Introductory Microbiology MMG 301
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MMG 301 Lecture 20 Respiration Questions for today 1 2 97 What is Respiration and how does it differ from fermentation What substrates are oxidized and which reduced aerobic vs anaerobic respiration How are electrons transferred from the electron donor to the electron acceptor How is E generated What are examples of respiration How do we calculate the energy available from respiration Overview of Respiration M substrates are required A amp B One substrate A organic or inorganic compound is oxidized The second substrate B 02 nitrate sulfate or other compound is reduced This is referred to as the final electron acceptor Electron transfer reactions drive ATP synthesis by Electron Transport Phosphorylation ETP little if any SLP A ataaa P e BaaQ Chemoorganotrophic Respiration an organic compound is oxidized typically to 002 and the electrons transferred to oxygen or another acceptor 002 wanna Carbon flow ATP a Proton Electron mauve flow tome Biosynthesis 02 504 Organice Aeroblcresplratlon acceptors so Anaerobic vespiralion Chemolithotrophic Respiration an inorganic compound is oxidized and the electrons transferred to oxygen or another acceptor s 02 N0 503 Biasynthesis Note the cleardifferences from the situation in fermentation Examples of Substrate Oxidation A Pyruvate oxidation Many organic compounds are converted to gyruvate eg glycolysis Each pyruvate is oxidized to 3 002 by the Citric Acid Cycle NM CGA Pyruvate three carbons ccz w Electrons reduce 4 NAD 1 FAD to form 4 NADH and 1 FADH2 that are used for ETP 1 GTP made by SLP used to form 1 ATP GTP B AcetylCoA oxidation Various organic compounds including fatty acids are decomposed to gt acetylCoA Oxidized via Citric Acid Cycle with electrons going to 3 NAD and 1 FAD C NonCitric Acid Cvcle oxidation of organic compounds Example Respiration of single carbon compounds s79 multiple electrons used for ETP D Oxidation of Inorganic substrates H2 gt 2 H 2 e39 used for ETP H28 gt 2 H 80 2 e39 used for ETP N0239 gt N0339 e39 used for ETP Fe2 gt Fe3 e39 used for ETP each pathway for C and D Will require a separate series of enzymes for the speci c oxidation reactions Electron Transport Chains Electrons derived 39om substrate are handed off through a series ofelectron c rn39ers39 A P shovm last time 2H and 2e39 avins Quinones Q lsaniiaxanne Mg a swan chrchC CHnlnH Ha N 0 a H H N N i w m mm m cm quot Ioi 0H EH mm u i Hz 0H NONOHOH Ironsulfur FeS clusters Cytochromes Cyt only electron transfer mo 94 mm wwm M mm These cofactors or proteins containing them are positioned in or on the cytoplasmic membr in a manner that allows for a bucketbrigadequot transfer of electrons while transferring protons across the membrane proton motive force PMF The electrons are nally used to reduce the nal electron acceptor substrate 02 aerobic respiration or nitrate nitrite sulfate fumarate ferric ion a other compounds anaerobic respiration Each pathway requires a distinct series of electron transfer proteins and a unique oxidase Example aerobic respiration in Paracoccus denitri cans and Escherichia coli Membranes allow buildup of drives ATP synthesis via ATP synthase I II I wow Putting it all together examples of Respiration Aerobic Respiration of Glucose Glucose A is metabolized to pyruvate via glycolyis and oxidized to 002 by the citric acid cycle NADH and FADH2 feed electrons into membrane bound electron carriers that result in protons being pumped Electrons oxidize oxygen B to produce water PMF drives ATP synthesis Aerobic respiration of other carbon compounds Alternative pathways are used for oxidation of the carbon compound to provide reduced NAD and FAD eg CH4 3 GallCatban 0 i H30 GHgDH GHEJS39r I HUGOquot M 39 39 39 1To C 103 Aerobic Respiration in Ll otrophs occurs by an analogous system An inorganic substrate is oxidized eg hydrogen sulfide nitrite ferrous ion Electrons feed through an electron transport chain to generate PMF Oxygen is reduced to water Hydrogen oxidation Nitrite oxidation nitrite oxidoreductase sul de oxidase Ferrous ion oxidation 398 F 4 not the detailed Aw pathwa s In Law a Anaerobic Respiration something other than oxygen is reduced eg nitrate sulfate Jmarate etc Denitri cation Energygenerating nitrate reduction in E coli vs Pseudomonas Note that some portions ofthe electron transport chain may be identical to that used for aerobic n resplratlo Dissimilative sulfate reduction Energy generation by anaerobic respiration of sulfate Lactic acid or other carbon compound is Lactate LDl ll OXIdIzed With A WWEI gate the electrons r reducmg sulfate to form sulfide Only carried out by one closely related group of microorganisms the sulfate reducers Other electron acceptors used for anaerobic respiration fumarate 2H 2e39 a succinate dimethylsulfoxide 2H 2e39 a dimethylsulfide ferric ion e39 a ferrous ion many others Measurement of Energy Direct calculation of AGquot Glucose 6 02 gt 6 002 6 H20 917 kJmol 0 394 kJmol237 kJmol AGO 63946 237 917 2869 kJmol 10fold that available via fermentation Find 38 ATP are made 2 during glycolysis 2 GTP from citric acid cycle and the rest via ETP Efficiency 100 3832 kJmol2869 kJmol 42 This approach also works when using different electron acceptors le types of anaerobic respiration and when using different substrates to be oxidized eg H2 H28 N033 etc However the values of free energies are not always readily available see table A1 1 Is there an easier way to calculate energy for these reactions culation of energy by AEA measured in V These reactions all involve two halfreactions donor substrate r product1 acceptor substrate product 2 E0 is the tendency of a compound to become reduced in units ofV AEO EO electron acceptor EO electron donor Using hydrogen gas as the electron donor Fumarate 0343 046 V Nitrate 04243 085 V Oxygen 8243 1 25 V Electrons flow down Guru ple t3 Fumarateisuccinate 003 2 e Cytochrome named mama 1 3 aws 02 1 a pH 7 2 Ubiqui oneomd 011 2 e Cyboo hmrne l3an 5125 1 B Cytochrome sumac 039 31 equot l martyr042 2 a Eul M 4350 a 4340 4330 D2D D1D 00 010 020 6 030 040 050 060 070 3 080 090 How do these values compare to AGO AGO nFAEO F 9648 kJmol n number of electrons So we can compare the three reactions AEO AGO H2 fumarate gt succinate 046 V 85 kJmol H2 N03 gt N02 H20 085 V 160 kJmol H2 12 02 gt H20 125 V 240 kJmol Can estimate the ATP available from such processes on the basis of 32 kJmol 016 V for a 2 electron process 1 0 Fluorescence microscopy AIs used to detect Gramstained bacteria BProduces high contrast 3D like images CIs generally used for extremely thin cells such as T reponema pallidum DCan be used with certain unstained cells as well as with specially stained cells E Requires staining with electrondense metal ions According to the following multiple alignment of partial rRNA sequences isolates C amp D are identical Which of the remaining isolates is closest to the C amp D pair Isolate A AAUCUGGGUCUA Isolate B AAACCGGGUCUA Isolate C AAAUCGUGGCUA Isolate D AAAUCGUGGCUA Isolate E AAAUCGAGGCUA AA BB C E DA amp B are equally distant to C amp D E B amp E are equally distant to C amp D MMG301 Dr Frank Dazzo Aguatic amp Wastewater Microbiology 0 Natural aquatic habitats for microorganisms include lakes ponds rivers springs oceans estuaries marshes o The concentration mixing and movement of nutrients 02 and waste products are the dominant factors controlling the abundance distribution and diversity of aquatic microbial communities Trophic levels and the microbial loop in aquatic ecosystems In contrast to soil phytoplankton algae and cyanobacteria are the predominant photosynthetic organisms in aquatic habitats Much of the organic matter synthesized by phytoplankton during photosynthesis is released as dissolved organic matter DOM x This DOMis consumed by Z onhnkhn bacterioplankton 79 J which become part of the suspen g a particulate organic Bl s uf matter POM pool col and manuals o A portion of these 6 bacteria are then consumed as food by protozoa predators Some ofthe nutrients immobilized in bacteria and protozoa are mineralized and then assimilated directly by phytoplankton without transfer to highertrophic levels eg sh in the a uatic ecosystem This is called the quotmicrobial loopquot arrows in red 02 plays a key role in microbial activity in aquatic habitats Photic zone oxic zone anoxic zone In lakes cammunity2 Community1 Cch zone PhO E zone Chemoorgarwtwphic L cyanobacleria and algae actena a 002 6 H20 CeHizoa 6 02 a l c Huoi 4 5 oz 5 coz is H20 Anoxic zone quot molhanogenia bacteria Eutrophic lake Oligotrophic lake Tree Leaves Rmmmmo s v 5020rganic matter pho nsynthe c activity water run 02 and organic nutrients are inversely interrelated in aquatic habitats Nutrientpoor oligotrophic A lakes recycle nutrients only within the water whereas nutrientrich eutrophic B lakes have major nutrient inputs from outside Oligotrophic lakes are typically Ozsaturated and have low microbial populations Eutrophic lakes develop high microbial populations that deplete the dissolved 02 by aerobic respiration during decomposition of abundant organic matter producing deep anoxic zones Temperature also impacts on the status of 02 and nutrients in temperate lakes Lag 3 Su ace 393 39 quotii 7 7 m I I 2 02 I Temperalune l 4 139 Epillmnian E 39 5 E I s I l l l I l I 14 i 1 I l l 1 Warrants 15 D 2 4 6 3 19 mg Qg iler a 1 2 a 4 5 mg Wit 0 4 a 12 1a 20 Temp Cl Anoxic conditions develop in the depths of the lake as a result of thermal stratification The cool bottom waters hypolimnion are more dense and contain H28 from anaerobic bacterial sulfate reduction The depth zone of rapid temperature change is the thermocline Typically as lake surface waters epilimnion cool in the fall and early winter they reach the temperature and density of the hypolimnionic waters and then they sink displacing bottom waters and the sediments affecting lake turnover and the redistribution of nutrients for the aquatic microorganisms Marine microorganisms eubacteria archaea and eukaryotes a True marine microorganisms are moderate osmiophiles requiring the salinity and ions esp Naquot of seawater for cultivation Most are psychrophiles 5 C for most of ocean volume Barophiles in deep sea hydrostatic pressures g 1100 atmospheres Most are oligocarbophiles adapted to the extremely low concentration of organic C in ocean seawater 12 mg C I liter Recent exciting find of endosymbiotic bacteria inside Riftia tube worms that develop on sides of black smoker vents at deep sea ocean floor a life sustained by geothermal energy rather than sun Black Smoker Hm vent 350 C Vna imnbum 1t Sinful minnowm9 axmhn Tmulwsuc39m cell NHEH39 Wain cycle Fiaim lCl lE rl gure at We lube Warm Bacterial ltdntinmluipt an A cummmilly nl39 rub wcium Mimi Wdlyrn ifa M IIquot Rm hydrmMmul vent sitciidtplh 1550 111 Each worth is mm than 1 malcr39m Imam and 1115 1 33cm gill plums db cl Sclmmaiic illusu alltin of hip anntmniml and Mysinlngital mguuzvlium ul ll39ie mine warm 39l lm animal 5 1 anchnrul intii c m pmluclivc mbc Im till 39u diilncl39l ml AL is antctiw ml is m remain on gill paumni insult the trunk V 39 ul tin worm is a lmphmsomc consisli Hg minwily nf mutinymlikmc hametia associan cells 11nd Mm 192 All d1 If T a pnmlinrcnd oiquot the animal 5 tin epistbmum whiiil meltnuts Lhawnrrn in in what div ltygmn inniton dimidc and 39 quot 4quot I hymen sum as alang 39Imzlugh Ilia gill plum and mponvd will trlmd cells m39tbc Hpqlmgm39i mi sul de is manual 1 like warm 395 lmmglbhin ilHSHbO 1 quotIii timed to me endosj39mbinm The bacteria midi the mme sul de and use 5mm oi lhr rel33ml eneim39 In x CU innu Cidaim We Same fraztium OEIIK milch wbun wmwunds smlmiml by the endasylmimu is mhmm In Eli Mi Imfs diam Concept of Biochemical Qxygen Demand The amount of biologically usable organic carbon in water is indirectly measured by its Biochemical Qxygen Qemand BOD It represents the portion of total carbon that can be oxidized by microorganisms in a 5day period under standard conditions It equals the amount of dissolved Oz needed for microbial oxidation of biodegradable organic matter in a water sample The effect of a point source discharge of an organic pollutant eg untreated sewage into a clean flowing river system is profound Hera ue k Of Jquot Algae and cyanobaclaa a cells or some 02 f G of nutrients quot 1 J a I ff t VIM1 f a a x f 39 Off2x m3 i quot 3939gt quot39 39 NHfandP fquot Bacteria organic cam and EDD Distance downsh39eam n input saw other waslewaters Heterotrophic bacteria organic carbon and BOD immediately increase at the pollutant input and correspondingly dissolved Oz levels decline due to the burst in microbial respiration Kills all aquatic life fish etc dependent on dissolved Oz Microbes mineralize and oxidize the organic N and P into inorganic nutrients NOa39 NH4 and PO4393 resulting in eutrophication with development of noxious algal I cyanobacterial blooms Further downstream selfpurification processes result in a decline of BOD the oligotrophic conditions and phototrophic microbial communities regain dominance and dissolved Oz levels replenish Microbiology of Domestic Sewage Wastewater Treatment I Sludge insoluble V Eigested sludge drying we as fertilizer or The treatment of human fecal wastes a organic matter plus many bacterial protozoan amp viral pathogens is one of the most important factors in maintaining an advanced healthy society Effects of discharging organic wastes into aquatic ecosystems can be drastic as described earlier Fecal pathogens are shed from patients with disease and from carriers maintain infection without expression of symptoms Conventional sewage treatment is a controlled intensification of natural selfpurification processes involving 1 2 and 30 treatment Flaw sewage Primary treatment removal of insoluble particulate materials from raw sewage by screening gravitational settling in tanks The resultant solid material is called sludge Secondary treatment microbial conversion of organic matter into microbial biomass and final decomposition products 90 95 reduction in BOD plus removal of many bacterial pathogens Tertiary treatment biological and chemical removal of inor ganic nutrients eg N and P to reduce eutrophication of receiving ecosystem virus removal or inactivation trace chemical removal 39 Primanr treatment Secnndary Mentath treatment incineration burial Tertiaryr Treatment Nutrient removal Treated ef uent to stream in secondarv treatment of domestic sewaoe Role In Activated sludge process Microbes eg Zooglea ramigera in a forced aeration tank form zooglea of activated sludge active biomass of suspended flocs that quotWe tram Dnma39y elment A ratlon tank Seuling m Adwamd sludge return Excess sludge lnpulelvlamlwm Helemlmvh WWW gmmm 39 Tricklin filter s stem a rotating arm of an aeration basin trickles wastewater over a bed of rocks Each rock develops a large microbial bio lm that sorbs and aerobically ecomposes the dissolved l mum organic matter in the Q m wastewater as it trickles over the rocks Dimbunon mm Rock Anaerobic anoxic sludge digestion process Anaerobic 3 The bioreactor has a lid cover to sludge digestor 139 maintain anoxic conditions A very I complex community of anaerobes actively decompose polymers by the processes indicated to final anaerobic metabolites dominated by methane CH4 and 002 as the 391 major products of anaerobic bio degradation The CH4 is burned or used to power the treatment plant 39 if Scum removal Supernatant removal l Hydrolysis by Fermenl39atlon Fermentation n H H305 bl Sludge outlet Cl MMG 301 Lecture 22 Bioenergetics Comparisons of Enerqv Mechanisms Oxygenic Photosynthesis ATP and NADPH are made in large amounts Requires a very complex machinery Produces oxygen toxic Anoxygenic Photosynthesis ATP made in large amounts NADP reduction requires an external reductant Fairly complex machinery no oxygen made Aerobic Respiration organotroph or lithotroph ATP and NADH are made in abundance Requires electron transport chain and oxidase Requires oxygen toxic Anaerobic Respiration ATP easily made and NAD easily reduced Requires ETC and terminal reductase Requires external electron acceptor Fermentation Little ATP no net NAD reduction no membranes MOST SIMPLE SYSTEM Multiple options in a single microbe Some microorganisms grow via a single bioenergetic mechanism eg only ferment or only carry out oxygenic photosynthesis Other microbes are more versatile eg a few microbes can grow by fermentation aerobic respiration anaerobic respiration and anoxygenic photosynthesis When capable of growing by multiple approaches the different systems are generally tightly regulated eg only make photopigments when growing phototrophically In these cases the mechanism used is typically the one yielding the maximal amount of energy under those growth conditions eg aerobic respiration gt anaerobic respiration gt fermentation How is the energy used Consider autotroghy 1 Aerobic phototrophs plants algae cyanobacteria and oh emolithotrophs nitn ers sulfur oxidizers x 0 2 using the a Vin ycle key enzyme nbulosebisphosphate carboxylase NADFH mm lighl leschanx or sometim es vevevse electron llowl h ese ATP Iran light mums enzymes are fou n d in an inclusion iag zm am called a as Camus am xys me Qlwevalex 12 Glycemlda wde s Ribuloss Gphosnhate 5phusphale las camans lan gamma 3 ATP 4 939 quotWWW lUGlyceraldeMde gramme Per c 02 an carbons 2 Two other less energyexpensive method for xing CO2 are found in Green Sul Jr Green Non sulfur and many other microbes Reverse Citric Acid Cycle W mulcvulkwwsuw k m up nnwmmmm Lquot AW N eed extemal reductant Hydroxypropionate Cycle Gimmi wymmymwwm Emu v 3 Some strict anaerobes x CO2 using the Acetyl CoA synthase pathway 4 H2 2 02 4 CH3000H4gt other building blocks Fm mwmm gmrpm Trg A mrx mum ngduc m39 afcozto mm H 0 my I 2 Ex I t Ra t li Jillm swam v cm mm cmymm lt up myquot 1 AYP cHrorO39 71 sew Acume ATP AcetyeroA e ATP is used then recovered so no net ATP Requires lots of an external reductant hydrogen gas Bottom line Some forms of autotrophy require huge inputs ofATP and NADPH Other Energy needs in a cell In addition to autotrophy energy is needed for synthesis of all other cellular components Consider Composition Percent d weight Protein 55 RNA rRNA tRNA mRNA 205 DNA 31 Lipid 91 LPS 34 Murein 25 glycogen 25 other organic 29 inorganic 1 Costs of biosym hesis from monomers Cmpd ATPg cell NADPHg protein 7287 11523 RNA 6540 427 DNA 1090 200 lipid 2578 5270 LPS 470 564 murein 248 193 glycogen 154 0 for E coli growing in glucose minimal medium from Neidhardt et al Physiology of the Bacterial Cell MMG 301 Dr Frank Dazzo Microbial Ecology PlantMicrobe Interactions Associations of Soil Microorganisms with Vascular Plants Topic areas General colonization phyllosphere rhizosphererhizoplane Specific beneficial associations root nodulation mycorrhizae Detrimental pathogenic associations crown gall tumorigenesis Plants secrete various organic compounds resulting in a nutritionally enriched environment favorable for microbial growth As a result plants are heavily colonized with a diversity of microorganisms whose reservoir is primarily the soil Microbes that colonize plants are called either epiph es colonize plant surface or endoph es colonize plant interior Microbial communities influence plants in direct and indirect ways commensalisms mutualisms amensalism and pathogenic consequences Phyllosphere aerial leaf surface of plants 0 Communities of microorganisms that develop on the phyllosphere are adapted to tolerate high irradiation and low humidity stresses 0 Many phyllosphere microorganisms antagonize airborne pathogens thereby protecting the plant I f gt A SEM of phyllosphere bacteria and fungi colonized on corn leaf surface Rhizosphere and rhizoplane colonization by microorqanisms 0 Plant roots secrete various nutrientrich compounds eg sugars amino acids vitamins organic acids into the surrounding soil This process called rhizodeposition can amount up to 25 of newly fixed photosynthates This nutritional enrichment around roots creates unique environments for soil microorganisms including the rhizosphere that volume of soil around roots influenced by root exudation and the rhizoplane the immediate root epidermal surface that interfaces the rhizosphere soil Epifluorescence micrographs of bacteria colonized on the white clover rhizoplane developing in soil Acridine orange laser scanning confocal microscopy Microbial communities that develop in the rhizosphere rhizoplane differ from microbial communities in bulk nonrhizosphere soil 1 Population sizes are higher in the rhizosphere 2 Dominant species rhizosphere dominated by fastgrowing predominantly culturable aminoacid requiring microaerophilic Gram negative rods e g Pseudomonas Bulk soil dominated by Gram positive rods I dwarf cocci predominantly non culturable or grow slowly with complex nutritional requirements satisfied by soil organic matter eg Arthrobacter Nzfixing Rhizobiumlegume rootnodule symbiosis 0 Several genera of soil bacteria form a symbiotic relationship with speci c legumes podbearing angiosperms that develop N2 xing root nodules Groups of rhizobial species or biovars that speci cally nodulate the same legume host are called crossinoculation groups Legume host Rhizobial crossinoculation group many clovers Rhizobium Ieguminosarum biovar trifolii peas vetch R Ieguminosarum biovar viciae common bean R Ieguminosarum biovar phaseoli R etli R tropici soybean Bradyrhizobiumjaponicum B elkanii R fredii alfalfa Sinorhizobium melilo ti lotus Mesorhizobium ot39 39 bium caulinodans esbania Ala 10 neptunia aquatic Allorhizobium undicola o This N2fixing symbiosis is of major importance to agriculture because N is the nutrient most commonly limiting plant productivity and legume crops can offset that limitation by forming an ef cient N2 xing symbiosis with Rhizobium ln Nature legumes are nodulated by both effective and ineffective strains of rhizobia Effective rhizobial strains symbiotically x N2 whereas ineffective strains don t o Legume crops inoculated with selected strains of rhizobia in a commercial inoculant help to ensure that an effective efficient N2fixing root nodule symbiosis results reducing the crop s dependence on chemical Nfertilizer to achieve high yields By proper placement and timing of inoculation on seedjust before planting the rhizobial inoculant gains a preemptive colonization ofthe root and successful competition for nodule occupancy Symbiotic rootnodule development involves various complex cell cell interactions defined at cellular and molecular levels 1 Roswmlnnn m anachmcnl lllnczJesln has ieuin 2 Excmimn 74 Nod rams w mum musing mm m cmng 3 invasion mnmmapenmralc m m and quotmuply mm inlechow Nud a ammmmcum Imam mm mm Vamps mmum mmau slimuln ed in am 5 Favmamu m bnclemid Stale mmquot mm om unmadel Yam m 5 Comme Mam m bilchle v dmsiar FIGURE me mm m the fonmhon oln mot nodulc m a gume mama by mm 0quot phohllukmgmph ofnn mum lhrmd m rug 5 77a 0 Molecular communication between the rhizobial and legume symbionts is mediated by signal molecules that activate expression of genes required for the symbiotic pathway Rhizobia a Plant Flhizobial quotaugmmm Plant a Rhizobia cup 0 m m willowquot cmmv o chH osoH cH o 0 H0 0 0 H0 504 H H0 504 HO 5m 0quot co co co cm 0 o ac R o ew an em m R momma a hnduum EH hizobial Nod factors u Chilolipooligosaccharides a sha e lue portion active ch l k The host legume root secretes phenolic compounds called avonoids These are taken up by the rhizobial symbiont where they activate expression of various symbiotic plasmidencoded nod Mulation genes Some ofthese nod genes encode enzymes to synthesize a special class of glycolipids chitolipooligosaccharides These signal molecules vary so in tructure but their non reducing end containing a Nacyl longchain fatty acid is bioactive in the plant host triggering root hair deformations and cortical cell divisions within the root leading to nodule formation 0 Within the root nodule the bacteria are released from infection threads into the host cell while still enclosed within a host derived membrane called the Eeribacteroid membrane They then divide and transform into enlarged pleomorphic bacteroids and make the enzymatic machinery which carry out Nz xation The entire endosymbiotic structure is called a symbiosome Infection muma To rum Metabolic reactions involved in Nz xation by rhizobia within legume root nodules Some rhizosphere bacteria promote plant growth plant growth promoting rhizobacteria PGPR by various mechanisms independent of root nodulation Inoculated Uninoculated a O39 O fixation and solubilization of nutrients so they can be utilized by plants eg N2 gt NH3 insoluble P gt soluble P production of bioactive growth stimulating hormones eg auxins gibberellins that expand root architecture see example so it is more efficient in uptake of plant nutrients from the soil reservoir antagonism of soilborne root infecting plant pathogens resulting in suppression of plant pathogenesis Most PGPR only colonize the rhizosphererhizoplane quotassociativequot interaction others are more invasive and establish an intimate quotendophyticquot interaction Examples Azospirillum brasiliense and wheat Acetobacter diazotrophicus and sugarcane Azoarcus and kallar grass Also Rhizobium and cereals eg rice rotated with legumes See Brock 10quot edition p 691 Heterolroph BNF Legume Root Nod ule ndosym biont Endulcalonizer Rhizobium life cycle in LegumeCereal Rotations Mycorrhiza Fungusplant root gymbiosis 0 Very common nearly universal roots of 95 of vascular plants are normally involved in mycorrhizal symbiotic associations Several different types most common are 0 Ectom corrhiza form a sheath around the root without penetration into plant cells normal case for many gymnosperms eg pine o VesicularArbuscular endom corrhiza invade plant root ce s associated with many angiosperms eg many agricultural crops 0 Distinguishing morphological features Ectomycorrhiza Vesiculararbuscular mycorr 39za endomycorrhiza Fungi establish mulually bene cial relationships with plam mols called myconhlzae Real cross t sections illus rate omerem mycorrhizal relationships 0 The plant provides a steady supply of photosynthetic organic nutrients to feed the mycorrhizal fungus o The fungus provides increased surface area for absorption of plant nutrients eg phosphate and water from the surrounding soil and provides them to the plant 0 The mycorrhizal fungus also protects the plant root from invasion by soilborne rootinfecting pathogens Major Plant Diseases Caused by Bacteria and Fungi smptoms Host amp D ease Pathogen acter Spots amp hllghts Bean halohlight Pseudomonas syringae Vascular wilts Apple wilt Erwinia amylovora anana wilt Bu holdana solanacearum Soft rots Potato black rot Erwinia caratovora 0 39 39 Pseudomonas l Canker Citrus canker Xanthomonas campestris Crown Gall Tumor numerous Agrohaderiun tumefaciens Fungh Puecinia gram Rusts W eat mst 39m39s Necrotl fa Ph ogh ro nfestans h 39c rot Potato mine rai several other plant diseases are caused by vlruses Crown gall tumors on tobacco made 39 MMG 301 Lec 34 Viral Diseases Questions for today 1 How does the life cycle of a virus infecting eukaryotic cells differ from that of bacteriophage 2 What are the major viral respiratory diseases 3 What are the major sexuallytransmitted viral diseases 4 What are other key viral diseases 5 What do we know about emerging viral diseases for more information see the Centers for Disease Control and Prevention web site at httpwwwcdcgov Life Cycle of Viruses that Infect Eukaryotes Most eukaryotespecific viruses are enveloped often with virusencoded proteins embedded in the lipid bilayer Envelope N TCapsid Enveloped virus Viral Attachment and Penetration Attachment involves specific interactions between typically protein components on the virus and the host cell In contrast to bacteriophage the entire virus typically enters the eukaryotic cell Entry may involve fusion between the host I membrane and the membrane surrounding the virus Alternatively the virus may enter by endocytosis 1 2 132 I Replication of viruses in Eukarvotes Typically much more complicated than for bacteriophage due to cell compartmentation eukaryotic DNA synthesis occurs in nucleus protein synthesis in cytoplasm Some viruses replicate in nucleus while other replicate in the cytoplasm Splicing and RNA modifications are often found Reverse transcriptase a polymerase using RNA as the template to make DNA is critical to some RNA viruses Effects on host cells w quotmy Vira Release from Host Cell Although lysis may occur a more common exit strategy involves budding envelope is from host Host cytoplasmlc membrane Wus Damcle Viral Respiratory Diseases Common cold Very All otherlnfeciinus diseases common Cases per on people per year About 50 of cases caused by the ssRNA rhinovirus remainder from other viruses Over 100 different serotypes varieties identi ed by antigenic properties each with varying degrees of pathogenicity Aerosol transmission from infected host to the next also through contact by hands WASH Infects upper respiratory tract to produce rhinitis in ammation ofnasal mucosal membranes nasal obstruction and discharges usually no fe Resi ta c 0 man immune defenses virus surface contains 4 basic antigens and 89 variants WOWC Seve a r drugs in development based on inhibiting virus uncoating and replication process lt39 don t memorize Influenza ssRNA enveloped virus that can undergo antigenic shift to elude host immune system Classified into A B or C groups based on the antigens in their protein coats Infection in upper respiratory tract usually through inhalation of droplets from another infected person Symptoms include fever chills aching Secondary bacterial infections can be a problem in infants and elderlv Responsible for annual minor epidemics and several pandemics worldwide epidemics 39 4 he if 391 ma Animals are common reservoirs as sources for new strains often new strains originate in Asia After a strain of influenza has moved through a population most people are immune New influenza strains develop because of antigenic shift segmented RNA genome allows shuffling and mutations of two major antigens that are on the surface of the virus envelope Can get annual vaccinations for newly developing strains epidemiologists try to keep track of which strains are coming in the next year or two Measles Mumps Rubella MMR vaccine lifetime immunity Measles rubeola virus cough fever nasal discharge eventually a rash develops Mumps as with measles is spread by airborne droplets Characterized by inflammation of salivary glands swelling of neck Virus spreads throughout body occasionally leading to complications like sterility encephalitis Rubella German measles milder symptoms than measles infection of fetus can result in stillbirth heart eye and brain damage Chickenpox Caused by varicellazoster virus a Herpesvirus highly contagious by airborne route Develops characteristic lesions on face and upper trunk erupt fill with pus rupture and covered by scabs Virus DNA can remain in nuclei of nerves and sensory neurons Viruses become activated later in life to develop shingles eg Dave Letterman Sexually Transmitted Viral Diseases Herpes type cold sores and fever blisters in and around mouth or lips Herpes type 2 genital lesions and blisters of penis cervix vulva vagina Transmitted by direct sexual contact Incurable treatment with drugs acyclovir Oral herpes no long term effects Genital herpes linked to cervical cancer Can be passed to newborn Acquired Immunodeficiency Syndrome AIDS Caused by Human Immunodeficiency Virus HIV Two types now recognized HIV1 responsible for 99 of all AIDS HIV2 similar to HIV1 but less virulence HIV infects host cells that have surface proteins called CD4 and CCR5 High CD4containing host cells found in immune system Lower CD4 cells in brain and intestinal cells The other host cell protein CCR5 is also involved in binding of the virus and fusion The viral protein gp120 binds to these host proteins HIV is a retrovirus thus it requires reverse transcriptase N o cur e several antiHIV drugs when used in combination can control virus multiple drug thera Progression Note the opportunistic infections a HOCH m N Oman 3 a mammal H ueoxynbose H C Reverse t inhibitors analog of DNA bases or inhibit in other ways Protease inhibitors pre sing of polyprotein to individual proteins and enzymes lt m E 392 AIDS is theoretically 100 preventable Today 40 million people are estimated to be living with HIVAIDS Of these 372 million are adults 176 million are women and 27 million are children under 15 An estimated 3 million new cases in 2001 including 11 million women and 580000 under age 15 95 of all AIDS victims live in developing countries Total AIDS cases and AIDS deaths Unile Slates Vaar Other key viral diseases Hepatitis hepaticus liver Eight types are currently known 5 are well characterized A B C D E We will focus only on one example here Hepatitis B serum hepatitis transmitted through blood transfusions unsterile drug users needles body secretions sexually Symptoms jaundice fatigue abdominal pain Hepatitis B vaccine available combined A and B forthcoming 300000 new casesyr in US 5000 deaths from cirrhosis of liver 1000 from liver cancer 200 million infected worldwide Viral gastroenteritis caused by rotavirus as well as others Mainly transmitted through contaminated food fecal oral route Symptoms include diarrhea vomiting Diarrheal diseases cause 510 million childhood deathsyr many are viral in origin lnfects infants 1 mo 1 yr in age 35 million rotavirus casesyr in US Emerging viral diseases CDC priority viruses Viral hemorrhagic fevers Ebola Marburg Hanz a viruses Are all RNA viruses Are dependent on animal or insect reservoirs Most cause severe life threatening disease Marburg virus Earliest cases seen when West German scientists became infected with a new virus from imported monkeys from Uganda Marburg viral hemorrhagic fever Symptoms included bleeding hemorrhaging as well as blood clots damage to retina Ebola virus First Ebola appearance 1976 infected 1000 and killed 500 a 1995 outbreak was more quickly contained Hanz a virus An outbreak in southwestern US in 1993 of a new type of hantavirus causing hemorrhagic fevers resulted in 30 deaths from hantavirus pulmonary syndrome due to destruction of lungs MMG 301 Lecture 21 Photosynthesis Questions for today 1Which microorganisms carry out photosynthesis 2 How is light energy absorbed 3 What is oxygenic Photosynthesis and how is ATP and NADPH made 4 What is Oxygenic Photosynthesis and how is ATP and NADPH made Overview of Photosynthesis who and how when not My enciosed oniy some members are phototrophs Archaea Some ofthe Extreme Halophiles use bacteriorhodopsin to pump protons Lec 5 Eukamotes Green plants and algae use chloroplasts derived by endosymbiosis to carry out oxygenic photosynthesis ie produce oxygenLec 6 eg Chlamydomonas Bacteria 4 groups 1 Cyanobacteria Gramnegative carry out oxygenic photosynthesis ancestor of chloroplasts eg Oscillat 39 2 purple bacteria dispersed within Proteobacteria Gramnegative carry out anoxygenic photosynthesis no oxygen made using c onents localized to invaginations ofthe cytoplasmic membrane called vesicles or lamellae Rhodobacter 39 3 green bacteriaquot two phylogenetically distinct groups sulfur and nonsulfur with the green sulfur bacteria often having granules of sulfur within or attach ed to the cell possess chlorosomes with nonunit Membranes eg Chlorobium 4 Heliobacteria Grampositive carry out anoxygenic photosynthesis using components localized to cytoplasmic membrane eg Heliobacterium Photopigments a colorful story Li ht abso tion b microor anisms different microbes absorb light energy at distinct wavelengths base on the pigments they contain This has important ecological implications Green Alga Purple Bacterium gamma me cumin m Wm pa 3 D7 summon mt n s l n 5 a n a p M Ahsomance n a mom son mu m sun son ammo sun eon 7m am Sun Wavelength my Wavelnngm mi Chlorophyll eukaryotes have chlorophyll a whereas different bacteria have various types of bacteriochlorophyll o CODCHS commitI39 79 Wquot mum 0 Ph l Chl a 3quot i I rum lam1 quot3quot il u1 Laquot 4315 47 Arvin H lm m absorption to 3 39 t u in u distinguish the 5 4 type of Bchl r W present as a way to identify l l I 11 the type of m l immm b 39 39V CHI quot5H1 4 14 4x I M an an DH I 39ii PR39Il lilu T I I mlcrobel NF n39lI r quotMNquot 39u1 aC H 43 cm r qr rum rR Wm I I Ludrmll Uh L l 391 1 ill rHnII IIEL d L cL AIL u H L L LIl r H mm m Imdaww A I Both Chi and Bchl are mem ranebound and have 9 dual roles light harvesting y ant nd a special pairquot 4 the photoreaction center Qgp Other pigments also are Q typically membrane bound Carotenoids diversity of compounds with extensive conjugation that are found in all phototrophs Betacarotene H30 H30 CH3 CH3 CH3 cH3 CHa H30 CH3 CH3 Depending on the structure these may be yellow red brown or green and again can be used a m to identify species Roles include light H r W harvesting and photoprotection NC Cyanobacteria and algal chloroplasts contain phycoerythrin red and W phycocyanin blue open chain 5 m ENDSquot03995 on 5 Anoxygenic purple bacteria 4 cm mm Finw Pmr 475 5 m 425 9 a m my Dwi cansuming no m t 1 705 1 i mm new I dgnms Hrs 502 gs star This process is o en termed cyclic photophosphorylation NT Fquot F 53 Key features Cyclic electron ow PMF PM F drives ATP synthesis V th drawal of electrons fo D energize electrons for NAD reduction How the protons are pumped Variations in other bacteria different components need for reverse electron ow mmmm m E39wniumrhiulnrimn mmm Oxygenic Photosynthesis Two photosystems P700 and P680 that are connected by an electron transport chain T n 75 cm mmquot m mm mm mm my mm mm w 631 m m M 52253 E E n 5 Pmuyneml ms 20 nD702H Mwugmuuanu WWWquot Key points to understand electron ow in PS amp PS II method of NADP reduction oxygen formation m Absorbs light energy at 700 nm Participates in cyclic electron ow tha generates a PMF Provides electrons for NADP reduction but this requires a source of electrons m Absorbs light energy at 680 nm Does not participate in cyclic electron ow Prov39d s electrons to PS ll while generating a PMF source of electrons an amazing reac 39 PC plastocyanin a 1e carrier Cu protein Oxygen formed at a 4Mn site MMG 301 Dr Frank Dazzo Microbial Ecology Methodology amp Soil Microbiology Some methods used to study microbes in natural habitats Microbial abundance o microscopy computerassisted image analysis measurement of cell constituents eg ATP muramic acid filtration I dry weight aquatic habitats viable enumeration techniques plating MPN membrane filtration quantitative PCR of DNARNA using phylogenetic probes Microbial viability 0 Combination of fluorescence microscopy using vital stains eg BacLite LiveDead and viable plating techniques Microbial activity Microscopy redoxsensitive dyes deox H e39 a de red Microelectrodes Enzyme activity assays Gas exchange eg uptake I production of 02 002 N2 CH4 Assess bacterial vs fungal contributions by use of selective antibiotic inhibitors eg chloramphenicol vs cyclohexamide Stable and radioactive isotope studies In situ rates of substrate utilization and product formation Microbial Community Structure 0 Computerassisted microscopy and image analysis eg CMEIAS o Polyphasic taxonomy amp physiological diversity of isolates 0 Various types of 168 rRNA analysis FISH RDPll bioinformatics a DNA amplification genomic fingerprinting functional genomics Epilluorescence microscopy techniques in microbial ecology 1 3 6 A I t 1 algal autofluorescence 2 d ct count acridine orange vital stain ct count DAPI stain for DNA cence using strainspeci c antiLPS t M 4 amp FITC mmunofluores a o y 4 bacteria on slide 5 bacteria on plant roo LSC 6 FISH39 escence in situ yb ization ritc165 rRNA oligoprobe viability stain Molecular Probes BacLight l Eluor 7 Live green dead red 3 ee n n sualllslul acylhomoserine lactone quorum signal red source green sensor Microelectrodes are used to measure the in situ distribution of 01 pH and H25 In hot spring microbial mats at submm resolution Depth mm 39i i Mmiama clmaesm slud mem aAooresampie 5 er mums Microbial mars and me Lhmugh e hol Spring 1 eyemueeiena henealh men are several layers o1 anox bacteria orange em yeiiow lsysrsi e Miompm les oicxygen e croeieolm I thmi mat Upper dark reen 39 lgenlc phniolropm same and pH m ma hol spring micvnolai mm me sumo ey mi es FIGURE 1814 Microbial community sampling using ribosomal RNA Microbial cummun39 y PCR Amplify 153 H A genes using genera or speci c primers Sampie i 2 a 4 T Allies i rRNAgenes Sample a I 1 2 3 A Excise bands Mam and cianeies 155mm r NAgeries genes DGGE Excise bands Sequence Sequence Bacillus subillis I 39 39 nknownEacilus species Bacilluscslsu s saciiius megaterium ovm low 60 grampositivequot n Unkn IE closti um histaMicum Unknown CIo strldium species Microbial Ecology of Soil A 39 ase o Solids consist of soil separates sand silt clay amp soil organic matter a 39 39 I 39 39 39 hartnria component affect ion lnutrient mobility buffer pH water retention porosity and gas exchange they form bacterial clay envelopes Formation and movement m of soil materials lead to L Emlun emmuns d discrete layers horizons plan ma mals A honmn viaoe m high m o bial W biomass and act In mmb a39 awMun this pro le varies In proporti n to the organic nutrient content highest BMW near the surface and 39 ixfs fg r 39 g g mlv decreases with depth so su aoe aecu uhhe 5 bass develops Ireclly m undalyl bedwck mammal mm may very mm A soil aggregate of sol 39 ponents by refuge in small pores Major groups of soil microorganisms and their significant activities Bacteria numerically abundant 1 09 cells lg soil but most nonculturable along with fungi most important decomposers of organic matter specialized groups participate in all biogeochemical cycles their extracellular polymers help bind soil particles into aggregates some form bene cial or pathogenic interactions with plants gt00 n z 3 o 3 n E m 5 specialized lamentous prokaryotes participate in decomposition of complex organic compounds produce many 2 metabolites eg antibiotics geosmins earth odor that give soil its characteristic distinctive aroma Fun o the major component of microbial biomass in soils 0 major participants in decomposition of organic matter 0 hyphal growth helps bind soil particles into stable aggregates 0 some associate with plant roots major plant pathogens beneficial symbionts increase nutrient uptake and decrease disease incidence a 0 major predators of soil bacteria grazing activities accelerate decomposition of organic matter in soil anobacteria and al ae reen al ae diatoms photoautotrophs form surface algal crusts important in H20 retention some cyanobacteria carry out freeliving and symbiotic N2 xation Viruses o Numerically abundant ecology not well de ned 0 Both lytic and lysogenic bacteriophage latter very common 0 Persistance and migration of human enteroviruses pose serious health issues with land disposal of sewage and fecal wastes C Special considerations for gasliquid relationships affecting microbial activity in the soil environment 0 Soil atmospherewater occupy the pore spaces in the soil matrix 0 Oxygen ux controls the type of metabolism aerobic vs anaerobic accomplished by the microbes Oxygen diffusion is 10 fold faster in gas than through water hence waterlogged soils quickly become anaerobic Microbes in discontinuous water films on the surface of soil particles have good access to 02 In contrast microbes in continuous water lled pores have limited 02 fluxes creating anoxic microenvironments Bacterial movement through gamma soil can occur when waterfilled spaces 39 pore spaces are continuous but ceases when they are discontinuous Oxygen concrv wlian npm invacnlnl Q7 How can one explain the detection of fermentative endproducts of microbial anaerobic metabolism even in sandy welldrained soils A Sandy soils still contain soil aggregates where radial 02 diffusion is restricted so Distance lmml e actively conducting anaerobic fermentative metabolism 3 o 3 Distance mm