Institute of Soil Ecology

Diversity of cbbL genes from autotrophic bacteria in differently managed agricultural soils

Draženka Selesi, Susanne Stein, Isabelle Pattis, Michael Schmid and Anton Hartmann

GSF National Research Centre for Environment and Health, Institute of Soil Ecology, München- Neuherberg, Germany

bIntroduction

Autotrophic bacteria of terrestrial environments may play a significant part in the conversion of carbon dioxide into organic matter and microbial biomass and may thus contribute to the global carbon cycling. Autotrophic microorganisms accomplish this metabolism by the Calvin-Benson-Bessham cycle, in which the key enzyme ribulose-1,5-bisphosphate carboylase/oxygenase (RuBisCO) catalyzes the first, rate-limiting step. The large subunit of form I RubisCO is encoded by gene cbbL (Kusian et al., 1997) and phylogenetically subdivided in two major groups, ‘green-like’ and ‘red-like (Fig. 1).

Objectives• to investigate the efficiency of cbbL as a functional marker for terrestrial CO2-fixing bacteria

• to assess the diversity of ‘green-like’ and ‘red-like cbbL genes in terrestrial habitats

• to elucidate variations in community composition on the basis of cbbL genes in differently managed soils.

Materials and MethodsTo gain insight into the genetic diversity of CO2-fixing bacteria in soil

habitats we developed PCR-based assays targeting the large subunit gene cbbL of the form I RubisCO. Based on the calculation of phylogenetic relationships we designed different primer sets with strong specificity for ‘red-like’ and ‘green-like’ cbbL sequences of selected terrestrial autotrophs. Bulk genomic DNA was isolated from agricultural soils with rye crop at different long-term fertilization as well as from a soil under clover/gras cover. RFLP and phylogenetic analysis of the amplified cbbL sequences were performed.

Results and Discussion

C. vinosum 2 (γ)

T. ferroxidans (γ)

C. vinosum (γ)T. denitrificans (β )Vent symbiont (?)

N. vulgaris (α )

H. marinus 1

P. hydrogenothermophila (?)C. vinosum 2P. marinus (C)

H. marinus 2 (γ)

Synechococcus sp. WH7803 (C)

Form I

„green-like“

„red-like“

Anabaena sp. PCC7120 (C)Synechococcus sp. PCC7002 (C)

P. hollandica (C)Synechococcus sp. PCC6301

Prochloron sp. (C)C. paradoxa (G)

C. reinhardtiiE. gracilis

1234

5

GreenPlastids

Form II

A. eutrophus ATCC17699 chrom. (β)A. eutrophus ATCC17699 plasm. (β)

A. eutrophus ATCC17707 chrom. (β)

R. sphaeroides (α)

X. flavus (α)

CryptomonasCylindrotheca sp. strain N1

O. luteus

Mn-oxidizingbacterium (α)

6

1. C. reflexa2. I. purpurea3. N. tabacum4. M. verticillata5. S. oleracea6. T. aestivum

C. vinosum 2 (γ)

T. ferroxidans (γ)

C. vinosum (γ)T. denitrificans (β )Vent symbiont (?)

N. vulgaris (α )

H. marinus 1

P. hydrogenothermophila (?)C. vinosum 2P. marinus (C)

H. marinus 2 (γ)

Synechococcus sp. WH7803 (C)

T. ferroxidans (γ)

C. vinosum (γ)T. denitrificans (β )Vent symbiont (?)

N. vulgaris (α )

H. marinus 1

P. hydrogenothermophila (?)C. vinosum 2P. marinus (C)

H. marinus 2 (γ)

Synechococcus sp. WH7803 (C)

Form I

„green-like“

„red-like“

Anabaena sp. PCC7120 (C)Synechococcus sp. PCC7002 (C)

P. hollandica (C)Synechococcus sp. PCC6301

Prochloron sp. (C)C. paradoxa (G)

C. reinhardtiiE. gracilis

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5

GreenPlastids

Form II

A. eutrophus ATCC17699 chrom. (β)A. eutrophus ATCC17699 plasm. (β)

A. eutrophus ATCC17707 chrom. (β)

R. sphaeroides (α)

X. flavus (α)

CryptomonasCylindrotheca sp. strain N1

O. luteus

Mn-oxidizingbacterium (α)

6

1. C. reflexa2. I. purpurea3. N. tabacum4. M. verticillata5. S. oleracea6. T. aestivum

Fig.1: cbbL based phylogenetic tree reflecting the affiliation of ‚green-like‘ and ‚red-like‘ sequences (modified from Watson et al., 1997)

References:Kusian, B. and Bowien, B. 1997 Organization and regulation of cbb CO2 genes in assimilation autotrophic

bacteria. FEMS Microbiol. Rev. 21: 135-155Watson, G.M.F. and Tabita, F.R. 1997 Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation. FEMS Microbiol. Lett. 146: 13-22.

Acknowledgement:

This study is supported by the Deutsche Forschungsgemeinschaft (SPP1090, Ha 1708/6).

Perspectives• Quantification of ‘green-like’ and ‘red- like’ cbbL genes by TaqMan-PCR

• Assessment of the cbbL transcript levels

• Effect of H2-treatment on the diversity of cbbL genes

800 bp

M 1 2 3 4 MM 1 2 3 4 M

1100 bp

Fig. 2: 1: soil with rye crop (Halle soil) without fertilization, 2: Halle soil with animal manure, 3: Halle soil with mineral fertilizer, 4: Nitrobacter vulgaris (positive control), M: MWM

Nitrobacter winogradskyi IFO

Nitrobacter winogradskyi AF UGREEN42 UGREEN7

Nitrobacter vulgaris

UGREEN22

UGREEN6 UGREEN15 UGREEN10 UGREEN13 UGREEN46

UGREEN3

UGREEN11

UGREEN41

HGREEN2 UGREEN9

Nitrosospira sp. TCH716Halothiobacillus sp. RA13

Nitrosomonas sp. ENI-11Thiobacillus intermedius K12

uncultured bacterium

Acidithiobacillus ferrooxidans

gamma proteobacterium MLHE-1Methylococcus capsulatus

uncultured bacteriumuncultured bacterium

Hydrogenophaga pseudoflavaNitrosomonas europaea

Thiobacillus denitrificansThiobacillus sp.

uncultured bacterium Hawaii Lo1uncultured bacterium Hawaii Lo2

Hydrogenovibrio marinus 1Rhodobacter capsulatus

Hydrogenophilus thermoluteolusendosymbiont of Pogonophora sp.

Nitrosococcus halophilusSynechococcus sp. WH 7805Synechococcus sp.

Synechococcus sp. WH 8103Synechococcus sp. WH 8101

uncultured Synechococcus sp. Synechococcus sp. WH 8103

uncultured Synechococcus sp.

uncultured Synechococcus sp.uncultured Synechococcus sp.

uncultured Prochlorococcus sp.Prochlorococcus marinus subsp. uncultured prochlorophyte 4DCH

uncultured Prochlorococcus sp.

Chloroplast uncultured cyanobaHydrogenovibrio marinus

Allochromatium vinosum

Thermosynechococcus elongatusSynechococcus sp.

Anabaena sp.Prochlorothrix hollandica

Synechococcus PCC7002

RED

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Efficiency of cbbLG amplification from soil

• successful detection of green-like cbbL fragments from soil• differences in cbbL amplification depending on the kind of fertilization were observed

RFLP analysis of cbbLG amplificates

• low variability of the RFLP patterns • low diversity of cbbLG

sequences• no detectable variation in pattern composition within differently managed soils

Phylogenetic analysis of cbbLG

• cbbLG sequences amplified from the different soil samples are closely related to cbbLG of Nitrobacter vulgaris and N. winogradskyi

Efficiency of cbbLR amplification from soil

• successful detection of red-like cbbL fragments from soil• no differences in cbbL amplification depending on the kind of fertilization were observed

RFLP analysis of cbbLR amplificates • high variability of the RFLP patterns • low diversity of cbbLR sequences

in halle soil without ferilization (1), high diversity in Halle soils treated with animal manure and mineral fertilizer (2)• significant variations in the composition of the cbbL-containing community in differently managed Halle soils

Phylogenetic analysis of cbbLR

Fig. 5: Phylogenetic tree based on ‘red-like‘ cbbL sequences

HaSMRED143 HaSMRED8 HaSMRED75

manganese-oxidizing bacterium HaKORED8

Bradyrhizobium japonicum HaSMRED139

HaSMRED20 HaKORED78 HaKORED36D

Nitrosospira sp. AFNitrosospira sp. 40KI

Nitrosospira sp. III2Nitrosospira sp. O13

Nitrosospira sp. A4 HaKORED32D

HaNPKRED1 HaSMRED2

HaNPKRED7 HaKORED15

HaKORED2 HaSMRED1 HaSMRED14

HaSMRED29 HaKORED7

HaKORED13, HaSMRED46

HaKORED6 HaSMRED12

HaNPKRED16 Rhodobacter azotoformansRhodobacter sphaeroidesSinorhizobium meliloti

HaKORED22 HaKORED21 HaNPKRED5

HaNPKRED20 HaKORED49b HaKORED5

HaKORED11 HaSMRED4

Ralstonia eutropha megaplasmid pHG1 Ralstonia eutropha

HaNPKRED17 Ralstonia eutropha H850Xanthobacter flavus

0.10

GREEN

Fig. 4:1: soil with rye crop (Halle soil) without fertilization, 2: Halle soil with animal manure, 3: Halle soil with mineral fertilizer, 4: Sinorhizobium meliloti (positive control), M: MWM

• cbbLR sequences amplified from the different soil samples are distributed all over the red-like phylogenetic group• discovery of yet undetected cbbLR sequences

Fig. 3: Phylogenetic tree based on ‘green-like‘ cbbL sequences

2)

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