes for H2 oxidation and carbon fixation.

Oxygenic photosynthesis

oxygenic photosynthesis uses a combination of a Q-type R and Fe/S-type RC Light energy excites P680 to P680*, which expels an electron and reduces Ph, a subunit of PSII. Ph reduces the mobile PQ which picks up protons from the cytoplasm. When PQ donates electrons to cut bf, protons are released into the per plasm to create PMF Protons are also pumped by cat bf as it passes electrons to PC, which delivers them to the RC of PSI. P700 is excited by light, and P700* expels an electron to reduce an FeS protein, which passes it to ferredoxin (Fd). Electrons on Fd can go one of two ways. 1) They can travel to a flavoprotein which reduces NAD+ or NADP to NAD(P)H This is a noncyclic phosphorylation. It generate both PMF and reducing power. 2) if the cell has abundant reducing power, then the electron of Fd can return to cut bf instead, and travel back through PSI, using more light energy. This is cyclic photophosphorylation, and it generates PMF ONLY, and no reducing power. (gerjneates PMF but not NADPH) Oxidized P700 is returned to ground state by electrons from plastocyanin. Oxidized P680 is returned to the ground state by electrons obtained from the splitting of water. Water is the electron donor, and these reaction occurring the oxygen-evolving complex, which is part of PSII.

Light harvesting pigments

pigments function in different types of light-harvesting complexes In purple bacteria, RCs are surrounded by light-harvesting complexes which absorb light and rapidly transfer excitation energy to the RC This enhances the rate of energy production by phototrophs, particularly in dim light or in the shade of other plants. Different spectra allow diff organisms to absorb light at different wavelength, which creates a variety of energetic "niches" in an ecosystem.

Glycolysis

the oxidation of glucose to pyruvate, which can be reduced to create fermentation products OR fed into the TCA (citric acid) cycle Stage I--> preparatory reactions, productions of glyceraldehyde-3-P Stage II--> generates pyruvate Stage III--> If respiration is possible, pyruvate enters the TCA cycle. If not, we proceed to stage III, where reduction reactions make fermentation products and regenerate NAD+

Chemolithotrophy Experiment

~1900 the Russian microbiologist Sergei Winogradsky conceived the idea of chemolithotrophy, the use of an inorganic compound for energy He observed large filamentous bacteria in water samples ONLY form H2S rich-springs, that grew without the addition of organic carbon. He enriched and observed the following: 1a) Bacteria grown with abundant H2S contain dark sulfur granules. 1b &c) If deprived of H2S, the bacteria gradually last their sulfur granules, but continue to grow (24h and 48 hr) After a long period without H2S, growth ceases. If H2S is resupplied, sulfur granules reappear. Concluded that H2S oxidized to S0, and when H2S is absent, S0 in granules can serve as an energy source instead.