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MICR 4280 (1)

Complete Lecture Notes.docx

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MICR 4280
Wendy Keenleyside

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Week 1 Readings for introduction ot the scope and current issues in microbial ecology: Class will be in the computer lab downstairs next - unless she posts questions you’re expected to know the Wednesday big picture Microbial Ecology The biosphere’s primary producers include microbes - Winogradsky, one of the first microbial ecologists - organisms who’s carbon source is carbon dioxide - stable community of organisms - bacteria, archaea,yeast, protozoa (non photosynthetic - nitrogen fixing bacteria, only way to convert atmospheric protists), algae nitrogen for everyone’s use - algae, photosynthetic bacteria, lithotrophs (vast majority of carbon fixing is done by lithotrophs) Microbial ecology according to the international society for - the biosphere’s consumers include microbes (they eat the microbial ecology primary producers) - biodiversity, many organisms can’t be cultured, bacteria - yeast, none of the multiceullar eukaryotes, bacteria, and archaea swap genes a lot archasea - exobiology, life of any kind that might be found on other - phototererotrophs, purple and green non sulphurs planets, physical and chemical limits to life - mixotrophs, group of organisms that is everywhere in the - ecology, Gaia is now a theory (was a hypothesis), the ocean, organisms that are heterotrophic but possess light entire earth interact with one another to give a stable activated proton pumps, do not have an ETC, need biosystem sunlight to generate energy from their organic materials - bioremediation, ocean iron fertilization, any environment - bacterial rhodophsins when they are hit with light they use is limited in iron and important for all life forms, some that energy to augment their proton motive force proposals to deal with global warming is to dump a ton of - mixotrophs with the exception of coastal waters there are iron in coastal waters to cause photosynthetic bacteria and not much nutrients so heterotrophs don’t survive well algae blooms and they will eat up all the carbon dioxide, no regulations at this point, no real studies on the impact and Microbial biogeochemical reactions whether it even works - without nitrogen fixation bacteria, life would cease on - Anthropocene era, named for humans changing the earth environment due to their activities - microbes can convert greenhouse gases to something safe and reverse reaction occurs too The beginnings of microbiology - organisms that use ammonia to produce nitrate and nitrite - bejinrinck coined the term microbial ecology produces nitirous oxide, a big greenhouse gas - microbes can somehow grab electrons from rocks on New technologies, new discoveries dump them on these minerals - Alvin was the first sub that was used to observe the deep sea vents Figuring out who’s there is complicated by “The Great - we need to know the temperature, gradients, salt level, Palte Count AnomalY” osmolairty, pressure (some of these microorganisms - there’s many different kinds of organisms on the plates, require high pressure, cannot be grown in normal levels) diversity what we know can’t be grow on plates alone - these organisms are the basis of the food chain in the - metagenomics, culture independent sequencing vents Metagenomics = Culture independent... 1980: Carl Woese - ex-stamp means we’ve got a pure culture of the - rRNA remains unchanged over time so we use the organisms sequence as a measure of evolutionary relatedness and time since the organism evolved Hiedelberg et al. - we need to turn it back to DNA before we can sequence it - we have a huge number of genomes for bacteria - it is so highly conserved that its not very useful to - number of problems of sequencing eukaryotes distinguish at the species level - ssuRNA, subunit RNA Next generation seqauencing - eukaryotic ribosomes 80S and prokaryotic 70S - metagenomics is sequencing a lot of genoomes straight - in the ssuRNA there is 1 rRNA, in eukaryotic its 18S and from the environment and assembling them prokaryotic 16S - genomics, pure culture to the sequence of the genome - we’re only sequencing portions of rRNA now because it - 454 is the only one we’ll be talking about later on gives a better picture - 454 sequencing is sequencing different from sanger - 454 sequecing, the cost of sequencing is more than 1985: Kary Mullis analysis, dollar per base is great than the cost of doing - quantification is very important in microbial ecology bioinformatics - how many organisms, different organisms, are affected - the other two is reversed by x and y, etc. - 454 generates longer sequences from a reaction - making sense of the data is the hard part these days Remote sampling - we are generating data faster than we can assemble and - machines can take in water and bring it back to the analyze it on the computers surface, take pictures, monitor currents, etc. Linking community structure with function - figure out who’s doing what, you use selective media to - bacteriachlorphyll bac have been found to be living in help you figure it out oxidized environments - metatranscriptomics, if a gene is identified in the - ammonium oxidizers they are finding now are anaerobic, environment it doesn’t tell you that its being used, but if end product is a highly unstable mnolecule (rocket fuel) you can sequence the RNA then you know which genes 2. – many sequences we’re searching for in the databases are in use are unknown so we need to go back to the lab and figure it - meta proteomics can produce several 1000 proteins and out if you can separate them out and identify them then you can study them Frst lovelock - ndobjection was that rocks and other things are “living” From biology’s next revolution..... - 2 thing is that this homeostasis is kept by spontaneous - horizontal gene transfer doesn’t happen often, evidence actions that’s happening occurring in bacteria and archaea, as we - evolution wasn’t built into the original hypothesis sequence more genomes and look at the bits of the chromosomes, some of these bits weren’t there originally, The Gaia hypothesis has since been revised.... E. coli O157:H7, recently evolved strain of E. coli, - know this for the exam - if more than 10% of 1 chromosome fails to anneal than - why? What causes the system to be homeostatic? that would be a different species - cosmopolitan, means its everywhere The connection between and the microbes - rhodopsin, light activated component, is found prevalent - people are trying to understand the changes that are in heterotrophic bacteria and arachea that lives in areas happening in many different levels including the microbial where energy sources are in low supply level - viruses, we don’t know much about viruses that attack - because they have been around since Earth was born archaea, bacteriophages is estimated to have billions per they are hypothesized that they affect the Earth’s ml of ocean water atmosphere - they are powerful agents of evolution because they kill many cells Free, A. And Barton.... - they are agents of horizontal gene transfer also - they are suggesting that homeostasis will continue, lifeforms might be different, stabilization might require From “what is community microbial ecology” millions of years Some critical/concepts... Proposal: self regulation arises.... - taxonomic diversity, measure of diversity - energy cannot be destroyed - functional divert, ecosystem stability, figure, know them - entropy can only move in one direction all. Will be on exam - G=free energy - richness meaning number of species, rich = lots of - if it is spontaneous then G is negative, exergonic, free species energy can be released, exergonic - ecosystem functionality, how many functions do we see in - S= entropy the environment - osmosis continues until G =0, reaches equiplibrium - around the same time the functions plateau you see other - cells have a low entropy and maintain it but expand a organisms doing it too, redundancy huge energy to do it - ecosystem stability starts when you hit redudncy and increases as redundcy continues Karnani, M. And Annilla..... - how do you measure stability, you change the - open systems meaning that there’s an exchange of environment a bit to see what organisms die off matter not just energy - resistance is how much stress can the system stand without see any changes in richness Continued - resilience is how much it can bounce back due to the - you can change the equilibrium but the change will stress, how long does it take for them to bounce to pre- continue to occur to bring it back to equilibrium stress levels back if at all Kleidon et al. 2010.... Introduction to the Gaia hypothesis - clouds and hydrology are impacted by microbes - Lynn Margulis proposed the endosymbiosis theory - algae produces a byproduct which evaporates and - things that are not in thermodynamic equilibrium is dimethyl sufloxide which ends up nucleating the formation basically alive, something’s affecting it, whereas dead of clouds impacting the weather things are in thermodynic equllibrium - Week 4 Week 2 TGGE Class discussion - molecules that move lower in the gel have a higher G+C 1. Madsen, 2011 and 2. Heidelberg, et al. 2010 and doesn’t denature as well 1. lots of new genes and enzymes we know nothing about - based on metagenomic data, there’s so little of what we Preventing the product from increasing... identified that there are more to learn - taqman PCR, qPCR, quantitative PCR - rhodopsin microbes were recently in class - how does using dUTOP and uracyl N-glycosylase solve - if you have a lot of stuff in the sample, it is difficult to get the problem of producing too much product, rid of all contaminants’ - cytosine can lose its amino group and the result is uracyl, - if your organism is living inside another organism you’ll this is a regular phenomenon, a mechanism fixes it in DNA miss the two before replication, uracyl N-glycosylase looks for uracyl, it will cleave that base and repair it, your product will have Analysis of sequence data using bioinformatics uracyl in it and it will be degraded, your probe is on the 5’ - contigs, overlapping data end and you are measuring the degradation of this probe - annotating, looking for ORFs, non coding sequences, regulating sequences, rRNA sequences, etc. Use Using fluorescence measurements to quantify target gene GenBank to get those sequences - blue is negative control - if you see that the sequences you find are 100% - at a particular point in the cycle you need to be able to tell homology then you know what organism is, if not then that the rise in fluorescence is not background, you label it depending on the homology you will figure out what it CT, cycle threshold, that is the degradation of the probe, if relates to have one copy of the gene in a ton of background DNA it takes a lot longer to get CT Bioinformatics: Insilico... - Blast, tool that looks at homology Principle of quantification - you need pure DNA, do separate reaction, make dilutions BLAST algorithms: of your gene - nucleotide sequence, translate to amino acid sequence, - understand how the technique works, the higher the and you look at protein sequence concentration of pure query sequence the lower the CT - sequence data on blast will show homology, conserved value, the more rapidly you cross the threshold of regions, and if some part of the sequence is totatlly resistance different it leaves it as blnak - just because you find homology to something else, mostly What might be the disadvantage... protein of unknown function, sequence of unknown - comparing 1 envrionment to another, how many say function, you will have to prove what you cloned and sulphur bacteria are in this environment compared to sequenced another? Genomics: determining a genome sequence from clone Figuring out who’s there after PCR amplification... libraries and Sanger sequencing: - you would pcr and clone the mass amount of DNA and - shot gun library, cut up the DNA with restriction enzymes, purify the bacteria and sequence it using the sanger clone every bit, sequence all the clones, purify the clones method until you think they collective represent the sequence of the - cloning, needs origin of replication, antibiotic resistance, original organisms cosmids (contains lambda consequences, plasmid), - laxa gene, blue white colour selection Other technical limitations - you can also use taq polymerase instead of restriction - during reaction, single stranded template, primer enzymes to add a bunch of Ts on the 5’ end hybrdizied, at 37 C, there’s nothing to stop DNA from - electroporation, shocks the cell membrane, dna goes in basepairing with itself, high G+C gives a hard stop - restriction enzyme bias Primer walking... - Mbp= double stranded - you look for a spot at the end that has good sequence, - draft genome, some discrepancy in sequence data use it to design your new primer, hopefully you’ll get another 1kb of good data from that point Next generation sequencing 454.... - multiplex, one run you can do 400,000 sequence Discussion on assigned reading: reactions - why viable real time PCR is required? - uses picolitre volumes, very fast, detecting the sequence - Doesn’t detect alive or dead cells, detects any sequences as the polymerization sequence that hybridizes to the probe - doesn’t require cloning - vPCR, use penetrating dye to interact with DNA and expose it to light and now it can’t be used as a template for 454 Sequencing – summary PCR (dead cells) - shear DNA through a syringe - two different dyes were used, PMA did not distinguish - adapters, it has biotin on one end of it, very small well between live and dead, it gets inside viable cells, false molecule, it has a very specific interaction, DNA capture negative results bead captures all the oligonutleotides, so you will only get - problems, biofilms, hard for light to penetrate through the DNA molecule captured by 1 bead, sequencing on a per layers and to the dye so the “live” cells will be sequenced bead basis - cells may be dead but they have intact membranes so the - amplify the DNA in each of those beads, each replicate of dye won’t be able to go in initial DNA have biotin on one end and gets recaptured by - some cells may be alived but their membranes are the bead damaged and eventually recover, the cells will look as - during the pCR you have one inital template on the bead “dead” because the dye will go in and your primers into there, when you get double stranded - spores, stable and nothing can get through the spore sequence and denature, you only recapture the copied coat, those won’t be detected strands with biotin - packing beads, reduces the reaction volume, enzyme Pedros Alio, C. 2006..... beads deliver enzymes like polymerase for example - 1970s, new molecular techniques were developing - sequencing by synthesis, real time observations as the - marine microbial ecology is important, half of the primary DNA is synthesized and tracking every single well when production on the planet is done by marine microbes, 95% the computer see light respiration is done by microbes, dominant nutrient cycling - one problem with this system, when you have a long sources, base of all food chains string of Ts (6 of them), distinguishing the correct number - genomics and metagenomics are powerful tools, opened of base up areas that are unaccessible, however we still need to culture these organisms 454 life sciences technology - having pure cultures allowed sequencing, comparative - much cheaper than sanger sequencing, extremely small genomics have revealed some interesting phenomons volumes - oxygenic autotrophic bacteria, proterococcus, most - know the advantages and basic method of dominant microbe in the world’s oceans, live at the pyrosequencing surfaces of these waters, genome sequencing of different isolates, depending on the niche (area + depth) their There are other single molecule... genomes differed substantially - reads=max number of nucleotides generated from - the strains depending on the depth had different sized reaction genomes - 400 bases of sequencing data are generated per reaction - genome streamlining where the top and bottom depth - the more information you can get from a single microbes dumped their genomes whereas the middle layer sequencing reaction the more understaning you will have had a bigger genome - stable concentration of nutrients and number of nutrients Press release: Sun Jan 15.... for the top and bottom layer - newest sequencer, size of a laser printer, $125,000, - the middle layer had a lot of changes in the nutrient level cheaper - when the nutrients are stable the microorganism don’t need to be as nutrient variable so they can afford to shed Bacterial artificial chromosomes some genes - 2 ways for metagenomics, pyrosequencing and make - prochlaoroccoccus, uses fewer nitrogen based bigger clones using BACs and fosmids compounds for the top and bottom layer - fosmids are like cosmids, based on E. coli F plasmid with - smaller genomes allows replication fasting, less energy the consequence, 54kb, advantage is that it is one copy used per cell (no toxic effects) - the less energy to grow the cell the better - BACs, no cos and lambda, contain F plasmid Origin of - should these different genome be called from the same replication species? - F plasmid can excised from the E. coli genome, stable at - ecotypes, organisms of the same species are different large sizes depending on their environment - they only replicate at one copy a cell - they found a gene that was similar to proteorhodopsin - 350kbp of insert DNA, introduce by electroporation - proteorhodopsin, in the plasmid membrane, when you hit - if you manage to get part or all of the operon then you it with light it actively pumps protons out, augments proton cna figure out who it is motive force, restricted to heterotrophs, related one in the halophiles that does the same thing, metagenomic Pedros Allo... sequencing identified that they are in many heterotrophs, - axenic culture, pure culture especially in the top layer (photic zone) - PRN= proteorhodopsin 2D gel electrophoresis - hypothesis, heterotrophs have this mechanism to help - first dimension, pH gradient, proteins will go through pH them generate energy when there’s limited nutrients graiden they take up net charge until they reach 0 net available in the ocean charge so they won’t move and you get a band - this photorophic ability allows them to grow more quickly - then you lay in on an SDS page gel, all the proteins are and efficiently linear, denatured, net charge same, now you are separating by molecular weight Culture independent analyses using isotropic labelling - you should be able to separate to individual spots - #1 article has typos - you can cut it out of the gel and do mass spec and - figures on the following slides are from #2 and #3 identify the protein theoretically, in practice the databases aren’t that great Detection techniques that allow.... - stable isotopes, normal mass of C is 12, radioactive Metaproteomics isotop is C 14, - not who’s there, may make some conclusions but not a - radioactive isotopes are heavier than normal C lot - radioactive C14 decays - metaproteomics, very limited currently - they are dangerous depending on the radioactivity - know what the advantages and things that we don’t know - stable isotopes are also heavier, don’t decay, C13, not from this technique dangerous - if you use these labels into the organism, who is Week 5 incorporating htis substrate? - then you will see that the parts of the organism will be - microautoradiography is produced on microscopes more dense - FISH-Mar is used to study relationships of organisms in - assimilatory reduction, nitrogen gas to ammonia to biofilms glutamine (amino acid), nitrogen fixation, glutamine - biofilm is fixed to the slide, probe, look under a confocal becomes part of the cell structure, scanning light microscope - dissimilatory nitrate reduction, always a reduction of a - the probes that are fluorescing on silver grains mean the terminal electron acceptor, in anaerobic respiration NO is2 organism they’ve incorporated it not use, H 2 is used as the electron acceptor - problems, need to know the sequence to make primers - if you have NO 2ut of N15 then you can ask which - you need a certain amount of cells to have fluorescing in organism have heavy DNA, RNA, phospholipids order to see the light - more informative to purify would be the DNA - Dopefish is a new technique, duel labelled - what’s the benefit of purifying phospholipids vs DNA? oligonucleotide probe, you can use more fluorescence Cells have to replicate their chromosome and divide, molecules to detect it replication time is long - who’s sitting side by side by whom? - whereas the phospholidis are quick, cell uses it up immediately to make phospholipids, substantial change in DNA/RNA-SIP followed by FISH density - accomplishes the same thing as FISH-mAR - who’s fixing nitrogen? Then do DNA or RNA - in conjunction with SIP - uses stable isotopes SIP experimental design - naphthalene, crude oil and gasoline contaminant - stable isotope probing - 2 different types of fluorescence, to confirm SIP - use radioisotopes to confirm your stable isotope findings - raman microspectrocopy, it can resolve 2 spots by 1 - microcosm on the right, mesocosom on the left micron (1 cell), it incorporates laser light and shines it on - do the incubation, use enough material so you get the sample, depending on the vibrating bond energy will incorporation, it needs to be levels that the organism is scatter the laser light which will produce a spectrum, the going to see in the environment shift in spectrum is a red colour (red shifts), use it to detect - if you add too much you will skew the microcosm studies the amount of C13 is in the cell because other microorganisms might dominate - at low levels pseudomonas at low levels doesn’t absorb - if you wait too long, the organism might release nitrogen any but at high levels it absorbs almost as much as the gas with this isotope and other organisms use it as a other one nitrogen source but it has no way of fixing it so your data is - raman is very expensive whereas FISH-Mar is cheaper messed up - PLFA, phospholipid derived fatty acids, you can purify or Another alternative method of confirming DNA/RNA-SIP do a mass spec, data.... - GCIRMS, isotope ratio mass spec - take ribosomal rna, tag it, hybridizing it to a phylochip, - you can use a density gradient gel electrophoresis for have microarray to detect fluorescence (find who were DNA until they hit the density gradient that matches their there), which ones have radioactivity, need a beta imager own and stops moving (microscope) - RNA is harder to work with, contamination issues - 3 types of radioactive, alpha is the lowest, gamma is the - High G+C DNA is more dense than unlabelled DNA highest - you can figure out which one is emitting your probe which SIP independent confirmation is required... is emitting radioactivity - FISH + autoradiography - incubate it and fix it on your slide, cryosection, freeze and Summary: culture independent slice - don’t need to read this - hybridize with fluorescent probe, use 16S probe - hit it with laser light to monitor fluorescent - after you figure out which organism has hybridized with it - focused extremophiles on Earth that might be in space Problems with Fish-Mar Why replace exogiolgoy with astrobiology - crossfeeding, the organism you’re trying to identify by using a stable isotope but if you wait too long it will How do we trace the evolutionary history of life on earth? produce waste products using that isotope and another - rRNA, evolutionary relationships organism will take it up thus it is a time problem - rapidly growing organisms have multiple copies of rRNA - when you are using phospholipid derived analysis, the and they can be different isotope doesn’t give you a lot for identification - each rRNA can evolve at its own pace - stable istope substrates are hard to make and find from - microfossils, bacteria and arcahe, negatively charged, suppliers positive cations on top of the organism and that forms a - the concentration of the substarte is critical, depending on shell/casing and that’s what is preserved the concentration different organisms will use that - biosignature molecule+isotopic...if we find orgain substrate, at low levels some will use it and at high levels molecules or other to be believed to be found in other others will dominate, also it might not be the most lifeforms combined with isotopic enrichment, which proves favourable substrate for the organism, that the life form once lived and produced these C12 - autoradiography is produced from x-ray film, silver precipitates on the film Evolution of life on Earth - Acretion of Earth 4.6 billion years ago, formation of the Mechanism of polymerization... planet (know this) - you need energy and water, more than likely happened in - Ga= giga annum, billion years ago water, stuck on the surface, perhaps on clay, if you provide - before we had life evolution we had chemical evolution the building blocks on clay and the energy you do get - 2 billions or more, no oxygen in atmosphere spontaneous changes - with evolution of oxygenic autorophs oxygen was - RNA, ribosomes, need to make proteins but which came produced first? - life forms changed the atmosphere - RNA can form complex shapes same as proteins - period where you have RNA but no DNA is called RNA Accretikon of earth... world - if you look at this chart you can see that there’s nore - it has been demonstrated that clay particles speed up the nitrogen and oxygen process of micelles - where did the nitrogen come from? Nitrogen was - LCA=last common ancestor probably in the earth’s crust - archaea, evidence that it evolved very early, branched off - the sun was 30% weaker in the early days, the earth’s shortly the bacteria, some things are more similar to surface was very hot but it started to cool bacteria (RNA and structure), transcription factors and - the average temp now is 13, the change is percentages metabolism are more similar to eukaryotes, are they of green house gas probably played a role in surface responsible for evolution of eukaryotes? We don’t know temperature - viruses, linear chromosome, very rare, linear telomeres - no oxygen = no ozone, protects us from UV, filters out UV aren’t found in bacteria, viruses have evolved rapidly, light suggested that viruses were here before or shortly after life on earth appeared Recent isotopic evidence.... - theory that maybe there was a linear double stranded - from 4.8G.a to 3.8G.a some have argued its not long DNA enveloped virus that gave rise to the nucleus enough for life to be born - some believe that others came from another planet (i.e. Also unknown: where did life first appear? meteorite) this is called panspermia - shouldn’t be on surface, too much UV - the meteorite, scientists discovered nano bacteria from - oceans or sediments, deep sea vents, deep waters, Mars, has been proven false now probably carried the first life - lost city vents, envionrment is alkaline and no super Paspermia, controversial claims, peer-review? heated water, perhaps this was where teh formation of organics and first life form occurred Chemical evolution of organics on prebiotic earth - organics in space dust Geological time cale with important evets.... - two scientists proposed the same thing suggested the - need to remember 4.6, the hadean, conditions on earth could have spontaneous spawned - 3.8 billion years, isotopic evidence for biological carbon, organics evidence of autotrophy, to make organics - where there’s oxygen there’s superoxide that can kill - 3.5 billion years, evidence of sulphur reduction, anaerobic anaerobes respiration, stramatotrophy evidence of first fossils that - carbon dioxide and methane are greenhouse gases that carried out photosynthesis are needed to produce organics - hydrogen sulphide, ammonia, and CO2 are important 3.5 - lots of different types of energy to make new types of - we have no discovered any photosynthetic bacteria building blocks - archae probably evolved before those photosynthetic autotrophs Miller and Urey provided.... - stromatolites are crystallized with cyanobacteria, cellular - the heat the flask, ocean part polysaccharide capsules, given the waters here had - gas and lightning, provided energy chemical reactions and that was how these stromatolites - all these gases when exposed to the energy could are formed potentially form organcs, condensed and then can isolate - stromatolites are 3.5 billion years old, isotopic evidence, from the “ocean” more than likely those first photoautotrophs were probably - in fact they did find a number of organics like organic precursors to cyanobacteria acids and lipids Week 6 Prebiotic earth: there is no consensus - not too sure when the meteorite impact ended (hadean Genomic comparison allow identification of core genes... era) - photosystem 1 is fed by electrons from photosystem 2 - if life formed at this time they should have died out from - photosystem 2 generates oxygen by hydrolizing water to the impacts obtain electrons - 1997 because we don’t know the conditions of back then - when light hits it turns the reduction potential to very we don’t know if they are spontaneously made or negative which allows the donation of electrons exogenously delivered by the meteorites or produced by - reducing power is needed to reduce CO2 for example to meteorites hitting the earth (energy) or the combination of make cellular materials all 3 - cyanobacteria hydrolyzes water, best reducing power - Hydrogen sulphide can be used also since it was also a - evolution of catabolic pathways, making molecules good supply smaller and smaller, goes hand in hand with the anabolic - this photosystem in genetic comparisons between pathways prochlorococcus and other organisms identified a gene core The bacteria and archaea are most... - first autotrophs were anoxygenic - bacteria and archaea can oxidized any molecule on the - photosystem 1 was probably the first system and planet photosystem 2 was developed along the way - most diverse, they’ve been around for a long time - bacteria and archaea are being carried by air currents, do Anoxygenic photosynthesis.. the environment select ones that survive? Are they - heliobacteria, gram bac, related to clostridium everywerhe and the environment selects or not? - all have a single photosystem - all use something other than water like sulphur From Woese and Goldenfeld, 2009 compounds for reducing power - lateral gene transfer doesn’t only mean from one cell to another, in eukaryotes it would also apply from the The great oxygenation event mitochondrial genome to the nucleus (same with - 4.6, 3.5, 2.45-2.2 remember these dates chloroplast) - in costal area there are iron oxide formations dates to - if you take the mitochondrial genome and compare to the around 2.4 billion years ago, before that it should be in the cyanobacteria you don’t find them in the organelle anymore reduced form but in the nucleus - Fe3+ is oxidized (lost 3 electrons), Fe2+ is reduced - this makes it better for coordination when the cell divides, - early days, atmosphere was reducing, iron was reduced more control, can control energy levels, etc. - abiotic formation of organics - cells cannot ATP or NADPH, so energy demand and - between first evidence of phototrophy and the evolution production must be balanced thus you need the nucleus to of oxygenesis there was methagenesis coordinate everything - know the order of things - we can see certain genes not be restricted brance of a - methanotrophy, organisms that consume methane tree, found in many different organisms from both bacteria - eukaryotes are aerobic and used the small amount of and archaea, but also you can tell its been acquired oxygen present during the methane period recently from the G+C part of the chromosome Evolution of oxic atmosphere: When is a tree not a tree? - all biogeochemical reactions changed due to oxygen - common pathways, would explained by lateral gene - ammonia plus oxygen, nitrification, nitrate, which can be transfer used as a terminal receptor so nitrogen gas is produced - cell structures must have been simple at worst - once oxygen accumulated, there was less and less - he’s proposing that evolution was communal, genes were abiotic formation of organics, organics are now produced spread horizontally in the begin and not vertically, by organisms branching occurred later on once geneology was stable - oxygen in the atmosphere produced ozone layer which - after swapping genes many times, at some point it would filtered out UV light, terrestrial colonization occurred cross the threshold to exhibit a root and vertical gene transfer occurs Wose 1990 - he is suggesting that bacteria crossed the Darwinian - proposed the last common universal ancestor threshold first, most basic, no similarities with eukaryotes - central dogma DNA, RNA, to protein - archaea crossed it soon after - glycolysis, is how many organisms make organics from - eukaryotes crossed it last - these 3 things are commonly found - endosymbiosis theory, mitochondria and chloroplasts in ancient times were living endosymbiotically with other Week 7 bacteria - Mitochondria, first symbiotic event because plants had Review them - tree of life might not be very accurate because 16S RNA - algae, precursor, had a symbiotic relationship with contains too little information to track changes that far back chloroplasts - evolution of the nucleus, unknown, perhaps some sort of Elongation factors archaea virus that gave rise to the nucleus - complexed system - computer models of ancient EF to look at conditions when The physiological diversity of bacteria.... life first evolved - cell membrane is more permeable than now - Tm= melting temperature - H was brought inside and used as a building block - figure out melting temperatures and figure out amino acid - eventually H would be depleted from having so many sequences, how are these thermophillic and how are these organisms then they would be forced to make F from G mesophillic? and they would eat F - Time 0 being now in the graph - through lateral gene transfer then they use the E enzyme - there’s a higher melting temperature in the past to modify D to F and they eat F - the darker line represents the predicted ocean temperature Can we find supporting evidence in the diversity of extant - the current record holder is -12 thermophiles? - they are saying that we should be able to look at the Psycrohpiles: optimal growth..... diversity of life on earth, especially the thermophiles - the lake in antartica will melt from time to time and a ton - if life evolved from harsh and hot conditions you should of methane will be released see diversity in those organisms - reason is because there`s no oxygen in water due to thick - y axis is temperature and x axis is pH ice, lots of methanogens in the water - there are no acidic cyclophiles, there are no organisms - lake vostok, fresh water lake, the last time that organisms that there are no acidic cold conditions so you can assume saw light was 15mil years ago that life did not evolve from these conditions - the drill would create a pressure and move the water up - there are no alklophillic cyclophillic organisms too and ice will form in this water and they will pick the ice up - survival at extreme conditions is not the same as and investigate the organisms without contamination replicating under those conditions - can we figure out how the psycrohpillic enzymes and - plasta.....was revived when the ice it was frozen in was thermophillic enzymes work and then make our own? melted, tolerant but doesn’t grow - there are lots of organisms in the hot acidic and basic 75% of Earth’s biosphere is cold conditions - average ocean temperature is 5 C o - there are a lot of support for theories of evolution from - one thing you must have is liquid water for life to occur deep sea vents which are hot and acidic - reactant and product, oxygenic photosynthesis - there are some support for lost city vents, thermal vents not quite as hot and alkaline conditions Pigthnted psychrophiles... - under alkaline conditions you can demonstrate - 4 century, they discovered pigemented psycrhophiles spontaneous organic molecules being formed that that coloured snow - cyanobacteria growing in the snow Bacterial strain GFAJ-1 from Mono Lake - liquid water is essential ingredient for life - arsenates in this particular lifeform can replace - pigemented partly to protect them from UV, alters the phosphates in the DNA backbone albedo of snow (drop freezing temperature), provides liquid - generated a lot of debates water to the cell, many of these organisms are halophiles - NASA claimed that this discovery will help find - lots of contaminants and salts which drops the melting extraterrestrial life temperature of water but making it very salty - discovery was picked apart - Redfield lab redid the experiments, they could not Biological/biochemical challenges reproduce the results - pscyrophiles, membrane gelling- loss of fluidity, - experiments of arsenate as components of the backbone transmembrane proteins can’t move in the lipid bilayer, any was flawed transport proteins, chemotaxis proteins, flagella proteins, - the bacteria can grow with very little phosphate but it still all of these need to move in the membrane, if you have a needs it gelled membrane, you will have trouble transporting - these vesicles are typical of cells that are dying which nutrients and solutes, chemotaxis will be affected was tale telling sign - cellular damage, ice crystals damage structures especially if they require any organization From Flakowski and Godfrey - increased tendency for cold denaturation, protein loses its - understand how organisms survive in extreme cold water shell, with protein folding all these proteins are conditions surrounded by a water shell, in cold temperatures water - hope to find life in space which is cold forms water water lattice to the point where it precipitates out and the protein loses the shell Microbial physiochemical adaptations to extremem - difficulty in protein folding, lack of thermo energy, if the environments water is not there and it becomes more viscous then the - extremophiles we’ve discovered on earth and the protein folding is interrupted undiscovered are probably the most weirdest we’ll ever - chaperone proteins help in folding to help the cell survive find - slow biochemical reactions, lower thermal energy, not enough for chemical reactions, as the environment gets Upper limits of terrestrial life colder it becomes more viscous, enzymes need the - barophiles, high pressure organisms substrate to diffuse into teh catalytic site, the substrate - when you look at one extreme environment, they are enters by diffusion, the enzymes also need to be able to exposed to more than one extreme conditions, for example move, with the cell becoming more viscous everything very cold conditions and very high pressure in deep sea, moves more slowly which impedes chemical reactions some cyclophiles are also halophiles (ice crystal molecules - temperature vs. growth rate graph, just reminding the veins with very high salt liquid water), hyperthermophiles range of which the different organisms can grow, it also are also in high pressure, remember that being tolerant represents enzyme activity (y axis), minimum temperature, doesn’t mean growing optimum temperature, maximum temperature, combination - we’ll be looking at organisms that grow at these of all three that defines the categories: mesophile, conditions (i.e. replicating at these conditions) thermophile, psychrophile Distribution of microbial life based on temp.... - know where the two bacteria on the page are found and - poychrophiles, an organism thao does not grow above how to grow it o 15 C and whose optimum is 5 C or lower - optimum temperature, no more than 15 C - maximum temperature, 20 C o - catalytic activity vs. temperature o - if they can grow more than 20 C they’re falling into the - red mesohpillic homolog (bottom one), blue psychrophillic mesophillic range - you can see that the activity is much higher in the psychrophillic organisms than mesophillic, the shape of the Cold adaptive components and processes curve you get this by being more flexible at low - lateral gene transfer by viruses even at lower temperatures temperatures have been observed - cold shock proteins, doesn’t just help protein fold/prevent Structural biology.... denatuartion, helps prevent RNA molecules to form - analyses of dynamic components of proteins, hard to do secondary structures, - lipid bilayer with (#4), polyunsaturated fatty acids, key to Experiment measure.... maintain semifluid membrane, the less molecule molecule - psychrophillic, tmesophillic, thermophillic attraction (kinks in the fatty acid chain), help maintain fluid - quenching versus acrylamide concentrations membrane - ratio of fluorescence at mesophillic temp vs. - compatible solutes, compatible at high concentrations, psycrhophillic temp amino acids (glycine), small molecules, outer environment - you add acrylomide to the enzymes, the acrlyamide will is highly concentrated in solutes, water diffuses freely diffuse to the catatlytic site and quench fluroesence, the through the membrane so water will move out, turgor more it diffuse the more quenching pressure is lost, to prevent this they pump compatible - if its more flexible at cold temperatures you’ll have more solutes in, made inside the cell, and flexible membrane quenching proteins pumps them out - the denominator becomes smaller, ratio becomes bigger - 8, these nucleate ice crystal formation outside away from the cell Bae E. and Phillips.... - 9, psychrophiles inevitably have this slime layer associate - adenylate kinase, take one atp and amp to produce 2 with them, in some cases has been shown to prevent ice ADP crystal formation, reduce freezing temperature, helps trap - small enzyme water, helps protect the flagella motor - primary sequence is highly homologous - globisporas, psychrophillic, subtillis, mesophillic, and Cold adaptive strategies stearophethermophilus thermophillic - methyl branched, introduce kinks in the fatty acids - the urve is showing temperature vs. activity, its a curve - cyclic, ring, takes up more room where the temperature for minimum activiating, always a - shorter fatty acids, less intermolecular interactions, bit less than optimum temp for growht makes membrane more flexible - 2 important temperatures to remember for psychrophiles, - desaturases, optimum less than 15, can`t grow above 20 - mesophiles also use these methods to survive if it finds - when you look at the enzyme optimum activity its always itself in these conditions, can’t grow but survives higher than growth optimum - cryoprotectants, prevent ice crystal formation - there are components in the cytosol, gel like, that - glycerol, has been used to free bacterial cultures, environment helps drops the temperature of optimum activity Compatible solutes - for mesophillic and thermophillic around the temperature - cold shock response, best known in mesophiles, induces optimum for growth many genes to make cold shock proteins, many are chaperones, others will protect secondary structure Crystal structures... formation in RNA and DNA, comparative genomics have
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