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by: Rubie Mertz


Rubie Mertz
Colorado School of Mines
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John Spear

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John Spear
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This 33 page Class Notes was uploaded by Rubie Mertz on Monday October 5, 2015. The Class Notes belongs to ESGN 586 at Colorado School of Mines taught by John Spear in Fall. Since its upload, it has received 22 views. For similar materials see /class/219636/esgn-586-colorado-school-of-mines in Environmental Science & Engineering at Colorado School of Mines.

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Date Created: 10/05/15
ESGN586 Lecture 11 Fall 07 Readings To Be Announced and Chapter 28 Key Points Wastewater Treatment Wastewater Treatment Wastewater A liquid derived from domestic waste sewage andor industrial wastes that cannot be discharged into a receiving water body without treatment due to public health environmental economic andor aesthetic considerations Water Has the potential to be the most common source of infectious disease Water Purification The single most important measure to ensure public health Ironically some of the best kinds of purification involve a host of microbiota Water Assessment Quality assured by a variety of chemical and microbiological methods Most of the microbiological methods involve traditional cultivation analyses THIS needs to change to routine molecular characterizations Pathogens As we have said 7 divisions of Bacteria contain pathogens There are many and it is impossible to screen for all pathogens in a water Instead Indicator Species Kinds are used Coliforms Organisms which inhabit the gut of humans and other animals Facultative aerobes G non spore forming rod shaped Bacteria that ferment lactose and make CO2 gas bubbles within 48 hours E coli Klebsiella pneumoniae and Enterobacter aerogenes technically not in the enteric group but is facultative Test developed 100 years ago and we still use it The Coliform Test Most Probable Number MPN Test and Membrane Filter MF Tests MPN Liquid medium in a test tube add an amount of drinking water any water growth in the medium 2 contamination MF At least 100 mls of water in question is passed through a 02 MM filter place filter on an EMB Plate eosin methylene blue medium medium is both selective and differential for lactose fermenting bacteria coliforms count colonies Ideally you have none Safe Drinking Water Act provides values for counts 1 lt1 per 100ml of all samples examinedmonth 2 4 per 100 ml in more than one sample if lt20 are examinedmonth 3 4 per 100 ml in more than 5 of samples when 20 or more samples are examinedmonth John McEncroe Any Thoughts POTWs Developed at the turn of the 20 h Century 110 years ago Large scale publicly operated wastewater treatment works also applied to drinking water works Both still in big use today however ON SITE wastewater treatment systems may be the wave of the future cheaper Wastewater treatment involves microbial bioconversion of carbonaceous biomass at the industrial scale Effluent water is then cleared for discharge into natural receiving waters Complete wastewater treatment involves physical chemical and biological treatment to remove andor neutralize contaminants 15 to 20000 POTWs in the US treating an average of lMGD The discharge from those is 35BGD The goal of wastewater treatment is to reduce the input of both organic an inorganic materials into receiving waters We have said that life needs 4 things Water Carbon an electron donor and an electron acceptor Wastewater contains all of that and more Reducing that allows the microbes in a receiving water to NOT go to town digesting that stuff depleting the O2 in the water in the first place and killing the fish BOD Biochemical Oxygen Demand a measure of the effectiveness of treatment the dissolved oxygen consumed by microbiota to digest the organics Typically a wastewater contains 200 BOD 1500 2000 BOD for industrial waste once discharged want lt 5 BOD Primary Treatment only Physical Methods used Separate solids particulate organic and particulate inorganic materials by screening settling and sedimentation By itself usually only locally done in small systems Worry about discharge Secondary Treatment Physical chemical biological methods used Anoxic Sludge Digesters and Bioreactors to remove high organic content A digestive and fermentative process that involved a stew of micorbiota Mainly produce CH4 and COz the CH4 can then be used as a fuel source at the plant or elsewhere Aerobic Microbiota under high O2 loading carry out digestion under low organic loading Common trickling filter and activated sludge methods used Trickling Filter Spray water over a rock pile What do you get a ton of biofilm Biofilm take the waste to CH4 COZ NH3 PO3 and S04 Activated Sludge High O2 minestrone of microbes Slime forming bugs Zooglea spp form flocs Other things protests fungi nematodes etc join the floc Flocs will settle in a clarifier cleaner water out recycle the flocs back to the aerator as activated sludge If the BOD has not dropped far enough can then send to an anoxic digester to finish the water Problems Cholera Vibrio cholerae causes extreme diarrhea 7 major pandemics across the world Current one is the V cholerae 01 El Tor biotype Another is the V cholerae 0139 biotype Comes from drinking contaminate water or ingestion of contaminated coastal shellfish It is not the V cholerae that kills you it is the effect of the enterotoxin on the cells of the gut that produce extreme dehydration through diarrhea Consumption of LOTS of water with salts and sugars is the preferred method of treatment Cryptosporidia Cryptosporidiumparvum a eukaryotic protozoa Grow as round coccidian and grow inside intestinal epithelia Interesting lifecycle but the most interesting is the oocysts they produce within the epithelial cells then shed into the intestine to poop to infect and share Oocysts are highly resistant to both chlorine and UV disinfection Diarrhea for 2 3 weeks let it run its course Electrolytes and sugars as needed Giardia Giardia intestinalis a flagellated eukaryotic protozoan Line and coat epithelia can give the fart burps Gives diarrhea treat with waterelectrolytes and or flagil Physical Problems Bulking and Foaming aka sludge bulking Filamentous Bulking The relationship between floc forming and filamentous bacteria is a critical parameter in wastewater treatment The three types of flocs are Normal Flocs balance between floc forming and filamentous strong floc hold integrity settle well Pin point Flocs No filamentous bacteria small flocs that don t settle well secondary effluent turbid Filamentous Bulking Too many filaments Filaments interfere with settling and compaction poor sludge settling as indicated by Sludge Volume Index SVI There are 25 known microorganisms that affect this Operators can control what kinds of organisms are in the plant by metabolic distinctions Floc Former Filament Maximum substrate uptake rate High Low Maximum specific growth rate High Low Decay rate High Low Resistance to starvation Low High Ability to use NO3 as ea Yes No The 25 filamentous bad ones One an Actinomycete called Nocardia spp aka Gordona spp can cause foaming Type 1701 Type 021N Type 0041 Thiothrix spp Nostocoida limicola are others By 168 on a couple of different sites I have found Big Thiothrix infestation and Rhizosphere soil bacterium filament problems A variety of causes Problem Cause Effect Dispersed Growth Microbes don t form ocs Turbid Ef uent Slirne Too Many Microbes Reduced Settling Compaction Zooglea spp No Solids Separation Pin Floc Small weak ocs Low sludge volume turbid Bulking Filaments High Sludge Volume clear Microbes extend from ocs gt 107 um mg suspended solids Rising Sludge Denitrification Scum of sludge on Clarifier N2 attaches to ocs rise Foaming Nondegradable surfactants Foam everywhere Or Nocardia spp What s in it Microbes Microbes then putrify Useful equation Sludge Volume Index SVI SVI V x 1000 MLSS V volume of settled sludge after 30 min ml L MLSS mg L SVI 2 ml g volume occupied by 1 g of sludge Low sludge volume index lt70 ml g pin ocs cloudy turbid ef uent High SVI gt150 ml g bulking condition clear supernatant Problem is the proportion of Normal Flocs to Pin point ocs to Filamentous bulking Factors that Contribute Waste Composition high carbohydrate wastes e g brewery waste conducive to sludge bulking Substrate Concentration filaments are slow growing Sludge loading and Sludge Age varies depending on CSTR or PFR In a CSTR increase sludge loading leads to a decrease in SVI and a decrease in filaments The MAIN operational design control mechanism Basically low sludge age filaments wash out pH optimum pH for an aeration basin is 7 75 Values lt 6 favor filaments Sulfide Concentration high dissolved HS in the aeration tank favors growth of sulfide oxidizers filamentous like Thiothrix spp Take HS to S DO Low DO favors filaments Aeration tanks should operate at a minimum of 2 mgml DO Nutrient Deficiency N P Fe trace elements may cause bulking Control of Bulkng Treat with Oxidants treat return sludge with chlorine or peroxide to kill filaments Bump up the Cl concentration in the return activated sludge line to 10 20 mgL Add occulants synthetic organic polymers lime iron salts Manipulate return activated sludge ow rate Biological Selectors have a tank upstream of problem basin that grows good microbes to ood out the bad in case the bad happens Conditions can be aerobic selectors anoxic selectors and anaerobic selectors Foaming What is a foam Undegraded surface active organic compounds Poorly bio degradable detergents white foams Scums due to rising sludge from denitrification N2 bubbles Brown Scums due to excessive growth of Actinomyctes a biomass laden foam Proteins from all the dying microorganisms Problems are more physical walkway coating worker coating wash out with effluent etc Prolific bugs in Brown Scum Foam Gordona amarae and Nocardia pinensis Foam Control Chlorinate the foam or return activated sludge kill off the organisms Increase sludge wasting Gordona for example takes 5 7 days to develop colonies Decrease the mean cell residence time Reduce air flow Reduce pH Water andor salt sprays Physically remove ESGN586 Lecture 2 Fall 07 Readings Brock Chapters 1 5 Chapter 1 5 Key Points What is a microbe Webster s A microorganism Brock A microscopic organism consisting of a single cell or cell cluster also including the viruses which are not cellular FYI A virus is a genetic element containing either DNA or RNA that replicates in cells but is characterized by having an extracellular state It is a nonliving particle Why do we care Responsible for SO many things Health amp The Pathogens but remember there are only 7 divisions of Bacteria that have pathogens and there are no archaeal pathogens Refrigeration Ex Hurricanes Katrina and Rita Packaging How we touch what we touch How we are social or not with each other Handle a baby Cleanliness of everything What is body odor Sleeping Bags Clothes Hands Teeth Other orifices How we taste How we smell How we hear How we feel Microbes are intimately involved in all of these senses Environmental Microbiology Here We Go H Microbial diversity reflects the different ways in which microbial organisms Obtain energy and nutrients Respond to the environment and other organisms Regulate genes and metabolism Grow and divide Survive adverse conditions N Microbial organisms have the same needs as macrobes Indeed most of biological diversity is microbial in nature historical focus aside most eucaryotes are microbial 9 Note the differing biological strategies manifest by macrobial and microbial phenotypes ESGN 586 Fall 2007 John R Spear Page 1 a Large creatures are comprised of different cell types differentiated from a common germ line that collectively and interdependently form the functioning community the macroorganism b The constituents of communities of microbial organisms are selected upon by the local chemistry and physical properties and collectively and interdependently form the functioning community eg a quotbiofilmquot In the macro case the organism carries the information for the required cell types in the micro case the environment selects for the suite of required cell types Different strategies of life stuff 4 Traditional large organism biology has focused on organism shape general biology texts usually paint prokaryotes as simple cells rods bacilli s bacillus note contrast with Bacillus a genus name cocci s coccus spirals misnomer really helices vibrios partial helices U1 But many other forms occur e g Long filaments gt 100 gm 2 01 mm common e g Beggiatoa Thiaploca Branched filaments eg Streptomyces Star shaped Stella Amorphous shaped e g Sulfolobus Flat looking eg Haloarcula 6 General texts often emphasize individual cells but aggregates are the common theme in nature Chains strepto eg streptococci Tetrads and higher regular clusters Rosettes ON 0 e g Planctomyces 6 O 7 Seldom do organisms in nature occur in isolation free living cell types Microbes form complex communities ensembles of different organisms in contrast to a population which refers to a collection of the same type of organism ESGN 586 Fall 2007 John R Spear Page 2 8 Most microbes in nature are attached to surfaces and associated with communities biofilms and mats containing interdependent quotsyntrophicquot suites of organisms syntrophs that often cannot be grown in pure culture Just like it is hard impossible to culture most human differentiated cell lines Note differentiated states vary in environment dependent ways e g Saccharomyces cerevisiae when starved for N becomes filamentous rather than yeast in form 9 Highly variable in size commonly 1 10um in diameter but some are visible to the naked eye e g Epulopiscium gets to 100nm X 700nm Others ca 50 of what you see in the environment by microscope are only 01 03um in diameter note that volume scales with 13 are they starved normal cells or truly small a Communities of microbes are commonly large e g meter size blobs and strata in sediments Little characterized b Stromatolites are fossil manifestations of ancient microbial communities 10 Size terminology funky nonstandard a nannobacteria sic 2 really small b Planktonic terminology microplankton 20 200 microns dia nanoplankton 2 20 microns picoplankton 02 2 microns etc H H Structural diversity a Many types of cell walls Gram negative Double membrane thin peptidoglycan wall periplasm Gram positive Single membrane thick peptidoglycan wall complex mesh probably periplasm like in many regards S Layers etc NOTE For historical reference you need to be familiar with the quotGram reactionquot see BBM pp 58 59 if you are not For all the historical import however it doesn39t have much quotphylogeneticquot significance The commonly used term typical Gram negative organism is an ESGN 586 Fall 2007 John R Spear Page 3 oxymoron People who use that term are usually referring to a representative of the bacterial division Proteobacteria b Many different external structures e g Flagella pili stalks and holdfasts capsules sheaths sometimes with multiple types of organisms etc c Many different internal structures e g Nucleoid bacterial nucleus spores inclusions of energy reserves eg sulfur poly beta hydroxybutyrate glycogen etc 12 Metabolic diversity switch hitters common capable of markedly different metabolisms a Chemoheterotrophs obtain energy and carbon from reduced organics e g Escherichia Bacillus b Chemoautotrophs energy from reduced inorganics eg H28 Beggiatoa Fe2 Gallionella carbon by fixing C02 c Photoheterotrophs carbon from organics energy from light eg Rhodobacter d Photoautotrophs carbon from C02 energy from light cyanobacteria plants the chloroplast is a cyanobacterium 13 Ecological diversity Microbial character is far more robust than the macrobial form a Extremes of ionic strength distilled H20 to NaCl saturated brines south SF Bay Dead Sea e g Haloarcula b Extremes of temperature ca 5 C to 113 C Pyrodictium to 121 C c pH pHlt0to 12 d All over the planet from deep subsurface to clouds in the sky any place there is liquid water and an energy gradient 14 Behavioral diversity a Motility and taxis chemotaxis phototaxis magnetotaxis b Developmental processes sporulation Bacillus developmental growth phases ESGN 586 Fall 2007 John R Spear Page 4 Caulobacter metabolic differentiation Anabaena heterocysts for N2 fixation c Communication between like cells Quorum sensing and unlike symbioses antibiotics 15 Evolutionary diversity the basis of it all a For all the differences between different organisms the underlying biochemistries are pretty much the same DNARNA based information transfer ATP NADP chemiosmosis based energy same general pathways for carbon metabolism and biosynthesis b All life on Earth is related ancestrally A Pretty Amazing Property of Life Describing Microbial Diversity the Changing Paradigm 1 Traditional taxonomy classification of microbes both prokaryote and eucaryote is in a mess but we are stuck with it for traditional reasons a A natural taxonomy would be based on evolutionary relatedness Thus organisms in same genus a collection of species would have similar properties in a fundamental sense b A natural taxonomy of macrobes has long been possible Large organisms have many easily distinguished features e g body plans and developmental processes that can be used to describe hierarchies of relatedness c Microbes usually have few distinguishing properties that relate them so a hierarchical taxonomy mainly has not been possible 2 Recent advances in molecular phylogeny have changed the picture a lot we now have a relatively non subjective quantitative way to View biodiversity in the context of phylogenetic maps evolutionary trees a Slowly evolving molecules e g rRNA used for large scale structure fast clock molecules for fine structure ESGN 586 Fall 2007 John R Spear Page 5 3 A culmination is The Big Tree a molecular coordinate system based on rRNA below a The literature language eg species and formal nomenclature of biology however remain solidly rooted in the tradition of Linnaeus at this time You have to call them something b First an overview of phylogenetic perspective on microbial indeed biological diversity then a brief look at other methods used to characterize microbes PHYLOGENETIC DIVERSITY 1 Phylogenetic relationships provide a natural classification of organisms Darwin s dream They are the only way to understand the evolutionary process a Note that phylogenetic relationships are predictive not only descriptive We can predict some properties of organisms based on the properties of their relatives e g We know a lot about Escherichia coli but relatively little about Chromatium vinosum 1 E coli is a chemoheterotroph whereas C vinosum is a photoheterotroph Classical physiological treatment would have considered these organisms wildly different From gene sequence comparisons we know they are fairly close relatives y group of proteobacteria 2 Thus even though one eats glucose and the other light we can predict that the biochemical underpinnings are similar protein synthesizing machinery antibiotic sensitivity DNA replication machinery amino acid synthesizing machinery nucleotide biosynthesis etc We expect to be able to swap many genes between these two superficially disparate organisms b Phylogenetic perspective allows rational selection of model systems sometimes you need a model organism to gain perspective on a more difficult system 1 eg Use of a model non pathogen can provide safe study of a pathogen if it is a close relative 2 Or can provide a simple system for a complex one e g for plants what to use as a model Chlorella Chlamydomonas Euglena Molecular phylogenetic studies say Chlorella or Chlamydomonas both of which are in the plant relatedness group clade Euglena is a trypanosome ESGN 586 Fall 2007 John R Spear Page 6 3 eg if want to study an uncultivatable symbiont identify a cultivatable free living form E g the Riftia symbiont c Phylogenetic perspective even on macrobes is quite recent say a century and on microbes only ca 20 years Many microbiology texts don t have it and many even most general biology texts get it wrong A Bit on the Evolution of Evolutionarv Thought 2 Prior to the late 19th century the concept of evolution was on the evolutionary ladder Man T Apes T Marsupials Reptiles T Amphibia T Fish T Invertebrates Plants T Fungi T Leewenhoek s animacules Thus we still deal in higher and lower eucaryotes I try not to use these terms they are dumb missing links and primitive organisms a In its milieu E coli is as highly evolved as are we E coli is simple 5gtlt106 bp genome we are complex 3gtlt109 bps complexity has nothing to do with evolutionary advancement b Lineages evolve by diversification quotradiationquot not progression c There is no such thing as a primitive organism alive today Simple yes but still a finely honed product of 4 billion years under the selective hammer of the niches that it and its progenetors have occupied 3 By the late 1800s the concept of evolutionary trees was on the ESGN 586 Fall 2007 John R Spear Page 7 table W e g Speeresquot was rst presented m Note torrgdoms of Plants mats Pr norrptarrts and am mlcrobxal arrd onemquot procaryotes m retrospect at base 4 Emst Haeckel 18 ousts mats mostly 55 Note that Darwm39s Cmgm of 1858 P Ildlprnnq himm rprz 2m nLJJHJnIIllatl39 M 124 4 ManuyhylInn SumatraL Dlpnlum a 1 quotWu m3 m h rs as the ve Idngdoms of lrfe39 here taken from a 1969 Scrence lead amcle ESGN 525 Fa 2m7 yomk Spear Ammalla Whmakl 155 MMH a sun mumzquot haeuena bthIs ume as Dngm pmgressmn up a laddemfsuns 12 Num mueh uthersuhecuvny e g whydn fung genu be a hngdnmquot and rule g Alveulates me euuues meansgeuaues Dr suamempues me mums Emun algae eemyeecesw n guess because mmhmnms are large semenmesv e Nam same subtleues by this tune 1 Chlumplas39s mangmmd as denvedfmm Blueaneen algae mwembaeuem 2 Mimhnmina thught pmbably aenveel mm same sun e baeuena had been m e the 1 Centuryquot urcumprehsnswe memew thhls hsimy and cantmversy see I Sapp Ewluunn by Assumaan A Hmury e Symbmsls quot 0mm 1994 Nam that endusymbmuc m1ng furthe nrganelles the an sme e1 sun many pmblems mm Lhs swry axon 525 Fan mm Ydm x Spear 1 Relationships among microbes both procaryote and eucaryote speculative at best 2 N 0 criteria to relate organisms between kingdoms even between eg phyla of animals a universal phylogeny was impossible 3 Implicit timeline remained procaryotes protists primitive 4 Suggestion that eucaryotic nucleus was derived from a procaryote progenitor turned out to be fundamentally incorrect the often cited date of 15 billion years ago for the origin of eucaryotes is BS 5 Studies in molecular phylogeny over the past two decades have changed the paradigm significantly ala Thomas Kuhn The Stucture of Scientific Revolutions a By comparing macromolecular sequences can extract evolutionary relationships evolutionary distances between organisms 5 The goal of molecular phylogeny is to relate molecules hence in principle organisms quantitatively so as to reconstruct their evolutionary histories e g as a phylogenetic tree a There are many ways to quotrelatequot molecules Some subjective ways are e g immunologically fractional gross reactivity e g DNA DNA heterologous hybridization more below But these are difficult impossible to precisely quantitate in terms of relationships b The best way is by direct comparison of sequences of nucleic acids or proteins This provides precise numbers for defining relationships between molecules and ideally organisms 6 For homologous of common ancestry nucleic acid or protein sequences A Consider OrganismA quot39AGCUGCCAGUquot39 ESGN 586 Fall 2007 John R Spear Page 10 X X X OrganismB quot39AAC UCCC AA Uquot39 TDNA OR RNA Sequence A is 70 identical to Sequence B Fractional identity is 07 Fractional di 39erence is 03 1 07 1 Note that the term homology is commonly and incorrectly used when identity is meant Note that gene sequences are not similar they are identical protein seqs on the other hand can be similar 2 You cannot meaningfully compare sequences unless they are quothomologousquot of common ancestry Homologous sequences are not necessarily identical identical sequences are not necessarily homologous e g promoters translation punctuation etc B Do difference 1 identity count for all pairs of organisms considered This difference count is a measure of the extent of evolution evolutionary distance separating the pairs of organisms eg with organisms ABCD and E C To build relationships construct a difference matrix for organisms A E A B C D E A 01 02 02 04 Fractional Difference B 09 02 02 04 C 08 08 01 04 D 08 08 09 04 E 06 06 06 06 Fractional Identity Can relate in a tree like figure a dendrogram ESGN 586 Fall 2007 John R Spear Page 11 Note that organism to node Fractioral distance is 12 of organism Di erenne Pl B c D E to organism distance on 01 02 ul newxxsarily the mu If the Hei Note that this or any other tree is a single dimension you can rotate around any node and have the same topology in an evolutionary sense 1 It is common to see sequence divergence presented in terms of time but this is not legitimate unless you have a fossil record with which to calibrate the line segment lengths Clock speeds of organisms vary the evolutionary clock is not constant 2 The root of the tree the line representing the common ancestral line may or may not be the deepest branch point Most properly the tree should be drawn UHEUCP 1 55 umed quot1mtquotabctve OR 3 E the outgroup quotrootsquot ABCD 7 What molecules to use for molecular phylogeny ESGN 586 Fall 2007 John R Spear Page 12 A Doesn t really matter so long as 1 Homologous molecule occurs in all organisms considered a More specifically you need to know that the molecules are orthologs not paralogs b Orthologs share ancestry and retain the same function in the different organisms c Paralogs result from an ancestral duplication with potentially different functions taken on subsequent to the duplication produce gene families have d For instance the 0L and 5 globins are a gene family they globins have evolved ancient common ancestry the OL type and 5 type independently since the ancestral 0L globins are orthologs 5 globins are orthologs 0L and 5 globins are paralogs duplication e The tree of the gene family would look like Human oc globin Mouse oc globin Frog oc globin Human S globin Mouse S globin Frog S globin f If you did not keep your orthologs and paralogs straight sometimes a tough call when you build the dataset you might get some most unexpected trees eg Human oc globin lt Frog OL globin Mouse globin 2 Need a sufficient number of nucleotides or amino acids to be statistically significant more is always better 3 Changes span evolutionary distance inspected ie compared sequences must not be randomized ESGN 586 Fall 2007 John R Spear Page 13 4 No lateral transfer the evolution of the gene must reflect the evolution of the organisms considered a Genes that are known to undergo lateral transfer are called xenologs If you are interested in metabolic genes there is a good chance at least among Bacteria that you are dealing with genespathways that can move b eg penicillinase and other commonly transferred antibiotic resistance genes 5 Note the evident impact of lateral transfers throughout evolution Much of microbial physiological diversity probably is dependent on laterally transferred genes a Note also that portions of genes can transfer eg with two component systems so that homologous blocks of sequence can show up in functionally unrelated genes Formation of intralineage quotgene familiesquot also results in mixing up functional modules of macromolecules B Considerable work done in past with protein sequences e g cytochrome C hemoglobin 1 But proteins hard to get and sequence it is now easier to isolate sequence genes 2 Most protein genes are shallow clocks eg E coli doesn t have hemoglobin 8 Choice of molecules for comprehensive all organisms phylogeny ribosomal RNAs rRNAs A Ribosome carrys out protein synthesis Small subunit I arge subunit GK 23S rRNA LSU 3000 5000nt 16S SSU rRNA 1500 2000nt 5S rRNA 120 nt ca 25 proteins ca 30 40 proteins B rRNAs present in all organisms and the major organelles mitochondria and chloroplasts C Highly conserved throughout evolution eg ca 50 identity between E coli and human SSU rRNAs over alignable nt ESGN 586 Fall 2007 John R Spear Page 14 1 Length variation between molecules may cause problems must consider only homologous nt more later 2 Note that rRNA while useful for distantly related organisms is not good for establishing resolving closely related organisms it is too conservative Consequently close relatives have too few differences to be reliable e g human apes eg E coli and Yersiniapestis causes Black Plague D Large enough for reasonable statistics E First used for all life phylogenetic trees by Carl Woese University of Illinois 9 Given sequences of multiple organisms ca 30000 now available Align seqs count number of differences is some measure of evolutionary distance Correct for multiple and back mutations Computer fit pairwise evolutionary distances to best fit overall tree topology More detail on all this below 10 The Big Tree emerges Outlines first seen by Woese in 1977 A Note that Woese did not have tree building methods now available In fact he did not have full sequences only short signature oligonucleotides more on signatures later Next page for Big Tree also see Pace Science article B In essence the tree is a quantitative map of evolutionary relatedness a comprehensive map of biological diversity 99 C Indeed a quantitative estimate of that slippery concept biological diversity for any particular molecule might be the summation of all unique line segment lengths in a comprehensive tree 11Some lessons from the Big Tree A There was a single origin for terrestrial type of life all lifeforms related B Three primary lines of evolutionary descent Domains ur kingdoms ESGN 586 Fall 2007 John R Spear Page 15 BACTERIA u a W W P W W WNW m s mp N5 953 w v w q o lt6 x EUCAR A w ESGNSXS mum mm m mm 1 Sometimes see referred to as kingdoms but usage in this context is probably not a good idea too historically loaded 2 You can inject quottimequot into tree but sequence change is not necessarily linear with time indeed probably it usually isn t Eucarya Bacteria Archaea quotTimequot C The eucaryote nuclear line of descent is as old as the quotprocaryotequot lines D Two primary lineages of procaryotes Bacteria formerly eubacteria and Archaea formerly archaebacteria try to avoid using term they aren t bacteria 1 The term procaryote is inappropriate in the light of the relationships 1 Are there still more domain level divergences to be discovered E Note that lines connecting organisms to nodes are not all the same length the evolutionary clock is not constant between different Haloferax vs Methanopyms Aquifax vs Bacillus representative of Archaea or Bacteria lineages eg Eucarya in general vs any 1 Rate of evolution not necessarily the same for a particular at all stages in the evolution of the line eg mitochondrion lineage Agrobacterium vs 2 Note domain level tendencies Eucarya fast clocks ESGN 586 Fall 2007 John R Spear Page 17 Archaea slow clocks Bacteria intermediate rates of evolution 3 Because of variable rates estimating time from sequence change is chancy even fatuous without some sort of calibration F Note that the phylogenetic space occupied by multicellular eucarya is shallow and limited but enormously diverse in morphological less biochemical phenotype A consequence of large highly plastic genomes 1 Note that the typical eucaryote is microbial and has a small genome e g Saccharomyces cerevisiae at 135 X 106 bps E coli at 42 X 106 bps Calothrix a cyanobact at 125 X 106 bps human at 32 X 109 bps Methanococcusjannaschii at 17 X 106 bps G The rRNA and other molecular data prove that mitochondria and chloroplasts were of bacterial origin bacterial divisions Proteobacteria and cyanobacteria respectively H Note how deeply divergent are Giardia Trichomonas and Vairimorpha in the eucaryotic line These organisms lack mitochondria so may have diverged from the main eucaryal line of descent before the mitos came in 1 It turns out they have a few bacterial genes but it is not clear where they got them 12 The three domain Big Tree is an unrooted tree you don t know where is the ancestral node You need an outgroup to root the tree and a universal tree has no outgroup A However the Tree can be rooted using paralogs that arose from duplication before the last common ancestor e g translation factors EF TU and EF G ATP synthase subunits 0L and 5 tRNAs met initiator and met elongator These paralogs occur in all three domains so presumably arose before the last common ancestor Each yields 3 domain tree upon analysis so can use tree with one paralog to root the tree with the other All concur e g Archaea Archaea Eucarya Eucarya Bacteria Bacteria ESGN 586 Fall 2007 John R Spear Page 18 indicating The root of the Big Tree is presumably deep on the bacterial line of descent B This means also that Eucarya and Archaea shared common history after divergence from Bacteria 1 This explains many similarities between archaeal and eucaryal machineries eg similar transcription machineries Archaea and Eucarya use TATA binding proteins whereas Bacteria use 0 factors for specification of transcription initiation eg Archaeal and eucaryal DNA synthetic machineries far more like one another than either is to bacteria for good overview Bernander Archaea and the cell cycle Molec Microbiol 29955 9611998 13 Note that the Big Tree shown is a limited set of specific organisms ca 30000 168 SSU sequences are now available A One database of rRNA sequences Ribosomal Database Project http www cmemsuedu RDP You can download trees carry out functions get programs etc 14 A few domain level trees for reference 15 Bacteria next page A This is a diagrammatic tree The wedge indicates multiple lineages the depth of the wedge the depth of the deepest branches These groups represent the phylogenetic divisions of bacteria referred to as kingdoms by Woese There is no formal taxonomic status of these divisions at this time ESGN 586 Fall 2007 John R Spear Page 19 9 93 lt o m 6 2 g 0 x 4 E 8 o s o O 6 ltgt 3 5 m P Q a K 0Q in c K a 0 BACTERIA 1987 639 m a a o P S A 9 9 w s e o O Q6 OS K i NfrOSpfa ACb39obacle re 006 0718 0 Fibrobacter OP 5 Marine group A 0P9 l Green sulfur Dictyoglomus r fer CyYOPhagaes Coprothermobac 77 ermUSD i m 9 ales em nococc rmotog bad Us The ulo O 5 00 0ch Themquot 9 98 i 9 a P9 Bacteria 2001 Archaea 03910 Eucarya B ca 35 divisions identified so far only ca 25 containing cultivated representatives black wedges open wedges have no cultivated representatives ESGN 586 Fall 2007 John R Spear Page 20 1 Uncultivated representatives identified in the environment by obtaining rRNA genes without cultivation environmental sample gt isolate total DNA gt clone rRNA genes gt seguence You know that the organism is there and get some idea of abundance How to get more information more later 2 Some of the environmental sequences that are abundant in the environment are poorly represented by cultivars or not represented at all eg Acidobacterium group contains only 3 cultivars but is very abundant in many environments 0P1 1 group is very abundant in anaerobic environments at low and high temperatures but has no cultivated representatives C Most of what we know about bacteria is based on studies of organisms representing only a few divisions Proteobacteria E coli Pseudomonas spp purple photosynthetic bacteria the classic quotGram negativequot group Low G C Gram Positive bacteria Bacillus Clostridium 1 w r r J High G C Gram Positive bacteria Streptomyces Mycobacterium Cyanobacteria D Note the expansion in known bacterial diversity over the past few years 16 Archaea A Three main lineages only two Crenarchaeota and Euryarchaeota have cultivated representatives 1 Crenarchaeota All cultivated types are hi gh temperature but uncultivated low temp types are abundant in the environment detected by cloning rRNA genes a Name cren from the Greek for spring or fount referring to the ostensible similarity of such organisms to the earliest ESGN 586 Fall 2007 John R Spear Page 21 life high temperature using geothermal compounds for energy eg H2 SOquot more later 2 Euryarchaeota methanogens extreme halophiles many heterotrophs more later a Name from Greek eury meaning varied referring to variable phenotypes compared to cultivated crenarchaeota 3 Korarchaeota only detected in high temperature environments by rRNA gene cloning Remain to be confirmed as a significant lineage ARCHAEA e 4g ARCHAEA 19237 39 6 I o 0 0a 52 a 04 86 Q S o e 27 ox o OC OO ethanOS 0 an arc Oiloo 0 5 Thermoplasma Crenarchaeota marine low temp Gp 3 low temp Archaeogmkigsg p Euryarchaeota bademo We e GD 90 err 70 JO 1 Korarchaeota 9 370 90 2 860 EUCARYA BACTERIA ESGN 586 Fall 2007 John R Spear Page 22 17 Eucarya Eucaryotes Crown Radiation 000 530 es Nmnes opw Stramenopllles N A Note that the microbial eucs are vastly more diverse than the popular three fungi plants and animals 18 The three domain tree is seen for all molecules in the central information processing machinery DNA RNA protein synthesis the core genes of genetic transfer When you consider molecules outside these core functions eg carbohydrate metabolism amino acid metabolism etc relationships can become weird ESGN 586 Fall 2007 John R Spear Page 23 A Expectation Coherence of domains ArchaeaEucarya to exclusion of Bacteria But Archaea in bold EUCARYA IN CAPS 1 Numbers at nodes are bootstrap valves the of trees in multiple solutions that give that particular node more later B These incongruencies with the Big Tree are generally considered to be the results of lateral transfer of genes between the vertical lines of descent For lots more discussion and the meaning in the larger context of biology see Woese Olsen 8011 Aminoacyl tRNA synthetases the genetic code and the evolutionary process Microbiol Molec Biol Rev 64 202 236 2000 19 What happened in evolution It looks as though the core genome reflected in e g rRNA had genetic continuity throughout evolution Most other things got scrambled A Endosymbiosis involved more than organelles 20 Differentiating features among the domains A The text version of this p 445 is the common stuff to see in texts but is misleading in many points and illustrates the egregious historical context of defining the entire domains on the basis of few not necessarily representative organisms coupled with shabby intellectual underpinnings ESGN 586 Fall 2007 John R Spear Page 25 Summary of major differentiating features among Bacteria Archaea and Eukarya 2 Characteristic Bacteria Archaea Eukarya Pmkaryotic cell structure Yes Yes No DNA present in covalently closed and Yes Yes No circular form 3 Histone proteins present No Yes Yes Membrane enclosed nucleus Absent Absent Present Cell wall Muramic acid present Muramic acid absent Muramic acid absent Li a Membrane lipids Ester linked Etherlinked Esterlinked Ribosomes 705 705 805 5 initiator tRNA Formylmethionine Methionine Methionine g lntrons in most genes No No Yes Operons Yes Yes No 439 Capp39 g and polyA tailing of mRNA No No Yes Plasmids Yes Yes Rare Ribosome sensitivity to diphtheria toxin No Yes Yes i RNA polymerases see Figure 1216 One 4 subunits Several 8 12 Three 12 14 7 subunits each subunits each Transcription factors required 0 Section 68 No Yes Yes l O 4 Promoter structure Sections 67 and 68 10 and 35 TATA box TATA box sequences Pribnow box Sensitivity to chloramphenicol streptomycin Yes No No and kanamycin Methanogenes s No Yes No Reduction of So to H5 or Fequot to Fe Yes Yes No H Nitri cation Yes No No Denitrification Yes Yes No n Nitrogen fixation Yes Yes No 3 Chlorophyllbased photosynthesis Yes No Yes in chloroplasts Li A Chemolithotrophy Fe 5 H3 Yes Yes No 5 Gas vesicles Yes Yes No Synthesis of carbon storage granules Yes Yes No composed of polyBvhydroxyalkanoates Growth above 80 C Yes Yes No u Note that for many features only particular representatives within a domain show the property Comments on table 1 Does this mean no nuclear membrane Can absence be a property 2 Some bacterial chromosomes are linear eg Borrelia Slreplomyces 3 This is a bacterial property Note that not all Bacteria produce muramic acid however eg Planclomyces 4 The nature of membrane lipids is an important distinction Where did eucaryotes get ester linked lipid biosynthesis Considering that Eucarya and Archaea are sister groups to the exclusion of Bacteria What are the two possibilities 5 S refers to size sedimentation rates Most eucaryotic ribosomes are bacterialarchaeal in size Only multicellular ribs are large ESGN 586 Fall 2007 John R Spear Page 26 6 Re ects conservation of translation apparatus AE to exclusion of B 7 Most eucaryotic genes do not have introns Note this refers to splicesomal introns and such introns are rare in microbial eucaryotes abundant only in multicellular genomes 8 This correlation is unclear e g nematodes have operons 9 Eucs are unique in capping but Bacteria and Archaea also add poly A tails The mechanisms for poly A deposition may be different from Eucarya 10 Fundamental difference between EA and B 11 NH3 gt N037 as E source 12 N037 as electron acceptor to reduced N or N2 13 Eucarya did not invent got from cyanobacteria this is evolutionarily speaking a symbiosis 14 Chemolithotrophic Eucarya do exist by virtue of bacterial symbionts e g hydrothermal vent associated animals BBM section 1612 read If this item is no for eucs so should be 13 15 In summary these quotdifferentiating featuresquot are anecdotes not fundamental properties 16 Although microbiologists had long hoped for a natural classification by the 1950s they had abandoned the possibility some even declaring it 39 quot 39 When J quot r ESGN 586 Fall 2007 John R Spear Page 27 methods became available it is ironic that the systematists were main hold outs in embracing the results For an interesting overview of the period of paradigm shift in the Kuhnsian sense check out Woese CR There must be a prokaryote somewhere Microbiology s search for itself Microbiol Rev 58 1 9 1994 7 For an interesting contrast between the way that the traditional systematist and the modern microbial biologist look at life from the larger view peruse Meyr E Two empires or three PNAS 95 9720 9723 1998 Mayr vs Woese Woese CR Default taxonomy Ernst Meyr s view of the microbial world PNAS 95 1 1043 11046 1998 Woese vs Mayer PDFs for both are on the Web site ESGN 586 Fall 2007 John R Spear Page 28


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