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Gerards S, Duyts H, Laanbroek J (1998) FEMS Microbiol. Lett. 26, 269-280

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Hooper AB (1989) In Autotrophic bacteria Schlegel HG, Bowien B (eds), pp. 239-281, Science

Tech Publishers, Madison, Wisconsin Hooper AB et al. (1997) Antonie van Leeuwenhoek 71, 59-67 Hyman MR, Arp DJ (1992) J. Biol. Chem. 267, 1534-1545 Hyman MR, Arp DJ (1995) J. Bacteriol. 177,4974-4979 Hyman MR et al. (1988) Appl. Environ. Microbiol. 54, 3187-3190 Hyman MR, Wood PM (1983) Biochem. J. 212, 31-37

Hyman MR, Wood PM (1984a) In Crawford RL, Hanson RS (eds), Microbial Growth on Ci

Compounds, Proceedings of the 4th International Symposium, pp. 49-52, American Society for Microbiology, Washington, DC Hyman MR, Wood PM (1984b) Arch. Microbiol. 137, 155-158 Hyman MR, Wood PM (1985) Biochem. J. 227, 719-725 Igarashi N et al. (1997) Nature Structural Biol. 4, 276-284 Juliette LY et al. (1993a) Appl. Environ. Microbiol. 59, 3718-3727 Juliette LY et al. (1993b) Appl. Environ. Microbiol. 59, 3728-3735 Keener WK, Arp DJ (1994) Appl. Environ. Microbiol. 60, 1914-1920 Killham K (1986) In Prosser JI (ed), Nitrification, pp. 117-126, IRL Press, Oxford Klotz MG et al. (1997) FEMS Microbiol. Letters 150, 65-73 Klotz MG, Norton JM (1995) Gene 163, 159-160 McLaren RS et al. (1991) J. Mol. Biol. 221, 81-95 McTavish H et al. (1993a) J. Bacteriol. 175, 2436-2444 McTavish H et al. (1993b) J. Bacteriol. 175, 2445-2447

Norton JM et al. (1996) FEMS Microbiol. Letters 139, 181-188 Rasche ME et al. (1991) Appl. Environ. Microbiol. 57, 2986-2994 Sayavedra-Soto LA et al. (1998) FEMS Microbiol. Letters 167, 81-88 Sayavedra-Soto LA et al. (1994) J. Bacteriol. 176, 504-510 Sayavedra-Soto LA et al. (1996) Mol. Microbiol. 20, 541-548 Semrau JD et al. (1995) J. Bacteriol. 177,3071-3079 Timkovich R et al. (1998) Biophysical J. 75, 1964-1972 Vannelli T et al. (1990) Appl. Environ. Microbiol. 56, 1169-1171 Vitousek PM et al. (1997) Ecol. Alications 7, 737-750

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Yamagata A et al. (2000) Biosci. Biotechnol. Biochem. 64, 1754-1757 10. Acknowledgements

This work was supported by DOE grant DE-FG03-97ER20266 to DJ Arp and LA Sayavedra-Soto.

DIVERSITY OF DINITROGEN FIXING AND DENITRIFYING BACTERIA IN SOILS ASSESSED BY MOLECULAR BIOLOGICAL METHODS

Botanical Institute, The University of Cologne, Gyrhofstr. 15, D-50923 Köln, Germany

Up until now, few analyses of the bacterial population have been performed with soils because of their complexity and variability in the chemical composition and the microbial life (for a review see Bothe et al. 2000). It has been stated that more than 104 different ribotypes exist per g of soil (Torsvik et al. 1990) of which only a few have been cultivated as yet. The study of microorganisms participating in the conversion of nitrogen gases in soils is of paramount importance. Soils are believed to be a significant sink and/or source for N2, N2O and NO, and the latter two gases have detrimental impacts on the atmosphere (Crutzen 1979). Both NO and N2O are produced by denitrification (the conversion of nitrate to N2 via nitrite, NO and N2O) and nitrification (the oxidation of ammonia via hydroxylamine to nitrite and then to nitrate with the concomitant release of small amounts of NO and presumably also N20). Dinitrogen and also N2O are reduced by nitrogenase in dinitrogen fixation. Little is known about the factors, which govern the uptake and evolution of the gases in soils and direct the life of bacteria of the N-cycle. Molecular biological techniques offer a new avenue for analyzing the population of N-converting bacteria in soils to get insights on their potential roles in the production and consumption of gases.

Initially, this laboratory analyzed the soil composition of denitrifying and N2-fixing bacteria by DNA-DNA hybridizations with 0.5-0.8 kb probes recognizing part genes coding for target enzymes of dinitrogen fixation (niftl of nitrogenase reductase) and denitrification (narG of nitrate reductase, nirS of the cytochrome cdj containing nitrite reductase, nirK of the Cu-nitrite reductase and nosZ of the nitrous oxide reductase). The probes specifically hybridized with DNA from numerous microorganisms of culture collections or of bacteria isolated from soils (Linne von Berg, Bothe 1992; Kloos et al. 1998). These investigations showed that the highest number of culturable denitrifying and N2-fixing bacteria occurs in the upper (5 cm) soil layer and decreases with the depth in several soils examined. The message obtained was, however, limited, as only a small percentage of soil bacteria, about 1% or even less (Amann et al. 1997) can be cultured in standard media. Therefore a protocol has recently been developed for the isolation of DNA from soils which allowed to assess the relative distribution of bacterial DNA hybridizing with the different probes in the soil horizons (Mergel et al. 2001 a,b). This study also showed that the upper soil layer contained the highest content of DNA hybridizing with the probes for denitrification and N2-fixation and that this content decreased with the depth of an acid soil of an oak-hornbeam forest in the vicinity of Cologne (Chorbusch soil; Mergel et al. 2001a) and of a strongly acid Norway spruce stand in the Black Forest near Villingen (Mergel et al. 2001b). Bacterial DNA content was consistently higher in soils taken from the vicinity of plant roots than from the bulk, plant free soil. However, there was no selective enrichment of dinitrogen fixing and denitrifying bacteria at the roots of the plants (assessed by hybridizing with the probes of genes specific for dinitrogen fixation and denitrification). The percentage of dinitrogen fixing and denitrifying bacteria was estimated to make up to 5-10% of the total population of bacteria in such soils.

The result that the highest numbers of dinitrogen fixing and denitrifying bacteria occur in the upper (5 cm) soil layer is somewhat surprising. The nitrate content did not decrease with the depth of the soil, particularly in the case of the oak-hornbeam forest in the vicinity of Cologne ("Chorbusch" forest). Thus denitrifying bacteria were expected to be enriched with the depth of the soil. Just the opposite was observed. In the upper soil layer the oxygen and nitrate contents are probably non-limiting. Therefore the counts of high numbers of nitrogen fixing and denitrifying bacteria in the upper layer raise the question about the selective advantage of the possession of denitrification and N2-fixation at this location. The stands examined are never waterlogged. Therefore one can only argue that specific microsites with favorable conditions for denitrification and N2-fixation to proceed exist in the upper layer of these soils. Perhaps physiological traits like denitrification and N2-fixation are only retained in the organisms which may have developed at other locations with selective pressures for these processes.

The DNA isolated from the two soils Villingen and Chorbusch was pure enough for PCR amplifications using conserved oligonucleotide primers for nifH and for denitrification genes (nirS, nirK, nosZ) and also for 16S-rRNA (as a general bacterial probe). For each of these genes, 16-60 PCR-products obtained with DNA from the upper soil layer have now been cloned and sequenced. The data obtained give first insights on the composition of the bacterial community being present in the upper layer of an acid forest soil. Data are presented here for the 16S-rRNA (Figure 1) and the nifíí (Figure 2) segments. Comparisons are given for the sequences deposited in the databanks and the own sequences from the oak-hornbeam forest Chorbusch (marked with C) and from the Norway spruce stand of Villingen, Black Forest (marked with V). The following general conclusions can be drawn from these sequence comparisons of Figure 1:

(i) All sequences obtained are new and not identical with any deposited into the databanks.

(ii) Many of the sequences cluster with Actinomycetes and the "Acidobacterium" phylum which are mainly non-culturable as yet. None of the sequences of PCR-products clustered with those of the Bacillus/Paenibacillus group.

(iii) Some of the PCR-products clustered on the sequenced range (328 bp sequenced out of the about 1.5-1.6 kb of the whole gene) with proteobacteria (e.g. C6A23 next to Azospirillum amazonense, C6016, C6021, C6060-62 next to y-proteobacteria).

(iv) None of the PCR-products gave identical sequences which might reflect the high diversity of bacteria in such soils.

(v) Culturable bacteria from the same soil depth of Chorbusch or Villingen (marked with A for grown in heterotrophic mineral medium, "Azospirillum medium", or with Y for YEM medium (see Kloos et al. 1998)) clustered within the Bacillus-Paenibacillus group.

When primers for nifH were used, only few different amplificates (Figure 2) were obtained compared to the situation with the gene probes for denitrification (not documented). However, identical sequences were obtained several fold (up to 9) from cloned PCR-products using nifH primers. As judged from the microscopic inspection also, it may be that only few N2-fixing bacteria, however, in higher copy numbers, occur in such soils. This impression needs to be verified by more detailed investigations. Some of the DNA amplified with the nifH primers came from organisms related to well-known N2-fixing bacteria like Rhizobium, Bradyrhizobium japonicum or Azospirillum (Figure 2).

Of course, a limited number of PCR-products only can be cloned and sequenced. The primers used in the present study have continuously been improved with the availability of more sequences in the databanks and provide PCR-products in the case of a wide range of culturable bacteria. This was the case for nifH and all genes assayed in denitrification (nirS, nirK, nosZ). However, as a large percentage of the soil bacteria is non culturable as yet, it may well be that their nitrogen fixation or denitrification genes are not amplified by the primers used under the conditions employed. The data set obtained until now may still be too small to represent the biodiversity of a bacterial soil population. However, this start of the characterization already showed that this approach allows access to a whole regime of still undiscovered bacteria in soils. Details will be published elsewhere (C. Rosch, A. Mergel, H. Bothe, in preparation).

Figure 1.

16S rDNA sequences of PCR products obtained with DNA from two different acid forest soils.

The sequence of Natro-nobacterium salstagnum was used as outgroup. 328 characters of the 16S-rRNA gene (N 357 to N 684 of the Escherichia coli sequence) were analyzed by the NJ algorithm (250 boot-straps).

Abbreviations: C = from the Chorbusch soil, V = from Villingen, Y = from colonies on agar plates containing YEM-medium, A = from colonies on agar plates containing heterotrophic mineral medium (= Azospirillum medium,). The clones marked with C6... only come from PCR products obtained by amplifying segments of the Chorbusch DNA using primers for the 16S-rDNA.

■ Natronobacterium salstagnum (Euryarcheota)

Clostridium ihermoautotrophicum DSM1974 Desulfotomaculatum gibsoniae - Desulfotomaculum 175 Desulfotomaculatum geothermicum

Eubacterium sp. C2

Clostridium sticklandii

Clostridium amlnophilum

Caldlceliulosiruptorsaccharolytlcus C6056s

■ Acidobacterium capsulatum

:6052s C6011 C6001 C6023

(isolate YC04)

Nltrospira moscoviensis Nitrospira marina

Coxiella burnetii

C6021 C6061

C6060 C6016 C6062

Pseudomonas butanovora

(isolate YC04)

Nitrospira Marina

Nltrospira moscoviensis Nitrospira marina

Legionella waltersil

(Cyanobacteria)

I"

3 ST

Legionella waltersil

cd O

Q3 O

(Cyanobacteria)

Flavobacterium okeanokoites Paenibacillus mcmeekinii Paenibacillus alkanoclasticus 1761— Bacillus halodurans

_?50 r Bacillus alcalophilus

'— Bacillus pseudalcaliphilus Virgibaclllus pantothentlcus Bacillus halodenitrlficans Bacillus marismortui isolate nrot-1"

Bacillus hatmapalus

Bacillus psychrosaccharolyticus isolate AC03 - - "Bacillus macroides" — isolate YC06 t- Bacillus sp. NIBHP3M26

1- isolate YC08

Bacillus simplex

cd s

Cr 05

57 54

- ALCALIGE NES FAECALIS

Glucona cetobacter diazotrophicus - R hodospirillum rubrum

Azospirillum brasilense Azos pirillum lip ofe rum C F 052 (9/16) VF 003 (3/5) CFY04 (3/6) (3/6)

rLI-YU'

— Herbaspirillum s eropedica e Rhizobium sp. ORS571 - CF051 (1/16)

rhizosphere bacterium (Accession AF216939) ■ C F049 (3/16) VF 009 (2/5)

Figure 2. Sequences of PCR products obtained for nifHfrom two different acid forest soils.

First number in brackets: amount of identical sequences obtained for this clone. Second number in brackets: amount of PCR-products sequenced at one location. The sequence of Alcaligenes faecalis was used as outgroup. 397 characters of nifH gene (N60 to N465 of the Sinorhizobium meliloti sequence) were analyzed by the NJ algorithm (100 bootstraps). Abbreviations mean: C = from the Chorbusch soil, V = from Villingen, F = for nifH, Y = from an enrichment culture grown in YEM-medium.

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