Nucleic Acids and Proteins in Soil

468 Pages·2006·4.62 MB·English

by  Blaž Stres

Soil Biology
8
Series Editor: Ajit Varma
Volumespublishedintheseries
Volume1
A.Singh,O.P.Ward(Eds.)
AppliedBioremediationandPhytoremediation
2004
Volume2
A.Singh,O.P.Ward(Eds.)
BiodegradationandBioremediation
2004
Volume3
F.Buscot,A.Varma(Eds.)
MicroorganismsinSoils:RolesinGenesisandFunctions
2005
Volume4
S.Declerck,D.-G.Strullu,J.A.Fortin(Eds.)
InVitroCultureofMycorrhizas
2005
Volume5
R.Margesin,F.Schinner(Eds.)
ManualforSoilAnalysis–
MonitoringandAssessingSoilBioremediation
2005
Volume6
H.König,A.Varma(Eds.)
IntestinalMicroorganismsofTermites
andOtherInvertebrates
2006
Volume7
K.G.Mukerji,C.Manoharachary,J.Singh(Eds.)
MicrobialActivityintheRhizosphere
2006
P. Nannipieri
K. Smalla (Eds.)
Nucleic Acids
and Proteins in Soil
With42Figures,2inColor
123
ProfessorPaoloNannipieri
DipartimentodellaScienza
delSuoloeNutrizionedellaPianta
P.ledelleCascine28
50144Firenze
Italy
e-mail:paolo.nannipieri@unifi.it
Dr.KorneliaSmalla
BiologischeBundesanstalt
fürLand-undForstwirtschaft(BBA)
InstitutfürPflanzenvirologie,
MikrobiologieundbiologischeSicherheit
Messeweg11/12
38104Braunschweig
Germany
e-mail:[email protected]
LibraryofCongressControlNumber:2005938057
ISSN1613-3382
ISBN-103-540-29448-1SpringerBerlinHeidelbergNewYork
ISBN-13978-3-540-29448-1SpringerBerlinHeidelbergNewYork
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Preface
Thisbookisdevotedtonucleicacidsandproteinsinsoil.Theintentionis
tocompileourpresentknowledgeandunderstandingoftheimportanceof
nucleicacidsandproteinspresentinsoileitherinsideoroutsideofmicro-
bialcells.Sincemicrobe-drivenfunctionsarekeyforanenormousrangeof
nutrientcyclingandgeochemicalprocessesinsoilitisexpectedthatabet-
terunderstandingofthestructuralandfunctionaldiversityanddynamics
of the microbial community will enable us to understand and eventually
predict or manage soil functions of importance. This is the reason why
therearesomanychaptersonsoilmicrobialcommunityanalysisinabook
entitled “Nucleic acids and proteins in soil”. The rather ambitious idea
behind the book was to link traditional soil microbiology, biochemistry
and chemistry approaches with the more recently developed ones in the
fieldofmolecularmicrobialecology,allaimingatabetterunderstanding
of soil functioning. Whereas only a few studies on the analysis of nucleic
acidsinsoilhadbeencarriedoutbeforethe1990s,thebibliographyonthe
extractionofenzymes,interactionsbetweenproteinsandsoilcolloidsand
quantificationofproteinNinsoilwasatthattimealreadyratherextensive.
The first chapter by Stres and Tiedje is entitled “The new frontiers in
soil microbiology: how to link structure and function of microbial com-
munities?”. Stres and Tiedje consider the attempt to link microbial com-
munity structure and function in a predictive way as a “Decadal Grand
Challenge” in particularbecause microbial communities in soils are very
diverseandcomplex,andvaryatverysmallscales.Severalchaptersofthe
bookdealwithrecentmethodologicaladvancesmadetouncoverthelinks
between structure and functions of soil microbial diversity. The basis of
most cultivation-independent approaches is the extraction of either nu-
cleic acids or proteins directly from soil. In Chaps. 3 and 11, written by
BakkenandFrostegårdandbyWellingtonetal.,thereaderwilllearnmore
abouttheongoingchallengetoobtainnucleicacidsfromthe“dirt”which
aresuitableformolecularanalysis.Thedifferentstepsrequiredfornucleic
acid extraction are shown, secrets and useful tricks are mentioned, and
thevariouspotentialbiasesarecriticallydiscussed.InChap.3,Bakkenand
FrostegårdcitealinefromoneofLeonardCohen’ssongs,“...thereisacrack
ineverything,that’showthelightcomesin”;abiasedmethodisproblem-
VI Preface
atic(cracks)untilweknowtheextentandthereasonsforthebias.Withthat
knowledge,experimentscanbedesignedtoexploitthebiastounravelnew
phenomena (“... how the light comes in”), and this rather philosophical
considerationseemsanappropriateapproachformanymethodsdiscussed
inthisbook.Themolecularanalysisofnucleicacidsextractedfromsoils
allowsusconversationwiththesilentmajorityofsoilmicrobes.Whilethe
analysis of polymerase chain reaction (PCR)-amplified rRNA genes from
TC-NAbycommunityfingerprintingtechniquesorcloningandsequencing
(Chaps.8and11)isnowadaysastandardtechnique,theuseofgenearrays
for structural and functional analysis of soil microbial communities still
remainsahugemethodologicalchallenge(seeChap.9).Whenthesetech-
niquesareappliedforTC-NAanalysisanideaofthekindsofnumerically
dominantmicrobialpopulationsandgenes presentisobtained.However,
in general, these approaches give us relatively little idea about the in situ
metabolicfunctions.Onepowerfultoolformicrobialecologistsproviding
a culture-independent means of identifying microbial populations con-
ducting certain microbial processes is stable isotope probing (SIP). The
potentialsandlimitationsofSIPareaddressedinChap.10byManefieldet
al. In recent years, the detectionand quantificationof gene expressionin
soilhasbecomefeasible,asillustratedinChap.11byWellingtonetal.The
obviousnextstepistheanalysisofthesoilproteome,asubjectdiscussed
inthechaptersbyOgunseitanandNannipieri(Chaps.5and4).Although
this methodological approach is still in its infancy it can certainly make
useofthelong-termexperienceinanalysingproteinsinsoil.
Onlyrecentlyhasthefullextenttowhichhorizontalgenetransferpro-
cesses drive bacterial diversity and adaptability become evident. Three
chapters of this book are devoted to this issue. How to assess the abun-
dance and diversity of mobile genetic elements (MGE) in soil bacterial
communities?Methodspresentlyused,theirpotentialsandlimitationsare
summarisedinthechapterbySmallaandHeuer(Chap.13).Acomprehen-
sive overview of our present knowledge on gene transfer by conjugation
in soil and the various biotic and abiotic factors influencing frequencies
and establishment is given in Chap. 14 by Van Elsas et al. Chap. 15 on
transformationbyMercieretal.makesthelinkbetweenfreeDNAinsoil
and bacteria with a particular emphasis on the transformation of bacte-
ria by transgenic or transplastomic DNA. As discussed by Nielsen et al.
inChap.7,theinteractionsofnucleicacidsandproteinswithsoilcolloids
protectthesemoleculesagainstenzymaticdegradation.Indeed,anintrigu-
ing aspect of soil as a biological system is the capacity of soil colloids to
adsorborentrapimportantmoleculessuchasnucleicacidsandproteins.
The origin of DNA in soil and the factors influencing the persistence are
addressedbyWackernagelinChap.6.Nucleicacidsadsorbedorboundby
clays are protected against degradation by nucleases. However, they can
Preface VII
be taken up by competent bacterial cells and can be integrated into the
recipients’genomebyhomologousrecombinationorhomology-facilitated
illegitimate recombination. A less microbe-focused and a broad view on
state, location and function of enzymes in soil is given by Gianfreda and
Ruggiero(Chap.12).
Itisamazingthatonaverageonly4%ofthetotalNinsoilispresentin
microbialbiomassNwhereasahighproportion(from30–50%)ispresent
as amino acids after acid hydrolysis. Thus it is reasonable to hypothesise
thattheamountofextracellularproteinNisalsoconsiderableanditismuch
higherthantheamountofintracellularproteinNinsoil(seeChaps.2and4,
respectively).Indeed,asdiscussedbyKögel-KnabnerinChap.2,theuseof
NMRhasconfirmedthatextracellularproteinNispresentinsoil.
The final three chapters of this book (Chaps. 16–18) concentrate on
marker and reporter genes and their use in soil microbial ecology. Al-
though these techniques rely on introduced microbes equipped with re-
spective marker and reporter genes, they allow researchers not only to
follow the fate and activity of introduced microbes, but also to discover
how they respond to environmental triggers. Thus these techniques con-
siderably contribute to a better understanding of the microenvironment
surrounding microorganisms inhabiting soil and to locally quantify the
activityofstimulators,inhibitorsandpollutantswithabetterlinkbetween
soilmicrobiologyandsoilchemistryandbiochemistry.
Generally,theinformationobtainedbyusingmoleculartechniquesare
consideredaspartof‘modern’soilmicrobiologyandbiochemistry,whereas
theinformationretrievedbyclassicaltechniques,suchassoilrespiration,
microbialbiomass,enzymeactivities,etc., areconsideredasblackboxes,
sumparametersandroughestimates.Perhapsforthisreasonthereisaten-
dencytoseparatethetwoapproacheswhichisconceptuallywrong.Inthis
book we have tried to overcome this separation because, in our opinion,
both approaches should be more often combined to gain better insight
into the microbial processes occurring in soil. The use of stable isotope
probing(seeChap.10)isoneofthemostfruitfulcombinationsofthetwo
approaches.
Firenze, PaoloNannipieri
Braunschweig, KorneliaSmalla
December2005
Contents
1 NewFrontiersinSoilMicrobiology:
HowToLinkStructureandFunctionofMicrobialCommunities? 1
BlažStres,JamesM.Tiedje
1.1 Introduction:
AFrameworkfortheStructure–FunctionGrandChallenge... 1
1.2 MicrobialCommunityStructure:
One-HalfoftheStructure–FunctionParadigm.................... 3
1.3 TheOtherOne-Half:
FunctionalTraitsofMicrobialCommunities....................... 6
1.4 NewerApproachesforLinkingFunction
withPhylogenyandStructure........................................... 9
1.5 FutureChallenges........................................................... 12
References............................................................................. 17
2 ChemicalStructureofOrganicNandOrganicPinSoil 23
IngridKögel-Knabner
2.1 Introduction.................................................................. 23
2.2 BiologicalFormsofOrganicNandPThatEnterSoils.......... 24
2.2.1 ProteinsandPolypeptides..................................... 25
2.2.2 AminoSugarPolymers ......................................... 27
2.2.3 DNAandRNA..................................................... 28
2.2.4 MinorN-ContainingMolecules.............................. 29
2.2.5 TeichoicAcids...................................................... 29
2.2.6 InositolPhosphates.............................................. 31
2.2.7 Phospholipids...................................................... 31
2.3 TechniquesToAnalyseSoilOrganicNitrogen..................... 32
2.3.1 Hydrolysis........................................................... 33
2.3.2 AnalyticalPyrolysisandThermochemolysis............ 34
2.3.3 Solid-State15NNMRSpectroscopy.......................... 35
2.3.4 X-rayAbsorptionNear-Edge
StructureSpectroscopy......................................... 36
2.4 FormsofOrganicNinSoilOrganicMatter......................... 37
X Contents
2.5 TechniquesToAnalyseOrganicPinSoils........................... 39
2.5.1 SequentialExtractionandSeparation...................... 40
2.5.2 31PNMRSpectroscopy.......................................... 40
2.6 FormsofOrganicPinSoils.............................................. 42
2.7 Summary....................................................................... 43
References............................................................................. 43
3 NucleicAcidExtractionfromSoil 49
LarsR.Bakken,ÅsaFrostegård
3.1 Introduction.................................................................. 49
3.2 LysisandExtraction........................................................ 51
3.2.1 CellRuptureDependsonCellTypeandGrowth........ 51
3.2.2 BeadBeating,EfficiencyandBias ........................... 52
3.2.3 Grinding............................................................. 56
3.2.4 Freeze/Thaw........................................................ 57
3.2.5 EnzymaticLysis................................................... 57
3.2.6 ChemicalAgents.................................................. 59
3.2.7 ExtractionforMetagenomeLibraries...................... 59
3.3 Purification.................................................................... 60
3.4 RNAExtraction.............................................................. 62
3.5 CellExtraction............................................................... 63
3.5.1 Dispersion .......................................................... 64
3.5.2 Separation........................................................... 65
References............................................................................. 67
4 RoleofStabilisedEnzymesinMicrobialEcology
andEnzymeExtractionfromSoilwithPotentialApplications
inSoilProteomics 75
PaoloNannipieri
4.1 Introduction.................................................................. 75
4.2 EvidenceforthePresenceofStabilisedEnzymesinSoil........ 77
4.3 ExtractionofEnzymesfromSoil....................................... 79
4.4 TheRoleofStabilisedEnzymesinSoilMicrobialEcology..... 82
4.5 Proteomics .................................................................... 83
4.6 SoilProteomics .............................................................. 85
4.7 Conclusions................................................................... 89
References............................................................................. 90
5 SoilProteomics:ExtractionandAnalysisofProteinsfromSoils 95
OladeleA.Ogunseitan
5.1 Introduction.................................................................. 95
5.2 RationaleandContextofSoilProteomics........................... 96