Soil Microbial Ecology
Soil Microbial Ecology ESPM 131
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This 48 page Class Notes was uploaded by Larry Bogisich on Thursday October 22, 2015. The Class Notes belongs to ESPM 131 at University of California - Berkeley taught by Staff in Fall. Since its upload, it has received 39 views. For similar materials see /class/226558/espm-131-university-of-california-berkeley in Environmental Science & Policy at University of California - Berkeley.
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Date Created: 10/22/15
GOVindarajula et a1 2005 l release NH ef ux into the interface between plant and fungus and upmke of NHX by plant cells Bold arrows show active 11pmke processes dashed an39ows passive cues From Leake et a1 2004 Leaks et a1 a Atmospheric N2 Hrgnly Lame m 4 Nu Organic Nitrogen Rem c ram Dissgrved organic N 7 From Leake et a1 2004 Atmospheric N2 nghly Lablle Organic Nitrogen Recalciirant m39ssowed organic N geachlng Figure 86 Metabolic zonation of expression of enzymes of N assimilation in the extra radical mycelium of ectomycorrhizas Expression of both glutamine synthetase GS and glutamate dehydrogenase GDH in the fungus is downregulated close to the root From Martin et 0 I992 with permission 3 0 g a b i a quot0 39 In a 2 h quot5 03 E Z 0 O39l gt i L 1 1 l 1 O l2 24 0 43 2 86394 Phosphorus applied 9 per poi Figure 56 The effect of phosphorus fertilization and inoculation with Glomus mosseae on dry weight of shoots of Trifolium subterraneum after 7 weeks39 growth a Superphosphate fertilizer b Cgrade rock phosphate O uninoculated control plants A plants inoculated with G mosseae From Pairunan et 0 I980 with permission Phosphorus nutrition of ectomycorrhizal plants 3 Figure 9I Transport of 32F through the extraradical mycelium of Suillus bovinus linked to seedlings of Pinus sylvestris and P contorta a Root observation chamber showing the mycelial connections between the plants and the site of feeding with 32F in halfstrength Melin Norkrans medium arrowed b Autoradiograph of the same chamber showing the distribution of 32F after 82 hours Label has accumulated in the rhizomorphs A mycor rhizal roots B and the shoots C There is also some accumulation in the advancing mycelial front D From Finlay and Read l986b with permission FUNGUS INTERFACIAL APOPLAST PLANT TREHALOSE H 39 SUCROSE T g I N 39 pH 45 v X X E R MAnnnoq Z i FRUCTE E T H s A X s E ATP ATP j gt H39 w ADP ADP GLUCOSE H H39O FRUCTOSE FPM PPM REioactiVi39fy calrmgper mi if 3m 248 Ectomycorrhizas Relative respiration rate a 0 Q 1 20 i V v1 2 O 10 20 24 48 96 124 Hours Figure 74 Effect of cutting the mycelium andhence detaching it from a source of carbohydrate from the plant on respiration of ectomycorrhizal mycelium The curves represent the relative respiration rate percentage of value before cutting for nine separate combinations of plant and fungal species From S derstrom and Read I987 with permission r d New 1 h 5 Phytologisr R862 7 Extramatrical ectomycorrhizal mycelium contributes onethird of microbial biomass and produces together with associated roots half the dissolved organic carbon in a forest soil Mona N Hogberg and Peter H gberg n r I I39 I 39 5 39i 39 C v A39i 39 39 39 39 39 V 39 17790135 Limes Sweden June Temperature 0 Sm espuanen mg 0 nr2 M wen 7 120 7 A a e Rootfree chambers Used to estimate respiration due to AM mycorrhizal ER mycelium fig from Leake et a1 2004 613 C o of 002 released 30 20 I 80 140 24 h min min Log Time of sampling after core removal Visualized carbon transfer in 14C labeled AM plants Each plant targets a Monotro es particular fungal p lineage BaSIdIOmycetes Ericaceae Cryptothallus is a nonphotosynthetic thalloid liverwort found below the soil surface in ectomycorrhizal forests And uses two Tulasnella species to acquire its carbon from surrounding pines or birch Bidartondo et al 2003 Proc R Soc London B Arachnitis grows in the understory of Andean forests and uses a particular Glomus sp to acquire its carbon from surrounding plants Bidartondo et al 2002 Nature 419 389392 a A DlSCOVERY IN THE DIRT IT l 0 39 NI DJ 439 D Mycormizal Diversity in an Abes cancelor Forest Fungal species in ranked order Work by Antonio 1220 Based on 36 soil cores from a total of 9 plots contained Within a 25 hectare region Assumptions of clone and sequence approaches No extraction bias No amplification bias No cloning biases Sequences retrieved were from living organisms Cloning and PCR artifacts are unimportant Phylogenetic placement is predictive of functional attributes Chimera formation Via partial extensions partial extension W m I denature W W Heterologous partial fragments anneal Extension creates chimera Further rounds of amplification create many copies Assumptions of clone and sequence approaches No extraction bias No amplification bias No cloning biases Sequences retrieved were from living organisms Cloning and PCR artifacts are unimportant Phylogenetic placement is predictive of functional attributes Proc Natl Acad Sci USA 93 1996 7981 GIOVannonl 6t 31 1996 Lepzonmn mm Syimchatm bujncnlifa u39ensis Bactzruides fragilis What can we say Fluvobnctzrium aqun lz Chlumbium m39bn39afnrm about unique sequences Chlamydia trachnmnris Deinucaccus India dumns GREEN NONSULFUR ACTERXA Herpetosiphon numntiacu Pirelluln stuleyl Planctamyces limnophilus Tharmumga mari Achenbach and Coates 2000 helm18mg pzrllitlum Mugnc taspirzllum glypIiswaldense Strain W1 MugIIetnSm ilum nmgnemmrurmu Magneloipzrillum SI strain AMBI Rllodoqvclm39 tennis photosynthetic Rludocyclus pulpm em Ferrilmvferium limlericum Fe reducing obligate anarobe Strain RC Non Fe reducing facultative anarobe Strain SILTL Strain CKB Strain PS 50 changes Strain LSOI From Achenbach and Coates 2000 Table 1 Examples of the unique and disparate phenotypic characteristics of closely related organisms in the Proteobacteria Organism Aerobicity Identifying metabolism Fermentation Electron acceptors utilized Fern39bacterium Strict anaerobe Fell reduction Nonfermentive Ferric iron nitrate Iimneticum fumarate Rhodocyclus tenuis gtllt Facultative anaerobe Phototrophy Ferments storage Oxygen polysaccharides Strain SIUL Facultative anaerobe Perchlorate reduction Nonfermentative Oxygen chlorate perchlorate Strain RCB gtk Facultative anaerobe Chlorate reduction Nonfermentative Oxygen nitrate Strain WD Microaerophile Perchlorate reduction Nonfermentative Oxygen nitrate chlorate Magnetospirilum Microaerophile Magnetosome production Not determined Not determined gryphiswadense Advantages and Disadvantages of Sequence approach to community analysis 0 Still is one of the best Produces an imperfect ways to identify a pool quantitatiVe PiCture of total unknowns 0 Doesn t tell you much about function 0 Cost and effort limit the number of replicate samples AH LIED D EMAMMLTAL NHLKDBKDUJGY Stpx 2005 p 554475550 Vol 71 No 1 00997224015508 0070 doi llZSAEMJL 554475550 2005 Capyngm 390 2005 Amman Socmy or Micmbmbgy An Riglus Rescn cd Fungal Community Analysis by LargeScale Sequencing of Environmental SamplesT Heath E O Brien Jcri Lynn Pancm Jasun A Jackson JcauAMaIc Moncahvo Rytas vilgalysI Duke T39mv ru39nl n and Cumman Mow Royal 0mm Mmm and Duganmem afBamny Uniqu of Toronto Tumnla Oxan chcimrd 10 November QHDMAMeplEd 15 Apnl mus Arr ENVIRON MICRoDioL O Brien et a1 2005 Baswdiomycma Asmmycma zygomymxa L Chymdmmycuta Glomeromycota m veoiana Cercozna Metazn a S saWES H subsmulmns I MIXEG Hardwood i Proportions of SSU sequences belonging to diiferent fungal phyla and other eukaryotie kingdoms The width of triangles corre lo the maximum amount of sequence divergence within clades see scale bars O Bn39en et a1 2005 Val 71 ms SEQUENCEVEASED DIVERSITY ASSESSMENT or SolL FUNG 5547 I swam I mam I Wm I mum I Fm 2 V V V In each lmncmlc group and hax wmm conespunds m we pmpomnn cl all sequences bcbnging m each library A Pmponinn uf all m Sequences belonglllg m eacll fungal kmgdom E Pmpomuu of Ascnmycuta ITS sequences b unglng m each subclass c Ymponiuu ul Easidiomvcmz Yrs 5 O Bn39en et a1 2005 Pine waed Hardwood Esnmaied Hchness 1 N0 of OTUS Obsewed richness lt gt I y I 39r m m I39m 70 m mquot N0 of sequences FIG 4 Speciesafar curves for obsered and estimated fungal OTU richness in each plot DGGE Denaturing gradient gel electrophoresis amp TGGE temperature gradient gel electrophoresis 0 Amplify portion of rDNA gene using a primer With a 5 GC clamp 0 Load pool of amplicons onto denaturing gradient gel 0 Slightly different products are separated by sequence differences that cause different levels of partial denaturation From Ward et a1 1998 M01 Biol Rev c A B39 Equot Mat 0100 urn 100200 um 200300 um 300400 um 400500 um 500600 um 600700 um 700800 um 800900 um 9001000 um damn 446 QIIH DGGE gel From Ward et a1 1998 M01 Biol Rev 4849 C 5357 5663 C 5957nc 6370 C 8472 0 AB39 ABI B A B39A AA Al39 E E E BB39 if B39 BI A39 A39 A39 3 A A A HeterodupleX formation a feature of all PCR reaction With complex mixtures of similar products i denature denature Reanneallng of strands i annealing of primers homoduplex mazzzzzzzzz hetemduplex extension extension m WW T RFLP terminal restriction fragment length polymorphism Amplify pool of sequences with one of the primers labeled E lllllllllllllllln Digest with a restriction enzyme Hil l Hi A B E a m g E Each amplicon B produces a single detected fragment An example of tiRFLP data from a very simple community m s 25 mm 52 a W mewssz 52 3 W was mums 5W mes maxing 7W mes mum same 52H7 24152m5me T RFLP analysis amp gel wmwmso 90100 30405060708090100 WM W l I if iii ii ll quot ll FIG 6 Dendrograms of community relatedness of four di erent complex communities based on two di erent bandbased coef cients the Jaccard end area sensitive coefficients Each community was represented by a phylogenetic signature constructed by stacking three individual 5 T RFLP patterns corresponding to the Hhal Mspl and Real digests by using image analysis software Area sensitive coef Jaccard coef Moeseneder et a1 1999 FIG 3 DGGE pallcm from the samplcs taken m the Aegan Sea a Band gt gt I 39 39 MK Rmnm In t uni d wuh a chargecuupled davice camua War a 27min expusurc period Longer integraxiun limes enhanced m signal mm weal bands mmby ovcrsatumling stronger bands h The DGGE gal was acquired with mac di erem Integranon drawn in a s emalic diagram Lines 1 nub 35 OTUs z msb39l as 01115 3 mnb 35 OTUs 4mnb1 33 OTUs 5 man 33 OTUs mnb4 23 OTUs similanty 040 060 UPGMA TRFLP rnnh4 mnb1 mnbz mnba msm msba mnbA mnb1 mnb2 man msb1 msb3 F MA dandrograms from TRFLP and DGGE analyses DIPCR FIGA 4 U G prOdIICIS of samples lakan m m Aegzan Sea Far he T RFLP the numbers of 01115 samples snaimd wilh from lhra separate restriction digests m paroled How can t RFLP analysis separate as many l6S sequences as DGGE Because many of the differences are based on indels rather than base substitutions in restriction sites What can t you do With t RFLP that you could do With DGGE or TGGE Retrieve the entire sequence by cutting the fragment out of the gel and sequencing it Single stranded conformational polymorphisms Amplify target 100 600 hp with one of the primers phosphorylated Digest products With Lambda exonuclease only phosphorylated strand is digested Separate remaining single stranded products on non denaturing gel Migration of fragments due to conformation rather than size SSCP gel from soil microbial community Schmalenberger amp Tebbe Mol Ecol 2003 12251262 Excised bands were cloned and sequenced and found to be complex pools of sequences Comparison of finger printing methods Method Unique advantages Unique Disadvantages DGGE TGGE Can excise clone and Specialized gel or sequence bands equipment needed heteroduplex bands Results difficult to reproduce between labs TRFLP Can be run on an Can not excise and automated sequencer sequence bands easily Highly reproducible size may Ot be very usefu estimates for protein coding sequences SSCP Can excise clone and Single fragments may sequence bands have multiple conformers Results difficult to reproduce between labs For higher resolution the same methods can be applied to the spacer region but no database exists I GarciaMartinez et al I Journal of Microbiological Methods 36 1999 55 64 57 Spacer 168 rJ r 23s ss M tRNAIle tRNAAla quot tRNA region Fig 1 Schematic representation of a 168235 spacer and organization of its functional regions shadowed boxes As indicated by brackets the presence of tRNA genes is not universal and their number and type may vary among species The ITS internal transcribed spacer region in fungi ITSHungal ITSI ITSS Size typically 540 950 bp Specific primers allow fungal sequences to be easily amplified from complex environments Usually highly variable between species groups Variation is often rich in IDELs ESPM131 Microarrays Enviromnental Genomics Quantification 506 Microarrays Wilson et al 2002 60000 probeschip now 500000chip hybridize uorescently labeled PCR pool or cDNA pool May miss total unknowns Reading differences in hybridizations within array may be tricky Currently expensive Enviromnental Genomics T erminology Shotgun cloning undirected cloning effort were everything is cloned and sequenced contig assembled continuous sequence derived from sequence reads of a single clone Scaffold assembled continuous sequences derived from multiple overlapping conti gs whose physical connection is inferred from sequence identity nX coverage mean number of times a region was sequenced from independent clones miniscaffold scaffolds assembled only by paired ends of overlapping contigs ie 1X coverage Bact Clones from environmental samples Delong 2002 Zeidner et al 2003 Iron Mt bio lm Tyson et al 2004 Pink biofilm growing at pH 087 simple system composed of 6 16S rRNA types Leptospirillium gr 11 High GC scaffolds with 10X coverage very low polymorphism 008 interpreted as evidence for one strain or species Leptospirillium gr 111 High GC scaffolds with 3X coverage contains the only nitrogen fixation genes in the system F erroplasma Type II 10X coverage scaffolds were 22 different from to FerI isolate even though the 16S gene was 99 identical average polymorphism within scaffolds was 22 interpreted as several strains with recombination ESPM131 Microarrays Enviromnental Genomics Quantification 506 Sulfobacillus partial 16 S recovered scaffolds for it if any may have been assigned to Leptospirillium group III Sargasso Sea Venter et al 2004 Some numbers 200 L of filtered surface water 01 3 um shotgun cloning of 2 6 kB inserts no PCR 166 million sequences obtained 246 mpb of unique sequence assembled into 64398 scaffolds ranging from 826 bp to 21 Mpb 1699 Mbp mini scaffolds and unpaired singleton reads 12 million protein coding genes 10X more that currently in databases 69901 novel conserved open reading frames 60000 16S rRNA sequences 148 of which are at least 3 different from known sequences Problem of assembly usually all unique sequences have equal probability of being sampled but in an environmental sample depth of coverage of a particular genome should be related to abundance of the organism Larger scaffolds sorted into organism bins based on similarity to known sequence and oligonucleotide frequency Estimates of diversity sampled at least 300 species sample Assuming homologous sequence gt 6 different belong to different species and using models based on Poisson distributions and 3 coverage based models estimates they estimate 1800 to 47000 unique genomes in the combined sample and minimum of 12X greater effort would be needed to have a95 of the unique sequences Populations are sequenced rather than an individuals Scaffolds with 14X coverage contained about 1 SNP10000 bases also insertions of bacteriophage sequences were common SAR 11 like sequences are abundant but very polymorphic suggestive of a population of related taxa that share a common 16S sequence Other results of ecological interest Evidence for patchiness in marine samples Sample 1 had and abundance of a Burkholderia and Shewanella not found in sample 2 Copy number bias of 16S sequences obvious figure 6 ESPM131 Microarrays Enviromnental Genomics Quantification 506 Estimations of species present based on coverage frequency classes Table 3 soil is still out of reach but maybe not for long Quanti cation via oligonucleotide probing hybridization to filters with blotted rRNA Stahl fig Specific hybridization universal what about unknown probe specificities Quanti cation via realtime PCR based on measure product as it accumulates see Mackay et al 2002 for a reviewLandeweert et al 2003 for ecological example Based on assaying the accumulation of product as it occurs Thermocylcer excites and detects orophores simplest way involves intercalary dyes ethidium SYBER green YO PRO l accumulation of double stranded product detected Only works for a quantification of one specific template at a time accumulation of specific product can be monitored with specific probes Usually based on FRET fluorescence resonance energy transfer either two orescent labels or one orescent label and a NFQ non florescent quencher several variations more all the time see fig 3 of McKay 539nuclease oligoprobes or quotTaqManquot probes based on 5 3 nuclease activeity of Taq polymerase A in figure hairpin probes or quotmolecular beaconsquot inactive until bound to template B more sensitive to mismatch than linear probes two adjacent probes C inactive until bound next to each other on template Sunrise primers D used as amplification primer hairpin is removed when primer is incorporated Scorpion primer similar to above but second stand synthesis is not required SNIP monitoring via melt profile Landeweert et al paper uses quantitative PCR to assay competition between two mycorrhizal fungi Paxillus and Suillus Probe design based on two head to head ESPM131 Microarrays Enviromnental Genomics Quantification 506 probes that uoresce when both are bound and specific amplification primers for ITS 2 regions of both Other quantification included visual counts of mycelium in single experiments PLFA estimates of mycelium which does not differentiate these species Three molecular methods used DGGE analysis of ITS region cloning and clone counts of ITS region real time quantification Bottom line all three show the same pattern Suillus increases over time as Paxillus declines References DeLong EF 2002 Microbial population genomics and ecology Current Opinion in Microbiology 5 520 4 Landeweert R C Veenman T W Kuyper H Fritze K Wernars and E Smit 2003 Quantification of ectomycorrhizal mycelium in soil by real time PCR compared to conventional quantification techniques FEMS Microbiology Ecology 45 283 292 Mackay I M K E Arden and A Nitsche 2002 Real time PCR in virology Nucleic Acids Research 30 1292 1305 Tyson GW Chapman J Hugenholtz P Allen EE Ram RJ et al 2004 Community structure and metabolism through reconstruction of microbial genomes from the environment Nature 428 37 43 Venter J C Remington K Heidelberg JF Halpern AL Rusch D et al 2004 Environmental genome shotgun sequencing of the Sargasso Sea Science 304 66 74 Zeidner G Preston CM Delong EF Massana R Post AF et al 2003 Molecular diversity among marine picophytoplankton as revealed by psbA analyses Environmental Microbiology 5 212 6
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