Institute of Soil Ecology Diversity of cbbL genes from autotrophic bacteria in differently managed...
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Transcript of Institute of Soil Ecology Diversity of cbbL genes from autotrophic bacteria in differently managed...
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
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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
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
0.10
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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