MICROBIOL ENGNRD ENVRON SYSTM
MICROBIOL ENGNRD ENVRON SYSTM ESGN 586
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ESGN586 Lecture 14 Fall 07 Readings To Be Announced Key Points Microbial Transformation of Toxic Organics Toxic Organics A whole class of compounds many natural many manmade that contain various compositions of C and H PAHs PCBs BTEX PCE TCE are a few of the many More than 100000 organic chemicals are produced commercially What is the fate of all of them Maybe we know pretty well about 100 of them We need to be concerned about the ones that are either produced or released in larger quantities are in widespread use or are an environmental or health concern For example there are gt1200 pesticides and 850 are in current production The organohalide DDT back in use Two particular problems common to microbial action break up of the benzene ring and vinyl chloride monomer CszCH Cl Example Gasoline what is in it 70 saturated aliphatics most of which should be octane 30 aromatic compounds 10 xylenes 47 toluene 33 124 trimethyl benzene 27 1 methyl 3 ethyl benzene 23 benzene 16 ethylbenzene 16 CH alkybenzenes 12 135 trimethyl benzene Rest methyl tert butyl ether MTBE Other industries Petrochemical plastics pesticides paint household electronics textile paper cosmetics metals wood preservation explosives TNT HMX RDX CL 20 TNAZ We have a long history of the chemicals and the problems A tipping point The Exxon Valdez In an oil spill a lot of the aromatics volatilize What is left is the long chain aliphatic goop When the carbon source is there microbes rapidly increase in number both in the water column and on surfaces 80 of the nonvolatile compounds in an oil can be oxidized within one year Some things remain longer branched chain and polycyclic hydrocarbons for example The Microbiology Clean up of petroleum hydrocarbon contamination in soils What microbes want to do is oxidize an oil hydrocarbon to CO2 Mineralization Clean up of Xenobiotics synthetic chemicals that are not naturally occurring Many xenobiotics are structurally similar to natural compounds Microbial Reductive Dechlorination of PCBs Bacterial Co metabolism some other C source present to provide energy of halogenated organics PAH Degradation Anaerobic degradation of nonhalogenated compounds Organopollutant degradation by Fungi Underlying theme Genetic change provoked by different environmental factors that get expressed Essence mutation to already existing pathways Criteria for Bioremedjation Organisms must have necessary catabolic activity to degrade compound at a reasonable rate to bring the concentration of the contaminant to a useful level that meets regulatory standards Target contaminant must be bioavailable not sorbed or bound as a residue Site must have soil conditions conducive to microbial and or plant growth Cost of bioremediation has to be less than or no worse than other technologies Principle of Microbial Infallibility For every naturally occurring organic compound there is a microbe or enzymatic system capable of its degradation Extended to synthetic compounds though some truly are recalcitrant Compound Degradation Example Methanotrophs normally chow methane Shown to able to chow oxidize TCE by the mutation of an enzyme called methane mono oxygenase MMO Diagram on board Example 2 Pesticide degradation Herbicides insecticides fungicides Some are suitable for carbon sources andor electron donors IF something can be used by a microbe it will be and it will be removed from a soil The rate at which this can happen is variable Example DDT 4 years Melathion 1 week 2 4 D 4 weeks Other processes can be occurring beside Bio volatilization leaching transport spontaneous chemical breakdown As such the bio effect can be combined with the chemical effect one relying on the other Organisms responsible or capable of mutation to metabolize compounds Methanogens 4H2 CO2 gt CH4 ZHZO Acetogens 4H2 ZHCO3 H gt CHSCOO 4HZO Sulfate Reducers 4H2 8042 H gt HS 4HZO All common in the subsurface which is where a lot of these processes take place andor evolve Also think about life in all 3 domains in the soil subsurface What are the nematodes doing for example Are they living off of microbial metabolites of toxics and or the organisms themselves ESGN586 Lecture 4 Fall 07 Readings Brock Chapters 10 11 Chapter 10 11 Key Points Bacterial Genetics First the Glossary go over the terms understand them A genome is what Genetic Blueprint composed of nucleic acids The blueprint sits in the cell as a large macromolecule wrapped up as a bacterial chromosome In the replication of the genomes mistakes happen Mutations occur a base substitution or infidelity of replication An inheritable change in the base sequence of nucleic acids in the genome If the mistake still leads to a viable protein that protein may be incapable of performing what was to be the coded function A new function can arise or the function may not be performed Either of which can be favorable or detrimental to the cell It does lead to inherited change in the next generation Typically that change is SMALL there are of course exceptions A strain that carries a mutation is a mutant It is different from its parent genetically and therefore its genotype nucleotide sequence of its genome is different However its phenotype the physical observable properties of the mutant may not be Typically things isolated from the environment are referred to as Wildtype and reflect in situ nature as is up to the moment of cultivation Genotype is coded as 3 lower case letters nifHI refers to a nitrogen metabolic gene of kind H mutant 1 for example Phenotype is reported as capital letters Ni T or NifH E coli for example Mutations can be spontaneous or induced Spontaneous can happen from chemical or UV or exposure to other radiation It physically alters the DNA Often these point mutations a base pair substitution occur as errors in replication Mutations can be selected for or directed for Grow something under a certain environmental condition long enough something gets used to that Combined with fast generation times of microbes a powerful genetic tool with which to study for example evolution You can screen lots of colonies to see what effects are present wanted or desired Mutations can be directed for by transposon mutagenesis A powerful tool whereby an insertion of a transposable element occurs with in a gene The process by which a gene moves from one place to another is transposition Naturally it is a rare event There are 3 types of transposable elements a molecule of DNA that can move from one site on a chromosome to another Insertion Sequences IS simplest type carry no genetic information other than that required to move about the genome Can be 1000 nucleotides long Transposons larger and contain other genes eg drug resistance Special Viruses incorporate their DNA with genomic DNA All 3 replicate as part of some other molecule of DNA Insertion sequences and transposons carry a gene to encode for tranposase the enzyme needed for transposition and they have inverted terminal repeats at their ends of the DNA molecule These can allow for intramolecular recombination Figure 1031 Transposition is critical for microbial evolution life evolution as it allows a built in experiment to happen Transposons bring in new genes which may or may not work Those genes may duplicate a cell s own genes or not The cell may still make its own gene product and mask that which was brought in by transposon Or the brought in gene may make a superior product protein that allows better fitness Read about Conservative and Replicative Transposition pgs 285 286 If an insertion site for a transposable element is within a gene insertion of the transposon will result in mutation of the gene Therefore you can create mutants with transposons 2 transposon mutagenesis That mutagenesis can be marked with things like antibiotic resistance markers they are part of the transposon Sitedirected mutagenesis is part of recombinant DNA technology whereby a specific mutation in a specific gene is made to happen You can change any base pair in a specific gene Procedure 0 39 an quot quot J primer 39 39 the desired base change Base pair primer to a piece of DNA with the complimentary strand Pairing is complete except for the spot where it is not Extend with DNA polymerase copy the rest of the gene Take double stranded product transform it into a host cell E coli select for mutation by a positive selection color change resistance etc Genetic Recombination is where genes possibly from multiple sources are recombined as one or brought together in an operational way to perform function in a cell that did not originally possess the function Typically that change is LARGE Homologous recombination genetic exchange between homologous DNA sequences from 2 different sources Homologous have nearly identical DNA sequences This is crossover in classical genetics Figure 109 Horizontal or Lateral Gene Transfer LGT is a process whereby microorganisms Bacteria and Archaea acquire a trait of functionality often related to survival and pass that trait through genetic transfer to another organism A whole gene or even an operational cassette of genes is swapped Best example antibiotic resistance Genes are transferred laterally from cell to cell verses vertical passage of genetic information from parent cell to progeny Both processes Mutagenesis and Genetic Recombination fuel EVOLUTION Transformation a process where free DNA is incorporated into a recipient cell and can confer genetic change Happens naturally cell breaks open big DNA molecule spills out 42Mbp breaks up in to 10 kbp chunks of about 10 genes each 1000bp gene Other cells have mechanisms for DNA molecule uptake A cell that can take up DNA is a competent cell and the ability to do it is genetically determined however you can alter that with chemistry and electricity Essentially ds DNA binds to cell surface it is either taken up or a nuclease degrades one strand and the other strand is then taken up Once inside a competence specific protein binds it A protein called RecA then incorporates the DNA in to the chromosome Other Notes 1 DNA gene transfer processes can have powerful effects on the phenotypes of recipient organisms Transfer of genes probably drives much microbial diversity in the environment clearly it has influenced evolution over the ages DNA seems to move around fairly freely between close relatives and with less frequency distant relatives Ways to transfer include Sex between like or even rarely remarkably unlike organisms Viral transfer Free DNA 2 Viruses in the environment an emerging field in microbial biology Most of what we know comes from studies of marine systems The open ocean blue water environment is pretty clean and it is easy to get a lot of it How many microbesviruses are out there A Can count directly using epifluorescence microscopy B Sample 9 stain with SYBR Green Intercalates into DNA and fluoresces View by epi uorescence you can see dots down to maybe 50nm but you can resolve only to about 02 micrometers C Cell virus ratio in the ocean is ca 1 10 and it stays there through out the water column D Essentially nothing is known about viruses of freshwater settings or of biofilms really hard to analyze E Note that viruses are really hard to track because to detect them specifically classically requires a host cell If we can t culture the organisms that are there we classically can t know their viruses What to do to study F Note that viruses probably are really important in driving ecological community changes There are few real measurements that I know of but some forward thinking workers believe that marine blooms are driven by the responding viral blooms To cartoon the notion viwr Mum 3 0r m Cause of crash or response to bloom 3 There is a lot of free DNA knocking about in the environment e g marine and fresh waters H20 gt lter through 02um filter gt filtrate concentrate and assay DNA 1 Ultrafilters Millipore TM cellulose acetate labyrinth of fibers Polycarbonate graded pores 2 Assay DNA Add ethidium bromide and read fluorescence at 590nm a EB intercalates into DNA changing spectral properties Absorbs light at 300 360 nm emits at 590 nm 3 Typical fresh water 5 20 ug DNAl sea water 1 10 ug DNAl a Note that this potentially would include both free and viral DNA how to distinguish DNase digest before EB test ca 50 of DNA resistant to DNase is protected somehow Viral coat Small cells DNA stuck to sleeze b How big is the enVironmental DNA sample gt solvent extract gt DNA gtAgarose gel electrophoresis i Solvent extraction e g With phenol partitions DNA into the aqueous phase since it is polar Whereas proteins lipids carbohydrates mostly partition into the organic phase or precipitate Phenol extraction is Widely used to purify albeit crudely nucleic acids but you get a lot of other stuff too Agarose gel profile 20 kbv 0 H M Wi va th A Average size 1 2 kb big enough to be a gene if it can get into cells B Free DNA in the environment is fairly stable even in aqueous conditions Introduce labeled eg 3213 into environment gt sample at times gt measure acid precipitable radioactive label 5 trichloroacetic acid TCA indicates is macromolecular Environ 12 life hrs Waste water 02 Fresh water 5 Estuarine water 5 P limited ocean 5 Not P limited ocean 40 80 Clay soil slurry 28 Dead cells in marine sediment 140 230 If dry DNA can survive for a very long time e g DNA on CroMagnon int blades 100K years e g in bacterial spores gt20 million years Raoul Cano has isolated Bacillus Sphaericus from bee guts dissected aseptically out of amber 2 How does DNA move Transformation conjugation and transfer factors BBM A Transformation free DNA incorporated into cell B Conjugation cell to cell transfer C Transduction Transfer factors DNA integrated into a viral genome or DNA packaged in donor cell probably usually using a helper virus to supply a coat for mispackaged DNA 3 Natural Transformation in contrast to artificial below A Phenomenon first recognized in 1928 Griffith in study of epidemiology of bacterial pneumonia read text pp308 309 1 Rough colonial type of not 39 f quot in mice r r 2 Smooth colonial type of S pneumoniae produces protective capsule polysaccharide highly infectious If mix killed autoclaved S type with viable R type and inject into mice disease results and S type cells are recovered from infected mice Ie R cells were transformed into S type B 1944 Avery MacLeod and MacCarty showed that transformng principle is DNA susceptible to DNase This was published in a medical journal and forgotten until 1952 C Transformation is an active process for both donor cells release DNA and recipients take up DNA 1 Widespread process eg Proteobacteria Azotobacter soil N fixation Haemophilus pneumonia Neisseria gonorrhea Rhizobium Plant N fixation T hermus Deinococcus Thermus aquaticusu Taq polymerase Deinococcus radiodurans radiation resistant Cyanobacteria Synechococcus Synechocystis Low G C Gram Positive Bacillus industrial subtilis disease anthracis 5 eg 39 39 Micrococcus spp skin indigents Others also but the survey has been limited I suspect that manymost organisms can docan be induced to transfertake up DNA 4 Natural transformation at reasonably high frequency involves a specific state of differentiation establishment of competence A Mainly studied in Streptococcus pneumoniae Bacillus subtilis Low G C Gram positive Division and H influenzae gamma group Proteobacterium What else is that kind of organism B Establishment of competence seems usually to involve adverse growth conditions in these organisms Experiment Inoculate culture eg Bacillus subtilis proline requiring mutant in proline containing medium 1 Monitor cell number with time growth curve 2 Mix samples with wild type pro DNA spread onto pro lacking medium count pro colonies Fraction competent usually 20 100 transformable This might make biological sense when you are in trouble grab some new genes C In other organisms development of competence may be independent of growth phase eg with Thermus Synechocystis Typically 01 10 competent throughout growth Such organisms also simply might be unhappy under culture conditions lots of creatures are unhappy in captivity and unhappy microbes tend to take up DNA eg E coli treated with ionic shock 1 Note that fraction of cells that are competent and phasing of competence may be artifacts implicit in growth curves can jury rig media in which Neisseria Haemophilis probably others have high competence throughout growth curve 5 Establishment of competence involves A Release Detection of a competence stimulating peptide typically 20 amino acids that induces other cells to become competent and at least in some organisms S pneumoniae release DNA Clumping of suspended cells tends to occur 1 Induces formation of DNA bindinguptake structures 2 Commonly sensed through histidine kinase two component system B Formation of DNA bindinguptake structures 1 In low GC Gram division What some organisms of this kind a 50 80 protein complexes deposited on cell surface b Complexes 2 8 10 proteins some homologous to pilus related proteins suggesting roles as pores for DNA uptake DNA binding proteins endonuclease probably also exonuclease autolysin open up wall to transfer or induce DNA release in the population c DNA binding considered non specific 2 In Haemophilus Y group Proteobacteria and Neisseria 5 group Proteobacteria a Formation of Transformasomes blebs off cytoplasmic membrane containing DNA binding and internalization proteins b Contains highly specific DNA binding proteins eg in Haemophilus only DNA containing an 11 bp sequence core of 25 30 nt specificity has low random probability of occurrence 1411 1 in 4 X 106 11 mers 01 X per genome 1 But there are 600 copies of this sequence in the Haemophilus genome randomly distributed in the genome Such organisms must like to take up only their own type DNA Safe sex in the microbial world 2 Note that all organisms also contain mechanisms to destroy foreign DNA the restrictionmodification systems QUE A In studied cases generally after binding to cell one strand of DNA is digested and the other is fed into the recipient cell as single strand for binding by RecA protein and undergoing integration text cartoon 914 Internalization prior to conversion to SS DNA also has been detected 6 Artificial Transformation A Natural hi gh efficiency transformation 1 100 requires competence but most organisms probably spontaneously take up DNA at low efficiency 10 10 10 7 transformants for plasmid added to normal growing culture of E coli or B subtilis 1 ie if a population needs a selective gene it probably will get into the population somehow 2 In essence anything that damages the cellular envelope promotes DNA uptake Molecular biologists make use of through artificial transformation B Getting DNA into cells with reasonable 10 8 10 4 efficiency This is the stuff of molecular cloning BBM 344 356 1 e g treatment of E coli with EDTA and alternatively Ca2 Rb eg use of very old cultures Such treatments make cells leaky probably creating microscopic disruptions of outer membrane wall cytoplasmic membrane integrity a Note that ionic balance of cell cannot be disrupted very much cell is a battery 2 eg Electroporation a Expose cellDNA or cell cell mixture to transient msec electrical pulse kvcm DNA drilled into and out of cells b Electroporation works with variable efficiencies with most organisms Bacteria Archaea and Eucarya 3 eg particle gun BBM Fig 915 a DNA on small particles e g gold is blasted into a paste of cells or target tissue Kinda crude but it works 7 Conjugation Gene transfer through cell cell contact A Very widespread and perhaps ubiquitous best studied in E coli BBM pp 319 324 1 Common in CC 5 Y Proteobacteria occurs in Streptomyces high G C Gram Division fungi almost everywhere examined in detail for conjugative plasmids plasmids that mobilize chromosome transfer This is a great field for discovery 2 Plasmids that recombine into chromosome can mobilize transfer of chromosome B Commonly shown theme see text pic 9 22 Note It is not clear that DNA transfer is through a cytoplasmic bridge It could probably does travel through pilus just as single strand phage DNAsRNAs do C DNA transfer is single stranded rolling circle propagation Note BBM pp 322 323 1nterrupted mating as a genetic tool Study text Figs 925 28 for cartoon of process 8 Transfer factors A e g viruses defective pick up host DNA or carrying integrated transposon or other insertion sequence B Transposons 1 Important genetic tools several types of insertion sequences IS elements e g as seen in Eco and other genomes 2 Transferred by phagechromosome transfers 3 Often carry antibiotic resistance genes 4 Probably drive the mobility of physiological islands 40 60 kb block of genes that act as cassettes for particular function 9 Physiological islands A E g Pathogenesis islands contiguous genes that code for invasion of host and pathogenesis e g adherent pili Type III secretion system mobilizing microbe transport mechanism for in uencing phagosome function e g paralyze phagosomelysosome fusion 2 Other eg Symbiosis island in Mesorhizobium cf Sullivan and Clive PNAS 955145 51491998 a Organisms of the rhizobia group eg Agrobacterium Rhizobium ia enter 39 39 with 39 plants fhzoba can 1 Recognmon and anachment micadesinrmediated 2 Excretion of rod factors by acterium causing root hair curiing 3 Invasion Rhizobia penetrate root hair and multiply within an quotinfeciion threadquot 4 Bacteria in iniection thread celis and ihose neavby are stlmuialed m divide 5 Formation of bacteroid state within plant cell Uninfecled rool hair 6 Commued plant and 39 bacienal celi dwismn b forming root nodules Inside the nodules a differentiated state of the bacteria carries out N fixation Text Fig 1676 we will return to c Sullivan and Clive mix Mesorhizobium loti strains one symbiotic but mutant biotin thiamin nicotinate and the other prototrophic nonsymbiotic but no special requirements 9 grow on agar lacking the vitamins 9 use what grows to infect plants and cause N fixation The symbiotic trait is acquired by prototrophs d Restriction sequence analysis 9 the strains acquired a 500 kb insert into the chromosome into a tRNA gene partially duplicated downstream of the inserted casette presumably the target of an integrase Indeed the insert has an integrase related to known phage and IS integrases and lots of genes of the types that might be expected to engage in the differentiation process e g NIF genes 3 Photosynthesis island in Rhodobacter Photosynthetic genes in this organism and other Proteobacteria studied are on a ca 40 kb contiguous block of DNA flanked by tRNA gene fragments that contains all the genes required for Photosystem II function and control 3 In contrast in cyanobacteria the PS genes are dispersed in the chromosome B Recent health related transfer C E coli 0157H7 serotype E coli containing a block of genes with shiga like cytotoxins and adhesins cause massive fluid losses 1 Codon usage of these genes is different than resident E coli indicating recent transfer 2 Note that E coli and Shigella dysenteriae are extremely closely related indistinguishable by rRNA 70 100 related by overall DNA homology analysis Ie these organisms are called different genera for historical not analytical reasons ala the Blind Men and the Elephant story 10 Some mobile DNA elements are honed to capture and express genetic information eg integrons text p 328 pieces of DNA that pick up other pieces of DNA The integron is contained in the genome or a plasmid and consists of Transposase gene catalyzes site specific recombination with the Attachment site where site specific recombination occurs by sloppy sequence recognition adjacent to a Transcriptional promoter fires into Att site turning on genes that happen to get recombined in A Integrons are a large related family distributed widely in at least gamma Proteobacteria E g E coli guts urinary tracts PS aeruginosa burns pulmonary infections Vibrio cholerae cholera How widely distributed phylogenetically What s the experiment B Integrons are responsible for the dissemination of muchmost drug resistance 11 In addition to genome perturbations by incoming elements structural stability is compromised by intragenomic rearrangements there are numerous sequences in genomes that are direct repeats gt gt or inverted repeats lt gt A Crossovers between such repeats can scramble and expand or contract the genome B Eg of sequence repeats that could cause flips and deletions in the DNA rRNA genes multiple copies of transposons or genes distributed regulatory information e g promoters transcription terminators paralo gs C Some consequences of intragenomic cross overs next p D They cause genomic rearangements Eg genomes of two Pseudomonas aeruginosa strains PA01 and DSM 1707 have been determined at 63 mega bps and are essentially identical except for a 15 mb inversion between two rRNA operons 12 Closely related organisms probably transfer genes and recombine frequently in nature A e g in the ECOR collection of E coli strains 1 72 distinct strains of Eco defined by antigenicity enzyme patterns electrophoretic and available as stocks 2 Many examined for sequence of Trp operon sequence heterogenieties show clear recombination between the strains B In rRNA analysis of natural populations substantial point heterogenieties are seen in sequences from same setting ie you don t see a singular sequence type rather a cluster of closely related sequence types at the 98 99 identity level 13 Gene transfer can even occur between the domains A E g Heinemann and Sprague Nature 340 205 1989 showed that E coli can mobilize transfer into yeast 1 Carry out crosses between E coli carrying plasmids YEp13 Sce leu2 andor R751 Eco transfer functions and yeast See leu 2 strain YEp13 E coli yeast Shuttle vector contains sites necessary for replication in both Eco and See R751 plasmid with Transfer functions Pilus and cell binding functions 14 Thus there are many ways that DNA moves around in the environment It is amazing that genomes are as stable as they are 15 Nonetheless genome content and arrangements of genes are highly fluid A See next page 11a for interdivision genome scrambling Mycoplasma genitalium low G C Gram vs Haemophilus in uenzae Proteobacteria 15 A good text for more detailoverview of these topics Josef and Guespin Prokaryotic Genetics Genome organization transfer and plasticity Blackwell 1993 ESGN586 Lecture 5 Fall 07 Readings Brock Chapters 17 19 Chapter 17 19 Key Points Metabolic Diversity This is an absolutely amazing thing in the microbial world Almost anything is possible and nothing should surprise you Microbial life is REMARKABLE in this regard Photosynthesis Light as an energy source ATP synthesized by light driven reactions and CO2 is fixed by cell Phototrophs the organisms that do it Most are autotrophs they fix their own C02 There are photoautotrophs and photoheterotrophs Quanta of light energy photons reach light reactive pigments chlorophylls to begins photosynthetic energy conversion to ATP Two things happen ATP Production 2 CO2 reduction to organic compounds For growth energy is supplied by ATP and electrons for CO2 fixation come from NADH Nicotinamide adenine dinucleotide Some phototrophs get reducing power from electron donors in their environment H2 H28 8 8203 etc Green plants algae and cyanobacteria split water as an electron donor a poor electron donor process The oxidation of H20 produces 02 as a by product and this is where all of the O2 in our atmosphere came from Photosynthesis that produces 02 is oxygenic photosynthesis Photosynthesis that does not produce 02 is anoxygenic photosynthesis Fig 172 page 533 Oxygenic phototrophs plants cyano s have chlorophyll Chlorophyll a absorbs read and blue light and reflects or transmits green Anoxygenic phototrophs have bacteriochlorophyll Chlorophylls are prophyrin ring compounds that contain a Mg atom instead of iron Cytochromes Bacteriochlorophyll s can be distinguished by different absorption spectra This property can aid in distinguishing who is in a particular community In Eucaryotes plants chlorophylls are packaged on stacked membranes called thylakoids inside chloroplasts In Bacteria and Archaea pigments are on some internal membranes The archaeon Halobium is a hetero photo organotroph That is it gets ATP from light but carbon and electrons come from organic sources Carotenoids A few words These are accessory pigments present in all plants and other phototrophs They are long hydrocarbon chains and absorb light in the blue spectrum They provide the amazing colors seen in the anoxygenic photosynthesizers the pinks and purples in a Winogradsky Column They do not function to make ATP but they do assist in electron transfer They can also be photoprotective to the cell and or chlorophylls Autotrophic CO2 fixation The ability to fix your own carbon is pretty amazing What if we could do it The Calvin Cycle named after Melvin Calvin Require NADPH and ATP and 2 KEY enzymes ribulose bisphosphate carboxylase RubisCO phophoribulokinase RubisCO catalyzes the formation of 2 molecules of 3 phosphoglyceric acid PGA from ribulose bisphosphate and CO2 PGA is then phosphorylated and reduced to glyceraldehydes 3 phosphate an intermediate of glycolysis Then glucose is formed by reversal of the early steps in glycolysis Figures 1721 and 1722 on page 546 are helpful Glucose Figs Some organisms Use the reverse citric acid cycle to fix C02 The green sulfur and green non sulfur bacteria Example green sulfur Chlorobium does this A mechanism of CO2 fixation found at high temperatures by members of the Aqui cales and some Archaea also do it Chloro exus a green non sulfur organism green non sulfur s thought to be deeply branching in the bacterial line of descent uses the hydroxypropionate pathway to fix C02 Thought to be an early form of autotrophy a deeply rooted form of metabolism and may be more widespread than currently recognized Chemolithotrophy The bulk of microbiota do this The use oxidation of inorganic compounds as energy sources Several can also fix CO2 so they are also autotrophs ATP synthesis is coupled to the oxidation of an electron donor These electron donors can be geologic biologic or anthropogenic in nature Reduction potentials for redox couples Chapt 5 are given in Table A12 back of the book many of those have inorganic components Hydrogen Oxidation Possibly the most widespread mechanism for ATP generation Oxidation of H2 by 02 H2 12 02 gt H20 AG 2 237 kJ An exergonic reaction that can support the formation of at least 1 ATP molecule Enzymes responsible hydrogenases Autotrophy in hydrogen bacteria 6H2 202 CO2 gt CHZO SHZO Oxidation of reduced sulfur compounds Most common electron donors H28 S0 and 820322 Final product of sulfur oxidation 8042 An 8 e transfer with a wide spectrum of applicability Generally occur in stages You can have S0 stored within cells as energy reserve Cells can also attach to colloidal sulfur particles and use them as an energy source One of the products of sulfur oxidation is H so acidification of the medium by H2804 can result Figures 1726 and 1727 page 552 useful Iron Oxidation Aerobic oxidation of iron from ferrous FeH Fe2 to ferric FeIIIFe3 is widespread A small amount of energy available one electron oxidation so large amounts of iron have to be oxidized The ferric iron produced is insoluble and drops out of solution as ferric hydroxide FeOH3 At acid pH ferrous iron is stable in water so most iron oxidizers are acidophilic Acidithiobacillusferrooxidans grows autotrophically using ferrous iron and is responsible for acid mine drainage Nitri cation and Anammox NH3 used as an electron donor and NO nitrite used as an electron donor Both oxidized aerobically by chemolithotrophic nitrifying bacteria in the process of nitrification Nitrosifyers Nitrosomonas Sp take NH3 to NOZ Another group Nitrobacter up take NO to N03 The complete 8 e oxidation is carried out by 2 groups of bacteria working together Thought to be strict aerobes Key enzyme ammonia monooxygenase Anammox anoxic ammonia oxidation NH3 oxidized under anoxic conditions by anaerobes Nitrite serves as the acceptor to NH N02 gt N2 2H20 AG0 2 357 kJ Members of the Planctomycetes are the only ones known to do this now Chapt 18 this stuff we are doing in class labs Some things we will not e g FISH so you should read about the things we don t do Chapt 19 A few key points Acid Mine Drainage Read about It is all over our state Read about the sulfur cycle and the iron cycle Microbial Bioremediation Turns out that a lot of microbes naturally react with both metals and organic compounds to transform Uranium for example is actively reduced from UVI to UIV forming the mineral uraninite U02 Could be that a lot of ore bodies in the Earth s crust were products of microbial metabolisms over time Now the question how can we use these naturally evolved processes to clean up human mess Bioremediation the clean up of elements metals oils organic compounds and toxic pollutants by microbiota Often this refers to oxidation state shifts for elements or compound shifts for organics For metals you can use microbial processes to leech metal from a rather low ore quality The metals form metal sulfides in the crust and those can be metabolized by microbial leaching Used to remove copper gold and uranium from low grade ores For copper microbially oxidize insoluble copper sulfides to soluble copper sulfates a very efficient leaching Metal Recovery in a Copper System Fig 1938 page 648 For Uranium You can have SRBs take UVI to UIV as a remediation scheme or you can have Acidithiobacillusferroxidans take UIV to UVI with 02 as an electron acceptor Solublize the U out of the ore May work in conjunction with Fe U02 Fe2SO43 gt U02s04 2FeSO4 UIV FeIII UVI FeII Gold is often found in nature complexed with arsenic FeAsSAu reacts with sulfuric acid produced by the A ferrooxidans to liberate both the arsenic and the gold Reaction pg 649 Reductive Dechlorination Chlorinated organic compounds serve as terminal electron acceptors under anoxic conditions Typically something like chlorobenzoate C7H402Cl 2H gt C7H502 HCL An important process in the removal of a lot of bad compounds Many organisms have the ability to do Can also have aerobic dechlorination of compounds and oxygen plays a much bigger role and the presence of oxygenase enzymes is critical ESGN586 Lecture 15 Fall 07 Readings To Be Announced and Brock Chapter 11 Websites httnWwwdenver 39 39 39 39 4752744 httpWWWtimecomtimemggazinearticleO9171155513200html httn llwwW time quot39 39 title09171 154128300html 4 I Key Points Origins of Life Origins Evolution is a random process in which hahazard genetic changes interact with random environmental conditions to produce an organism somehow how fitter than its fellows After 35 billion years of such randomness a creature emerged that could ponder its own origins and revel in a Mozart adagio music in a leisurely manner Time httmIWWW time quot39 39 39 39 title09171154128300html Some terms Abiogenesis the study of how life may have evolved from non life sometime between 39 and 35 GYA May include some thoughts on extra planetary panspermia and extra terrestrial origins If you are going to think about this you need to think about the overall evolutionary processes of the universe from the Big Bang at 137 GYA Origin of Life where did we come from The mechanisms by which non life became life are at best elusive Life 2 The quality that distinguishes a vital and functional being from a dead body An organismic state characterized by the capacity for metabolisms growth reaction to stimuli and reproduction Also spiritual existence transcending physical death Webster s Charles Darwin kind of grew up with some thoughts on Natural Selection and Evolution from his grandfather On 1 February 1871 he wrote a letter to his friend Joseph Hooker that life may have begun in warm little pond with all sorts of ammonia and phosphoric salts lights heat electricity etc present so that a protein compound was chemically formed ready to undergo still more complex changesquot He went on to explain that quotat the present day such matter would be instantly devoured or absorbed which would not have been the case before living creatures were formed quot In other words the presence of life itself prevents the spontaneous generation of simple organic compounds from occurring on Earth today a circumstance which makes the search for the origin of life dependent on the sterile conditions of the laboratory Basic compounds needed for and origin of life CH4 NH3 H20 H28 CO2 and PO43 And maybe life went from Primordial Soup first synthesized organic compounds to spontaneous phospholipids formation of lipid bilayers cell membrane to polymerization of nucleotides into random RNA molecules that could have been ribozymes to selection pressures for catalytic efficiency and diversity that formed small proteins and gave birth to the ribosome to proteins that out compete the ribosome for catalytic activity whereby nucleic acids then are reserved for genomic coding use A theory of universal common descent based on evolutionary principles was proposed by Charles Darwin in his book The Origin of Species 1859 and later in The Descent of Man 1871 This theory is now generally accepted by biologists and the last universal common ancestor LUCA or LUA that is the most recent common ancestor of all currently living organisms is believed to have appeared about 35 billion years ago Rock Record Look for isotopically light rocks carbonaceous materials that contain primarily 12C not 13C as well as other element contributions Stromatolites fossilized microbial mats formed by filamentous microbes Banded iron formations BIFs fossilized iron oxidizing communities First kinds of life Based on Early Earth Environment This organism is referred to as the Universal or Common Ancestor It would have had the following characteristics because of the environment in which it evolved it would have been anaerobic it would have been hyperthermophilic and halophilic it would have been a chemolithoautotroph obtaining both energy and carbon from inorganic sources using H2 or reduced sulfur compounds as electron donors and CO2 or oxidized sulfur as electron acceptors to provide energy and fixing CO2 as their carbon source Chemolithoheterotrophs would have evolved later in this scenario as quotopportunisticquot consumers of organic matter formed by autotrophic producers There is also a hypothesis that the first living organism was heterotrophic but this could only have been true if the prebiotic broth contained significant concentrations of abiotically produced organic molecules which is not likely especially from the point of view of continuous supply Modern chemolithoautotrophs Organisms thought to be similar to these first chemolithoautotrophs have been isolated in the last few years from what we would call quotextreme environmentsquot These organisms are isolated from hot sulfur springs on the earth s surface or hydrothermal vents quotblack smokersquot on the ocean floor where these organisms form purely prokaryotic ecosystems Conditions in these environments are thought to mimic those present on the early earth ie high temperature high sulfur anaerobic high salt These organisms grow optimally under anaerobic conditions in high salt at 80 1 10 C in fact they grow completely independent of oxygen and sunlight they could even grow on another planet if water was available They may be relatively unchanged genetically and structurally from the first living cells This is borne out by sequence analysis of 16S rRNA genes and other genes which are highly conserved during evolution ie ancestral genes ESGN586 Lecture 3 Fall 07 Readings Brock Chapters 6 7 Chapter 6 7 Key Points Microbial Growth Binary Fission Figure 61 DNA Replication Cell Elongation Septum Formation Completion of Septum with formation of cell walls Two separate Cells Notes Can be quick can be slow Fast Generation Time The Beauty of Microbial Study Clonal daughters progeny re ect prior generations Growth Patterns The interval for the formation of two cells from one is a generation The time required for the formation of a generation is Generation Time the time required for the cell population to double Sometimes doubling time When doubling occurs at a regular interval exponential growth Can measure that with a spectrophotometer and get a cells ml value Exponential Growth N NOZN and generation time g 2 tin Where N 2 final cell number No initial cell number n number of generations that have occurred g 2 generation time t 2 duration of exponential growth in dayshours minutes Page 142 Example Growth Cycle Lag Phase Cell inoculation into a medium Lag Phase turn enzymes on wake up etc Basically a period of adjustment Exponential Phase Cell divides forms two cells which form 2 more cells Usually the healthiest state of the cells Stationary Phase Limitation on growth by essential nutrient used up or waste products accumulate No net increase in cell number Cell function continues just limited or no growth Death Phase No nutrients to support cellular function Can have cell lysis Measure Microbial Growth Count em Viable Count You want to know what is Viable Plate Count or Colony Count but has pitfalls Not all things grow Spread Plate and Pour Plate Can also do CSTR or Chemostat Growth Microbial Environments The short end of it Everywhere Can be from Cold Psychrophiles to moderate Mesophiles to hot Thermophiles 65 to 95C and Hyperthermophiles gt 90C Each organism has a minimal an optimal and a maximal temperature the Cardinal Temperatures Molecular Adaptations to all Cold more oc helices in proteins than S sheets secondary structures in functioning enzymes More polar less hydrophobic AAs in protein assemblages A more exible protein assemblage Cytoplasmic membranes more unsaturated fatty acids maintains more exible cells a more semi fluid state at low temps Biotechnology what could you do with cold adapted enzymes Hot more amino acid cross bonding very few needed to stablize More ionic bonds between AAs More saturated fatty acids in cell membranes make them more hydrophobic Other 02 Low 02 and No 02 environments Halo and other osmotic effects DNA 1 Replication 1 double helix goes to 2 double helices 2 Transcription DNA to Protein through an RNA intermediate mRNA 3 Translation sequence of amino acids in a protein is CODED by DNA codon The Central Dogma of Biology DNA to RNA to Protein All of life does this except some Viruses Heat up DNA denatures Cool down oligonucleotide primers present in abundance Anneal to DNA DNA Polymerase Extension in the presence of deoxyribonucleotides Heat up again a Products of one PCR cycle can serve as template in the next b 23quot possible pieces in 30 cycles P9P Usually Heat gt 1min Heat 30 sec Anneal 45sec Extend 1 min Go to 2 29X Extend Cool ESGN586 Lecture 9 Fall 07 Readings Suggested Readings Walker et al Nature and To Be Announced Key Points What is Geobiology From Wikipedia Broadly defined It is an interdisciplinary field of scientific research that explores interactions between the biosphere and lithosphere or atmosphere Investigators from numerous fields are involved in geobiologic research including but not limited to such disciplines as paleontology microbiology mineralogy biochemistry sedimentology genetics physiology geochemistry organic and inorganic and atmospheric science One major sub discipline of geobiology is geomicrobiology an area of study that focuses on investigating the interactions between microbes and minerals Another related area of research is astrobiology an interdisciplinary field that uses a combination of geobiological and planetary science data to establish a context for the search for life on other planets One example of geobiological research in a modern context is the study of bacteria that quotbreathequot metals such as manganese and uranium These organisms use metals as terminal electron acceptors in the same way that humans use oxygen These processes hold promise as tools for environmental bioremediation I I x I 39x quot Geobiology also includes investigations of 39 J J 1 throughout Earth39s history as preserved in the sedimentary rock record One example of such an interaction is the introduction of oxygen into the atmosphere by photosynthetic bacteria This oxygenation of Earth s early atmosphere may have resulted in the precipitation of banded iron formations Main kinds of organisms Chemolithotrophs organisms that utilize inorganic energy sources limited to a few divisions or phyla of Bacteria with the exception of the hydrogen oxidizers which are widespread environmentally and biologically H2 oxidizers link the oxidation of hydrogen by a class of enzymes that are hydrogenases most reversible to some electron acceptor Many kinds of organisms have this class of enzymes so it is likely a widespread metabolism Not limited to oxic or anoxic environments occurs in both Sulfur oxidizers usually oxidize hydrogen sulfide elemental sulfur or thiosulfate Often found at interfaces between oxic and anoxic conditions Can be acidophiles in low pH environments as well as in neutral environments Example Thioploca AP Accumulate nitrate up to 500 MM intracellularly at one level of oxic water then dive down to use H28 as an electron donor in an anoxic water at the expense of the nitrate Iron oxidizing Bacteria mentioned previously but they are in competition with chemical oxidation at neutral pH the chemical oxidation of ferrous to ferric is rapid In acid as in acid mine drainage there is abundant FeII and Thiobacillus can use that as an electron donor Acetogens organisms like Clostridia Acetobacterium Acetogenium and others use H2 as a donor to fix CO2 into acetate which they then use for growth Microbe Rock Interactions There are many From Walker et al look at what can happen in the pore space of rocks Things live at their light optima Much like the endoevaporite the light follows a tortuous path through granite limestone sandstone and clays Mineralizations There are many but we are going to focus on one magnetite Several magnetic iron minerals are biomineralized by different microbes Iron oxide magnetite FO4 Fe2 Fez O4 and the iron sulfides greigite FS4 and pyrrhotite FeqSB are a few Can form in 2 ways biologically induced mineralization BIM and biologically controlled mineralization BCM In BIM the biomineralization is not controlled by the organism The mineral particles form extracellularly are of broad size range and lack a regular crystalline morphology In BCM the organisms control the mineralization minerals are of uniform size and often deposited intracellularly Magnetotactic bacteria form magnetites by BCM Dissimilatory iron reducing and dissimilatory sulfate reducing bacteria use BIM processes to form magnetite and greigite respectively Dissimilatory 2 reduction or use of iron and sulfate as terminal electron acceptors for energy Assimilatory 2 iron and sulfate would be incorporated into cell material Many kinds of organisms use FeIII as a terminal electron acceptor and produce extracellular magnetite The 2 most studied are Shewanella putrefaciens and Geobacter metallireducens Typically there is no known use for magetites produced by BIM and excreted extracellularly BUT there is great ecological evolutionary and physiological significance to magnetites produced by BCM The biomineralization of fine grain magnetites lt 1 pm in sediments can be useful to understand the paleomagnetic record and paleoclimate of a place but you would want to know how these minerals were produced Formation of these compounds can lock up Fe for a long time so this can affect what comes later And as magnetic compounds they can serve to scrub or scavenge metals and other species from solution IN BCM there are all kinds of bacteria that produce intracellular magnetite to be magnetotactic the amazing thing is they know direction Typically they are all motile don t like oxygen live in aquatic habitats are found at the oxic anoxic transition zone OATZ or the redoxocline and they contain magnetosomes The magnetosome contains a single magnetic domain crystal of a magnetic iron mineral surrounded by membrane Usually that crystal is magnetite or greigite The magnetosomes are arranged in chains to maximize the magnetic dipole moment of the organism MagnetotaXis is used in conjunction with aerotaXis to maintain position in the OATZ zone WHY Presumably in a vertical concentration gradient it increases the efficiency of the organism at finding and maintaining position relative to the vertical gradient in 3 D space it turns a 3D problem into a 1D problem There are probably a host of cellular magnetotaxis chemotaXis functions in the microbial world The Deep Subsurface It is likely true that members of all 3 domains of life occupy life at depth in the deep subsurface For Eucarya it is protozoa fungi lichens and algae Interestingly algae and some protozoa lay down CaCO3 as intracellular support structures For Archaea it is the thermophiles halophiles and methanogens all in the subsurface Often to great depths 7 km Methanogens taking CO2 4H2 gt CH4 ZHZO CH3OH gt 3 CH4 C02 2H20 C1 Metabolism CH3COO H20 gt CH4 HCO3 C1 Metabolism C1 metabolisms typically have a C02 or a methyl carbon is reduced Methanogenesis pathway involves seven steps and has been fairly well characterized In the subsurface methanogens often associated with syntrophs bacterial species which provide C l or C 2 compounds after having fermented longer chain fatty acids excreting H2 and the C 1 compounds needed for the methanogens ESGNSI ham 6 m 3907 Methiillin mm Saphylozonu Wm Math1 m xs 5 mmw spamumbmmmm Andaman am pnmlhn class mme mt ma cammanafa m mammanymaxe pmx mn me Dnlgsfm mumm gr mm mamquot cephalzxm uclaxamlhn and dwlnxmllm A 3 drugs hzlp m Inhle m ramm afbacteml cell Whammy cmsslmkmg afppndnglcan layzxs m G bmm n dues thls bybm a m enzymz tnnsppndase which ls m enzymz xespuns ule fax m crass hnkmg afthe was Drdmylralamm m ppndnglyun synthesis Methiti m mt used In pumms Nbducdlm us 50 m anuhmnc used m m Mammarth canl39mn 951mb m m cultivated ample banana a mz39hcdlm 15 passed amnnd mismme cammnnmzs pmbahlymnsdybyhnnmmal ax wequot gen myquot HGT ax LGT af m mnA gen passed an a larger assent afgems callzd m aphylacaccal assent chmmnsamz ax scam hlcxlhn quotmum maphyzomu mm 15 than m a have caused max um 94 um s n W happn In any cammnnnyat large schnals ka aces w wnhbad hygxem mamas and mm cammnn expusmi nl39ectmns 1974 2afa 5taphmfecnans wm MESA 1995 227 Occurs fnquzndym m ewley m ymmg hunk cm wuxkzxs and African Amman presumhlybecause ah mm m afchmnlc muss m Afncm Amencans4e n ectmn af pun hunk cm mm L157 Sometimes MRSA can appear as a simple rash or zit on the skin of a healthy human You can be a carrier of MRSA yet your body keeps it in check The Mode 01 Modes of Transmission Those who carry MRSA are the most common source of transmission Then human hand contact is the most prevalent mechanism Think about microbes when you shake hands For example For the time beingiMRSA is still treatableiwith the newest kinds and classes of antibiotics Question is How long will they last And where do we get new antibiotics Surveillance of antimicrobial resistance is a critical issue Is it bigger than bird u You decide CDC actively follows patterns of surveillance on over 15 bacteria molds viruses and parasites ESGN586 Lecture 6 Fall 07 Readings Brock Chapters 20 21 and 26 Chapter 20 21 and 26 Key Points Microbial Growth Control This is an absolutely necessary to understand how microbes affect both their environment and you What is sterilization for example What is community What is individual What is species Things that kill bacteria are bacteriocidal things limit growth are inhibitors and are bacteriostatic Decontamination removal of nutrients that can feed microbes thus less microbiota Disinfection target pathogens but may not eliminate all microbes Both share the same goat to reduce the total microbial load Heat Sterilization Microbes have a maximum temp for growth Death from heating is an exponential first order function You have to adjust both time and temperature to sterilize properly One thing to kill live vegetative cells Another to eliminate spores for example Autoclave Pasteurimtion works by controlled heat It does not kill everything it does not sterilize At 71 C for 15 seconds followed by rapid cooling most pathogens are eliminated Radiation Lots of possibilities X Ray y Ray UV Microwaves even IR can all have damaging effects to microbiota UV good for surfaces does not penetrate The others produce ionizing radiation which causes penetration of destructive particles these entire help to destroy cells Filter Sterilization Simply create a physical barrier to remove microbes OK but what size What material Chemical Compounds can be bactericidal fungicidal or viricidal To quantify an antimicrobial compound has a minimum inhibitory concentration MIC the smallest amount needed to inhibit growth of a test organism Industry uses A LOT of different compounds to protect their products Ex pressure treated wood TimberSill Antimicrobials can be synthetic or natural often bacterially produced compounds Synthetic Compounds The Sulfa Drugs created right before during and after WW II Syphilis Streptococcus have been subjected to sulfa drugs The drugs themselves often had negative side effects allergic reactions Usually with a synthetic compound you want to target a pathway inon a microbe that it has but its host does not The book used folic acid production as an example Problem resistance to most sulfa drug compounds is widespread Isoniazid blocks mycolic acid production in the Actinobacterium Mycobacterium tuberculosis the mycolic acid is essential for cell wall formation Problem if the infection is in your lungs you have to get the drug to all parts of the lungs That requires a long drug treatment and you cannot miss any part of it Broad Spectrum Antibiotics These are effective at killing both Gram negative and Gram positive organisms These have wide medical use because of their killing power What is it to be G or G Think about the nature of polymicrobial disease Treat first with a broad spectrum than go in with a narrow spectrum drug like Vancomycin kill off most of everything than kill off more resistant G things Antibiotics can target protein synthesis initiation elongation modification reaction blockings Antibiotics can target transcription block RNA formation then no coding for proteins Book discusses the effects of different ones and different kinds Antifungals more tricky Why Eukaryotes like us Kill them kill us Antimicrobial Drug Resistance read the sidebar page 694 Microbes and Humans Terms important and you should know them all The Skin Look at Fig 212 page 703 What do you see there What do I see there G vs G a factor The Mouth Over 600 described species in the mouth Biofilm formation complexes are amazing Tooth decay often attributed to lactic acid producing like Streptococcus mutans regular production of lactic acid from sucrose table sugar causes etchingerosion of the tooth enamel Consume honey what happens Dental Plaque basically geobiology in a nice environment Biomineralization processes Several Streptococci form laminations of biofilm layers with layers of glycoproteins Filamentous F usobacterium embed in the glycoproteins to thicken and strengthen Spirochetes Borrelia come in next and then anaerobes can be well adjusted with members of the Actinomyces colonizing The Borrelia and Actinomyces are 2 of the many things that can lead to pretty strong halitosis bad breath Dental Caries Organic acids produced from local biofilms decalcify the tooth enamel Therefore tooth decay is an Infectious Disease caused by microbiota It is the gingival crevice that is often the source of problems where tooth meets gums Fluoride can be incorporated into the calcium phosphate crystal matrix as calcium fluoro hydroxyappetite which is more resistant to acid decalcification Thus uoride in water and in toothpastes Caries dependent upon genetics diet sucrose fructose hygiene inoculation patterns The Human Gut The Alimentary Canal An amazing organ Figure 218 be familiar with Except the mouth should be included Digestion begins in the mouth Colonization of your body begins in your mouth Helicobacter pylori is in your stomach Stomach pH is low yet these organisms generally thrive but in susceptible individuals can colonize the stomach wall and produce ulcers General gut flora composition begins with diet Turnover can be fast and high depending on genetics diet nutrient loading and disease For example eat meat have a higher content of Bacteroidetes they secrete more proteolytic enzymes than other organisms As you move through the regions of the small intestine bacterial archaeal numbers increase When the small intestine joins the large via the cecum numbers really increase and by the time you are in the colon you can have 1010 1012 cells g of poop The colon is a big fermentation tank Many things live on the surfaces of the lumen and process things as they go by A few facultative aerobes like E coli can be present however the colon is mostly anoxic and dominated by anaerobes In the gut nutrients are consumed products are made communication is happening and many other things occur simultaneously Vitamins B12 essential for methionene biosynthesis and K essential for blood coagulation are made by microbes for your benefit Biles and steroids I 1quot secreted by the liver aid in lipid digestion and are 1 n J by the Flatus N2 from where CH4 CO2 and H2 are the dominant ones You excrete a lot per day The dump the mass is r J of 39 J39 quot r elements waste products and about 13 is actually microbial cell mass Constant replacement is going on in the colon and the colon is actually functioning much like a chemostat Other Parts Respiratory System Think about what you breathe in Upper respiratory tract is the scrubber but is it Book says lower respiratory tract has no resident microflora Does it Urogenital Tract Think of the possibilities Bladder is sterile but the urethra and ureters are generally lined with many things including enterics Vagina typically lined with many things but dominantly Lactobacillus acidophilus to maintain low pH Infection Think polymicrobial verses mono microbial Think about Routes of infection Exposure has to happen break in skin all skin including mucous membranes Something you may not think about intercourse weakened immune system etc Adherence has to occur microbe has a machine in place to adhere to epithelial cells to then gain entrance Invasion then occurs where the process of pathogenicity is initiated Might be cellular take over destruction or general colonization Colonization allows the organism to gain control of your body Might be local might be systemic Measure Infection by Virulence LD50 the dose of an agent that kills 50 of the animals in a test group Not the best measure because some pathogens are way more virulent than others S pneumoniae very Salmonella lyphimurium less so Keep the organism in culture long enough may loose its virulence capacity an attenuated strain Can put back in the host and get an immune response so that when the pathogenic form arrives if ever your body has a defense Read about Salmonella virulence page 714 716 Chapter 26 Notes For your own enlightenment read the whole chapter ESGN586 Lecture 10 Fall 07 Readings Suggested Readings To Be Announced and Chapter 30 Key Points Subsurface life DUSEL The Deep Subsurface Cont Last lecture Lecture 9 Notes we mentioned C1 metabolisms as a possible carbon source for subsurface life What else is there Example The Henderson Mine Empire Colorado Cenote Zacaton central Mexico A Brief PowerPoint Presentation on the place Industrial Microbiology What we are talking about is The Modern Era of Microbiology Mostly the 20 11 Century The field of Microbiology split into two directions Applied Microbiology Medical Microbiology Agricultural Microbiology Aquatic and Marine Microbiology Microbial Ecology and the bulk of industrial work and Basic Microbiology Both Clinical and Environmental both enhanced by Molecular Biology greatly Microbial Genetics led to the explosion in Molecular Biology Knowledge However the feedstock for Applied is the Basic The one most important thing for the transition of knowledge gained from Basic Microbiology to Applied Microbiology the discovery of restriction enzymes came the ability to introduce foreign DNA into a bacterial host and control its replication Typically E coli What came next was Biotechnology About the same time Mid 1970s nucleic acid sequencing got easier to do which then led to molecular genetics and phylogeny evolutionary lineage revealed in DNA sequence IF you have not read the Woese Papers yet now is the time This then led to Genomics a whole field onto itself of the study of organismal genomes and the comparative analysis of genes between organisms Genomics generates A LOT OF INFORMATION This is fueling advances in both Clinical and Environmental Microbiology and both Applied and Basic Microbiology As a sub discipline of genomics Proteomics involves the study and manipulation of organismal gene products through gene expression Industrial Microbiology basically seeks to capitalize or enhance a naturally occurring metabolic pathway Overproduce a product of interest say an antibiotic Microbial Biotechnology differs in that gene manipulation is used to create new microbial products most if not all of which are not natural Biocatalysis is the word used to describe the metabolic processes optimized Basically it all started with alcohol Humans wanted to ferment more to drink more From the very early beers banana beer in parts of Africa to wine then spirits humans have capitalized on Eucarya based processes From this knowledge then came the ability to manipulate large quantities of microbes for things like antibiotics food additives amino acids in excess enzymes for laundry detergent butanol fuel and vitamins The strains used are often quite different from Wild Type They are manipulated by mutation and recombination to produce XXX in high yield Both organisms and processes are patented You can for example insert extra genes or whole gene pathways into an organism to overstimulate production Gene Amplification To go industrial an organism must Grow in culture Form product in culture Spore former maybe allows for easier manipulation Grow rapidly on a cheap medium Eg mixed corn liquors ethanol whey Produce product rapidly Non pathogenic contamination is everywhere on big scale Be mutateable There are Primary Metabolites substances produced during cell growth exponential phase eg ethanol Secondary Metabolites substances produced during stationary phase eg tetracycline Not essential for growth Dependent on growth conditions Produce several related compounds Overproduction possible hard to do with primaries only so much ethanol Terms Fermentor The tank in which the organism grows can be 1 L to 500000 L They can be aerobic more add ons more work heat build up and control for example and anaerobic heat build up but little control necessary Fermentation Any large scale microbial process may not necessarily be fermentation Fermenter the microorganism involved Control of the process comes from controlling heat to the measure of cells and product to the alteration of environmental parameters Data acquisition of all data points has to be real time Scale up is necessary to take the process from a bottle on a bench to CSTR to big big CSTR If you can be a master of these you can make a lot of money The Bene ts Antibiotics Def Compounds produced by microorganisms that kill or inhibit the growth of other microorganisms Typically for industrial purposes they are produced by Fungi and Bacteria Archaea possible in the future Typically they are secondary metabolites require a lot of steps to produce their complex structure Discovery screening and modeling Growth once accomplished in a fermentor you then have to purify the compound This can involve removal by ion exchange solvent extraction absorption or chemical precipitation all of which add cost and complexity Ideally what you want is highly purified crystalline compound that exhibits the biological effect at hand Book talks about Penicillin and Tetracycline Processes Enzymes Japanese Attack Laundry detergent filled with Proteases Vitamins and Amino Acids Book pg 953 Aspartame Nutrasweet Brand Sweetener An amazing compound A lot of money Aspartic Acid Phenylalanine linked with one di peptide bond It is aspartate a methyl ester of phenylalanine Structure Class Alcohol Most folks like it Where does it come from Fermentation of vegetative materials with naturally occurring yeasts Alcohol and CO2 produced as by products