Molecular Biogeochemistry, Lecture 4 - MIT OpenCourseWare · Institute of Biogeochemistry and Marine Chemistry, University of Hamburg, Bundesstrasse 55, 20146 Hamberg, Germany Received

Molecular Biogeochemistry Molecular Biogeochemistry

• Hopanoids – Different structures and known bacterial sources,

• C30 • C35 • CompositeComposite • Unsaturated • Methylated

– Biosynthesis • SSquallene hhopene cycllase • Beyond shc • Genes and taxonomic distribution of hopanoids

– Function • Localization • Membrane permeability • Stress responses • Novel functionsNovel functions

1

Lecture 4

Hopanoids

• First recognized as a class of C30

pentacyclic triterpenes found in ferns pentacyclic triterpenes found in ferns,

mosses and dammar resins

• ‘Hopane’ named after the

Dipterocarp plant genus Hopea, itself

after botanist John Hopeafter botanist John Hope

• Biosynthetic kinship to sterols,

tetrahymanol & oleanoids viatetrahymanol & oleanoids, via

squalene recognised in 60’s

© Gaines, Eglinton & Rullkötter 2

Hopanoid Structures: C30 Hopanoids

diplotene

diplopterol

• Diploptene found in all hopanoid producing bacteria

• Diplopterol detected in most hoppanoidp p producers

• Biosynthetic intermediates only?

3

Side Chain VariationsSide Chain Variations

Z

X Y

30

31

TETRA: X=OH, NH2, composite; Y = Z = H

PENTA: X = OH, NH2, composite; Y = OH, Z = H X = OH, Y = H, Z = OH

HEXA: X = NH22; Y = Z = OH

Composite

NH2

O OH

OH OH

NH2

HO OH OH

H N NH2

OO OH

O OH

O OHOH

COOH

4

Analysis Of BiohopanoidsAnalysis Of Biohopanoids

Hi hl ti d hi hilli• Highly ffunctionali lized, amphiphillic

• Not amenable to conventional GC‐MS

• Side chain cleavage (Rohmer et al., 1984)

– Periodic acid/sodium borohydride

– Product structure directly related to number and position of functional groups in side chain

• Specific nature of functional groups lost

5

Periodic Acid Oxidation

TETRA O

OH

OH OH OH O OH H5IO6/NaBH432 OOO O 32 hopanol32‐hopanol

OH H

H5IO6/NaBH4 OH OHPENTA OH

31‐hopanol 31

OH OH OH

HEXAHEXA OH OH OH OH H5IO6/NaBH4 30‐hopanol30

OH OH NH22

Topic #1: How was analysis of functionalized (i.e. C35) hopanoids improved? (Helen Talbot papers)

6


HO HO

OH

HO

OH bacteriohopanetetrol

• Thought to be most common hopanoid produced

• Found in most but not all hopanoid producers:Found in most but not all hopanoid producers:

• Cyanobacteria

• Gram‐positive heterotrophs

• Gram‐negative heterotrophs Gram negative heterotrophs

• Obligte/Faculative methylotrophs

• Purple nonsulfur bacteria

• Sulfate reducing bacteriaSulfate reducing bacteria

7


bacteriohopanepentols

HO HO

HO

OH

HO

OH HO

OH OH

OH

OH

• h d h h d l ( f lC35 hopanoids with 5 hydroxyl groups (pentafunctionalizedd))

• Only observed in cyanobacteria

8


AminohopanoidsAminohopanoids

aminobacteriohoppanetriol NHNH2

aminobacteriohopanetetrol NH2

HO HO

OH

HO HO

OH

HO

OH

HO HO

OH OH

HO HO

aminobacteriohopanepentol NH2

9



aminobacteriohopanetriol HO

HO

NH p

OH NH2

3 h d l i h i C 35• 3 hydroxyl groups with amino group at C‐35

• Type I/II methylotrophs

• Nitrogen fixing bacteria

• Beijerinckkia

• Purple nonsulfur bacteria

• Rhodopseudomonas

• Actinobacteria

• Streptomyces

10



HO HO

OH OH

• 4 hydroxyl groups with amino group at C‐35

NH2 aminobacteriohoppanetetrol NH

• Type I, X, II methanotrophs

• MethylosinusMethylosinus

• Type II facultative

• Methylococcus

•• Type X obligate Type X obligate

• Methylomonas

• Type I facultative

11



HO HO

HO

OH OH

• 5 hydroxyl groups with amino group at C‐35

NH2 aminobacteriohoppaneppentol NH

• Type X methanotrophs

• MethylococcusMethylococcus

• Type X obligate

• Methylocaldum

•• Type I/X obligate Type I/X obligate

12

Methylotroph vs Methanotroph

• Type I, X, II methylotrophs

•• Methylotrophs: aerobic bacteria use C 1 compounds as Methylotrophs: aerobic bacteria use C‐1 compounds as carbon and energy source

• Methanotrophs: subset of methylotrophs; can use methane

13

as carbon and energy source

• obligate and/or facultative

Type distinction made primarily on how they assimilate formaldehyde:

• Type II

•

•

• Serine pathway

• All α‐Proteobacteria

Type I • b l h h ( )ribulose monophosphate (RuMP)

pathway

• β‐Proteobacteria (no CH4 oxidation)

•• Proteobacteria (CH xidation) γ‐Proteobacteria (CH4 oxidation) o

Type X

• subset of Type I

• RuMP + some serine pathway enzymes

• Can grow at higher temperatures

• Usually have a higher G + C content

• γ‐Proteobacteria

Hanson and Hanson, Microbiological Reviews, 1996: p. 439‐471

14

Aminohopanoids as proxies for aerobic methanotrophy?

• Biogenic methane from methanogenesis is severely 13C depleted; reflected in biomass from methanotrophs

• Aminohoppanoid identification couppled with stable isotoppic analyysis ggood indicator of methanotrophy in given modern environment

HO HO

OH

HO

NH2

OH HO

HO

HO

OH NH2

OH

aminobacteriohopanetetrol aminobacteriohopanepentol

15

Unexpected occurrence of hopanoids at gas seeps in the Black Sea

a,* b bVolker Thiel , Martin Blumenberg , Thomas Pape ,b Richard Seifert , Walter Michaelis b,*

a Geowissenschaftliches Zentrum der Universität Göttingen, Goldschmidtstr. 3, 37022 Göttingen, GermanybInstitut für biogeochemie und Meereschemie, Universität Hamburg, Bundesstr. 55, 20146 Hamburg, Germany

Received 12 July 2002; accepted 1 October 2002(returned to author for revision 20 August 2002)

Occurrence of unusual steroids and hopanoids derived from aerobic methanotrophs at an active marine mud volcano

a,*Marcus Elvert , Helge Niemannb,c

aOrganic Geochemistry Group, Department of Geosciences, University of Bermen, Leobener Strasse, D-28359 Bremen, Germanyb Max Planck Institute for MArine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germanyc Alfred Wegener Institute for Polar and Marine research, D-27515 Bremerhaven, Germany

Received 26 July 2007; received in revised form 5 November 2007; accepted 15 November 2007 Available online 22 November 2007

Aerobic methanotrophy in the oxic-anoxic transition zone of the Black Sea water column

*Martin blumenberg , Richard Seifert, Walter MichaelisInstitute of Biogeochemistry and Marine Chemistry, University of Hamburg, Bundesstrasse 55, 20146 Hamberg, Germany

Received 8 February 2006; received in revised form 17 AAugust 2006; accepted 30 August 2006 Available online 30 October 2006

16

Hopanoid Structures: Composite Hopanoids

• Hydroxyl or amino group at C‐35 linked to diverse complex moieties

guanidine substituted bacteriohopanetetrol cyclitol ether bacteriohopanetetrol cyclitol ether

• Cyanobbacteria • Methylobacterium organophilum • α‐proteobacteria

• α‐proteobacteria • Acetic acid bacteria •• Type II methylotroph Type II methylotroph • Type II methylotrophs

• β‐proteobacteria

• Burkholderia

• γ‐proteobacteria

• Azotobacter

17

Hopanoid Structures: Composite Hopanoids

• Hydroxyl or amino group at C‐35 linked to diverse complex moieties

• Cyanobacteria

• αα‐proteobacteria proteobacteria

• Acetic acid bacteria

• Type II methylotrophs

• Zymomonas mobilis Zymomonas mobilis

• β‐proteobacteria bacteriohopanetetrol glycoside • Burkholderia

18

11 known ring systems

Ring Variations

Δ11

2

3 Δ6

19

Hopanoid Structures: Unsaturated hopanoids

Unsaturated bacteriohopanetetrol cyclitol ethers

• Acetic acid bacteria most abundant producersproducers

• Also produce Δ6Δ11 double unsaturation

• Recentlyy discovered in Burkholderia

• Other unsaturated BHPs found in small amounts in

• Cyanobacteria

• Methylosinus

• Methylocaldum

20

Hopanoid Structures: Methylated hopanoids

• Cyanobacteria • Methylation at C‐2 • Produce all of these structures

• α‐Proteobacteria

• Only methylate some of these

HO HO structures

• Varies between bacterial classes OH

OH OH

• Rhodopseudomonas H3C HO • Bradyrhizobium

• Methylobacterium

• Beijerinckia

HO

OH OH

H3C

OH

21

Hopanoid Structures: Methylated hopanoids

• Methylation at C‐3 • Acetic acid bacteria

• Type I andd Type X methhanotrophhs

Methylhopane biomarker hydrocarbons in Hamersley Province sediments provideevidence for Neoarchean aerobiosis

a,b,* aJennifer L. Eigenbrode , Katherine H. Freeman , Roger E. Summons ca Department of Geosciences and Penn State astrobiology Research Center, The Pennysylvania State University, University Park, PA, 16802, United States b Geophysical Laboratory, Carnegie institution of Washington, Washington, DC 20015, United States c Department of Earth, Atmospheric, and planetary Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States

Late Archean molecular fossils from the Transvaal Supergroup record the antiquity of microbial diversity and aerobiosis

a b,1 cJacob R. Waldbauer , Laura S. Sherman , Dawn Y. Sumner , Roger E. Summons b,*

a Joint Program in Chemical Oceanography, Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, Cambridge, MA 02139, United Statesb Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United Statesc Department of Geology, University of California, Davis, CA 95616, United States

*

22

Isotopic Signature of 3‐Methylhopanoids

Distribution and C‐isotopic fractionation in hopanoids of M. capsullatus as ffunctiion off growthh

stage

Summons et al., GCA, 1994 23

Tetrahymanol

• Not a hopanoid

• Discovered in ciliated protozoan TetrahymenaTetrahymena pyriformispyriformis

• Also found in

• Other ciliates

•• An anaerobic rumen fungus: An anaerobic rumen fungus: Piromonas communis

• A fern: Oleandra wallicii

• Two α‐ProteobacteriaProteobacteria::Two α

• Rhodopseudomonas palustris

• Bradyrhizobium japonicum

• Proposed to function as sterolProposed to function as sterol surrogates; particularly in anaerobic unicellular eukaryotes

24

Hopanoid and Sterol Biosynthesis

squalene

lanosterol

Enz‐AH+

squalene hopene cycllase Enz‐AH+

oxidosqualene cyclase

l h

hopene

?

bacteriohopanetetrol

cholesterol

25

Hopanoid Biosynthesis

squalene hopene cyclase (Shc)

26

erol viewed as a side

Squalene hopene cyclase

• Most well studied hopanoid biosynthesis protein

• Purified from 7 organisms

• Crystall structure ffrom A. acidocaldarius

• Catalyzes one of the most complex enzymatic one‐step reaction enzymatic one step reaction

• Shc can generate several minor hydrocarbons in vitro

• Diplopterol (1) viewed as a side(1)Diplopt product

• Tetrahymanol (2) catalyzed from squalene not by Shc but Stc

• Loose substrate specificity (Table 2)

• More so than the oxididosquallene cycllase

27

Loose substrate specificity of Shc

28

Phylogenetic analysis of Shc

Welander PV, et al. PNAS, 107: p. 8537‐8542

• Phylogenetic tree of bacterial species

• Blue bar = presence of Shc

• Before genome analysis of Shc, it wasore genome analysis of Shc, it was Befthought about 50% of bacteria made hopanoids

• Lipid surveys of approx. 90 strains

• BLAST l i f Sh h th t i BLAST analysis of Shc shows that is onlly about 10%

• Shc found in

• Firmicutes

• Actinobacteria

• Thermotogae

• Cyanobacteria

• Planctomycetes

• Acidobactria

• Proteobacteria

•• δ α β γ δ, α, β, γ • Not ε

29

Evolutionary link between Shc and Osc?

30

Hopanoid Biosynthesis: Beyond Shc

31

HpnH: Generating the Adenosyl hopane Intermediate

32

HpnH: Generating the Adenosyl hopane Intermediate

• The hpnH gene identified in Methylobacterium extorquens

• HppnH annotated as a radical SAM protein

• Transfers adenosine ribose to diploptene to from adenosyl hopane

• Mechanism not experimentally verified

• BLAST analysis of HpnH shows that all Shc containing genomes containall Shc containing genomes contain this protein

• Leads to idea that all hopanoid pproducers can make functionalized hopanoids (i.e., C35 hopanoids)

• Questions the use of adenosyl hopane as a biomarker for soil bacteria – just an intermediate produced by all hopanoid producing bacteria

33

HpnG: Removal of adenine

34

HpnG: Removal of adenine

• The hpnG gene also first identified in Methylobacterium extorquens

• HpnG annotated as a nucleoside hydrolase

• Removes adenine nucleotide to form ribosyl hopane


• BLAST analysis of HpnG inconclusive

• High similarity to other nucleosides not involved in hopanoid biosynthesis

• Presumably all Shc and HpnH containing genomes would have thiscontaining genomes would have this protein as well

35

Conversion of ribosyl hopane to formyl hopane

• Equilibrium between open and closed form of riboseform of ribose

• Hypothesis is that no enzyme needed to catalyze this step

36

HpnO: Addition of amino group

37

HpnO: Addition of amino group

• The hpnO gene identified in Rhodopseudomonas palustris

• HpnO annotated as an aminotransferase

• Presumably adds amine group to formyl hopane


• BLAST analysis of HpnO shows limited numbber off aminohopanoid id producers i h d

• Confirms presence in strains known to make aminohoapnoids

•• Demonstrates potentially new Demonstrates potentially new aminohopanoids producers

38

HpnO: Phylogeny

39

Unknown biosynthetic steps

• Gene specifically needed to produce p y p bacteriohopanetetrol not discovered yet

• No composite hopanoid biosynthesis genes known

40

Enz AH

Methylation at C‐2: R. palustris Experiments

eutB hpnP hpnN2 4266 4258 4257 hpnH ispH hpnN1 hpnOshchpnC hpnE hpnG hpnQ

eutC 4265 hpnD 4259 4252 4251

squalene

Enz‐AH+

hopene 2‐methylhopene methionine


41

hpnP encodes for the C‐2 methylase

Wild type diploptene

diplopterol 2‐methyldiploppterol y p

2‐methyldiploptene

25.00 29.0028.0024.00 26.00 27.00

ΔhpnP diplopterol diploptene

24.00 25.00 26.00 27.00 28.00 29.00

Time (min) Welander PV, et al. PNAS, 107: p. 8537‐8542

42

Enz AH

HpnP: Methylation at C‐2

eutB hpnP hpnN2 4266 4258 4257 hpnH ispH hpnN1 hpnOshchpnC hpnE hpnG hpnQ

eutC 4265 hpnD 4259 4252 4251

• HpnP annotated as a B‐12 binding radical squalene SAM

• Uses S‐adenosylmethionine radical to add CH to C 2CH3 to C‐2


Enz‐AH+

hopene 2‐methylhopene methionine


43

Phylogenetic analysis of the HpnP methylase 0.2

C a ndK o rib a ct er v e

Thermosynechococcus

7425

102

R palu

Rpalustris H

R pal ustris Bis

Nitrobacter sp

Nb31 og

rads

kyi N

b255

am

burg

ensis

X14

r sa ti lis E lli n3 4 5

s elongatus BP1

Cyan

othe

cesp

PCC7

Nos

toc pun

ctifo

rme PC

C7310

Gloeobacter violaceus PCC7421

PCC7822

other Rhizobiales

M2831A53R palustris BisB18

R palustris TIE1

alustris CGA009

HaA2 sB5

11A

Nw

ino

Nha

Cyanothece sp PCC782

Cyanothece sp PCC7424

Mradiotolerans JC

M28

M populi BJ001

M extorquens AM1 M extorqu

Mex

CM4

60ovor

ans O

M5

icum

USD

A110

rhizo

bium

spBT

Ai1

Bradyrhizobiumsp

ORS278R palustris BisA

5

rquens PA1

xtorquens DM

4

Mch

loro

met

hani

cum

C

ethy

loba

cter

ium

sp44

6

Mno

dula

nsO

RS20

60

Beijerinckiaindicyl

ocel

lasi

lves

tris

BL2

Olig

otro

pha

carb

oxid

o

Bja

poni

Brad

yrB

Cyanobacteria Acidobacterium

caA

TCC9039

Met

hy Me

Methylobacteria

Welander PV, et al. PNAS, 107: p. 8537‐8542 44

Toppic 4: Are 2‐methyylhoppanes ggood biomarkers for cyyanobacteria and/or O22 ‐pphotosyynthesis?

45

Methylation at C‐3: M. capsulatus experiments

HO

OH NH2

OH

HO

methionine

A I

HO

II III IV

Wild type

18 20 22 24 26 28 30 32

I B

ΔhpnR

II

18 20 22 24 26 28 30 32

III I

ΔhpnR + pPVW100

C

II IV

18 20 22 24 26 28 30 32

Time (min) Time (min)

I and II: desmethyl aminohopanoids III and IV: C‐3 methylated aminohopanoids

46

Methylation at C‐3

HO HO

OH NH2

OH

HO

methionine

• HpnR also annotated as a B‐12 binding radical SAM radical SAM

• Uses S‐adenosylmethionine radical to add CH3 to C‐3

•• Mechanism not experimentally Mechanism not experimentally verified

• Very low sequence identity to HpnP

• Although share the Bshare the B‐12 binding and Although 12 binding and radical SAM motifs

• Raises evolutionary questions about the similarity of these two methylations

Topic 5: What is the C‐2 and C‐3 methylation mechanism proposed earlier in the literature? How is it different from the use of radical SAM chemistry?

47

Phylogenetic analysis of the HpnR methylase

• Very few bacteria with HpnR in their genomes have been tested for hopanoid production

•• Two have been tested (*) and no Two have been tested (*) and no 3‐methylhopanoids reported.

• If HpnR is correlated to 3‐methyylhoppanoid pproduction in other organisms:

• Expands diversity of 3‐methylhoapnoid producers beyond methanotrophs and acetic acid bacteria

• Actionbacteria

• α, γ, andd ββ‐P tProteobbactteriia

• Nitrospirae

• Acidobacteria

• U lUnclassifi ifiedd organiism

48

Functional Role of Hopanoids?

R

R

K

ohmer & Ourisson, 1976

ohmer et al., 1979

annenberg & Poralla, 1980

Many lines of evidence show an association of hopanoids with association of hopanoids with

cellular membranes

But majority were in vitro studies. What about in vivo studies?

49

Hopanoid localization in Nostoc punctiforme

• Hopanoids localize to the outer membrane; none to the cytoplasmic

• Also observed in M. capsulatus

• Akinetes are resting state structures that do not do oxygenic photosynthesis

• Functional role not involved in oxygenic photosynthesis

50

Outer membrane versus cytoplasmic membrane

LPS

Outer membrane

Cytoplasmic membrane

• Gram‐negative bacteria have outer membrane in addition to cytoplasmic membrane • Studies are finding that hopanoids localize to this membrane • Hopanoid membrane studies were all done in cytoplasmic membrane models

• Do they apply in vivo? • Currently no in vitro system available to model the outer membrane

51

Other in vivo studies: R. palustris shc mutant

tB h P h N2 4266 4258 4257 h H i H h N1 h Ohh C h E h G hpnQeutB hpnP hpnN2 4266 4258 4257 hpnH ispH hpnN1 hpnOshchpnC hpnE hpnG hpnQ

eutC 4265 hpnD 4259 4252 4251

squalene

squalene hopene cyclase (Shc)

hopene

52

Δshc strain no longer produces hopanoids

Wild type

tetrahymanol

triglycerides

BHTs and 2‐MeBHTs Wild type

diploptene aminoBHT

20 25 30 35 40

Δshc squalene triglycerides

20 25 30 35 40

Time (hours)

53

TIE 1

shc

TIE 1

shc

Sensitivity of hopanoid mutant to pH

unbuffered medium MOPS buffered , pH 7 medium 0.4 -Wild type

0.2

0.3

OD

600

Δshc

0

0.1

O OD 6

00

0.6

0.5

0.4

0.3

0.2

0.1

0

-Wild typ

Δshc

e

100 150 200 100 150 200 00 5050 100 150 200 00 5050 100 150 200

Time (hrs) Time (hours)

pH 7.22 pH 8.2 pH 7.0 pH 7.0

54

wild type

Δshc

wild type

Δshc

0.2 Δshc

Membrane integrity of the Δshc mutant is compromised

Aerobic Growth 30oC Aerobic Growth 30oC NNo bilbile sallts 0 5% bil lt0.5% bile salts

0.5 0.5

0.4 0.4

0 3 0 3

Wild type

Δshc

0 20 40 60 80 100

Time (hours)

0.3

OD 6

00 0.3

Wild type

0.1 0.1

0 0 0 20 40 60 80 100

Time (hours)

0.2 OD

600

55

Porin

Sensitivity to bile salts is indicative of a permeable OM

OM

CM

Periplasm

Bile salts

LPS

Porin

56

Membrane integrity of other hopanoids mutants is NOT compromised

Does this indicate a novel function for amino and methylated hopanoids? Does this indicate a novel function for amino and methylated hopanoids?

0.5 No bile salts

OD

600

OD

600

wild type

Δ hΔshc

ΔhpnH

ΔhpnO

ΔhpnP

ΔhpnN

0 20 40 60 80 100

0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 00 0

0.5% bile salts

0 20 40 60 80 100 Time (hours)

ΔhpnH: Only C30 hopanoids produced ΔhpnH: Only C30 hopanoids produced

ΔhpnO: No amino hopanoid production

Δh P N th l t d h id d ti ΔhpnP: No methylated hopanoid production

ΔhpnN: No hopanoids in outer membrane

57

Functional role for 3‐methylhopanoids in stationary phase survival?

Growth experiments with M. capsulatus hpnR mutant

Growth over time at 37°C Cell survival assay 0.16

1 0E+10 1.0E+10 M. capsulatus ΔhpnR

0.12 1.0E+08

0.08 1.0E+06

0 0.5 1 1.5 2 2.5 Day 2 Day 7 Day 14

Opt

ical

DDen

sity

(600

nm) 1.0E+04

0.04

0

Col

ony

Forrm

ing

Uni

ts 1.0E+02

1.0E+00

0 16 0.16

0.12

wild type

ΔhpnR

1.0E+10 R. palustris ΔhpnP

1.0E+08

1.0E+06 0.08

0.04 1.0E+04

1.0E+02

0 1.0E+00 5 7.5 12.5 1500 2 52.5 5 7 5 1010 12 5 15

Day 3 Day 7 Day 14

Time (Days)

58

3 meth

Functional role for 3‐methylhopanoids in stationary phase survival?

Growth experiments with M. capsulatus hpnR mutant

Cell survival assay

1 0E+10 1.0E+10 M. capsulatus ΔhpnR

1.0E+08

1.0E+06

1.0E+04

1.0E+02

1.0E+00 Day 2 Day 7 Day 14 3‐methyls? yls?

1.0E+10 R. palustris ΔhpnP

1.0E+08

1.0E+06

1.0E+04

1.0E+02

1.0E+00 Day 3 Day 7 Day 14

Col

ony

Forrm

ing

Uni

ts

Methylococcus cyst 59

Diagnostic Bacteriohopanes?

OH OH 19

1

R3 OH

21 32

17

R2 2

43

10

11

19

25

29 14 16

35

R1

22 diagenesis maturation

4

cyanobacteria cyanobacteria δ13C ‐20 to ‐35 ‰

R3

OH

OH

OH 21

32 1711

19

25 35

O

22

NH

diagenesis maturation

1 OH

2

43

10 29

14 16 OH NH2

methanotrophic bacteria methanotrophs δ13C ‐45 to ‐80 ‰

• Will the physiological and biochemical data reveal that certain hopanes are better proxies for microbial processes rather than a specific bacterial group?proxies for microbial processes rather than a specific bacterial group?

60

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