MICROBIAL DIV EVOL
MICROBIAL DIV EVOL MIBO 4300
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The abundance of prokaryotes on earth Indirect evidence of the abundance of prokaryotes 1 The earth s atmosphere is composed of gases made by prokaryotes Biologically formed of air prokaryotic processes that forms the gas gases found in air N2 78 1 Denitrifrcation 02 209 Photosynthesis primarily eukaryotic C02 360 ppm respiration CH4 18 ppm methanogenesis N20 03 ppm nitrification Major other gases are the inert or noble gases ie He Ar etc 2 Through nitrogen xation prokaryotes are the major source of organic nitrogen for all organisms 3 Prokaryotes can be found virtually everywhere on earth from 77 high in the atmosphere to the subsurface The percentage of prokaryotes in various habitats on earth habitat of total marine subsurface 66 terrestrial subsurface 26 soil 48 seawater 22 freshwater and saline lakes 00043 domesticated animals 0000080 sea ice 0000074 termites 0000012 humans 00000072 domesticated birds 0000000044 Most prokaryotes are free living and in soil the oceans and the subsurface Prokar39yotes are abundant on modern earth Number and biomass of prokaryotes in the world environment no of prokaryotic Pg of C in cells x 1028 prokar yotes aquatic habitats 12 22 oceanic 355 303 subsurface soil 26 26 terrestrial 25 250 22 215 subsurface TOTAL 415 640 353 546 Pg 1015 g Relationship of plant and prokaryotic biomass to primary productivity ecosystem net primary total carbon content Pg C productivity Pg Cyr plant soil and subsurface aquatic prokaryotes prokaryotes terrestrial 48 560 26 22 215 marine 51 18 22 303 Comparison of plant and prokaryotic nitrogen ecosystem total nitrogen content Pg N plant soil and aquatic subsurface prokaryotes prokaryotes terrestrial 10 62 53 52 marine 014 053 73 Assuming a CN ratio in marine plants of 125 Isotope fractionation a signamre of life in the fossil record huqu lmnhum m HHJJHH llu39nmnnf Cm n Wu mm Mnawl Hicm wn m Hwhmnyhmc H m u39 k m mwuxwmh Ciurbnmlc mwmmummummmymmum mm m mm H qum qum hrkmmlc39 muHrh vmlmlh mm a n Mm mm m 4 mm Anvan ma Hmmu m 4 m o I Lul PnB Remquot am aquot rmiznluxgznic m innxganic czrhunztzs urban mm the uxgznic mman ispmhahly dismmdheczuse ufcllzngzsinlllz u m m gmchzmialp mags mmquot m inugznic mm quotmm is cunn39lmd n be mu quotm u Cundug39nn39 m hhmzss fnc nn ufcarhun in circulzl39nn in an hinq lzn has h an 2iner c nslznl furhillinns uryms llwzspmhzhly myme u m zvullninn ufeuhmtzs Model for the early evolution of prokaryotic life Prokaryotes probably rst appeared on earth 38 Ga ago Soon thereafter they achieved a high biomass This model is supported by microfossils and stromatolites F Stromatolite A rounded multilayered sedimentary structure up to about 1 meter across formed by microbial mats in which a rocklike layer of either sand or precipitated minerals is also present Fossil stromatolites constitute our earliest and most pervasive direct record of life on Earth and have been found in rocks dating back at least 28 billion years Although many stromatolites are fossils there are a number of locations on the modernday Earth Where stromatolites are still forming as shown in the illustration From David Darling Encyclopedia of Astrobiology l I I39 Astronomy and Space ight httpWW 39 u V 39 I Laminae of stromatolites are very distinctive and H a make them easy to recognize even in ancient rocks Laminae are formed by cycles of deposition of V a different materials during biogenesis image from 1 httpWWWfossilmallcomScienceAboutStromatol itehtm Micrufnssil mm m acdmlculury m S s 3 S muhl ml um Mass or lprusunmhl r rml Ih pal munum AGE in O W ESPREAD MUNMNT MIERDFUSSILS AEUNMNY STRDMMHLIIES mama FOSSILS 2 o N o z m o z u mcaoam smcmmuzs WIDESW EM 539 39a Fuufzscus 5m I mean wow n39 a a was onequot B awznmw wow a W chcm mm gunmanLs mum y rm xmw sznmzuwv mos m u lml n Eww r 4va m w p M mum r m h My qu mm mm a 4mm mmnw quotan um um mm mmm u a mm m1 mm pmrrnlh mm hm V m m A mm H w m u n 4 mvkunmluml mm m vlw uh M 3 5 Origin of oxygenic phototrophs cyanobacteria An 39 1 L Cambrien Precambrian cutoff is 05 153 r v billion years 2 393 4 Multicellular plants and animals 20 m 1 E g 10 5 g 4 lorigin of modern eukaryotes E E Endosyi nbiosis E g 2 Development cgf ozone shield 1 8 ID I a o a 01 N 2 o 3 Oxic a 4 OJ E 4 4 First traces of cellular life Chemical Prebiotic synthesis Anoxic evolution of bromolecules Formation of ghee rm 4 46 x 109 years before the preseh t 0 Figure 118 Major landmarks in biological evolution and Earth39s changing geochemistry One gigayear GYJ is one billion years Note how the oxygenation of the atmosphere due to cyano bacterial metabolism was a gradual process occurring over a period of about 2 billion years Although full 20 oxygen levels are required for animals and most other higher organisms this is not true oi prc karyotes as many are tacultative aerobes or microaerophiles cf3 Section 615 and Table 64 Thus pmkaryctes respiririg at reduced 02 levels may have dominated Earth for the period of a billion years or so before Earth s atmosphere reached current levels of oxygen From Madigan and Martinko 2006 Prokaryotic diversity What is diversity and how is it measured One can imagine many types of diversity morphological functional evolutionary etc Each type of diversity might be of interest for different reasons Morphological diversity early editions of Bergey s Manual classified organisms according to shape because this characteristic could be easily measured Functional diversity in the study of pathogenesis one is interested in the functional diversity the ability to cause different types of diseases in different hosts Plant pathologists frequently name species of bacteria according to the plant they infect Similarly microbial ecologists frequently discuss groups of prokaryotes according to their role in geochemical cycling ie nitrifiers metliylotrophs Evolutionary diversity considers the ancestry of the organisms Implies that organisms whose last common ancestor is more ancient and more diverse Unlike other types evolutionary diversity can lead to a quotnaturalquot system of classification Systematics the scientific study of the kinds and diversity of organisms and of any and all relationships among them the classification of organisms in an ordered system designed to indicate natural relationships Classification the ordering of organisms into groups on the basis of their relationships genetic phenetic or phylogenetic Taxonomy the theory and practice of classifying organisms Identification the process of placing an individual into a given group Taxon Taxa a taxonomic group of any rank that is sufficiently distinct to be worthy of being assigned to a specific category Advantages of a natural classification it is explanatory it is predictive it is conceptual ie it follows from and adds to our concept of what is a prokaryote Phylogeny the evolutionary history of an organism The phylogeny of prokaryotes has been difficult to study until the recent advent of molecular techniques Currently it is the basis for prokaryotic systematics The library metaphor Imagine that each prokaryote is a book in a library and your goal is to learn the contents of every book If the library is organized arbitrarily then you have to read very book to learn the contents of the library If the library is organized in a natural system you only have to read all of some books and only parts of the other books The remaining contents of the library can then be inferred from the location of the books The downside is that you also have to know the organizational principles Numerical Taxonomy Based upon the principles of Adanson an 18th century botanist A phenetic analysis in which taxa are de ned on the basis of overall similarity according to all possible tests of all possible strains where the results of each test are weighed equally number of tests lt40 not statistically signi cant 100 200 usually optimal number of strains upper limit usually about 300 characteristics for classi cation care must be taken to choose characters representative of many properties of the cell ie morphology biochemistry cell wall chemistry nutrition etc unit character a taxonomic character of two or more states which can not be subdivided logicallyftest are designed to delineate the unit characters Table 21 Characters for classifying micro organisms Sneath 1978b Class of character Examples Morphological Physiological Biochemical Chemical constituents Cultural Nutritional Drug sensitivities Serological Genetic 39 Number of agella Shape of spores Ability to grow anaerobically Oxidase activity Acid production from galactose Presence of lysine in the cell wall Usual appearance of colonies on a de ned medium Ability to grow on acetate as sole carbon source Requirement for thiamine Sensitivity to benzyl penicillin Agglutination by an antiserum to a reference culture Presence of a speci c precipitin band in a gel ltration experiment Percentage 39of GC in DNA Ability to be transduced by a given bacteriophage preparation Extent of pairing with a reference sample of DNA l A FLOW CHART OF NUMERICAL TAXONOMY i I I CHOICE OF SPECIMENS 2 DISCOVERY IMEASUREMENT 0F CHARACTERS 9 CALCULATION OF AFFINITY SIMILARITY BETWEEN SPECIMENS 5 CLUSTERING OF SPECIMENS INTO PHENONS akin 5a Vr fl 6 EXTRACTION OF DATA ON TAXA r 1 WM k pa 7 IDENTIFICATION OF SPECIMENS Limitations of numerical taxonomy 1 For routine classification the process is very time consuming 2 Not truly objective due to dif culty in determining unit characteristics and in sampling a representative population 3 Sensitive to error in test design Advantages of numerical taxonomy 1 Knowledge of the properties of an organism is useful for other purposes besides classification 2 Many of the techniques are accessible to most microbiologists L Table 24 Phenotypic characteristics of ten hypgtbe l bacterinl clones Colony Presence Spore Spore V V Sporangial texture b of spore shape position shape g1 roughsmooth yesno round I central 1 swollen mUCOId oval 39 terminal I normal lClone 1 Rough No NC NC NC 1 Clone 2 Smooth No NC NC NC gClone 3 Smooth Yes Round Central Swollen Clone 4 Rough Yes Oval Terminal Swollen lClone 5 Smooth Yes Oval Terminal Normal Clone 6 Rough Yes Round Central Swollen Clone 7 Rough Yes Round Central Normal Clone8 Mucoid Yes Oval Terminal Normal Clone 9 Smooth Yes Oval Telmin 39 Swollen gt Ch 10 MW N0 NC NC 3L NC 1 Codlng of 0010112 texture rough I l 0 0 Table 25 Binary for colony texture and presem 33572 smooth 1 0 3 L Colony texture 739 Premise of spore mucoid NC 1 Clone 1 1 o o o 1 Clone 2 NC 1 0 0 r Clone 3 NC 1 o 1 number39 6 l 4 l 1 39 z 5 NC 1 g 1 different 111133er Clone 6 1 0 0 l 9 Clone 7 1 o o 1 rough vs 1 2 7 Clone 8 NC NC 1 1 Clone 9 NC 1 o 1 smooth l Clone 10 7 NC NC 1 0 I rough vs 1 1 1 M 39 j mucoid r Table 26 Bluwy eohg r morphological properties fanes and Sporangia I I smooth vs 1 1 mucoi 1 Spore shape Spore position Sporangial shape d gone 1 NC NC 39 NC lone 2 NC NC NC Clone 3 l l 1 Clone 4 0 1 Clone 5 0 0 0 1 None 6 l l 1 Clone 7 1 l 0 lone 8 0 0 0 lone 9 0 0 I l 1 Clone 10 NC NC NC Table 27 Binary coding the phenotype listed in Note that the of spores is Table 24 39 39 quot quot welghted Spor Colony Presence of Spore Spore 39 angial texture spore shape position shape 2 AClone l l 0 0 0 0 0 0 NCNC NC NC NCNC 3 Clone 2 NCl 0 0 0 0 0 NC NC NCNC NC NC Clone 3 NCl 0 l l 1 l l 0 1 0 l 0 Clone 4 l 0 0 l 1 1 1 NC 1 39 NCl 1 0 Clone 5 NCl O l 1 l l NCl NCl 39 NCl A iCloneo 1 0 0 l l l l l 0 1 0 1 0 Clone 7 l 0 0 1 l l l l 0 k l 0 NCl Clone 8 NC NC 1 1 l l 1 NC 1 NC 1 NC 1 Clone 9 NC 1 0 l l l 1 NC 1 NC 1 l 0 0 0 0 0 NC NC NC NC NCNC Clone 10 NC NC 1 Calculation of similarity OTUi 1 0 1 a b OTsz 0 c 1 simple matching coef cient Sij a d a b c d J accard s coef cient Sij a a b 0 different coef cients are not necessarin jointly monotonic ie they can give different results J accard s coef cient wanid he preferred if a large number ofw but 390 ed bl 31 Table 31 Hypothe ka dam set for four Table 32 Simzlarity cw CM data m Ta e quot OTUS on ve binary Chamaers Simple matching coef cient Jaocard s coef cient 39 similarity matrix similarity matrix Character 2 4 5 39 010 1 2 3 4 I OTU 1 2 3 4 1 l 1 0 0 1 DTU 2 1 o o 0 0 1 10 l 10 3 1 l 1 0 1 2 06 10 2 033 10 4 0 0 0 1 1 3 08 04 10 3 075 025 10 0 a4 04 02 10 4 025 00 02 1 1 l A l 39 57 I 2 0 7 l 3 5 0 0 I 4 5 0 0 0 I 7 0 1 0 I 0 l 0 s 0 c 0 0 a 1 I 0 I 0 10 0 I 0 9 40 0 55 3 100500055 l0 0 0 0 0 0 I 0 quot5200530 l29507000 l4 5 5 I 7 0 0 0 245 007500 5000005 68000003 I00 077750 200500055 235500090 I 5553050 2 4 5 0 7 0 0 0 2571005310 t700770017 139 07000000 1l oouoos Icenab oouu 51704015 1 0 SIMILARITY STRAIN IDENTITY 39 56 SIMILARITY STRAINS IDENTITY 3 TO 90 3900 I DO I Unmtiilld Gram l Wm 670m 5 l motive red I M tlvo rod 2 3 Preview In MW 7 Chum I 5 Cumquot 0 Loam torWu no Clu u 2 39 I a Lachelm up I0 Claim 2 ll I2 I 24 I5 I 6 VINIG m 20 CIUIMI 3 23 5 mm pawnmi 2 CMquot 3 22 25 l7 l9 l l w FIG 1 Presentation of output from a numerical taxonomy analysis of bacteria in which a sorted similarity matrix has been calculated A the numbers have been rounded up to one decimal for convenience From this information a shaded diagram B dendrogram C and simplified dendrogram D can be prepared Example of numeriCal taxonomy of strains of Photorhabdus These are bioluminescent bacteria symbiotically associated with insectpathogenic and other nematodes Also some human clinical isolates found in this group From Akhurst et a1 1996 Inter J System Bacteriol 461 1034 1041 39 TABLE 3 Phenotypic characters that discriminate Photorhabdus W 9 u 8 g 13 Assimilation of an ssiaasg a g V H AEE Isolate f g ggggg ag g 58 0 HM gl gig sswsggaggg a a i iai iiiii ggsgga asggsgiagiiiiiii i i giiiga lt2 ac39g ag39n39a 5 Egt55 5 oEEm ngsiacg 45 artg 396 Hbl ATw w w w Hb2 w Tw w 39 w Hml wI HmZ 1 in to w HV161 395 5 w HV162 r NC1621 val I I L In iv NC1622 w wwv a C H w HIl Eg32m ww HIZ w l V mam2 ww Kggg 4 ns22H ww K r 016 2 ww K811 w imam2 r e uang 4 K802 euan hH2112 ww 11 4 XIUtZ D12 g jz sni39 ww C84061 rern C84062 012 HP881 132an w HP882 w HF851 T3371quot ww HF852 ww w mg 4w HL811 w erUt1 ww Hlez w w HFR861 v HFR862 w HW791 w HW792 w w ww XLNach1ww XLNach2 w w w XLLit1w w XLLitZ w Bubanal w ww Habana2 39 ww NZH31 w NZH32 w 4 Meg1 gt Meg2 w T3271 w 73272 ww FIG 1 Numerical analysis of phenotypic data for Photorhabdus isolates showing the signi cance of phaserelated characters Similarities calculated With the Jaccard ooe icient were clustered by using the furthestneighbor strategy t airman ts km Numerical taxonomy leads to one concept of a species Liston Wiebe and Colwell 1963 J Bacteriol 85 10611070 Hypothetical Median Organis described by the collection of characters typical of most of the strains of a species all of the strains will have a similarity coef cient of gt 75 to the hypothetical median organism The median organism describes the typical strain not the type strain TABLE 4 39ulrulnn vl rummu 0 IIIIl39I for I m mImmmrs mirummw hlntin lique ed Slum rovl Round or slightly tnr Litmrs mill acid and he pen0d cml peptonizod 3 5331659 I lDC 1 Single and pairs NH from l f39llitllll 1 31quot 2 50v Motilc Imlnr fluttl39llu N0 y 5 i 9 9 39 5m in t lHl lrr u w 7 k hr 39 triuiltluvvnt 39ll Mmlinm 39nlml 2 In 5 tall gt 39l4wl 39mnt vix Tu39aloicmverce r gl di 039 33 Silums lllllll Entire 4ng Km Tlitlilsilvlv prawn lllm rmrrnt pipnmnl Pvllit39lv nirulrrun39 won turliirlil 39itrnItanL itnn Hugh and Ltii mnr nm39oliic glucosw positivm l Inicillinuw lnmlurml l HridirIixheihltivr iru lll in 37 Agar not llL39t39llll rm Fl 3 Frtqurllr y ojipnircd hararlrr mtnrrcnrv Unmznmm 7m inrlquL i w s C a 14 l7 539 l3 MENz so I A 50 iquot a SW at Pnsmvg numerous rm 2 Imulrmvx of Pscudamnnrzx acruginnsn 81mins arrordiny tn nmnlmr of pnsili39rr I39lmrm39trr39s Limitations of numerical taxonomy 1 For routine classi cation the process is very time consuming 2 Not truly objective due to dif culty in determining unit characteristics and in sampling a representative population 3 Sensitive to error in test design in a very complex fashion Advantages of numerical taxonomy 1 Knowledge of the properties of an organism is useful for other purposes besides classi cation 2 Many of the techniques are accessible to most microbiologists 3 Provides a description of the organism DNAzDNA hybridization Methods for determining the ability of genomic DNA from different organisms to hybridize to each other Used as a measure of relatedness of the organisms Typically this involves these steps 1 Genomic DNA is isolated from each of the organisms to be tested 2 The DNA is sheared to about 500 bp in length and then melted or denatured to obtain t e monomers 3 The melted DNAs from each organism are mixed and allowed to reassociate under optimal conditions usually 25 C below the melting point of the duplex 4 Under these conditions DNA this identical rehybridizes quickly DNA that is similar rehybridizes but the complexes are less stable DNA that is dissimilar doesn t hybridize 5 Typically DNA from two related organisms will be a contain segments of DNA that are almost identical and hybridize well segments of DNA that are similar but not identical and hybridize slowly and form less stable complexes and segments of DNA that fail to hybridize at all 7 To compare the relatedness of genomes from different organisms one can determine the stability of the heteroduplexes determine the fraction of DNA that hybridizes TM or TSDH the melting temperature or the temperature at with 50 of the duplex DNA melts An indication of the stability of the duplexes DNA the is less closely related melts at a lower temperature Also depends upon the mo GC DNA with a higher proportion of AT pairs also melts at a lower temperature g DNAspeciesA HornnduplexWA e n I Denature into Measure singlestrands l i l l heb DNAspecies B and mix de aturatia as temperature Heteroduplex W3 is raised Mis matched region Singlestranded DNA 3 2 3 Ti 1 Tern peratu re C D Report ofthe Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics Inter J System Bacteriol 37 1987 463464 established three criteria for definition ofa prokaryotic species AT Members of the same species I would possess gt70quoto DNA hybridization 2 with lt5 C change in the melting temperature ATm of DNAzDNA hybrids 3 have similar phenotypic properties Thermal stability ATm is measured as the difference in temperature required to melt half of the hybrid duplex versus half of the homologous duplexes The thermal stability ofthe DNAzDNA duplexes is correlated to DNA sequence similarity not the extent of hybridization AT increases about 1 C for every 17 quot0 base pair mismatch Caccone et 211 J Mol Evol 27 212216 Fig I Graph of data in Table 1 Numbers correspond to left most column in Table 1 Bars indicate 1 standard error Regression line is drawn using all points without constraint of passing through the ori 39nt See Table 2 for allcmative analyses l t t l I I I I 1 2 3 4 5 6 1 3 2 hp mismatch Genospecies species de ned on the basis of DNA sequence similarity or other genetic characteristics such as criteria 1 and 2 above A second species concept if two organisms are similar the DNA sequence of their chromosomes should be similar DNA DNA hybridization attempts to measure the extent of this similarity by measuring the ability of denatured DNA from one organism to reanneal to DNA from another organisms or fomi duplexes The extent of duplex formation hybridization is an indirect measure of the extent of DNA sequence similarity M 1 I I m pman usml m dilterem mLmuus nuy ymm mme BuLlunul U 017 EU m n mu punm e Z 2 lt z n m Hni m mu m mu 39 Mrurnr muHAnnv 1m pmmr m null m u m u u rcsuls Sec Brennacl m Mb lll Mum 1un Wm M quotmm Inn mm m I m m m Wm m mm w y rmw m 1 WWW am I y w m 7 my my mm M W m 1 Hum My WM 4 mm u h my mlvmwmnm n Wm m m y UM W m m INHWWW w m mm M mm M Hm hm u w H m mm m w whn m H W m Universal small subunit rRlVA rec green hncrcri arlerln BACTERIA rlcln Thermnmgnlcs cxlrcnlc hulaphnn Iclhunuguns ARCHAEA L39xlrcllu mermuphilcs plums EUCARYA rungl animals cilinlus cellular dime mulm agellum Fig 1 u unmulcd unmml phyhlgcncuc mu Th mm was pmdurcd from m cvnlulionury lllsmncc malr ix dunvcu I rum an allgnnlcnl ul mull suhnnll rR sequenccx nly Hum plmnuxlc m m nllgnmcnl Judgl l nu be homologous among all llm lulnullls wcm used In the calculanon Dclallcd dcsmpuuns ul Ihusc pmccdurcs can be found In Wocsc lus nm rulm 39nucs cued llwrmur From R Wuesc 1997 Prokaryolic syslcmalics lhc evolution ol39sclcnue In The Prokuryotes pp 318 Tun ideas follow from this 1 Any organism can he uniquely identi ed by 1lsnls IS around 60 94 Z l mkulymic diversily exceeds eukaryolic divcrsily Rucugnillon ol lwo pmkaryolic domains Archaca and the Bacle u very diffcrcm phylogcnclic groups where each group is abuul as related to lhc olhcr as each group is as related In the cukaryolcs Even in the cukmymcs mos oflhe diversity is found in lhc prolisls 39placu 0n lhu Irccr Nola the quoto S for all living Because the 1 Changes slowly it is most useful for detcnnining the higher taxonomic levels Although these are applied with 1 great deal ot39llcxibilily some guidelines for prokuryotcs lire at the species level iFrRNA sequence Similarity 45 is lt98 quotn evidence ol dil39fereiit species But there is no correlation between WAS and DNA hybridization il ilS is gt98 quot0 see below in this case some other technique like DNA hybridization must be used ul the genus level ims is lt93 05 quotAl evidence ol dii l ercnt general it the family level ifS is lt 8891 quotIl uviilcncc ol diffcrent Families ul the phylum level iquotquotl39lS is lt80 3954 evidence ol39dil39l cl39cnt phyla 39nlnpurison iftaxunoniic ranks with S ill eukaryotes compiled by Mark Wise 497 W worm lrult Fly Oyster Lamprey Killilish A C Frog Rabbit Rat Human Kingda Kingdom Kingdom Kingdom Kingdom Kingdom Kingdom Kingdom Fruit Fly 7039 Kingdom Kingdom Kingdom Kingdom Kingdom Kingdom Kingdom Oyster 7296 8015 Kingdom Kingdom VKII39IngDl39I39I Kingdom Kingdom Kingdom Lampley 7344 7353 8532 K Phylum Phylum 39 Phylum Phylum Phylum Klillllsh 7355 7757 33 49 I 9118 Phylum Phylum Phylum Phylum 39 A Frog 72 86 7E 74 8456 91 90 9510 hylum Phylum Phylum Rabbit 71 4B 75 41 8142 l 8808 9044 92 76 f 39 is K Hal 7320 7854 8362 9135 9291 9517 9535 Class Human 7353 7856 8373 9175 9352 9556 9608 9925 Upper triangle matrix lowest common taxonomic level based on D K P C 0 F G S nomenclature Lower triangle matrix similarity between 185 RNA gene Organisms in separala Phyla 703985329 similarity Organisms in separate Classes BEDS9556 similarity Dragnlsms In separate Orders 95359925similallty 39mnpurisnn of rRNA sequeni e similarity with DNA hybridization Slziekchl39zlliill and Goebel 1994 ntulz l Systcm Buetcriol 44 8464549 showed that 168 l39RNA sequence similarity was l39ClHlCd to DNA hybridization zit sequence homology values below 075 1 it is unlikely that two organisms have inure than ii to 70 quotll DNA similarity and hence lllill they are related at the species levelquot DNADNA reassociation u in 20 30 do so so 70 so so we L it I 39 a x 1 x 1 ix 05 xx x a m X o g 3 W g 0 lt 2 z i m c mu rcliwuu39lullun ullm avmhul and mum it my mm m l liil m l itrmlurnlll li nu mum lll 1 renalumtilvn m muhhulm i mm rill mu m initHK tllc um mmhulu mm m 5pm delineull m w l us in lmtillllngy um m ml l u mrmhmnl mll39 melliliu ll V 51 nuclcm mum Polyphasic taxonomy seeks to assemble and assimilate many types of information from genotypic to physiological and biochemical to ecological to yield a multidimensional taxonomy or a consensus of approaches A major advantage of this method is it avoids errors caused by artifacts associated with anyone particular method For instance in some lineages the 168 rRNA gene has unusual indels insertions and deletions For these organisms the rRNA sequence similarity is very misleading From Vandamme Pot Gillis De Vos Kersters and Swings 1996 Microbiological Reviews 60 407438 GENCODTYPHG HNFQRMATKDN T OTAL DNA 0 BASE SEQUENCING MOL G c o LMW RNA PROFILES o RESTRICTION PATTERNS RFLP PFGE o GENOME SIZE 0 DNADNA HYBRIDIZATIONS DNA SEGMENTS 0 PCR BASED DNA FINGERPRINTING RIBOTYPING ARDRA RAPD AFLP 0 DNA probes 0 DNA sequencing 1L mTM quot o ELECTROPHORETIC PATTERNS OF TOTAL CELLULAR OR CELL ENVELOPE PROTEINS 1D OR 2D o ENZYME PATTERNS MULTILOCUS ENZYME ELECTROPHORESIS L L EMOTAXONOMIC MARKERS EXPRESSED FEATURES o CELLULAR FATTY ACIDS o MORPHOLOGY o MYCOLIC ACIDS o PHYSIOLOGY Biolog API 0 POLAR LIPIDS o ENZYMOLOGY APIZYM o QUINONES o SEROLOGY monoclonal polyclonal o POLYAMINES 0 CELL WALLS COMPOUNDS 0 EXOPOLYSACCHARIDES PHENCDTYPHC HNFQRMATHCQN FIG 1 Schematic overview of various cellular components and techniques used RFLP restriction fragment length polymorphism PFGE pulsed eld gel electrophoresis ARDRA ampli ed rDNA restriction analysis RAPD randomly ampli ed polymorphic DNA AF LP ampli ed fragment length polymorphism LMW low molecular weight 1D7 2D7 one and twodimensional7 respectively Family enus pecies train 5 U vvrv Technique Restriction fragment length polymorphism RFLP Ribotyping DNA amplification AFLP APPCR repPCR DAF RAPD ARDRA Phage and bacteriocin typing Serological monoclonal polyclonal techniques Zymograms multilocus enzyme polymorphism Total cellular protein electrophoretic patterns DNADNA hybrid izations GC tDNA PCR Chemotaxonomic markers polyamines quinones Cellular fatty acid fingerprinting FAME Cell wall structure Phenotype classical API Biolog rRNA sequencing DNA probes DNA sequencing FIG 2 Taxonomic resolution of some of the currently used techniques Abbreviations are de ned in the legend to Fig 1 Nomenclature of prokaryotes Prokaryotes are named according to the binomial system of naming plants and animals All species are named by a binary combination consisting of the genus name and the speci c epithet All species are based upon a type strain A type strain is made up of living cultures when possible of an organism descended from a strain designated as the nomenclatural type when the species name is first proposed The strain should have been maintained in pure culture and its characteristics should closely agree with those in the original description The type strain does not have to be and frequently is not typical of all the strains of the species It serves as a reference point if the taxonomy is later revised When the type strain is lost a neotype strain may be proposed The neotype strain should have properties similar to those of the original description All higher taxa have a type chosen from the lower taxa For instance a genus has a type species and an order has a type genus In this way all the taxonomic ranks are ultimately connected to a biological specimen or type strain However the species is the only taxonomic rank which is de ned by a consensus of workers in many different elds Prokaryotic taxonomy is governed by the International Committee on Systematics of Prokaryotes ICSP formerly the International Committee on Systematic Bacteriology ICSB which is a committee of the International Union of Microbiological Societies IUMS The IUMS itself is a loose federation of national societies like the American Society for Microbiology or ASM The International Journal of Systematic and Evolutionary Microbiology IJSEM formerly the International Journal of Systematic Bacteriology IJSB is the of cial publication of the ICSB Prokaryotic nomenclature is governed by the International Code of Nomenclature of Bacteria 1990 Revision also called the Bacteriological Code Only taxa that are validly published have any of cial standing in the Bacteriological Code Taxa described before 1980 were validly published in the Approved Lists of Bacterial Names For taxa described since then the original proposal must either be published in the 1 SB or if published elsewhere appear on the Validation List of 1 SB The effective publication date is the date of validation Taxonomic ranks denotes ranks commonly used for prokaryotes subspecies often used to make important functional distinctions species genus phylum division kingdom domain not widely used outside of Microbiology not a formal rank Modern populations of prokaryotes Modern populations of prokaryotes have been studied by multilocus enzyme electrophoresis This methods enables investigators to distinguish between closely related strains Method 1 prepare a water soluble extract of about 10 cells 7 2 electrophoresis of extract on starch gels 3 detect individual enzymes by activity staining Reference Selander RKs DA Caugant H Ochman J M Musser MN Gillner and TS Whittam 1986 Appl Environ Microbiol 51 873884 Starch gel electrophoresis separates proteins on the basis of chargeand molecular weight Electrophoresis of proteins with a known amino acid sequence suggests that 8090 of the amino acid substitutions will produce differences in mobility Allozyme or electrophOretic variant is an orthologous protein with a different electrophoretic mobility It is roughly equivalent to an allele for a structural gene Activity staining in a mixture of proteins it detects a single protein on the basis of its enzymatic activity by the choice of substrates and reagents example for malate dehydrgge 1135 g soak the gel in a solution containing malate MgCl2 NAD PMS phenazine methosulfate pMTT dimethylthiazol tetrazolium a er incubation at the appropriate temperature the location of the band containing malate dehydrogenase turns blue Similar to numerical taxon omy allozymes could be considered unit characters they are homologous and have the same weight in closely related organisms Example of analysis of multilecus an quot TABLE 9 Hypothetic al example showing electrophoretieftiype for ve isolates Isolate no Electromorph allele at enzyme locus A B C l 1 3 2 1 3 3 339 l 3 5 4 l 2 l 5 1 1 2 mun NH U TABLE 10 Allele frequencies in sample of ve isolates Enzyme locus Frequency of allele 1 2 3 39 4 s B 020 020 060 f C 020 020 020 020 0amp3 D 020 040 040 quot See Table 9 11 Genetic distance D between pairs isolates TABLE Strain 1 2 5 I 439 3 is 1 000 quot 2 025 000 3 050 050 000 4 075 075 075 000 5 075 075 075 050 0000 quot Genetic dista Table 9 nces are based on the alleles at the four enzme lmii39e 7 d 39 an 39 quot on is 0 7 Y n FIG 1 Gels illustl39atlnn elemrophoretie variation in three en zymes A Mannitol 1phosp arrow hate dehydrogenase in Eeoli18 isolates B Glucose 6bhesphate dehydrogenase In meningili dis 19 isolates C Malawi cle vdrogenase in fair M Holmes i Anodal direction ol migration Item the origin is indicated by the Generic dis fance 75 25 O 39 239 39 l I 4 I L FIG 2 Dendr39omm g liei tanee D in Tablel 1 j Do prokaryotic species exist in nature Three points of view 1 Species are real entities that are discovered or revealed by classi cation 2 Nature produces individuals species have no actual existence Species have been invented so that we can refer to great numbers of individuals collectilvely 3 Species are phenotypically similar groups that represent adaptivepeaks Thus species are formed by an interaction betWeen phylogeny and ecology Clonal model of population structure Populations of bacterial species are basically clonal Even though the number of individuals may be on the order of 102 these individuals are recently descendents of a much smaller 102104 group of ancestors population a group of related individuals in a speci ed area clone a group of genetically identical or nearly identical cells that owe their similarity to a recent descent from a common ancestor in the absence of chromosomal recombination or genetic exchange Evidence for a low rate of recombination 1 In chemostats the rates of phagemediated and conjugative plasmidmediate recombination are very low about equal to the rate of spontaneous mutations 108 per cell generation 2 In natural populations the variations in allozymes is consistent with very low rates of recombination If recombination was equent the distribution of allozyme see below would be random 0 o 300 0 0 observed 0 0 land om o 200 0 Lo IOO of pairs Number I iise re uo iz Of 0 Number of differences Flaunt 4n Distribution of differences at E loci in 13quot pairwise comparisons of 53 ii39l s V Caugant DA 1211 Levin ananKf sdimderjl981 Genetic diversity 39 V theE coli39population oftrhehuman39fhhs GMc89814674901 Vi 14 7 Numbtrolclombl mmor aeapn umpugggm V ri ip Dat andnumberof clonesanalvud 1 39 7 39 W T 517 520 39823 112 113 11439 11511 317118 119 1301 121 122 4 34 29 21616 37 1 3 11 21 2o 16 20 20 20 14 19 20 39202 20 20 13 1 320 50 74 39 3935 was 35 r P 1 00 a 11 OQVO MOWIO 1o 39 usale 12 17 14 16 2039 7 13 20 39 2 neu39mhs 3 1 3 12 9 2 7 15 19 14 v 11 1 39 15 39 1 1 m 1 17 39 39 as 19 20 nan wpmmu g 399 ambiti 11 amgwu A 23 39 1 V 1 39 r 51 39 39 22 172553734 124 21939 2161 351 Mam 20 18 91 3920 so 74 as 35 35 51 520 841 1 121713 114 115 11 339117 118 119 120 121 1 1139 21 1 16 20 2o 20 14 20 19 20 20 so J 393 FEE3999quot 0i 03 a u 0quot Am u mbuauo 39NI gegpg uana amp 39 Full fecal sample FFS1fecal probe FF 51 faker by ivyb S Example of linkage disequilibrium Hypothetic allelic frequencies Enzyme locus Frequency of allele 1 2 3 A 040 040 020 B 033 033 033 Two models for evolution For the population allele at locus allele at locus B 1 40 1 33 2 40 2 33 3 20 3 33 If alleles are in equilibrium the distribution of the alleles in individuals of the population might be if allele at allele at if allele at allele at if allele at allele at locus A is locus B is locus A is locus B is locus A is locus B is 1 B1 33 33 33 B2 33 2 33 33 B3 33 33 3 33 If the alleles are in linkage disequilibrium an example of the distribution of the alleles in individuals of the population might be if allele at allele at if allele at allele at if allele at allele at locus B is locus A is locus B is locus A is locus B is locus A is 1 B1 80 0 0 B2 20 2 60 0 B3 0 40 3 100 Expected f 39 of ET in either case Distribution of ET in r r 39 quot ET Equilibrium Disequilibrium 11 0132 032 12 0132 008 13 0132 000 21 0132 000 22 0132 024 23 0132 016 31 0066 000 32 0066 000 33 0066 020 V0 Low High I A Low High How general are these results Inch of Association l VDNEl VU obsen39ed variance belween eleclruphorelie types ET VE expccied variance between electrophorelie types In populations ofprokaryotes reproducing by binary ssion 1 depends on the relative rates of 1 divergence oflincages lltroiigli xation ofnew mutations and 2 transfer ofgenes between lineages by recamhirmn an lA U is evidence for a high relative rale ofrecombinallon D i mean genetic difference per locus between strains Smith JM NH Smith M O Rourke and 86 Spran 1993 How clonal are bacteria Proc Natl Acad Sci USA 90 4384 quot 8 FIG chrgsenu uns of populllion quotmatures A Ind B hrnncnns The poyIliaLion menu in A is email 1 All levels luck that the dendm i In Yolulio no re mhinaLian m Howevzr Mmlon ly highly Iuccessful incnascx rapidly in mummy u pmdnc m epidemic clone Tlhl l 11 Del in bultria No or No of No of A ism HTS only Bacleria isolates ETs loci D n Rd N guwniwm 217 39 9 0109 M14 o15 m7 1 N menmguidl 688 331 15 0507 0547 9 Haemophilu influenan 2209 56 17 0226 0534 27 Division Iquot 1117 42 17 0186 0452 ivlsian 2quot 92 1 X7 036 0654 Salmontllu H95 6 1 0355 0366 23 p n a u 2i mm 0138 S paranphi 118 14 Z 0074 0163 S phimun39um 140 17 2 0035 01 S pawnphi C X 9 24 0056 014 S chaltrusui 174 l 24 0036 0173 RMmbium mclilnli 232 50 H 0738 0195 N Dlvlsiun A 208 34 14 0101 0233 39vision 2 15 M I 219 02 9 Legmnella 170 62 22 0359 01le 6 L pneumayhilu 143 50 22 0152 0312 quotSpec 3 lquot 24 9 0107 0151 Bardrnlla branchiseplica 304 21 15 0097 0240 W Pundammuu yringu 13 0 26 0M7 0686 1 P ryringae lomam 17 I 26 0076 0150 L60 3 067 P ryn39ngar ryn39nga 6 6 0480 0480 112 2 057 n D Sundnrd ermrs In for Lil null hypothesis 0 independence belween loci m 39 npuhhshed dam Fn yrsix clnslers identi ed by Musser u 41 27 If recombination is common with prokaryotic species can it be observed by sequence comparisons Compare gene tree versus organismal tree Gene tree represents the phylogenetic relationships of an individual gene in a group of strains Organismal tree represents the phylogenetic relationships of the whole organisms Dykhuizen DB and L Green Recombination in Escherichia coli and the de nition of biological species J Bacteriol 173 72577268 FIG 1 Elfect of lecombination on the apparent by the transfer ancestor of emit B 4004 70 E 134 mm 2 ampA IMJSE mtmzm u mznul RM9 whiz wummc 39 I I I mwzou non non we now avian nice nice do e50 m m Genete Dillon 0 6 1920th luaue Iydujvoas e p554 aker39oe fk p cfd e e e h I huonshtp of three strains The gene tree of gene X end C together whereas the gene tree for any other gene will place etnms A end 3 together This di 39erence in the gene tree is ceased 39 ofgenextromenenceetorofeminctmoen amazon gamut gum gar2 g RMZWT gamma Emmott 04091 gun amen Gaelic Dimtee D 0310 new not aim no not min 06 aim non eooo gamer and mRMZOIC 05094 l numzm nuance gamut autumn Eamon 31012 o u39e zz Dino Genetic Dew D 1 f yfbfhan AbW JIo gang 0M Vulural mm mm nf proknrynlcs I 7 39 39 run m luthch p sex 39 mm L uwm Wmwwwmwmymummm HunHA1 numb y W 1w mm mwmmnm m 1mm Hum HuhL um 4 WW m mm mm m u uuw mm A w rm m 3w quot L Mm mnuh h Wm n M m m u n m mm m mu mm h m m n m m Em 39 In is put g dry Velglu ofsui V39Im k Illr d unity unlu Irnkuryuru 39 I mi ansvxk Il xl I39I mJ lml r 39nnhiol 17 I70 39 V quotgmumcmc 39 meu nrnNA Ana Immml dmummlmn muuD me allow Ihe DNA In rcussn U n m clmvcr he mussntimion in higher he camplcmy ml m Inrgn um gennmc my DNA rmquot 3 commumly mgn complcxnv rcsulls fmm Ile prescnm anny vunumcs m umuan species VHmhL r m gamma mmplcxuy nfcnmmuml DNAcumpmiuv nfa reprcscm livc genom luwm mm m w m mm W 1mm u m xm quotMumquot m gtMmm w v i a r y m 07 mum Wm m Awu m MW 7 mmmmuuxm n x w 1 H mm um um x u x 39 mum w mu M n V NUI mm H quot39 quot Jquot 39 l nmm dmmu Nubmumw W4 7 man mum M mm Ath m u u U HM Hulmuumlummu mum 1w VM K uvuv wn nnxw u m NW WWWquotw Hm W W n m x m m mm A m mm mm nu mywdmm 414mm m H mm Mm mmmumm w ur w mm a L mhm W H mm um VLqmwun m L m rmquot n m w p vWMwunm urmmm m r PCR of Environmental Samples Most common application is to amplify rRNA genes with the goal of determining what organisms are present in the community Community composed of many organisms Lyse cells and extract DNA to produce a mixture of genomic DNA Ideally all organisms should lyse with the same efficiency but what about spores Also if sone cells lyse very easily their DNA might become sheared during severe lysis procedures PCR ampli cation of 16S rRNA genes from genomic DNA This step increases the concentration of rRNA genes relative to the other DNA We would like all the rRNA genes to amplify at the same rate so that the proportion is the same as the genomic DNA in the original mixture rRNA genes are cloned so that the proportion of clones is the same as found in the PCR products Because the genomic DNA is at a lower concentration and does not have 3 A overhangs with Taq it is cloned at a much lower efficiency Screen the clones to identify the membeis of the original community Common methods for screening include sequencing and restriction fragment length polymorphisms RFLP PCR artifacts and biases for a review see Wintzingerode et a1 1997 FEMS Microbiol Rev 21 213229 Chimeras Formation of chimeric PCR products following an amplication of a mixed template some of the products appear to be formed from a fusion of the starting templates Wang and Wang 1996 Microbiology 142 11071114 showed that fraction of chimeras was related to the cycle number and relatedness of the mixture of templates but was independent of the presence of sheared DNA Composition of the rRNA gene may affect PCR ampli cation Secondary structure of rRNA may prevent successful ampli cation by blocking the action of the DNA polymerase rRNA from hyperthermophiles dif cult to amplify without the addition of denaturant such as 5 acetamide Reysenbach et al 1992 Appl Environ Microbiol 58 34173418 Thermus thermophilus rRNA genes amplified poorly compared to the genes of mesophiles Farrelly et al 1995 Appl Environ Microbiol 61 27982801 Mol GC for E coll Bacillus subtilis and Pseudomonas aeruginosa rRNA genes 5355 For T thermophilus the mol GC is 657 Presumably the high GC content inhibited complete denaturation and successful amplification Ampli cation of a mixture of closely related templates produced a variety of errors Speksnijder et a1 2001 Appl Environ Microbiol 67 469472 Most of the errors are single base substitutions Expected mechanism was formation of heteroduplexes in later cycles These would be repaired upon cloning in E coll producing chimeras of the starting sequences In the end the artifacts introduced sequence with 0212 sequence difference from one of the starting sequences RNA heterogeneity with a single organisms Most organisms contain more than one rRNA operon In some taxa the sequences of these operons are not always the same Usually these sequences possess very high sequence similarity but occassionally it can be as high as 1 For instance a 120bp nucleotide sequence containing the hypervariable region of the 16S rRNA gene was examined from 475 Streptomyces strains Display of the direct sequencing patterns revealed the existence of 136 heterogeneous loci among a total of 33 strains All of the substitutions conserved the relevant secondary structure The 33 strains were divided into two groups one group including 22 strains had less than two heterogeneous bases the other group including 11 strains had five or more heterogeneous bases Ueda et a1 1999 J Bacteriol 7882 See also Nubel et al 1996 Appl Environ Microbiol 178 56365643 for studies of Paem39 bacillus spp VCRdarken nu m fur delermhnug snll dh enhy mmqu on rlllml I punh DNA mm sail nrmhuruumm sumpk gt V 39 umylny gum 39 msmm l mlmnm Hn A mun a vlnuthLJmlw 4v 39 pm 391 m m ulguml mum IIX39IcaKnm mum n 39Lqmm a v ul39 llmZHU mdn idum chm an u mmnnhlmncl Imh cm lugll dcgwbnnlh39c u msuilpmknryulcsnnd man nmcl MnLI mam uan u Illnngcul 2m Annltxmmn Mmoth 121 ms mu H mm mm m 7 7 W W W h M I mum w l w H w h u w u w u M w H y u v m w m u h NW mwmw g n a y H u MW um u m h w u w u m Hum W m m i w nWWWm m Trrpldl mm uxso um sum ol Ilwsc phyta sugguls q 2M 031 1qu I cum rqu h kquot t h39l m b mewpluhu m Inu chmbml 1 rhgrmuphihr SmImnmI m ltdplnlngrunnIrrnrrudunl mm mm IIMAI frum mu sax mp almmmn u m r a mm m drew m m uvwh lmrnm mm mull chum n n sumam mwuzv CA a trldlum unu 5ixld I unchuxnud x 1 Chllmydxl Vomuamntabx mu 1 nu Hanapun MM hm A m M WM wa r W WNHW Wm m mm my ruhmmw m m m wquot H mm H 1 mx l39mhqvn xmuw mwhhummnh m M mm H m 4 M h m m um mum m 4 an HI MmhHrkhhuhwdu WWW mm M Lm mu m w W m n m mmumd mm mm mmwm Compare the soil habitat to the large intestine of humans Total number of cells in feces is about 3 x 10H cells per g wet weight Extensive culture work has been performed in the 196019805 Most are strict anaerobes Estimates ofculturability number ofCFUsnumber ofcells vary from about 1090 See Suau et al 1999 Appl Environ Microbiol 6547994807 Results of PCR libraries 284 clones examined contained 82 molecular species with gt98 sequence similarity within each group 95 of the clones were representatives of three groups Bacieriodes C lostritlitmi caccoirles subgroup and the C lostridium leptum subgroup All of the remaining clones were related to previously characterized organisms to a much greater extent than found in soil 76 of the molecular species did not correspond to culture organisms Note This study was perfomted on a fecal sample from a single 40 year old male Presumably there is a large variation between individuals diets etc Likewise earlier studies suggested that there was little or no overlap between the human intestinal micro ora in terms of species and that of other animals 001 1 clone UB 3 324 1 clone Clostridium spamsphaeraldas 1 2 clones 1 clone Ruminococcus flavelaciens 1 Huminococcus llavelaciens 2 058 2 clones 275 1 clone 371 1 clone Huminococcus bromii 309 8 clones M 69 1 clone Eubacterium plaunl Clostridium viri e ubactarium desmolans 2 w 32 3 clones 3 90 K so UFlB RFN7 O 395 70 FIG 3 Phylogenetic tree derived from partial 165 rDNA sequence data for m members of the Claslrirlium L39plllm subgroup Bar represents 2 sequence dI B VCYgLHCC Designations clones 21 t e ey organism used to name the g t E 50 an in bul lace type The tree wzu constructed with the SlM39llJleTY and 2 4o NEIGHBOR programs Bootstrap values are based on SUI replications m w gt E 20 E 10 8 o a 150 200 250 3C 0 50 100 Number of Sequences FIG 4 Estimation of the hiodivenily which was obtained by direct cumula nity analysis of a fecal sample Th1 cumulative number of Cl Us is ghen as a function of the number of clones sequenced Clones were randomly used mummic pnpulnlinn ofsubglngivnl plague nl39 39 from 3 mlivduxl This smdy Menn ed n Luge number ofnovcl phylulypes m plague many mm m M7and Deferrilmmares A large number ul pulemia palhugens wcru also idcnnned F mu 7 Dwain u yknn mu Wm m mm m Mutated m u 4 mm m m damn mm Hum m 21 l39unuhwmn m m Kduumy I m wmmmm Ily w a y u 4 gt mv 1 u mm 2 51 er IS acute necroxizing uxcmm gmgmlis mm 51 v IerenlT Banana l83L20U1H77IH7SJ LE P mpmwunwmmnrums mm Mm mm mm mm m pmlaxypu man lrum mm nnn vm m gm m my mm mm m nuummn m mu my n MW u WW 7 n 7 7 a 7 77 e 7 7 n 7 a 1 MM mmxmmg H3111 7 7 7 11 gm 1mm p 1mm 7 1 Wm m mm 7 7 H mm M WWW NMMW 7 x mm 7 77 7 m w mum 7 u mummy 8mm 7 m HleIHIM mmxrm 5 WWW 7 7 7 7 7 mm 7 mm 15 MW 7 3 WWW 7 77 7 1 WWWquot m w v2 WWWHumum7 H 7 M mum x mm m 7 n WWM 7 7 n maximum 7 7 5 WWW mum1W 7 7 1 quotWm WNWWW 7 n WWW us MW 7 7 7 m Iwrmmm m I39thrrnmvmfvnmulu 7 7 a mm m m 74m munmumpch m mm m mm W L m mm mm m mm 7 Species or phylarype swim or done ID Gen unk secession total 1 clones or isolates Stregtacoccus oralismitisquot AF 003929 42 Iquot 0 V V VV 39 Oral isolate 7A AY005040 1 Deteeled in subjects with I Refractory periodonu39tls n1 1 O Periedou tis n ANUG 114 Oral clone AA007 AY005046 2 0 Streptococcus sanguis ATCC 10556 AF003928 12 9 9VV V Stregweaecus cristatus NCTC 12479 AB0083 13 1 l H quotV V V V Streptococcus gardanii ATCC 10558 AFGO3931 3 9 V Streplucuccus sulivarius ATCC 13419 M58839 14 Stregtrwuccus intermedius ATCC 27335 AF 104671152quot II I VVV S constellatus ATCC 27823 AF104676 20 NH 9 Srregmcaccus angjnusm ATCC 33397 AF104678 17 V Srreprococcus cricerux NCDO 2720 1P X58305 Streptococcus sobrinus NCTC 12279 M243966 1 Enrerococmsfaecalis ATCC 19433 Y18293 1 O 1 Abiotroghia adiacens ATCC 49175 D50540 8 W Abiotrophia elegans DSM 11693AF016390 11 Abiolrophia defectiva ATCC 49176 D5054 3 O Lacmbacillus pontis LTH 2587 X76329 A Hrv n2 v Health F5 Bucilli Lactabacillus brevis ATCC 14869 M58810 1 Oral clone BXOOS AY005049 2 Sraplrylococmrs wamerr ATCC 27836 L37603 11 Camella Strain 93388 Y13366 2 Gemclla haemolzsans ATCC 10379 M58799 11 O 100 Mycaplasma salivarium ATCC 23064M24661 2 7 Mycoplasmafaucium DC333 AF125590 5 En sipeZahrix familiarum ATCC 43339 ABOI9248 Solabacterium mom2i clone K010 AYOOSOSZ 4A Luctabacillus catenaforme ATCC 25536 M23729 3 Illallicutes 39 Clostridia 3 1 1 FIG 41 Phylogenetic tree of the phylum Finnicurex identi ed from clone libraries The phylum Fr mu39curex is presently divided inlu three elaxies Bucilli Mallicuux and Clarrrizliaf39 This phylum represented the largest group detected 15 species or novel phylulypes were identi ed Table 4 Slrepmcuccux urulis and S mink could 1101 be differentiated based on sequence comparisons Consequently the two species are grouped together The marker bar represents 1 5 di crenee in nucleotide sequences ralauvu prann uumsnencs mzeol my eonsemuun mm FISH nunresesmln sin hybridh n emethod eol olrgohueleoude probes to label whole eells 1n atyplcal h 39 165 rRNA ztin uses uorescendy label h an m s m s The be ls r flxed eells speclflc rRN 4 types ofcells dlfferent eolors Posslble to use wuh mRNA m some eases Advantages l Deter mlnes eell humber expllcldy 2 r eommumues where posruohal lnformauon may be lmponant Disadvantages l Sensluvlty ls low forrestmg eells wuh a small number ofnbosomes The rrbosome content ofcells mer es wlth the gromh rate For slowly growrhg eells the osome content beeomes very low and the probe slgnal beeomes very small poruohs are avallable for probe blndmg Fuchs at al 1998 Appl Ehvrroh Mrerobrol 64 49734982 3 le cult to use for eells oflow abuholanee many elds may have to be countedto see only afew eells Compansoh ofrelatlve average eohseryauoh gray of parueular allgnment posruon mo am am um am am m m nu ma mm mm um um 15ml umts E mdsealel eohseryauoh au where low values mllpnmlnn ow evoluuohary Fish from Orphan et al 2002 Proc Natl Acad Sci USA 9976637668 View natural populations from anaerobic sediments near methane seeps In other experiments mass spectroscopy was used to show that the archaeal cell masses identi ed by FISH also contained a very low 13C content suggesting that they were obtaining their cell carbon from methane These cells are candidates for the anaerobic methane oxidizers believed to be abundant in these sediments but never isolated Fig 1 Individual cells and cell aggregates of ANMEl and ANMEZ archaea from ERB sediments visualized with uorescentlabeled oligonucleotide probes Unless otherwise indicated scale bar represents 5 ppm A An ANMEl archaeal lament stained with Cy3 labeled ANMEl862 probe portraying the characteristic features of the ERB ANMEl group including a segmented ultrastructure and square shaped cell termini B Tightly associated cluster of ANME l rods stained with the same ANMEl oligonucleotide probe C Color overlay of archaeal ANMEl rods visualized with the ANMEl862 probe labeled with uorescein in green and Desulfosarcina spp stained with the DSS658 probe labeled with Cy3 in red D Large archaeal ANMElsulfatereducing Desulfosarcina aggregate showing an apparently random association of the two groups Scale bar 10 ppm E Color overlay of a layered ANMEZDSS aggregate showing a core of ANMEZ Archaea hybridized with EelMSMX932 probe surrounded by sulfatereducing Desulfosarcina hybridized with DSS658 probe imaged by laser scanning confocal microscopy F Large aggregation of loosely associated individual microcolonies of ANMEZ EelMSMX932cy3 and Desulfosarcma DSS658FITC G Monospecies aggregate of archaeal ANMEZ EelMSMX932cy3 not af liated with a bacterial partner H Color overlay documenting an association of the ANMEZ archaea with a bacterial partner not affiliated with the sulfatereducing Desulfosarcina The aggregate was simultaneously hybridized with three oligonucleotide probes including two FITC labeled probes for the ANMEZ EelMSMX932 and Desulfosarcina DSS65 8 in combination with a Cy3 labeled probe for bacteria EUB33 8 In this aggregate microcolonies of the ANMEZ were stained with FITC but the bacterial Desulfosarcina were not detected Instead clusters of an unidentified Bacteria in red hybridizing with the general bacterial probe labeled with Cy3 were associated with the archaeal ANMEZ cells stained in green l However as me urearformamlde concentration rnereases the DNA denatures aners moblllty deereases sharply y a n gradlent across the gel TGGE Advantage 1 Can be usedto sereen a large number of samples Bands ean be eluted from the gels for sequenclng andldenu catlon Disadvantage eyeles to vlsuallze the products TGGEfrom Wlelandetal 2001 Appl Envlron Mleroblol 67 584975854 1711 1 mnl were taken a er r em 9 respectlvely a er 8 weeks from contamers vmln bean lanes 10 to 13 and a er 11 weeks fr 14 to l om a er RT r RN R1336 Lanes 1 anle Show separaadn dune productmlx used as amarker RTVPCR reverse transcnptase R copies nbosomal RNA drreeuy to serve as Used PC RT template forPCReondmons 35 cycles at 94 c for 10 see 56 cfdr 1 mln 68 c for 1 mln pnmers are L1346 and UQSSVGC at 0 4 11M
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