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279 Cards in this Set
- Front
- Back
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microbiology
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science of microorganisms, very small, unicellular organisms or cell clusters
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microbiology began with?
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robert hooke describing fruiting structure of molds in 1664
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microorganisms include
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bacteria, archaea, algae, fungi, protozoa, viruses, prions
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why study microbiology (6)
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1. disease: medical, veterinary, agricultural
2. food production and preservation 3. cycling of nutrients 4. energy/environment 5. biotechnology 6. insight into life processes common to all life forms |
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why better condition of microbial diseases on humans? (3)
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understanding of microbes, improved sanitary practices, anti-microbial agents.
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agriculture (4)
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N2 fixation, nutrient cycling, animal husbandry, use of rumen to breakdown cellulose to animal protein
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food (5)
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food preservation, fermented foods, food additives, food spoilage, food-borne contamination.
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biotechnology (3)
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genetically modified organisms, production of pharmaceuticals like insulin and other human proteins, gene therapy for certain diseases with correct genetic lesion.
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energy/environment (3)
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biofuels like methane, fermentation
bioremediation for spilled oil or organic pollutants microbial mining |
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why study microbiology? 3
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microorganisms were the first life forms on earth
microorganisms created the biosphere that allowed multicellular organisms to evolve multicellular organisms evolved from microorganisms. |
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causative agent of black plague, transmission thru?
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yersinia pestis, transmission via fleas/black rat
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yellow fever people
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finlay and reed
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Antoni van Leeuwenhoel: around 1684
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dutch amateur microscope builder, refused to sell his microscopes to others, hindering microbiology. around 1684
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francesco redi 1668
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hypothesis: maggots come from flies and do not arise by spontaneous generation.
maggots when open jar with meat, no maggots when sealed and when netted. |
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2 prevailing hypotheses existed at the time about where do microbes come from?
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spontaneous generation and microbes obtained from the air, grow in culture.
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lazzaro spallanzani
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experiment to test whether spontaneous generation or microbes from air.
criticism: 1. heating destroys life-generating substances 2. oxygen may be necessary for spontaneous generation. |
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louis pasteur major contribution
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french chemist:
refuted criticism 1: heated, capped, opened cap: microorganisms grow in liquid: remove cork and culture grows. refuted second one: poured nonsterile liquid into flask, neck of flask drawn out in flame, liquid sterilized by heating, air forced out with open end. After liquid cooled, dust and microorganisms trapped in bend so liquid remained sterile for many years. however, if flask is tipped so microorganism-laden dust contacts sterile liquid, microorganisms grew in liquid. Demonstrated: 1. spontaneous generation does not occur 2. organisms found were present in the air. |
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other contribution
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sterilization, wine production, pasteurization, vaccines, silkworm industry rescue, germ theory of disease
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robert koch
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germ theory of disease: study bacillis anthracis, and tested "are microbes the cause of or correlated with disease?"
developed postulates for proving that specific type of microorganism causes specific disease. Koch's postulates: procedure for defining the agent of any disease: 1. microbe present in diseased animals and absent from healthy ones 2. isolate microbe in pure culture 3. when inoculated into susceptible animal, disease results. 4. able to re-isolate organism and pure culture must be shown to be the same as the original organisms. |
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other contribution
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discovered bacillus anthracis, tuberculosis, cholera.
invented nutrient broth plating techniques: agar as medium |
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MRSA
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drug resistant microbes in our skins. used to be bad with hospitals with bacteria, antibiotics. but now it's bad with gyms, sports teams, maybe worse than aids.
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tuberculosis
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drug resistant tuberculosis: MDR-TB (multi drug resistant) vs XDR-TB (extreme drug resistant)
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what else is drug resistant?
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gram negative!
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swine flu
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people read about it, thought they had it, hospitals overwhelmed. of more virulent, would have been bad.
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features common to all cells (4)
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nucleic acids, plasma membrane, cytoplasm, ribosomes.
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prokaryotes: (2 imp facts)
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lack nucleus (nucleoid)
generally lack membrane bound organelles often: flagella, pili, vesicles, spores, cell wall, peptidoglycan, 1 chromosome. |
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eukaryotes (3 key facts)
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nucleus, membrane bound organelles, cytoskeleton
others: flagella, cilia, peroxisome, lysosome (vacuole) |
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rod shaped bacteria size
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1 micron 3 microns.
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euk cell size
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20 microns across
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simple microscope
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single lens (objective)
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magnification
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enlargement: size of image/size of object
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resolution
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ability to distinguish two adjacent points: 2 points within object can't be distinguished, points are unresolved. 2 points appear as overlapping disks, they are only partially resolved. two points are visible as separate entitites, they are fully resolved.
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compound light microscope, and critical because?
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2 lenses. focuses light, sample diffracts light. it's flipped 3 times. can have inverted scope: good for live cells without putting on slide.
critical for development of microbio. |
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magnification for compound light microscope?
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objective x ocular
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why oil?
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oil aids in light gathering ability of lens by rays emerging from specimen at higher angles.
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limit of light microscope
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mag: 1000x
res: 0.2 micro |
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bright field microscopy (3)
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microscope detects light scattered by cells compared to surrounding media
bacterial cells are particularly difficult to see due to low diff in contrast. stained/pigmented cells scatter light better. |
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staining samples. Why and how? (5 steps)
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aids in viewing samples by bright-field.
1. spread culture in thin film over slide 2. dry in air 3. pass slide thru flame to fix (now use fermaldehyde) 4. flood slide with stain, rinse and dry 5. place drop of oil on slide, examine with 100x objective. use cationic dyes (crystal violet, methylene blue) to stain negatively charged cellular components. |
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differential staining
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1884 Hans Christian Gram. differential staining exploits differences in cell wall composition of gram+ and - bacteria.
1. flood the heat-fixed smear with crystal violet for 1 min. all cells purple. 2. add iodine solution for 3 min. all cells remain purple (iodine:dye complex) 3. decolorize with alcohol briefly for 20 seconds, gram positive cells are purple, negative are colorless (EtOH destain) -> complex can't get out of positive, but can get out of negative. 4. counterstain with safranin for 1-2 min, gram positive cell are purple, gram negative are pink to red. |
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phase contrast
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1. unstained cells scatter light poorly
2. phase contrast microscopy changes wavelength of undiffracted light by 1/4 wavelength accentuating the diff between undiffracted and diffracted light (darker image on lighter background) 3. phase contrast microscopy provides better detail of structures, can be performed on live cells in absence of staining. Changes 1/4 in incident, 1/4 in diffracted so now 1/2. darkest spot is now light b/c wavelength 1/2 change, kicks it back into phase. |
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dark field microscopy
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light modified to reach specimen from the sides
only scattered light reaches the lens (light image on dark background) |
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differential interference contrast
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polarized light passed thru prism, giving 3D-like appearance. can see internal structures: nuclei, vacuoles, unstained cells.
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fluorescence microscopy
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used to visualize specimens that fluoresce (emit light of one color when light of another color shines upon them)
fluorescent protein: GFP (green fluorescent protein. can use diff combos of colors to see diff colors), YFP, RFP immunofluorescence assay (IFA): antibody coupled to fluorochrome used in combo with phase, BF, DIC. 1. first barrier filter: lets thru only blue light 2. beam-splitting mirror: reflects blue light, transmits green. 3. second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescein. |
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direct immunofluorescence assay
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fluorochrome and antibodies combined. sticks to cell surface antigen molecules.
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indirect immunofluorescence assay
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1. add unlabeled primary antibody (mouse anti-surface protein)
2. wash away unbound antibody 3. add Fl secondary antibody (binds Fc region, like goat anti-mouse) advantages: use 1 labeled secondary ab for all mouse primary ab's. also amplification signal because more than one can bind. |
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confocal microscopy (3)
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laser source instead of light
precise "optical sections" 3-D models |
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electron microscope - transmission EM (7)
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1. light microscope: 0.2micro
2. EM: 1000x better resolution. 3. electrons are used instead of light rays. 4. internal structures detected with high resolving power. 5. thin sectioned samples treated with specialized stains for improved contrast. (diamond knife, has to be really thin) 6. can be deceiving: mito doesn't look like peanut, usually cutting and seeing barrel, can cut next to nucleus so don't see it. 7. has to kill everything. |
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electron microscope, scanning EM (3)
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used for revealing external features
specimens treated with heavy metal (gold) really high resolutions |
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what events led to microbial diversity that we see today? (2)
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lots of time
adaptations of microbes to changing environments via mutation of genomes |
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RNA world (5)
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life evolved from RNA
because RNA has coding and catalytic activities (Self replicating) lipoprotein vesicle: trap protein RNA codes protein (proteins take over catalytic functions) DNA came around: central dogma, replaced RNA. |
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bacterial speciation: adaptive mutation
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ecotype 1: cell containing an adaptive mutation, adaptive mutant survives. original ecotype 1 wild-type cells outcompeted. repeated many times, new species of ecotype 1.
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why small number of prokaryotes?
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1. hard to find, most dependent on one another
2. nonculturable because we don't know how to grow them 3. we don't chase them if doesn't cause diseases. |
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methods for classifying microbial diversity
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1. morphological diversity: features, structures (key advance microscopy)
2. metablic diversity: biochemical (key advance: enzymology) 3. ecological diversity like extremeophiles 4. genetic diversity: gene sequences (key advance: molecular biology, DNA sequencing) |
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morphological diversity (2)
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sufficient for distinguishing prok/euk and some obvious prokaryotic differences.
insufficient to distinguish microbe types that appear the same. |
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metabolic diff can help because all organisms need?
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E and carbon.
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energy
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chemotrophy:
organic chemicals: chemoorganotrophs inorganic chemicals: chemolithotrophs (restricted to prok) phototrophy: phototrophs. |
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carbon
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organic: heterotrophs
CO2: autotrophs. |
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extremophiles live in crazy (4)
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temp: -5-118C: hyperthermophile and psychrophile.
salinity: halophile pH: 0-11: acidophile and alkaliphile pressure: barophile. |
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pyrolobus
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hyperthermophilic: found in undersea hydrothermal vents, grows best just above 100C
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halobacterium salinarum (5)
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high saline environments (4M): salt evaporation ponds and salt lakes
osmotic balance: cells pump high [K+] into cytoplasm. many enzymes requiring high K+ cells actually require salt for cell wall stability. cell wall glycoprotein stabilized by sodium ions bacteriorhodopsin: light mediated synthesis of ATP. |
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2 kingdom model:
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1. plant (cyanobacteria)
2. animal (bacteria) |
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5 kingdom model: Whittaker
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bacteria: Monera
Protozoa and Algae: Protista plantae fungi animalia |
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genetic diversity (3)
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DNA sequencing allowed comparison of genes from diff microbes
alteration in DNA sequence allow for construction of molecular phylogenetic trees why gene should one choose as a "molecular clock?" 1. universally distributed 2. functionally homologous. does same job, because if different, then have different pressure and mutations 3. slow to change in general sense (like bacteria/fungi general comparison) 4. right size, not too big, unreasonable. |
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why extremophile gene important?
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genes we can express for bioengineering like tac polymerase. detergent process chemical.
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so example of a molecular clock gene? who thought of this?
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by Carl Woese! ribosome!
All organisms contain ribosome. rRNA excellent chronotometer: highly conserved functionally constant. 16S and 1500 nt just right size because can see changes throughout evolution loops and stems are highly conserved. if looking at similar species, look at loops that are diff. so sequenced the 16S rRNA from a broad array of microorganisms |
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ribosomal RNA gene sequencing and phylogeny: (5 steps)
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1. pure culture or environmental sample cells are lysed and DNA isolated.
2. gene-encoding ribosomal RNA is isolated and amplified by PCR 3. amplified rRNA gene is sequenced 4. obtained sequences aligned by computer: pairwise comparisons: tree 5. tree depicts difference in rRNA sequence between organisms analyzed. |
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length of line in tree proportional to?
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evolutionary distance!
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3 domains of life discovered how? (2)
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woese compared rRNA of methanogens to bacteria and eukaryotes.
saw 2 distinct taxa of prokaryotes! |
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what did carl woese find out about 3 domains of life? (4)
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1. bacteria and archaea separate domains of prokaryotes
2. archaea more closely related to euk than bac 3. mito and chloro branch with bacteria 4. root of tree: universal ancestor! |
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other stuff to support 3 domains?
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1. RNA polymerase: B has 4 subunits, euk has 10-12 subunits. they purified polymerase for B, E, and A, separated subunits by SDS-PAGE, visualized subunits by staining, and showed 8 or more subunits present in archaea.
inhibitors of translation: sensitives to inhibitors suggest A translation is more like E than B. phospholipid bilayer: ester linkage to fatty acid for B and E. 1. linkage to hydrophobic side chain and 2. type of side chain diff in A. -B and E has ester linkage bonds for fatty to glycerol while A has ether linkage bonds side chain to glycerol -A has isoprene instead of fatty acids. archaeal lipids: phytanyl bilayers and biphytanyl monolayers: phytane (4 isoprene molecules), hydrophobic molecule of A lipids, glycerol diether, while biphytanyl monolater is hyperhthermophilic archaea and resistant to high temp peeling. also signature sequences shows diff in all 3 domains, FISH probes to exploit signature sequences to identify groups of microorganisms. |
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what did they find out from phylogenetic analysis of bacteria, 16S? (2)
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largest group of bacteria known is proteobacteria.
ENV: not cultured organism by environmental sequences |
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what did they find out from phylogenetic analysis of archaea, 18S?
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2 major subgroups: euryarchaeota and crenarchaeota
some hyperthermophiles, methanogens, halophiles, acidophiles, more ENV. |
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what did they find out frm phylogenetic analysis of eukarya, 18S?
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early-branching lack mitochondria and other "euk" organelles
many early-branaching euk (protozoans) are pathogenic parasites of humans or animals |
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mito and chloroplasts
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mito and chloro evolved from bacteria:
16S rRNA, circular chromosomes, 70S ribosomes, bacterial-like lipids (cardiolipin) those lacking mito probably lost rather than never acquired bulk of bacterial DNA from these organelles escaped from nucleus, can transport back in if we need them (necessary proteins, use hydrophobic that can make in membrane) |
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how can we make microbial blooms?
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add iron
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venter and sargasso sea
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"shotgun" sequencing of ocean water microbes. tons of new base pairs, new genes, new species, new data.
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mechanical isolation of cell
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tweezers, pick put. hard to do.
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general enrichment (4)
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1. mix some leaf litter, filter paper, cardboard, etc.
2. calcium carbonate and calcium sulfate 3. pond mud or pond water (source of microbes) 4. put in cylinder and place on window shelf |
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winogradsky column
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-chemical gradients are formed to give many different micro-environments, plastic wrap covering top reduces evaporation
-with these many micro-environments, you get many bacterial types: top layer: consisting of pond water is aerobic with cyanobacterial and algae middle layers of mud are anaerobic, but increasingly aerobic toward top of column. nonsulfer photosynthetic bacteria and photosynthetic bacteria. bottom layer of mud, which contains sulfate and carbonate salts and cellulose is completely anaerobic. decomposition of organic matter creates sulfide-rich environment: photosynthetic bacteria. |
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streak plate
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sample enrichment
streak on nutrient plate incubate visualize colonies can get some interesting cells but it may be difficult if desired type of microbe is present in low numbers |
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pour plate or spread plate
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inoculate by pouring in soft agar, or spread on surface.
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selective enrichment method
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uses defined medium of energy, carbon and nutrients.
then inoculate, incubate, visualize. then streak cells, incubate, re-visualize. |
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direct isolation method
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sample environment directly, streak, incubate, and visualize!
need to design a special medium: like single carbon compound, carbonate, or single energy source like hydrogen sulfide. works best only when desired cell type is abundant. |
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the development of methods to isolate single cells:
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1 cell -> 1 colony
needed a solid surface vs. liquid: potato slice (only starch), gelatin (melting point), and then the "agar" by Fannie Hesse in 1882, which has high melting point and hardened gel when cool. not attacked by many organisms, nutritional. agar was the new technology after 200 years. |
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how do we know more diversity than 6000 strains known?
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from isolated DNA of 16S RNA genes, compared, way more out there!
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what is the "New Technology"?
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environmental DNA sequencing
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how to design a cell culture medium
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to culture a microbe in lab, we just need to provide each element in the correct amount, and in a usable chemical form.
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"synthetic medium" is chemically defined, to design one... Provide 4 things
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1. appropriate carbon source
2. appropriate energy source 3. all essential minerals and nutrients 4. any needed environmental conditions (temp, pH, moisture, ionic strength) see what grows! we can demonstrate tremendous diversity. |
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diversity of microbe necessities
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medium might contain only few carbon sources, few minerals, or it could be very complex!
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regardless of cell type, cell composition are...?
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similar: 90% water, carbon, oxygen...
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cells also need ...?
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trace minerals.
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"non-defined medium." also, why use it?
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not characterized by chemical type or amounts
ex: plant or animal extract and water (yeast extract, beef extract, tomato juice). it provides the carbon and energy sources along with minerals and various nutrients. provide any needed environmental conditions, like temp, pH, moisture, ionic strength. why use non-defined medium? easy to make and very inexpensive! it won't work for many types of microbes. |
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fig trees
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mostly grow on fruit sugars: chemo-heterotrophs like bacteria, yeasts, and fungi.
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uptake of nutrients (4 facts)
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variety of nutrient uptake systems
some highly specific, some are general sometimes dozens of diff transporter types in a cell synthesis is often inducible. |
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passive diffusion
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gasses, water
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facilitated diffusion (4)
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aided by protein
generally molecule specific allows movement in either direction depends on molecule gradient. |
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carrier-mediated transport
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uptake rate is fast!
system can saturate (exponential and logistic when graph is external concentration of solute vs. rate of solute entry) while simple diffusion is just gradual flat incline on graph. |
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3 classes of active transporters
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ion driven transporters: ion gradient dependent
group translocation systems: PEP-dependent ABC transporters: ATP-dependent. |
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ion-driven transporters
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fueled by proton, potassium, or sodium ion gradients
uniporters (facilitated), symporters and antiporters (active) |
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uniporters
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imports a single substrate
driven by an ion gradient (K+) inport of potassium ions |
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sym-porters
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imports substrate while also importing a charged molecule (proton gradient driven H+)
import of nutrient like lactose |
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anti-porters
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imports its substrate, while exporting a charged molecule (usually proton gradient driven H+) like export of sodium ion or waste product.
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energy for ion-driven transporters is directly or indirectly provided by?
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electron transport
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lactose type transporter (lac permease)
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accumulates solute against a concentration gradient
proton driven symporter 12 membrane-spanning alpha helices many other permeases are structurally similar, but differ in substrate specificity. |
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ABC transporter class (primary transporters) (3)
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requires cooperation of 3 types of proteins
requires ATP to drive uptake process accumulates the solute against concentration gradient. |
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3 parts of ABC type transporter
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substrate binding protein:
has specific binding specificity has high affinity for substrate located in periplasm of - cells tethered on outer surface of + cells membrane channel protein: form selective channel and can only work with its cognate SBP ATP hydrolyzing protein: provides energy to drive solute uptake can only work with its membrane partner. |
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aquisition of iron
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aerobic conditions, FE is in FE3+ state (insoluble form as FeOH3)
since equilibrium is far to right (more FE3+), little iron is free in solution! how do cells get iron? they produce chelator compound that binds FE3+ (enterochelin or enterobactin) |
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iron ABC-type uptake system (5 steps)
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1. SBP protein delivers Fe3+ chelate to membrane
2. the membrane protein forms a selective channel 3. ATP binding protein provides energy to drive solute uptake 4. ferric reductase causes release of iron 2+ inside cell 5. ferrous iron is then incorporated into proteins. |
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group translocation (or phospho-transferase systems) (4)
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requires 5 types of proteins (HPr, EI, EIIa, EIIb, EIIc)
energy provided by phosphoenolpyruvate (PEP) nutrient is modified upon cell entry (phosphorylated) examples in E.Coli: transport of glucose, fructose, mannose |
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how PTS works
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phosphate from PEP to pyruvate, to enzyme I, to HPr, to Enzyme IIa, to Enzyme IIb, to glucose that is going thru enzyme IIc, then glucose is phosphorylated to make G6P.
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how many transporters in e.coli?
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150
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what are transporters used for? (3)
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1. uptake of nutrients (organic, inorganic)
2. elimination of waste products 3. maintaining correct ion levels (K+, Na+, H+) |
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how are transporters efficient?
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most transporters are made only when needed, their genes are highly regulated.
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facilitated versus active
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solute conc: outside equals inside for facilitated, inside is much higher for active.
no energy requirement for facilitated, ions, ATP, or PEP for active. active can increase concentration of in by 100 to 1000 vs out. in facilitated, consumption of substrate inside cell allows diffusion of additional molecules into cell (by glycerol facilitator) this uptake in active is needed to fuel many enzyme reactions that have Km values in a mM range where nutrient levels outside the cell may be microM or less. |
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active transport system are molecule-
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specific
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synthesis of transport system is?
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inducible, along with production of "downstream" enzymes
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what can be inducer of gene expression?
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substrate
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cost of ____ is far less than cost of _____
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transport, biosynthesis.
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although microbes exhibit different cell morphologies...
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they grow and divide in similar ways
cell reproduction yields two identical progeny division is preprogrammed but the speed can vary |
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binary transverse fisson (3)
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asexual process
events are programmed in temporal and spatial context cell mass doubles: proteins, membranes, cell envelope, and small molecules. |
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cell division apparatus is called...? and 6 details?
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divisome,
-multiprotein complex -composed of many Fts proteins -FtsZ binds to the membrane, and then forms a "ring" -GTP hydrolysis follows -it contracts to form the septum. -takes 20 min for E Coli |
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what needs to happen before cell splitting?
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DNA replication, carefully timed. at beginning, DNA attached to membrane, replicated parental DNA needs to be replicated.
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the speed of binary transverse fission depend on (3)
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the species
nutrients (medium composition) environment (culture conditions) |
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ways to describe microbial growth (2)
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plot cell numbers vs time in linear scale or log scale
derive generation time g |
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generation time, g, td
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time required for the number of cells to double during the exponential growth phase.
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k
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growth rate 1/td or 1/g
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4 phases of cell growth
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lag phase: cells were on plate (low on nutrients) and moved to flask with new nutrients, so they have to see what they have. no cell growth occurs. make proteins to process. begin uptake of nutrients, start-up machinery, make new ribosomes, enzymes to obtain energy. enzymes for biosynthesis and macromolecular assembly. gearing up factory to start replicating. cells shift from an unbalanced state to balanced growth.
exponential growth phase: after preparation, begin elongting by binary transverse fission. log is linear, so exponential growth. cells are making all enzymes needed for each cell to make all components and in balanced amounts as needed. continue to detect environmental changes. growth continues until environmental conditions change. keep going til nutrients run out or waste is too much and poison. they individually growth, no communication. stationary phase: metabolism continues by at a reduced rate. cell does maintenance reactions to repair damage and maintain ion/pH balance (tries to stay alive). there could just be 1 low nutrient, and rest are still available. storage materials can still be accumulated. sit and be able to last as they use up this resource to live. different storage materials depending on species. cell maintenance can function even if limited nutrient. (can accumulate poly-phosphate, starch, poly-alanine, other polymers) death phase: cells continue to detect environmental changes. (exponential) can no longer do maintenance reactions. can't maintain ion balances. cells start dying. some lyse and release contents, then might be taken up by other bacteria. cell death rate is different in all conditions and species. (soil bacteria are generally much better survivors than pathogenic microbes, why?) |
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characteristics of cell growth in batch culture (4) and examples
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system is closed: no addition of new material, no removal of waste material, no removal of cells.
examples: yogurt production, beer fermentation, antibiotic production, blood infections |
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yogurt
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inoculate milk with lactic acid bacterium, milk is then fermented to make yogurt. lactic acid doesn't allow microbes to grow anymore. so cell content never gets too high.
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budding
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small protuberance (bud) forms on the mother cell and enlarges as growth proceeds.
when bud is sufficiently mature, it separets from the mother cell. the mother cell produces another bud from the same location on the cell surface and daughter cell produces first bud. most of new cell is new material (binary: daughters are equal in new and old material. budding: new material in new cell) |
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polar growth
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newly synthesized cellular components are distributed unequally between two daughter cells.
grow COMPLETELY then break off. |
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ways to monitor cell growth
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1. total cell count
2. viable cell count 3. turbidity estimate of cell numbers 4. cell dry mass |
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total cell count (a direct method)
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counting chamber is used to physically count cells.
make cell dilutions use a known volume count cell number get cells/volume calculate concentration. very accurate but it doesn't distinguish between live and dead cells. |
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viable cell count
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(also a direct method)
count living cells only make cell dilutions and plate use "spread" or "pour plate" incubate, then count colonies (one colony=one cell) calculate cell concentration |
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turbidity estimate of cell numbers
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(indirect method using light scattering)
see how much light is scattered, tell content. graph and standard curve. standard curve is first prepared by a plot of turbidity vs cell number. the cell number for any unknown sample is then easily determined by measuring the turbidity and extrapolating the value. the curve differs for each type of organism, because cells of diff species may differ in size, shape, texture, etc. don't distintuish between live and dead, can tell total |
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when graphed, viable count and turbidity similar in 3 of 4 phases...
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different in death because turbidity doesn't tell which are already dead.
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cell dry mass (4)
|
(indirect method)
1. cells are filtered thru a pre-weighed filter that has sub-micron size pores (retains cells) 2. filters are then dried and weighed 3. dry mass is proportional to cell number 4. you need a standard curve (takes a lot of cells to get an accurate measure) |
|
|
for dry mass, standard curve must be prepared for each type of organism. why?
|
cells of diff species may differ in size and shape. each strain exhibits a different standard curve.
|
|
|
from dry mass and cell number, you can calculate...?
|
average mass of cell
|
|
|
how to determine temperature range for cell growth (3)
|
1. grow individual flastks at different temperatures
2. calculate cell growth rate 3. plot vs temperature |
|
|
what happens to microbes at higher temp or lower temp than optimal?
|
low: membrane gelling: transport processes so slow that growth can't occur. membrane solidify at lower temp, so can't work as well. above optimal, protein denaturation, collapse of cytoplasmic membrane, thermal lysis and don't work well anymore.
|
|
|
highest thermophile known
|
118C
|
|
|
k is expressed in...?
|
hours
if g is 30 min, k=2 if g=1 hour, k=1 if g is 2 hours, k=0.5 |
|
|
4 kind of temperature bacteria
|
psychrophiles, mesophiles, thermophiles, hyperthermophiles.
|
|
|
why such huge range of temp?
|
because evolved for long time, adaptation.
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|
|
pH
|
1-11, at pH 7, H+ and OH- are 10^-7.
bell shaped curve. optimum growth different ion concentration necessary to function and survive. curves similar, just shift between left and right. keep pH at optimum by pumping in or out right substrates. |
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|
acidophiles and alkaliophiles
|
pH outside 1-6 and 8-11
|
|
|
unifying feature of bacteria and archaea in pH (4)
|
1. internal cytoplasmic pH is always about 7
2. acidophile and alkaliophiles have each evolved strategies to maintain their internal cell pH 3. they balance entry of any protons by pumping out protons and by uptake of K+ to maintain internal pH 4. cytoplasm buffering ability can also be aided by synthesis of glutamate and polyamines. |
|
|
effect of oxygen on cell growth. 5 kinds!
|
strict aerobes: requires oxygen (top)
strict anaeorobe uses no oxygen (bottom) facultative anaerobe prefers oxygen (more on top, less on bottom) aerotolerant species grow throughout (don't care, don't use O2 to advantage) microaerophile needs less oxygen (all bundled kinda near top) kinda like pathogenic microbes in intestines. |
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|
oxygen can be toxic!
|
O2 can give rise to radicals that damage cells. O2 can react chemically (nonbiological) with metals and make reactive stuff, and can alter structures and be harmful to organisms. for example, O2 can react with FE2+ or reduced flavin molecules to generate:
superoxide (O2-) hydrogen peroxide (H2O2) Hydroxyl radicals (OH.) |
|
|
some cells make one or more protective enzymes
|
1. superoxide dismutase
O2- + O2- +2H+ -> H2O2 + O2 2. catalase (for hydrogen peroxide) H2O2+H2O2 -> 2H20+O2 3. peroxidase (for hydrogen peroxide) H2O2+NADH+H+ -> 2H2O + NAD+ 4. spontaenous (hydroxyl radical OH.) OH. + e- + H+ -> H2O |
|
|
what do you use for catalase to see if it's working?
|
spot test: hydroperoxide.
pick up colony with wire loop, and put in, see tons of O2. microbe made catalase, broke down to O2. |
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|
effect of osmolarity on cell growth
|
table salt (NaCl), Mg/Na chlorides, sulfates, borate, and carbonates. (certain microbes adapt and grow at over 3 M salt!
S SF: evaporate and salts left on coast, can be sold as table salt or put back into ocean. |
|
|
the 4 degrees of salts for microbes
|
non-halophiles
halotolerant obligate halophiles extreme halophiles |
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|
osmoregulate (5)
|
1. cells maintain a constant "turgor pressure"
2. internal pressure is adjusted to match the external force. 3. this prevents the cytoplasmic membrane from busting and deflating efflux of water occurs if cells encounter high osmolarity (cells shrink, contents don't work well) influx of water occurs if cells encounter low osmolarity (cells may burst) |
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|
how do cells compensate or do osmoregulation?
|
they balance the osmotic pressure exerted on the membrane
cells uptake or synthesize "compatible solutes" (also called osmoprotectants) solutes are either charged or neutral molecules: 1. K+ ions, alpha-glutamate, proline (charged) 2. betaine, ectoine (zwitterions) 3. sucrose, trehalose, glycerol, mannitol (no charge) |
|
|
osmotic changes exerted by sugars
|
some microbes can adapt and grow at medium sugars
but none can grow at high levels of sugar (jams, jellies, and honey) |
|
|
1 atm
|
14lbs/sq inch
|
|
|
microbe descriptors regarding growth and pressure (3)
|
barotolerant
barophiles obligate barophiles |
|
|
high pressure can affect what in microbes?
|
membrane stability, ribosome function.
|
|
|
pressure ranges from ...?
|
0-1000 atm!
|
|
|
continuous cell culture
|
in natural habitats there are many examples where cell grow well below their potential (optimal growth rate)
continually profused, intro of new nutrients, remove waste and organisms. |
|
|
examples of continuous culture
|
microbes in stream/river
human intestines LAX sewage treatment facility surface of leaf or rock |
|
|
how do we keep continuous environment in lab?
|
use chemostat. machine controls cell growth rate. In culture vessel or growth chamber, metered growth medium is put in, stirring motor keeps it stirred, there's airline for constant oxygenation, and collection vessel that gets waste.
|
|
|
chemostat always operated under ____________?
|
nutrient limiting conditions (rate of nutrient addition or inflow limits cell growth rate)
only one essential nutrient needs to be limiting. cells lack sufficient nutrient and thus must grow slower. all other nutrients can be in excess abundance!! KEY: vessel inflow rate = vessel outflow rate |
|
|
what to limit?
|
limit carbon source, nitrogen source, PO4 source, sulfer, iron, etc.
|
|
|
you cannot ask cells to...? (2) because? (2)
|
grow faster than their genetic program will allow
grow slower that they can maintain basal metabolism. otherwise: chemostat conditions violate cell ability to grow cells can't "keep-up" and are "washed out" of the vessel. or cells just don't get enough food to survive, and are "washed out" |
|
|
how to use chemostat experimentally?
|
example 1: how many ribosomes are in the cell?
you can manipulate cell growth rate using a chemostat. at different rates, measure ribosome numbers per cell. ask, if and how cell composition may change. |
|
|
does the protein factory always have the same capacity? or does it vary the ability to convert raw materials into finished product (proteins and new cells)?
|
for E coli growing a different rates, number of ribosomes per cell increases when cell doublings per hour increases.
|
|
|
do ribosome number vary in batch culture cell?
|
no, cell growth rate does not vary in batch culture, so ribosome numbers remain constant during log growth. protein synthesis is maximized for cells in exponential phase.
|
|
|
2 other example of using chemostat experimentally
|
example 2: do enzyme levels vary at a given growth rate? (effect of substrate availability on gene expression)
in principle, you can examine if a gene is expressed, and if so, what conditions are optimal. example 3: for two diff bacterial strains (which one has a selective advantage under a given condition?) how to test this idea: inoculate a chemostat with equal numbers of 2 diff strains. allow cells to grow for several days to weeks. each day, count the number of cell type present. what is observed?: the organism present in the highest numbers is better able to compete under the conditions provided. the other organism will likely disappear with time (called a wash-out) |
|
|
batch verses continuous (7)
|
1. system is closed versus open
2. no addition of materials versus constant additions 3. no removal of materials versus constant removal 4. cell growth rate is maximum, cell growth is limited 5. cell density changes versus once at equilibrium, cell density remains constant. 6. nutrients are depleted vs nutrient levels stay constant 7. waste products accumulate versus waste products are constant. |
|
|
cytoplasmic membrane
|
1. a lipid bi-layer ~8nm thick
2 retains cell contents (proteins, metabolites, ions) 3. has selective permeability (movement of nutrients and wastes) 4. location of specialized proteins (energy generation, cell envelope synthesis, nutrient uptake, cell motility, and signaling) |
|
|
membrane phospholipid heads made of?
|
glycerol phosphate.
|
|
|
bacterial lipids
|
C14-18 fatty acids
|
|
|
archaeal lipids
|
C20 or 40 isoprenoids, depending on if they're bilayers of two layers of glycerol diethers or monolayer of diglycerol tetraethers.
|
|
|
role of bacterial cell envelope (3)
|
1. determines cell size, shape, rigidity
2. provides for attachment of cell appendages 3. acts as a selective barrier: to hydrophobic compounds, hydrophilic compounds, toxic compounds. |
|
|
+
|
cellular membrane
peptidoglycan (penta-peptide) teichoic acids and lipoteichoic acid chains also have vertical cross-linking of the glycan chains |
|
|
-
|
cellular membrane
peptidoglycan (DAP) outer membrane lipoprotein (LPP) Lipopolysaccharide (LPS) has lipid A and polysaccharide. can have porin |
|
|
peptidoglycan layer
|
1. also called murein, glycan, cell wall
2. backbone chains of repeating sugar types (NAG, N-acetylglucosamine and NAM, N-acetylmuramic acid) 3. amino acid side chains cross-link the sugar chains at NAM 4. provides for the characteristic cell size and shape of bacteria 5. protects against osmotic lysis and turgor pressure: thin in -, thick in + |
|
|
peptidoglycan in + and -
|
+:
L-alanine, D-glutamate, L-lysine, D-alanine. pentaglycine crosslink -: L-alanine, D-glutamate, L-diaminopimelic cid (DAP), D-alanine direct cross-link |
|
|
teichoic acids
|
class of molecules with similar properties
poly-glycerol or poly-ribitol. (diversity) goes through the layers of peptidoglycan, cross sections peptidoglycan. joined by phosphate. sometimes decorated on glycerol or ribitol side chains for diversity, so keep from being attached by enzymes with variety and also gives extra charges, makes envelope stronger barrier to environmental molecules. lipoteichoic acid goes through the peptidoglycan layers and into cytoplasmic membrane to anchor. RIGID. |
|
|
what's good about the fact that there are D-amino acids?
|
resistant from enzymes that normally break down L-aa's.
|
|
|
+ and - diverged when?
|
long time ago
|
|
|
properties of the outer membrane (6)
|
1. lipid bilayer consisting of phospholipids, proteins, lipo-proteins, and lipopolysaccharide. (thicker than CM)
2. membrane is about 12-15 nanometers thick 3. provides a permeability barrier to many molecules 4. enhances cell stability 5. LPP: small lipoprotein anchors the outer membrane to peptidoglycan layer. 6. LPS decreases permeability to some molecules (like bile salts) and acts as an endotoxin. |
|
|
lipopolysaccharide 3 parts, and where is it?
|
1. strain-specific O-polysaccharide (hydrophilic)
2. core polysaccharide 3. lipid A (hydrophobic): 4 FA side chains, liked to sugars. huge diversity of sugars sticking off between lipid A and core, to be distinguished by antibody response. recognition! on outer leaflet. |
|
|
Lipoprotein
|
inner leaflet
3 FA to N terminus of LPP polypeptide and anchor to the outer membrane. N terminus of LPP is linked to glycerol moiety by thioether bond at cysteine. the C terminus of LPP is linked to cell wall at D-nitrogen of lysine. anchor outer membrane to peptidoglycan and give rigidity |
|
|
periplasmic space of the cell envelope (4)
|
1. space bw/ cytoplasmic and outer membrane
2. contains as mucha s 10% of all cell proteins 3. gel like consistency (highyl hydrated) 4. many protein types (binding proteins, hydrolytic enzymes, cell wall, and outer membrane assembly proteins) |
|
|
what transporters may be present in gram - bacteria?
|
ABC transporters.
|
|
|
outer membrane proteins (OMPs) (6)
|
1. porins
2. allow outer membrane to serve as a molecular sieve or permeability barrier 3. specific for certain substrates (like iron), some general. 4. always folded into B-barrels whereas proteins of the plasma membrane found into alpha-helical segements within the bilayer. 5. small molecules across only 6. trimers: 3 pores. |
|
|
how to rupture cell wall gram + and - bacteria? (2)
|
1. mechanical press
2. enzyme attack |
|
|
mechanical press
|
1. put bacterial solution into cell
2. apply pressure to 20,000 psi 3. slowly open valve 4. bacterial walls rupture upon release of pressure changes to 14 psi 5. chemical bonds of the cross-linked peptidoglycan are broken |
|
|
2 molecules not found in archaea or eukarya found in bacteria? why important?
|
n-acetylmuramic acid and diaminopimelic acid. important because enzymes only affects bacterial cells because eukaryotic cells don't have these parts.
|
|
|
enzyme attack
|
human tears contain enzyme lysozyme, attacks cell envelope, digests, peptidoglycan backbone, cells round up, membranes can then rupture.
in low solute solution: lysozyme digests wall, water enters and lysis. in isotonic solute solution, lysozyme digests wall, bacterial cell comes out of wall and makes protoplast. |
|
|
cell division
|
has to happen without cells bursting, so careful synthesis of new material so these cells aren't vulnerable at weakest part of life. so some antibiotics inhibit formation of bonds. these ones have no effect on bacteria that's not growing, so if stationary, can't kill. only at exponential when making new peptidoglycan.
|
|
|
4 kinds of antibiotics and functions
|
1. lysozyme: cleaves crosslinking
2. penicillin: inhibits crosslinking 3. vancomycin: inhibits glycan linking 4. bacitracin inhibits precursor formation. |
|
|
summary of bacterial cell envelope (6)
|
1. forms rigid structure
2. protects membrane from rupture (osmotic control) 3. not a selective barrier to small molecules but selective barrier to larger molecules 4. repels attack by many hydrophilic and hydrophobic molecules 5. structure may differ from organism to organism 6. location of many types of proteins needed for biosynthesis and assembly, protein secretion, energy generation, nutrient uptake, appendages for motility and contact. |
|
|
archaea (4) and some examples
|
1. cell envelopes function in an analogous way
2. but they differ in biochemical makeup 3. some species contain a pseudo-peptidoglycan (has same physiological function for the cell) 4. other species has "S-layer" protein coat or sheath (composed of glyco-protein subunits) examples: extreme thermophiles, halophiles, methanogens. |
|
|
pseudo-murein (3)
|
1. present in archaeal species only
2. glycan of NAG and TAL 3. backbone structure is B1-3 and B1-4 lysozyme not effective because not NAM. |
|
|
s-layer protein sheath
|
S-layer has the paracrystalline: not crosslinked but has sugar moiety on outside: can make hydrophilic barrier on outside. (hexogonal symmetry)
|
|
|
phosphoryl-donor molecule must...? examples
|
have sufficient energy to make synthesize ATP. PEP, 1,3BPG, acetyl-phosphate, butyryl-phosphate.
|
|
|
where does SLP occur?
|
glycolysis (humans to bacteria)
and fermentation (lower euk, bacteria, archaea) |
|
|
RLP (3)
|
1. some chemo-heterotrophs, and in ALL chemo-autotrophs
2. ATP always made via electron-transfer reactions (respiration) 3. involves ATP synthesis driven by proton motive force |
|
|
PLP (3)
|
1. occurs in all photoautotrophs and in all photoheterotrophs
2. ATP always made via electron-transfer reactions driven by light energy 3. ATP formation consumes a proton motive force |
|
|
what's going on with smoky maneur?
|
chemotroph: carbon goes to making ATP and NADH. but some lose to heat. reach temperature of 100F, chemical activity intense, heat.
|
|
|
fate of organic carbon on earth
|
for every naturally occurring organic compound found in nature, some microbe has evolved a way to degrade it. as a source of cell energy, as a source of cell carbon.
|
|
|
fate of a carbon compound
|
polymers -> oligomers, monomers -> cellular intermediates -> energy extraction.
|
|
|
polymers must...?
|
be first degraded outside the cell
enzyme are secreted. those exo-enzymes then attack the polymers. then small molecules are then taken up across the cell envelope. |
|
|
molecule uptake occurs by active transport systems that:
|
are usually molecule specific, usually inducible, require energy.
|
|
|
upon entry into the cell...
|
molecules are further degraded. Energy is then harvested (ATP, NADH, proton/ion gradients)
|
|
|
some bacteria and fungi specialize in woody plants
|
lignin, hemi-cellulose strands, cellulose fibers: numerous thick strands, each consists of many smaller polymer strands, molecules tightly packed, 1000 glucose molecules each.
|
|
|
lignin and cellulose provide for rigid plant walls (roots, shoots, and stems) how do lignin and cellulose interact
|
lignin molecules wrap around the cellulose.
|
|
|
cellulose and lignin remaining
|
white rot fungi (cellulose) and brown rot fungi (lignin)
|
|
|
lignin (5)
|
1. extremely difficult to attack and degrade
2. initial steps are probably non-enzymatic 3. generates many aromatic compounds 4. subsequent steps are enzymatic 5. energy rich source (if you can do it) |
|
|
cellulosome (6)
|
1. extracellular machine!
2. anchoring protein to keep it close 3. hold with scaffolding 4. cellulose binding protein: literally clamp on to be close 5. dockerin domains too 6. then cellulose degrading enzymes. |
|
|
cellulose strands different, so...?
|
enzymes have different efficiency for specific types (wheat, grass, etc.)
|
|
|
polymer degradation occurs by (4)
|
1. hydrolytic attack at bonds joining monometric units
2. water molecule is consumed 3. bond energy is not conserved 4. rather, energy, it is lost as heat. |
|
|
hemi-cellulose
|
5 carbon sugars including xylose, arabinose, etc.
quite easy to attack and degrade. |
|
|
chitin and starch
|
all NAG, crab and insect shells.
amylase and amylopectin. |
|
|
phospholipases
|
A1 and A2 remove long chain fatty acids
C removes phosphoserine D removes serine. |
|
|
fate of glycerol and fatty acid moieties
|
1. glycerol is recycled into central metabolic pathways (either recycled or degraded)
2. free fatty acids are degraded by beta-oxidation pathway to give acetyl-CoA 3. acetyl-coa is then degraded by TCA cycle 4. very high energy yields in NADH, ATP, and ion gradients. |
|
|
cell metabolism is... addition of what 3 things?
|
catabolism+anabolism+maintenance rxns
|
|
|
some species of bacteria can degrade...
|
only single compounds
or family of related compounds (aromatic or aliphatic) like mono-cyclics (benzene, phenol, toluene) bicyclics (naphthalene) tricyclics (anthracene) cells cannot easily metabolize these molecules |
|
|
aromatic ring breakdown by (2)
|
mono-oxygenase or di-oxygenase
|
|
|
microbial growth on benzene (2)
|
cell must first accomplish "ring activation": requires energy input (NADH) and requires oxygen
then subsequent reactions yield energy. |
|
|
going backwards, but how do microbes break down oil droplets?
|
bacteria secrete surfactants, break down into globules, increase SA, bacteria live at oil water intermediate and break down oil.
|
|
|
monooxygenase (1)
|
1. one molecule of oxygen goes into ring
2. other molecule goes to water (then comes back in to attach to molecule) 3. uses NADH. |
|
|
after initial ring attack and ring cleavage, subsequent breakdown reactions (5):
|
1. generates NADH during oxidations
2. give C2, C3, and C4 organic acids 3. these are then activated with CoA 4. oxidized to CO2 via TCA 5. generate more NADH + H+ and ATP. lots of E made here |
|
|
di-oxygenase enzyme
|
both molecules of oxygen go onto ring, followed by NADH generation steps as before.
|
|
|
breakdown of aliphatic compounds (C2 to C50+)
|
1. again initial molecule attack by oxygenase (mono or di) to activate it
2. subsequent dehydrogenation steps to give a carboxyl group at one end 3. CoA activation of the acid using ATP 4. beta oxidation pathway gives acetyl-CoA's 5. TCA cycle oxidation to CO2 |
|
|
energy released is based on...?
|
-larger compound, more energy
-oxidation state also affects energy content: fully reduced, most usable E. partially oxidized, some usable E. fully oxidized, no biologically usable E. |
|
|
Ys
|
cell growth yield: amount of cells made per mole of substrate used is usually expressed as grams of cells (dry weight) made per mole of substrate consumed.
Ys varies for diff compounds. |
|
|
respiration linked phosphorylation definition
|
oxidation of reduced organic and inorganic compounds dependent on added electron acceptors
|
|
|
TCA cycle
|
1. two C3 -> 2 ac-coA and 2CO2 yields 2NADH
2. two ac-coa -> 4CO2 yields 4NADH, 2FADH, 2GTP. |
|
|
who doesn't do electron transport system?
|
archaea. does something different.
|
|
|
electron transport pathway is in...?
|
all mito (nearly every euk has one)
delta proteobacteria, many gram positive bacteria, etc. |
|
|
e.coli diff because?
|
cytochrome o oxidase is diff. missing second system. different stoichiometry of protons. different proton motive force.
|
|
|
cytochrome oxidase evolution
|
only 1 made it to euk, but at least 6 present.
|
|
|
general RLP scheme, 4 steps, and equation
|
1. oxidize DH
2. reduce A 3. produce pmf 4. make ATP DH+A -> D+AH |
|
|
coupled electron-transfer chains (5)
|
1. can be simple (2 enzymes)
2. can be complex (4 or more enzyme complexes) 3. considerable diversity in nature cell may make diff enzyme modules to insert into membrane for using diff substrates principles of ET are conserved. |
|
|
e.coli uses alternative electron acceptors, so...?
|
has diverse respiratory pathways (oxygen, nitrate nitrite, DMSA, TMAO, fumarate, so cytochrome oxidaze, nitrate reductase, etc.)
cell makes diff enzymes upon demand. nitrate reductase: NO3- -> NO2-. H+ + NO3- to NO2- + H2O, ultimately, electrons pass to O2 to form H2O. |
|
|
how much energy is released by the reactions? equation
|
G=(-nF)E
n=number of electrons F=faraday constant E=(E acceptor-E donor) |
|
|
e.coli has dehydrogenases for ..? So?
|
NADH, formate, hydrogen, lactate, glycerol, succinate.
to use these alternative electron donors, it just makes yet more enzyme modules upon demand. |
|
|
Q loop
|
protons are picked up on the cytoplasmic side by MQ.
the protons are then deposited on the periplasmic side. |
|
|
electron carriers (3)
|
1. quinones (Q or MQ): small lipophilic molecules, transfer both electrons and protons. ubiquinone in aerobes (Q), menaquinone in anaerobes (MQ), caldariella quinone in some archaea (CQ). lipid tail to be able to move thru membrane as e- carriers.
2. cytochrome c: small protein with a c-type heme transfers electron only - the iron, contained within porphryrin ring structure can carry single electron. 3. ferredoxins: small proteins with iron sulfur centers transfer electron only. FeS- proteins can have diff types of iron sulfur centers. iron-sulfur groups are carriers of electrons only. as nonheme iron protein accepts 1 e- from flavoprotein, a H+ is released into the periplasm or cytoplasm. |
|
|
forming proton motive force (3)
|
proton consumption
proton pumping Q-loop mechanism. |
|
|
mito has 1 kind of what?
|
cytochrome oxidase.
|
|
|
vinegar bacteria
|
ethanol is oxidized to acetic acid to generate NADH
ethanol -> acetaldehyde -> acetic acid use 2 NAD+'s. NADH is then reoxidized with O2 as the electron acceptor. electron transfer modules are NADH dehydrogenase cytochrome oxidase. |
|
|
general scheme for electron transport
|
1. use specific dehydrogenase
2. use specific oxidoreductase 3. couple to form pmf 4. use pmf to form ATP |
|
|
energy from anaerobic iron 3 reduction
|
pretty good, chemoautotrophic.
|
|
|
iron Fe2+
|
iron Fe2+ oxidation is coupled to oxygen reduction.
inorganic electron donor (DH)=Fe2+ electron acceptor = O2. enzymes modules are rusticyanin, cytochrome c, cytochrome a1 oxidase. tiny energy made, but can make living. |
|
|
definition of fermentation
|
metabolism without added electron acceptors (or anaeorobic re-arrangement of organic compounds without net oxidation or reduction)
Energy (ATP) is derived solely by SLP |
|
|
where does fermentation occur?
|
in natural and man-made anaerobic environments:
fridge, food, beverage, humans, fruit, sewage, landfills, lake sediments, oceans, etc. |
|
|
what types of molecules can be fermented? how do microbes choose these molecules?
|
almost all!
microbes specialize in polymer types ability to utilize depends on their genetic blueprint need to consider temperature, pH, and salt ranges. |
|
|
general strategy
|
1. activate molecule
2. partly oxidize it 3. generate NADH 4. generate ATP 5. reduce intermediates 6. excrete reduced products make 2 ATP, no net oxidation or reduction. energy harvested by SLP. |
|
|
sugar based fermentation with any type of sugar as substrate... end product?
|
short chain alcohols, short chain organic acids, gasses.
|
|
|
lactic acid bacteria (homolactics)
|
per one C6, two C3's.
energy yield 2ATP |
|
|
where do ATP's come from in fermentation?
|
protein kinase reactions in glycolysis.
|
|
|
how do phosphorylations work in fermentation?
|
bond energy is stored as phosphorylated intermediates, then they are then used to make ATP. then cells reduce carbon intermediates to use up the NADH (like pyruvate to lactate)
|
|
|
during fermentations, something often limits growth before nutrients are depleted.
|
lactic acid production stops cell growth.
|
|
|
homolactic acid bacteria
|
one C6 to 2 C3 by the glycolysis pathway (2 ATP)
|
|
|
heterolactic acid bacteria
|
one C6 to one C3, one C2 , and one CO2 by the oxidative pentose phosphate pathway (2ATP)
glucose is cleaved to make 1 ATP. 1 pyruvate is cleaved to acetaldehyde and CO2 by pyruvate decarboxylase. 1 pyruvate is reduced. NADH's are recycled so get 1 lactate, 1 ethanol, 1 CO2. pentose phosphate pathway is used. |
|
|
fermentation energy yields for if glucose is fermented to lactic acid vs if it were completely oxidized to CO2 and H2O
|
33% harvested E (like maneur pile) versus 2% harvested: lactate is dumped.
|
|
|
making silage why?
|
for preservation and storage when plants don't grow. cover silage so no oxygen gets in there. drop pH enough so that doesn't decay. bacteria makes proteins to ferment, but this E preserved when eaten.
|
|
|
kimchi
|
heterolactic fermentation. don't want to squeeze jar too tight because of CO2.
|
|
|
why can some fermentations go bad?
|
because microbial populations can give different fermentation results. be careful with what bacteria to use. if use heterolactic fermentation on pickes, can have bloater or floater because of gas.
|
|
|
how are microbial fermentations named? examples
|
by their end products:
lactic acid bacteria ethanol producing microbes, etc. |
|
|
ethanol fermentations examples
|
industrial ethanol: medicines, gasohol, mouthwash...
sugar source: corn, sugar cane, sugar beets. beverages: beer, wine, etc. sugar source: wheat, barley, rice, grapes microbe types: special Saccharomyces cerevisae strains |
|
|
fermentation of glucose by yeast
|
glucose cleaved to pyruvate: make 2ATP and 2NADH
pyruvate is cleaved to acetaldeyde and CO2 by puryvate decarboxylase. 2 NADH are then consumed. ethanol formed: alcohol dehydrogenase. |
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wine fermentation
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grapes trap yeast cells on their waxy surfaces. wineries can use this natural flora or inoculate with a pure "starter" culture. biologists need to make sure right bacteria, nutrients, temp because heat would be produced and bacteria would kill themselves.
inoculate to make sure we know what bacteria and to make sure it's totally fermented. |
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ethanol fermentation by bacteria "zymomonas mobilis", examples
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1 glucose -> 2 ethanol + 2CO2 + 1ATP
tequila: blue agave juice... glucose is cleaved to make 1 ATP, 1NADH, 1NADPH. pyruvate is cleaved to acetaldehyde and CO2 (pyruvate decarboxylase). NADH/NADPH are recycled and get ethanol at the end. (Entner-Doudoroff or ED pathway is used) |
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white bread
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yeast: S. cerevisiae
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cheese vary in (5)
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type of milk
type of starter culture salt content moisture content aging process |
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cheese production (4)
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1. add starter cultures (Lctobacillus sp.). incubate! (Lactococcus sp.)
2. depending on type of cheese, add other cultures 3. treat milk: rennin to clot, remove curds, press and age 4. depending on cheese type, add more cultures. |
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soft cheeses
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fungi produce white spore tubes
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propionic acid bacteria
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swiss cheese:
propionibacterium sp. (a gram + bacterium). starting product is lactate from the lactic bacteria (not sugar). inhabits cow rumen. complex pathway leads to propionate. propionate and acetate give flavor. carbon dioxide makes the holes. fermentation of lactate by propionic acid bacteria: 3 lactate: 2 propionate, 1 acetate, 2 CO2. very complex! |
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fermented meats and fishes
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fish, shrimp, beef and pork (sausages, bologna, salami)
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anaerobic habitats in mammals
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humans and other warmblooded animals: small and large intestines, teeth, toenails, vagina.
animals: rumen, intestines of all grass eating animals. |
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enteric bacteria
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mixed acid fermentations
glucose, makes 2NADH, 2 pyruvate makes acids, alcohols, and gasses after making 2 NAD+, so no net oxidation. |
