MMG 301 Fall 2010 Study Guide for Exam 3

Bacterial Genetics [Lecture 20] Vocabulary: Phenotype= depends on what genes are expressed Genotype Homologous Holiday junction= formation of a crossed-strand structure Heteroduplex DNA= a double-stranded region of DNA that contains one or more base matches Mutation Auxotroph: a mutant with a nutritional requirement for growth Conjugation Pilus Hfr IS sequences= F plasmid can integrate in several specific locations in the genome called insertion sequence oriT= origing of transfer Gene Complementation, Inversions Translocations Reversions= a mutation that returns a mutant allele back to the wild-type allele Second site reversions= mutation at a different site restores the wild-type Merodiploid: two copies of a gene within a single cell Concepts:

1. Review the 4 types of point mutations; the types of mutations/rearrangements involving many base pairs Point mutations: substitutions involving one base pair o Silent: change in DNA seq., but new codon codes for same AA (no change in protein seq.) o Nonsense: results in a stop codon;shorter protein bc transl. is stopped before maturation o Missense: results in a different AA o Frameshift: results from insertion or deletion that changes the triplet reading frame o Reversions: point mutation giving restoration of a wild-type phenotype; can occur in 2 ways Same site reversion: base pair mutates back to original genotype Second site reversion: mutation at a different site restores the wild-type phenotype
These mutations involve larger segments of DNA

Insertions: new stretches of DNA are added Translocations: large segments of DNA move to another location on the genome Inversions: orientation of a segment of DNA is reversed 2. What is the Ames test; how is it performed; A standard test for carcinogens (mutations that give rise to cancers) suspected compound is exposed to a liver extract that contains enzymes that could metabolize seemingly harmless chemicals into mutagens (liver extract is added to stimulate the effect of metabolism, as some compounds are not mutagenic themselves but their metabolic products are) rapid, fast and accurate method to test for carcinogens
What organism is commonly used; what phenotype is observed, how is it interpreted commonly used organism is Salmonella enterica His- auxotroph; looks at back mutation (reversion) to His+; potential mutagenic compound + liver extract is added to the His- auxotroph; resulting bacteria is spread on complete medium minus His; only revertants (His- to His+) can grow

3. Review the sequence of steps involved in homologous recombination; what 2 proteins promote strand invasion
steps involved: 1. Nick- one of DNA molecules 2. SSB- nicked strand detaches from other strand with help of SSB 3. Strand displacement- nicked strand displaces homologous region of recipient DNA (Rec A is responsible for strand displacement) 4. Crossed-strand exchange- formation of a crossed-strand structure (a crossover or a Holiday junction) 5. Resolution- cutting of DNA strands to yield new DNA with heteroduplex regions- segments originating from different DNA molecules

4. What is natural competence, how can artificial competence be induced

organisms must be in a competent state in order to uptake DNA Natural competence: organisms that have natural transformation ability (Haemophilus, Streptococcus, Bacillus) Artificially induced competence: bacteria can be induced to uptake plasmid by chemical treatment E-Coli, cells are suspended CaCl2 to promote transformation by plasmids Alternately, exposure to electric currents results in competence and uptake of plasmid DNA (electroporation) plasmid uptake detected by antibiotic resistance

5. Review transformation, transduction (generalized and specialized), conjugation (F plasmid transfer and F
plasmid + host genome transfer); how is generalized different from specialized transduction Transformation- uptake of DNA from the external environment- natural or artificially induced; organisms must be in a competent state in order to uptake DNA Transduction: bacteriophage-mediated cell-to-cell transfer of genes Generalized: random pieces of DNA are transferred by a bacterial virus (phage) particle Specialized: Chromosomal segments adjacent to integrated phage DNA are transferred. Only occurs at certain sites in the chromosome

6. What are Hfr strains of E. coli; how can one map the relative positions of genes by interrupted mating
Regulation of Gene Expression [Lecture 21] Vocabulary: gene= segment of DNA that codes for a polypeptide, tRNA, or rRNA operon= contiguous set of genes transcribed as a single mRNA under the control of a single regulatory region regulon= spatially separated genes regulated by a common regulatory molecule feedback inhibition= the final product inhibits an enzyme that acts early in the pathway constitutive gene= a gene that is not regulated and has essentially constant levels of expression over time allosteric regulation= an effector molecule binds to a noncatalytic site on a protein and causes a conformational change that regulates its function proteome= the collection of all proteins that a given cell or species can make shuttle vector= a cloning vector that can propagate in two or more different species, such as E. coli and yeast denaturation (of DNA)= breaking the H-bond between dsDNA Concepts: 1. how are sigma factors involved in transcriptional regulation sigma factors recognizes specific DNA promoter site; aids binding of RNA polymerase; is released as transcription begins different sigma factors are used for simultaneous regulation of many genes (global regulation)

2. review regulation of by negative control with a repressor protein: arg operon (repression by arginine), lac operon
(induction by lactose) arg operon (repression by arginine) [arg] high, genes for arg synthesis shut down arg binds to free repressor protein argenine-repressor protein complex can then bind to operator and block transcription lac operon (induction by lactose) [lac] high, gene for lactose utilization turns on induction= de-repression inducer binds to operator-bound repressor protein repressor protein released from operator site and transcription proceeds they involved in the stringent response ppGpp and pppGpp can act as alarmone RelA protein synthesizes them amino acid starvation turns on alarmone synthesis downregulate growth and division and increases AA synthesis

3. what compounds act as alarmones; what enzyme synthesizes them, what turns on alarmone synthesis, how are

4. review positive control regulation with an activator protein: mal operon (induction by maltose)

 activator protein binds inducer, genes for maltose utiliziation are transcribed

5. what are the roles of a sensor kinase and response regulator protein in signal transduction
sensor kinase binds signal molecule and phosphorylates itself, phosphate is transferred to the response regulator molecule, phosphyralted response regulator then binds to operator on DNA

6. how is catabolite repression involved in diauxic growth using glucose and lactose as carbon sources
catabolite repression= if a more favorable carbon source (glucose) is available, then genes for utilization of other carbon sources (such as lactose) are repressed observed in cultures grown with glucose and lactose in the medium as diauxic growth cAMP works as an inducer glucose high= cAMP low= lac operon off glucose low= cAMP high= lac operon on thus for growth on lactose, two conditions must be satisfied: high cAMP and lactose must depress the lac operon

7. what is antisense RNA and how can it control translation

antisense RNA=an RNA strand that is complementary to a strand of mRNA it binds to 5 end of gene A and blocks translation Hfq protein aids in antisense RNA binding

8. how do riboswitches control translation

mRNA changes conformation in presence of metabolite 5 end of the mRNA is unavailable for ribosome binding and translation is prevented If metabolite concentration becomes low, mRNA returns to normal translation-competent conformation

9. what is proteomics and how can it be used to observe changes in gene expression
proteomics is the study of protein function at the genome level. It involves the study of many proteins simultaneously Biotechnology I Cloning, Genetic Engineering, Genomics [Lecture 22] Vocabulary: recombinant DNA cloning vector= plasmid library, blunt ends = the end of a DNA fragment resulting from the breaking of DNA molecule in which there are no unpaired bases, hence, both strands are of the same length sticky ends, agarose gel electrophoresis annealing template DNA, hybridize, electroporation= the use of an electric pulse to enable cells to take up DNA ORF= open reading frame: a genetic sequence that does not contain stop codons artificial chromosomes, expression plasmids, shuttle vector= has origin of replication for eukaryotic organism (yeast, insect cells) and an origin of replication for E.coli pyrosequencing= a type of next-generation DNA sequencing motif= the name given a domain or AA sequence that functions in a similar manner in many different proteins Concepts: 1. look at the animations to review important concepts 2. how is a library of genomic clones made; what are restriction endonucleases; what does DNA ligase do A large population of heterogenous fragments (called a library) can be joined into a single type of cloning vector (basically you can make a library of all genes in genome by introducing them in a clone) cut DNA with restriction enzyme; add vector cut with same restriction enzyme; add DNA ligase to form recombinant molecules; introduce recombinant vector into a host restriction enzymes: recognize a 4-8 base pair (bp) sequence in dsDNA; make a cut in both strands of DNA, cut may be staggered (sticky ends) or blunt DNA ligase joins DNA fragments into cloning vectors; use ligase from phage T4

 sticky ends base pairing stabilizes joining and facilitates ligation

3. briefly review polymerase chain reaction; what occurs at the 95, 55, and 73C steps during PCR; the links to
the animations in ANGEL may be helpful uses of PCR: sequencing/evolutionary relationships- 16S RNA sequencing diagnostics- detection of microbial infections Detection of viable but non- culturable microbes cycles are repeated 12-20 times 95 C: denaturation 55 C: priming 72 C: annealing or extension 4. what are the differences between shotgun and top down sequencing of microbial genomes shotgun approach: DNA extraction, DNA fragmentation, Clone into vectors, transform bacteria, grow, isolate vector DNA, sequence the library, assemble contiguous fragments to side down sequencing: start with large, ordered clones of the DNA to be sequenced. Ordered clones are mapped to their location on the DNA

5. what methods are used for genome analysis of environmental microbial communities;
two approaches : community sampling and total community sequencing community sampling: amplify single gene, for example gene encoding 16S rRNA (sequence and make a genetic tree) total community sequencing: restriction digest total DNA and then shotgun sequence, OR sequence directly (without cloning) using a high throughput DNA sequencer what types of information does each approach provide community sampling: phylogentic snapshot of most members of the community; identification of novel phylotypes what is comparative genomics; how can this method be used to search for genes responsible for pathogenesis community sequencing: identification of all gene categories, discovery of new genes

6. what steps are involved in using DNA microarrays to measure gene expression levels
Biotechnology II: Products and Processes [Lecture 23] Vocabulary: fermentation: anerobic respiration, produces ethanol in yeast, extremozymes=an enzyme (created by extremophiles) that can function under extreme environmental conditions such as very high pH, very low pH, high temperature, high salinity or other factors that would otherwise denature typical enzymes. must= mashing= a mixture of powdered malt and hot water, which is stood until the sugars dissolve to form the wort wort= the sweet infusion of ground malt or other grain before fermentation, used to produce beer and distilled malt liquors bioconversion= the conversion of organic matter, such as animal or plant waste, into a source of energy through the action of microorganisms Streptomyces, high fructose corn syrup Concepts: 1. review key concepts of primary and secondary metabolites primary metabolites: substances produced by the microbes during the primary growth phase (kind of metabolite that is directly involved in normal growth, development and reproduction secondary metabolites: are excreted into the growth medium, are produced near or during stationary phage (wird nicht aus dem grundlegenden Stoffwechsel wie Atmung, Wachstum, oder Reproduktion hervorgerufen) in contrast to primary metabolites absence of secondary metabolites does not result in death secondary metabolites have ecological functions humans use them as medicine, flavoring and recreational drugs

2. what are the main features and process controls of a fermentor

fermentor: an apparatus that maintains optimal conditions for the growth of microorganisms , used in largescale fermentation and in the commercial production of antibiotics and hormones mixing, aeration, and oxygen monitoring (getting enough oxygen is a major problem), temperature control, sterilization, sampling and harvest ports (Ernteausgaenge), pH control types of fermentors are based on size laboratory scale (1-10 liters) pilot scale (100-3000 L) industrial, production scale (up to 500,000 L)

3. how can antibiotic-producing microbes be identified from environmental samples using agar plates
First, isolate the bacteria that produces antibiotics spread a soil dilution on a plate of selective medium allow incubation (colonies of streptomyces or fungi species will form) overlay with an indicator organism (organisms that will not grow in vicinity of antibiotics) zones of growth inhibition (aka clear zone) = antiobiotic producing organism Second, test against several bacterial (activity spectrum) streak antibiotic producer across one side of plate incubate to permit growth and antibiotic production (produced antibiotics will diffuse into agar) cross-streak with test organisms incubate to permit test organisms to grow clear zones (inhibition zones) indicate antibiotic sensitive organism

4. what 3 types of microbes are most often used for antibiotic production
Spore-forming Gram positive bacteria (bacillus) Actinomycetes (streptomyces is the most common source) Fungi

5. what is strain optimization and how can it be used to increase yields of microbial products; how was this
accomplished with Brevibacterium flavum strain optimization is increasing yield products of antibiotic organisms for industrial production there are two methods to do that genetic methods= plasmid cloning of antibiotic production genes optimization of growth conditions to obtain high-yielding variants Example of Strain Optimization for Antibiotic Production: screening process for increasted production of an antbiotic by genetic strain optimization initial microbial antibiotic-prodcing strain is discovered and characterized mutagenesis using chemicals, UV, radiation; Genetic manipulation (gene amplification, metabolic engineering) Screen for variant strains producing higher quantities repeat the cycle until strain has desired production Genetic strain optimization: Industrial use of Brevibacterium flavum to make lysine: mutation of aspartokinase gene is triggered to eliminate feedback inhibition of biosynthetic pathway other beneficial mutations: altered cell membrane to allow for secretion of amino acid product; example: Corynebacterium gutamicum for glutamate production

6. how is penicillin production by Penicillium chrysogenum cultures maximized

different precursors (precurser I,II, and III) are added during fermentation Penicillin fermentation using penicillium chrysogenum production increases as culture enters stationary phase

 extra feeding with carbon and nitrogen sources at precise rates and times maintains production longer for
greater yields precise feeding needed for maximal production

7. what is metabolic pathway engineering and how is it used for production of microbial products
genetic alterations for addition or mutation of specific steps in a metabolic pathway designed to enhance the production of desired substances; enzymes are added by incorporation of genes from other organisms 8. how can large-scale growth of microbes be used for bioconversion reactions

9. review microbial production of vinegar; what is ethanol converted to; what type of bacteria do this

10. what is the malting process and how is it used in making beer; review the steps in beer fermentation and
production

11. review wine making; what are the must and pomace; what are sulfites used for

Biotechnology III: Microbial Leaching and Bioremediation [Lecture 24] Vocabulary: xenobiotic= a completely synthetic chemical compound not naturally occurring on earth (man-made compounds not found in nature) biodegradation= dehalogenation= is a critical ability for microbes to metabolize many xenobiotics cometabolism= the metabolic transformation of a substance while a second substance serves as primary energy or carbon source mineralization, petrophilic periplasm= the area between the cytoplasmic membrane and the outer membrane in gram-negative bacteria microbial leaching= the removal of valuable metals such as copper from sulfide ores by microbial activities bioremediation= use of microorganisms to remove or detoxify toxic or unwanted chemicals in an environment Concepts:

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a) how are microbes used to extract copper from ore; by microbial leaching b) what reactions do they perform in this process; Direct bacterial oxidation of the copper (cu1+ to cu2+), e.g. Cu2S to CuS Indirect oxidation of the copper by chemical reaction with Fe+3 c) what are two organisms that do these reactions; Acidithiobacillus ferrooxidans and Leptospirillum ferooxidans (iron oxidizer) d) why are acidic conditions needed for this process

2. how does the mercuric reductase system eliminate toxic mercury; what do MerA, MerP, and Mer T do
the mercuric reductase system converts Hg2+ to Hg0 which then evaporates MerP binds mercury MerT transports Hg2+ into cytoplasm (MerA) reduces Hg2+ to Hg0 note: Hg is bound to enzyme by thiol groups; Hg0 is volatile and evaporates

3. what is the most common mechanism used by microbes for heavy metal resistance
Efflux of the metal ion

4. what toxic herbicide is degraded by Burkholderia cepacia

2,4,5 trichlorophenoxyacetic acid

5. what are the 2 types of microbial dehalogenation reactions and how do they differ
reductive or oxidative

 reductive: no oxygen- anerobic oxidative involves O2

6. review the beneficial and harmful effects of microbes in the petroleum industr
beneficial microbes can aid in petroleum recovery bioremediation of petroleum spills harmful in storage tanks, microbes need water, so organisms grow at oil-water interfaces; under anoxic conditions hydrogen sulfide (H2S) is produced, which is corrosive Biofilms can plug rock pores and reduce fluid flow in petroleum recovery

7. what is in-situ bioremediation and how does it accelerate removal of xenobiotics by microbes
Microbial Ecology and Biogeochemical Nutrient Cycles [Lecture 25] Vocabulary: anoxic zone= anaerobic organisms live here photic zone= sun rich regions of the ocean where most photosynthetic organisms live oxic zone= oxygen rich region in the ocean (aerobic organisms live here) allochthonous= enters the system from outside microenvironment= the immediate small-scale environment of an organism or a part of an organism, esp. a distinct part of a larger environment mutualism= when 2 organisms live together and benefit from each other commensalism= one organisms benefits, the other organism is neutral antagonism= aka amensalism; unidirectional release of an inhibitory compound by one organism that harms a susceptible microorganism (e.g. production of antibiotics or bacteriocins (antibacterial proteins) syntrophy= cross-feeding; one species lives off the products of another species populations= individual cells of the same type multiply to form populations guilds= metabolically related populations community=mixture of different guilds conducting complementary physiological processes interact to form microbial communities ecosystem= interaction between communities of macroorganisms and the physicochemical environment Concepts: 1. what are the key features of the global carbon cycle; review what processes happen in oxic vs. anoxic parts of the cycle; where is methane formed; in both processes CO2 is converted to organic material and organic material is converted back to CO2 Oxic: CO2-> organic matter : oxygenic photosynthesis and chemolithotrophy organic matter-> CO2 : aerobic respiration Anoxic: CO2-> organic matter: acetogenesis, anoxygenic photosynthesis (example of syntrophy) methane formed by methanogenesis; anaerobic; CO2 + H2 + acetate-> CH4

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N cycle: know meaning of nitrification, denitrification, nitrogen fixation, ammonification; how can denitrification be inhibited in agriculture nitrification: NH3-> NO2 (nitrite)-> NO3 (nitrate) denitrification: NO3-> N2 Nitrogen fixation: N2 + 8H+ + 2e -> 2NH3 + H2 ammonification: organic compound -> NH3 denitrification is a problem in agriculture bc NO3 is provided in soil for fermentators; bacteria turn it back to N2 into atmosphere; solution: denitrification enzyme inhibitors what are the two major protein components of nitrogenase; what is the pathway going from pyruvate to ammonia; how much energy is expended for each N2 Dinitrogenase reductase and Dinitrogenase pyruvate is source of electrons for reduction Pyruvate passes electrons to flavodoxin that passes electrons to the first enzyme of the nitrogenase complex (dinigrogenase reductase); that enzyme passes the electrons to the second enzyme with use of energy (ATP) It takes 16-24 ATP molecules for each N2 molecule enzymes that do the N2 fixation are destroyed by oxygen; therefore N2 fixation occurs in certain cells called heterocyst (differentiated cell which doesnt have O2)

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what is the anammox reaction anammox = anaerobic ammonia oxidation NH4+ + NO2- --> N2 + 2 H2O carried out by Brocadia anommox contributes up to 70% of the nitrogen cycling in the worlds oceans useful method for removal of ammonia and amines from wastewater anammoxome enzymes surrounded by membrane ATP is gained during the process S cycle: what is sulfur oxidation, sulfur reduction, sulfate reduction, sulfate assimilation, desulfurylation Aquatic and Marine Microbial Ecology & Wastewater Microbiology [Lecture 26] Vocabulary: Oligotrophic= nutrient poor (mainly photosynthesis), low microbial populations, O2 saturated Eutrophic lakes = nutrients inputs from outside, high microbial populations, low O2 (depleted by microbial and animal population Mesotrophic= intermediate between oligo and eutrophic Epilimnion= the top-most layer in a thermally stratified lake, occurring above the deeper hypolimnion. It is warmer and typically has a higher pH and dissolved oxygen concentration than the hypolimnion. Being exposed at the surface, it typically becomes turbulently mixed as a result of surface wind-mixing. It is also free to exchange dissolved gases such as O2 and CO2 with the atmosphere. Thermocline= (aka metalimnion) is a thin but distinct layer in a large body of fluid (e.g. water, such as an ocean or lake, or air, such as an atmosphere), in which temperature changes more rapidly with depth than it does in the layers above or below. In the ocean the thermocline may be thought of as an invisible blanket which separates the upper mixed layer from the calm deep water below. Hypolimnion= the colder, more dense, and often anoxic bottom waters of a stratified lake. It is the layer that lies below the thermocline. Typically the hypolimnion is the coldest layer of a lake in summer, and the warmest layer during winter. Being at depth, it is isolated from surface wind-mixing during summer, and usually receives insufficient irradiance (light) for photosynthesis to occur. Sludge= residual, semisolid material left from industrial wastewater, or sewage treatment processes. It can also refer to the settled suspension obtained from conventional drinking water treatment, and numerous other industrial processes. The term is also sometimes used as a generic term for solids separated from suspension in a liquid; this soupy material usually contains significant quantities of interstitial water. Activated sludge= the process for treating sewage and industrial wastewater using air and a biological floc composed of bacteria and protozoans. Purposes: oxidizing carbonaceous or nitrogenous matter, removing phosphate, and driving off entrained gases (CO2 , ammonia, N2) Oligocarbophile= adapted to the extremely low concentration of organic C in ocean seawater (1-2 mg C/ liter) Microbial mats= thickly layered and stratified biofilms found on surface around vents. A multi-layered sheet of micro-organisms, mainly bacteria and achaea. Microbial mats grow at interfaces between different types of material, mostly on submerged or moist surfaces but a few survive in deserts. Zoogloea= a special state of bacterial cells when their capsules become slimy and form gelatinous structures or films. Consists of polysaccharides, sometimes with an admixture of nitrogenouse compounds. It is inherent only in some bacteria, particularly in the Zoogloea ramigera genus. Benthic = attached to sides or bottom (e.g. free-living organisms)

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Concepts: Review populations, guilds, communities, ecosystem Populations: assemblies of individual cells Guilds: metabolically related populations Communities: sets of interacting guilts Trophic levels in aquatic ecosystem: cyanobacteria and algae are primary producers in OXIC envts. Phytoplankton are floating or free-living algae. Much of the organic matter synthesized by phytoplankton during photosynthesis is released as DOM (dissolved organic material). This DOM is consumed by bacterioplankton which becomes part of the suspended particulate organic matter (POM). A portion of these bacteria are then consumed as food by protozoa predators. Microbial loop: some of the assimilated nutrients in bacteria and protozoa are mineralized and then assimilated directly by phytoplankton without transfer to higher trophic levels (e.g. fish) in the aquatic ecosystem.

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Why are lake nutrient content and dissolved O2 inversely proportional O2 and light play key roles in microbial activity and distribution in aquatic habitats Higher nutrient content provides higher microbial and animal populations which deplete the oxygen How does thermal stratification result in anoxic conditions in the hypolimnion; what is seasonal turnover In the hypolimnion dissolved O2 levels are low. Thus hypolimnion is an anoxic environment often containing H2S from sulfate reducing anaerobes Turnover occurs in the fall and spring when changing seasonal temperatures allow cooling the epilimnion. Temperatures of the hypolimnion temporarily are higher than in the epilimnion as a result the warmer water rises Complete mixing of the water column results in redistribution of nutrients the entire lake becomes aerobic for a brief time

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O2 and nutrient distribution in rivers most rivers are well mixed because of flow turbulence However, from the input point of sewage or organic waste, there are significant changes in downstream nutrient levels rise in bacterial activity-> decrease in O2-> NH4+ rises (from sewage input) but then falls due to nitrifying bacteria (NH4+ -> -> NO3) -> photosynthetic algae and cyanobacteria growth increases O2 Thus there is a self-purification process that results in oligotrophic conditions and replenished O2 levels Sewage and organic waste pollution of aquatic ecosystems can be measured indirectly by biochemical oxygen demand (BOD) (higher levels of organic pollution= greater demand for oxygen by microbes) Microbial distribution in marine habitats moderate halophiles, most are psychrophiles (5 C for most of ocean volume), most are oligocarbohphiles review the symbiotic relation of Rifta tube worms and bacteria; what is the ultimate energy source endosymbiotic bacterial live inside of Riftia tube worms (at deep sea ocean floor) in trophosome organ (symbiosis occurs here) life sustained by geothermal energy rather than solar energy (chemosynthesis rather than photsynthesis) Microbial life in marine environments Deep sea and hydrothermal vents microbial mats: thickly layered and stratified biofilms found on surface around vents (also found in many other habitats: hot springs, shallow marine basins, rivers) Remember: most deep ocean habitats have low temperature (except near thermal vents), high pressure, and low nutrient concentrations what bacteria and algae are the primary producers in open oceans; where is Pelagibacter found Major phototrophic primary producers bacteria: Prochlorococcus: an extremely small photosynthetic bacterium (small size= high surface/vol ratio) Trichodesmium: a photosynthetic cyanobacterium; produces compounds very toxic to humans; is a nitrogen fixer Eukaryotic unicellular algae Ostrococcus: cells smaller than most bacteria- 0.8 micrometers Major non-phototrophic microbes Pelagibacter; found in pelagic zone; an oligophile know differences between piezotolerant, piezophilic, and extreme piezophilic organisms Piezotolerant: can tolerate high pressures (piezo=baro) Piezophillic: prefer high pressure Extreme piezophiles: can only grow at high pressures why is BOD proportional to the amount of dissolved organic pollution in a water sample review the characteristics of O2, bacteria (+ organic carbon and BOD), algae, as they change downstream of a

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pollution entry point in a river

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why is sewage treatment important; what happens in the primary, secondary, and tertiary treatments sewage includes organic matter, bacterial, protozoan, and viral pathogens treatment of sewage is our most significant method of disease prevention Primary treatment: removal of solids from raw sewage by screening and gravitional settling in tank. The resultant solid material is called sludge Secondary anoxic and aerobic treatment: Microbial conversion of organic matter into harmless cell biomass and final decomposition products (90-95% reduction in BOD) and removal of many bacterial pathogens Tertiary treatment: biological/chemical treatments to decrease nutrients (P, N) and inactivate viruses how do the activated sludge and trickling filter methods work; why are biofilms important for trickling filter there are two ways to do the secondary treatment: Activated sludge method: uses aerated tanks containing slime-producing bacteria (Zoogloea) as well as other bacteria and protozoans; zoogloea produces a slime that results in flocculent material that serves as growth stratum for protozoans and other organisms; the flocs and suspended solids in this process are termed activated sludge Trickling filter method: wastewater is trickled through bed of small rocks where protozoans and other organisms attach to a microbial biofilm on the rock surfaces. Biofilms are important because the microbial biofilm digest the organic matter into harmless products what happens during the anaerobic sludge digestion process; what are the gas metabolic end products solids are converted into gaseous products by fermentation of anaerobes sealed container, only a small opening for escape of gas; final anaerobic metabolites dominated by methane and CO2; the methane can be burned or used to power the treatment plant. what organisms are used indicator microbes when testing for water quality; what makes a good indicator organism Fecal coliforms: Gram (-) non-sporulating facultative anaerobic rods from the intestine of warm-blooded (most common method) Fecal streptococci: useful indicator of fecal contamination in estaurine and marine waters, where it survives better than E.coli Lytic bacteriophages of E. coli: viruses survive chlorination better than bacteria Animal-Microbe Interactions [Lecture 27] Vocabulary: Cellulose = Acetogen, Ruminant = A mammal that digests plant-based food by initially softening it within the animals first stomach, then regurgitating the semi-digested mass, now known as crud, and chewing it again. The process of re-chewing the cud to further break down plant matter and stimulate digestion is called ruminating. Rumants include cows, goats, sheeps, as well as giraffes, buffalo, Ilamas, and elk Omasum, Abomasum = The fourth and final stomach compartment in ruminants. It secretes rennin- the artificial form of which is called rennet, and is used in cheese creation. It serves primarily in the acid hydrolysis of microbial and dietary protein, preparing these protein sources for further digestion and absorption in the small intestine Rumen = Aka paunch, forms the larger part of the reticulorumen, which is the first chamber in the alimentary canal of ruminant animals. It serves as the primary site for microbial fermentation of ingested feed. Volatile fatty acids = Are fatty acids with a carbon chain of six carbons or fewer. They can be created through fermentation in the intestine. Ex: acetate, propionate, butyrate Trophosome = An organ in deep-sea tube worms that is colonized by bacteria supplying the host worm with food and energy Pyogenic =Bacterial infections that make pus. The pus is mostly composed of dead neutrophils that are destroyed by bacteria such as Streptococcus pyogenes and Staphyloccus aureus trhough the release of leukocidins. Prion = An infectious agent composed primarily of protein, unlike viruses which consist of two or three parts: a helical molecule, protein coat and sometimes a viral wrapper Concepts:

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how is the termite dependent on gut microflora for survival; what is difference between lower and higher termites; what do some spirochetes do to aid certain lower termites; Termite is completely dependent on its gut microflora to provide its C + N. Nutrition is derived from an anaerobic degradation of the wood particles. Cellulases are enzymes that degrade cellulose, they are produced by termites, via cellulolytic prozoans symbions, or by ingested cellulose-containing fungi. Lower termites- populations of protozoans and other microbes in the gut anaerobically degrade cellulose ->-> acetate (which is absorbed into termite) and CO2 and H2 certain spirochetes fix N2 that the termite uses as N sources. Higher termites- includes bacteria, and few archaea in hindgut; can digest wood by salivary or ingested cellulases

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review how the rumen is involved in the digestive system of certain animals; what is the major type of carbon nutrient that is absorbed through the rumen wall; what are the gas products of this anaerobic process; Rumen contains microbial community that digest cellulose anaerobically Rumen microbes decompose the ingested plant material into volatile fatty acids (such as acetate, propionate, and butyrate) which are absorbed and utilized by the anima plus gas fermentation products that the animal expels (CO2 and CH4) Wolbachia-Insect symbiosis Wolbachia is the most successful parasite in earth- infects 20% of all insect species, as well as certain shrimp, worms and other invertebrates infects the developing sex cells of its hosts and gets passed on from mother to child in the egg in certain wasps, Wolbachia alters sex life of the host, so it gives birth only to females which then no longer need males to reproduce genome sequence of fruit has showed that almost the entire genome of Wolbachia could be found within the fly genome- eukaryotic acquisition of a bacterial genome how do symbiotic bacteria aid the African Honeyguide and Hoatzin birds, Olavius worm, and pea aphids Nutritional symbiosis of Buchnera-Pea Aphids: Aphid food source (sap) is low in amino acids. Bacteria living inside specialized cells (bacteriocytes), surrounded by host-derived membrane provide excess amino acids to insect. Insect provides energy, carbon and nitrogen sources to the bacteria. Olavius worm: A worm that has no mouth or digestive system, which lives in sediments around the Mediterranean Sea. Has two symbiotic bacteria living under his skin. They provide organic compounds, amino acids and vitamins to the worm. Microbe- bird symbiosis: African honeyguide: lives on beeswax- intestines contain Micrococcus and Candida to digest wax Hoatzin- leaves are major food source; contains a rumen-like crop where bacteria aid digestion of cellulose viral diseases: review animals affected, transmission routes for: Eastern Equine Encephalitis, West Nile virus, rabies West Nile virus animals affected: horses, birds and humans insect vector= mosquito symptoms: weakness, ataxia (uncoordinated muscle movements) 30-40% fatal in horses; 99% of affected mammals are horses; high fatality in birds Horse vaccine available since 2001 Eastern equine encephalitis virus (EEE) affects all equine animals (75-90%) normality insect vector= mosquito rarely affects humans: 33% fatal symptoms: fever, loss of appetite, weakness, nervous system disorder Rabies caused by Rabies virus (a Rhabdovirus) can affect many mammals but mainly found in bats attacks nervous system 100% lethal in humans what is Epizootic Hemorrhagic Disease; what is the type of infectious agent; what is major animal affected Epizootic Hemorrhagic disease is epidermic in mold animals usually fatal viral disease of wild ruminants transmitted by bitting midge insects characterized by extensive hemorrhages from various organs

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symptoms appear after about 7 days- death in 8-36 hours Avian influenza: how are subtypes determined; review characteristics of the virus Avian (bird) flu (H5N1) caused by influenza A viruses that occur naturally among birds. There are different subtypes of these viruses because of changes in certain proteins (hemagglutinin [HA] and neuraminidase [NA] on the surface of the influenza viruses and the way the proteins combine are ssRNA enveleloped viruses; segmented genome can infect humans there are 3 important antigens present, the haemagglutinin (HA), neuraminidase (NA) and the nucleocapsid proteins (NP) NP comes in 3 forms: A,B,C ->provides basis for the classification there are 16 HA subtypes (H1-H16) There are 9 NA subtypes (N1-N9) Influenza A viruses occur in humans, birds, and horses and pigs Influenza C infect dogs, pigs, or humans (very mild or no symptoms- not a source of epidemics) Influenza B only affects humans antigenic differences of HA and NA antigens of influenza A viruses provide the basis of their classification into subtypes bacterial diseases: review animals affected, organism causing disease, transmission routes for: Salmonellosis, caused by salmonella affects birds, wild and domestic animals, humans spread by ingestion or direct contact with contaminated poultry eggs, dairy, meat, or other animal byproducts infects gut of animal and usually causes diarrheal illness Bovine tuberculosis, caused mainly by Mycobacterium bovis but some from Mycobacterium tuberculosis Respiratory infection of lungs Animals affected: cattle, deer Lyme disease caused by Borrelia burgdorferi; infects humans begins as rash but spreads through body- fever, lameness transmitted by tick can lead to paralysis, involvement of organs what are Type C and E botulism toxins; what is the bacterium Types C and E Botulism in waterfowl transmitted by Clostridium botulinum that lives in anoxic sediments of lakes Type C- animal ingests spores during feeding on aquatic shores Type E- organism is ingested by mussels -> fish -> birds causes paralysis and death thousands of waterfowl died in 2010 outbreak Soil Microbiology/Plant-Microbe Interactions [Lecture 28] Vocabulary: FISH, soil separates= specific ranges of particle sizes (sand, silt, clay (smallest)) soil subsidence, signature sequences, phyllosphere= aerial leaf surface of plants endophytes= colonize plant interior epiphytes= colonize plant surface rhizosphere= area adjacent to the root but not in contact with the root rhizoplane= the root surface legumes= pod-bearing angiosperms (peas, soybeans, beans) commercial inoculant= bacterial cells that are specific for whatever you are planting and that produces more inoculants lectin, Nod factors,

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nodulation, flavonoids, bacteroids, peribacteroid membrane, infection thread, endosymbionts, leghemoglobin Concepts: a) name the 3 phases found in soils; solid, liquids and gas (solids predominate) the greatest number and highest diversity of soil organisms near the surface b) review the four vertical horizons found in mature soils O horizon: layer of undecomposed A horizon: surface soil (high in organic matter, dark in color, is tilled for agriculture; plants and large number of microorganisms grow here; microbial B horizon: Subsoil (minerals, humus, and so on), leached from soil surface accumulate here; lttle organic matter; microbial activity detectable C horizon: Soil base (develops directly from underlying bedrock; microbial activity generally very low) bedrock 2. how does water affect oxygen in the microenvironments around soil particles microbes in discontinuous water films on the surface of soil particles have good access to O2 microbes in continuous water-filled pores have limited O2 fluxes, creating anoxic microenvironment (water-saturated soils have lower O2 transfer rates) 3. how is fluorescence in situ hybridization used for identification and enumeration of microbes 4. what are the three main locations for associations of microbes on plants Phyllosphre: aerial leaf surface of plants Rhizosphre: region adjacent to the root Rhizoplane: the root surface

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what are 3 ways plant growth promoting rhizobacteria increase plant productivity and yield Solubilization and fixation of nutrients Production of growth-stimulating hormones Antagonism of soil-borne plant pathogens

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why is N2-fixing symbiosis important to agriculture; review order of events in development of root nodules Nitrogen is the nutrient most commonly limiting plat productivity, and legume crops can offst that limitation by forming an efficient N2-fixing symbiosis with Rhizobium order of events in development of root nodules the host root excretes flavonoid compounds flavonoids are taken up by bacteria and expression of nodulation genes (nod) commences some of the nod genes synthesize nod factors (chitopooligosaccharides) that are reconized by the root hairs Root hairs begin deformation, curling and subsequent formation of a nodule infection thread forms; cells divide and enter into interior root cells formation of bacteroid state how do plants and mycorrhizal fungi benefit from each other The plant provides a steady supply of photosynthetic organic nutrients to feed the mycorrhizal fungus The fungus provides increased surface area for absorption of plant nutrients (e.g. phosphate) and water from the surrounding soil and provides them to the plant how does Agrobacterium tumefaciens cause crown gall disease what plasmid mediates this process, what is T DNA

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involves transfer of T-DNA from a bacterial plasmid (the Ti plasmid) to a plant cell infection of plant wound by Agrobacterium tumefaiens; Attachment of Agrobacterium cells to plant cells; Processing of T-DNA from Ti plasmid; Transfer of T-DNA to plant cell and integration into plant genome; Tumorigenesis and production of opines what is FISH; how is it used for microbial ecology; briefly review how microscopy, genome sequencing of microbial communities, and rRNA sequencing can aid our understanding of microbial community structure FISH= Fluorescent in situ Hybridization; used to examine microbial communities synthesize oligonucleotide (probe) specific for the organism you are looking for; probes can be general (for Archaea or Bacteria) or very specific (certain species) chemically attach a fluorescent dye molecule to the oligonucleotide treat sample containing cells to make membranes permeable to the oligonucleotide + dye molecule examine sample using fluorescence microscopy; only cells in which the oligonucleotide has correctly base-paired (hybridized) will be fluorescent Examination of microbial community structure by genome sequencing (see lect. 23) 16S rRNA gene sequencing to elucidate microbial communities A PCR-based method: 16S rRNA methods are amplified Short regions of rRNA that are unique to certain groups of organisms are used for design of phylogenetic oligonucleotide probes for identification of newly isolated organisms

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