molecular analysis of ammonia oxidation and ... · nia oxidation [10]; nitrogen cycling in coastal...

Molecular analysis of ammonia oxidation and denitri¢cationin natural environments

Hermann Bothe a, Gu«nter Jost b, Michael Schloter c, Bess B. Ward d,Karl-Paul Witzel e;*

a Botanical Institute, University of Cologne, 50923 Cologne, Germanyb Baltic Sea Research Institute, Warnemu«nde, SeestraMe 15, 18119 Rostock, Germany

c GSF-National Research Center for Environment and Health, Institute of Soil Ecology, Ingolsta«dter Landstr. 1, 85764 Neuherberg, Germanyd Department of Geosciences, Princeton University, Princeton, NJ 08544, USA

e Max-Planck-Institute for Limnology, P.O. Box 165, 24306 Plo«n, Germany

Received 10 April 2000; received in revised form 17 August 2000; accepted 18 August 2000

Abstract

This review summarizes aspects of the current knowledge about the ecology of ammonia-oxidizing and denitrifying bacteria. Thedevelopment of molecular techniques has contributed enormously to the rapid recent progress in the field. Different techniques for doing soare discussed. The characterization of ammonia-oxidizing and -denitrifying bacteria by sequencing the genes encoding 16S rRNA andfunctional proteins opened the possibility of constructing specific probes. It is now possible to monitor the occurrence of a particular speciesof these bacteria in any habitat and to get an estimate of the relative abundance of different types, even if they are not culturable as yet.These data indicate that the composition of nitrifying and denitrifying communities is complex and apparently subject to large fluctuations,both in time and in space. More attempts are needed to enrich and isolate those bacteria which dominate the processes, and to characterizethem by a combination of physiological, biochemical and molecular techniques. While PCR and probing with nucleotides or antibodies areprimarily used to study the structure of nitrifying and denitrifying communities, studies of their function in natural habitats, which requirequantification at the transcriptional level, are currently not possible. ß 2000 Federation of European Microbiological Societies. Publishedby Elsevier Science B.V. All rights reserved.

Keywords: Nitri¢cation; Ammonia oxidation; Denitri¢cation; N-cycle; Molecular ecology; DNA probing of N-metabolizing bacteria; Antibody ofN-metabolizing bacteria

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6742. Nitri¢cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674

2.1. Taxonomy and distribution of nitrifying bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6742.2. Physiological aspects of ammonia oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6752.3. Enzymes involved in ammonia oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6752.4. Analysis of the community structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676

3. Denitri¢cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6783.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6783.2. Serological approaches to study denitri¢cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6803.3. Detection of denitrifying bacteria in soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6833.4. Denitri¢cation in aquatic habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684

4. Conclusions and future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685

0168-6445 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 4 5 ( 0 0 ) 0 0 0 5 3 - X

* Corresponding author. Tel. : +49 (4522) 763-265; Fax: +49 (4522) 763-310; E-mail : [email protected]

FEMSRE 704 3-11-00 Cyaan Magenta Geel Zwart

FEMS Microbiology Reviews 24 (2000) 673^690

www.fems-microbiology.org

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685

1. Introduction

Nitri¢cation (the oxidation of ammonia to nitrate vianitrite) and denitri¢cation (the reduction of nitrate to mo-lecular nitrogen via nitrite, nitric oxide and nitrous oxide)are essential steps in the global nitrogen cycle. In manyecosystems, these processes counteract natural and man-made eutrophication, and they are used to reduce N con-centrations in sewage treatment plants. The importance ofboth processes is re£ected by numerous reviews: ecologyof denitri¢cation and dissimilatory nitrate reduction [1] ;autotrophic nitri¢cation in bacteria [2] ; aspects of denitri-¢cation in soil and sediment [3] ; factors controlling deni-tri¢cation [4] ; probes for phylogenetic and functionalgenes of nitri¢cation and denitri¢cation [5] ; enzymologyof ammonia oxidation [6] ; cell biology and molecular basisof denitri¢cation [7] ; enzymology of the nitrogen cycle [8] ;nitrifying bacteria and aquaculture [9] ; anaerobic ammo-nia oxidation [10]; nitrogen cycling in coastal marine eco-systems [11]; dissimilatory nitrate reductases in bacteria[12]; and inorganic nitrogen metabolism in bacteria [13].

For both nitrifying and denitrifying bacteria, substantialknowledge has accumulated on the physiology, biochem-istry and the molecular regulatory mechanisms of only afew selected species (e.g. Nitrosomonas europaea or Pseu-domonas stutzeri). This has provided the initial informa-tion from which to develop molecular approaches for en-vironmental studies. However, the current state ofinformation on the ecology of these organisms is inad-equate. Only a few thousand bacterial species have beencharacterized and deposited in type culture collections, yetthere is evidence that 1 g of soil contains 4U103^104 dif-ferent bacterial genomes [14,15]. Only about 1^10% of soilbacteria [16] or even less have been cultivated so far andare thus available for taxonomic characterization. Molec-ular techniques may be powerful tools to investigate bac-teria in their natural environment, especially those that arenot yet culturable (see [17]). We are starting to recognizethat for nitri¢ers and denitri¢ers the real bacterial world isalso much more complex and diverse than ever thought.Those bacteria which have been studied extensively in purecultures are probably not the major `players' in most nat-ural habitats. Thus, molecular techniques have given ac-cess to a whole world of yet unknown nitrifying and de-nitrifying bacteria.

The scope of this review is to stress the recent achieve-ments in the ¢eld of nitri¢cation and denitri¢cation ob-tained by applying molecular techniques, with emphasison soil and natural aquatic environments. Engineered sys-tems, like sewage treatment plants, are mentioned onlyoccasionally, when they provide examples of recent, inno-vative approaches.

2. Nitri¢cation

2.1. Taxonomy and distribution of nitrifying bacteria

Nitrifying bacteria are ubiquitous in soil, freshwater andmarine environments. They have been found even in ex-treme habitats such as building sandstone [18], extremelyalkaline soda biotopes [19], Antarctic ice [20], hot springs[21], or in association with marine sponges [22]. They arefascinating and ideal study objects, partly because most ofthem are of monophyletic origin and have a unique me-tabolism, with many enzymes that have been found onlyin this group of organisms.

Never since their discovery by S.N. Winogradsky at theend of the 19th century has this group of bacteria receivedso much attention as in the last 5^7 years. Even today onlya few experts worldwide are able to isolate pure culturesand maintain them inde¢nitely.

Nitri¢cation is a two-step process which involves twodi¡erent groups of bacteria. Ammonia-oxidizing bacteria(AOB) oxidize ammonia to nitrite, and nitrite-oxidizingbacteria (NOB) oxidize nitrite to nitrate. So far, no auto-trophic bacterium is known to oxidize ammonia directly tonitrate. Traditionally, AOB were classi¢ed by cell mor-phology into the ¢ve di¡erent genera: Nitrosomonas, Ni-trosococcus, Nitrosospira, Nitrosovibrio and Nitrosolobus[23]. Recently, on the basis of 16S rRNA sequence homol-ogy, Nitrosospira, Nitrosovibrio and Nitrosolobus were pro-posed to be combined into one common genus Nitroso-spira [24]. With the exception of Nitrosococcus, all generarepresent closely related organisms of the L subclass ofProteobacteria [25]. The genus Nitrosococcus is phyloge-netically not homogeneous. N. mobilis is a L subclass or-ganism, while N. oceani, and probably also N. halophilus,are Q-Proteobacteria [25]. So far, only a few ammonia-ox-idizing Q-Proteobacteria have been isolated from marinehabitats, and none from soil or freshwater habitats. Ingeneral, AOB are obligatory chemolithoautotrophs,although some of them can use organic compounds (ace-tate, pyruvate) for mixotrophic growth. NOBs will not bediscussed in this article.

Some heterotrophic bacteria and fungi [26] can also ox-idize ammonia and/or reduced nitrogen from organic com-pounds to hydroxylamine, nitrite and nitrate. Whilst Noxidation is the only energy-yielding process in autotro-phic nitri¢ers, nitri¢cation in heterotrophic organismsseemingly does not contribute signi¢cantly to their energymetabolism. Heterotrophic nitri¢ers are known for theirability to nitrify and denitrify simultaneously [27]. Morerecently, it was shown that the autotrophic AOB N. euro-paea and N. eutropha could also simultaneously nitrify anddenitrify when grown under oxygen limitation [28].


H. Bothe et al. / FEMS Microbiology Reviews 24 (2000) 673^690674

2.2. Physiological aspects of ammonia oxidation

Until recently, ammonia oxidation was thought to be astrictly aerobic process, requiring molecular O2. Usually,AOB can cope better with low O2 than with low ammoniaconcentrations. In a chemostat fed with water from ahighly eutrophic wastewater reservoir, nitri¢cation was ob-served at O2 concentrations down to 0.05 mg l31 [29].Under anoxic conditions N. eutropha and N. europaea,and probably also some other AOB, can oxidize ammoniain the presence of pyruvate, and with nitrite as electronacceptor [30] or with NO2 gas [31]. Thus, AOB can deni-trify endogenously produced nitrite to N2O, NO and N2.Both groups of nitri¢ers can survive under anoxic condi-tions for months. Therefore, high numbers of nitri¢ers canbe observed in anoxic hypolimnia [32] or sediment [33] ofeutrophic freshwaters or wastewater basins [34] even underconditions that do not permit active nitri¢cation. In soiland sediments of eutrophic freshwaters with areas of peri-odically low O2 concentration, chemolithotrophic AOBare probably adapted to nitrify under microoxic condi-tions [35]. It seems that this is due to physiological adap-tations, such as higher a¤nity for oxygen or alternativeelectron acceptors, rather than to a change in the compo-sition of the nitrifying community.

2.3. Enzymes involved in ammonia oxidation

In autotrophic ammonia oxidizers, two key enzymes arenecessary for energy conservation during the oxidationprocess, ammonia monooxygenase (AMO) and hydroxyl-amine oxidoreductase (HAO). In vivo both enzymes areco-dependent because they generate the substrate and elec-trons, respectively, for each other. AMO catalyzes the oxy-genation of ammonia to hydroxylamine:

NH3 �O2 � 2H� � 2e3 ! NH2OH�H2O

The two electrons required in this process are derived fromthe oxidation of hydroxylamine to nitrite by HAO:

NH2OH�H2O! NO32 � 5H� � 4e3

One of the oxygen atoms in NO32 derives from O2, the

other one from water. Two of the four electrons generatedby HAO are transferred via the tetra-heme cytochromec554 [36], either to AMO, or diverted into an electrontransport chain [37,38], where many of the carriers in-volved and intermediates have not been completely char-acterized [6]. As these reactions have been studied mainlywith N. europaea, alternatives to this pattern may be dis-covered when a broader range of organisms from diversehabitats are examined.

2.3.1. Ammonia monooxygenaseIndirect evidence suggests that the enzyme AMO in N.

europaea, and probably some other autotrophic AOB ofthe L and Q subclasses of Proteobacteria, has three sub-

units, AMO-A, AMO-B and AMO-C, with di¡erent sizes,structures and arrangements within the membrane/peri-plasmic space of the cells. The enzyme catalyzes the oxy-genation of a broad range of substrates [6]. Due to itsessential function in the energy metabolism of AOB, theenzyme is probably constitutive.

The only puri¢cation of an AMO as active enzyme hasbeen achieved from the heterotrophic nitri¢er Paracoccusdenitri¢cans [39]. This enzyme consists of only two sub-units (not three as the AMO from autotrophic nitri¢ers)and has several features in common with the enzyme fam-ily which includes not only AMO, but also the particulatemethane monooxygenase (pMMO) from methanotrophs.From another heterotrophic nitri¢er, Pseudomonas putida,a DNA region has been sequenced which showed partialhomology to the amoA gene of N. europaea [40]. Therewere also indications that AMO is expressed in this organ-ism [40].

The three subunits of AMO from autotrophic AOB areencoded by the genes amoC, amoA and amoB of the amooperon [41,42]. All three AMO genes have been clonedand sequenced from several AOB [43^47].

PCR primers used to amplify sequences of the amo op-eron from di¡erent environments have been designed totarget amoA, encoding the subunit that carries the activesite of this enzyme [48^50]. Within amoA, the region en-coding the C-terminus appears to be a suitable target sitefor primers and probes to discriminate between AOB ofthe L and Q subclasses of the Proteobacteria as well asbetween ammonia and methane oxidizers within the Q sub-class [42].

Between the three amo genes, intergenic spacer regionsof variable, but strain-speci¢c length have been observed[42]. A spacer between amoA and amoB was observed onlyin the Q subclass proteobacterium N. oceani [42]. This fea-ture may be used to screen for the survival rate of aspeci¢c strain or to describe the population dynamics inenvironmental samples by PCR with primers £ankingthe variable intergenic regions [42]. Because of the keyfunction of AMO in the energy metabolism of these bac-teria, all three genes encoding the subunits of the enzymemust have evolved under high functional pressure, whereasthe intergenic spacers may provide a selectively neutralregion.

2.3.2. Homology of AMO and pMMOThe enzymes AMO of nitrifying bacteria and pMMO of

methanotrophic bacteria are homologous and share manyfeatures [51,52]. The structures of their operons are almostidentical. The high similarity of the nucleotide sequence ofthe genes is indicative of a common evolutionary origin[53,54]. At the DNA and amino acid sequence level, sim-ilarities between amo/AMO and pmo/pMMO of the Q sub-class of the Proteobacteria are higher than within the amo/AMO of the Q and L subclass AOB [42,53]. It has beenproposed that amo from the L and Q subclass AOB and


H. Bothe et al. / FEMS Microbiology Reviews 24 (2000) 673^690 675

pmo from the Q subclass methane-oxidizing bacteria sepa-rated into three lineages early in evolution [42].

Methanotrophic bacteria can oxidize ammonia to nitriteby the combined action of pMMO and a unique HAO,which is di¡erent from that in AOB [55,56]. Methanotro-phic and ammonia-oxidizing bacteria occupy similarniches in soil and aquatic habitats, at counteracting gra-dients of O2 and methane or ammonia, respectively. Theirinteractions are complex and poorly understood so far.

2.3.3. Hydroxylamine oxidoreductaseHAO is an unusual enzyme with a highly complex struc-

ture, located as a soluble enzyme in the periplasmic space,but anchored in the cytoplasmic membrane. Each subunitof the trimeric enzyme contains, as probable componentsof the active site, seven c-type hemes and one residue ofthe unusual heme P460 [57,58]. The molecular and bio-chemical characterization is given in [59^61]. Soluble,non-heme-containing hydroxylamine oxidases from theheterotrophic nitrifying bacteria P. denitri¢cans and Pseu-domonas strain PB16 [62,63] are very di¡erent from theHAO of autotrophic AOB and may be responsible forthe inability of these heterotrophs to grow autotrophicallyby nitri¢cation.

2.3.4. Multiple copies of genes encoding electron transportproteins

Several of the genes involved in ammonia oxidation arepresent in multiple copies per genome. It has been sug-gested that the ratio of the number of copies of di¡erentgenes could be responsible for maintaining a certain ratioof the gene products [64]. Between two and four copies ofthe amo gene have been observed in the AOB of theL subclass [41^43,45,65]. Additional copies of amoC some-times occur outside the operon. N. oceani (ATCC 19707),a member of the Q subclass, and another unnamed strain(C-113) have only one copy of the entire operon [42]. Allof the multiple copies described so far are highly similar,and it is not yet clear whether they have di¡erent func-tions. Studies with insertional mutants of N. europaea, inwhich each copy of amoA alone was inactivated, showedthat both copies were expressed. Inactivation of only onecopy resulted in slower growth, less full-sized mRNA, andreduced AMO activity (measured as ammonia-dependentO2 consumption and [14C]acetylene incorporation). Thus,each copy had a distinct metabolic function, but was notessential for growth [66]. As most of the multiple genecopies are nearly identical whilst much less similarity existsbetween amo from di¡erent species, it is unlikely that theyoriginated from horizontal gene transfer, they probablyarose by a gene duplication event [65,67] that occurredrelatively early in the evolution of this lineage. It is ofparticular interest for evolutionary ecology that the high-est multiplicity of amo genes is found in organisms of theNitrosospira/Nitrosolobus group, whereas the two AOB in-vestigated so far from the Q subclass have only one copy.

Three copies of the genes for HAO (hao) and cyto-chrome c554 (cycA) have been identi¢ed in the genome ofN. europaea [44]. No other proteins with substantial sim-ilarities to HAO were found in a database search[64,68,69].

2.3.5. Regulation of enzyme functionAmmonia regulates the activity of AMO at the tran-

scriptional [70], translational [71,72] and post-translational[72] levels. Using RT-PCR, products of all three amo geneswere detected in the same transcript, indicating that amoCis part of the amo operon [41,47]. In N. europaea, tran-scription of amo as well as hao was induced by ammonia[70] but mRNA of these genes disappeared within 8 hunder conditions of ammonia starvation.

In N. europaea cells that had been starved of ammonia,HAO remained stable for up to 72 h, as indicated byWestern blot analysis and activity measurements [73^75].In nature the key enzymes AMO and HAO should remainstable even under conditions of £uctuating ammonia con-centrations. No indications have been found for the di¡er-ential expression of the amo operon.

Ammonia limitation caused speci¢c inhibition of theAMO but not of the HAO activity within 24 h, whereaslittle change in the ammonia oxidation activity occurred ina medium without ammonia [74]. Under long-term (342days) ammonia starvation of N. europaea, the activity ofAMO and HAO remained stable, and the cells maintaineda high level of the enzyme. After the addition of ammoniaor hydroxylamine, there was an immediate response, mea-sured as nitrite production, without initial protein synthe-sis [76].

2.4. Analysis of the community structure

Due to their special requirements, which are often un-known, and low growth rates, autotrophic AOB are di¤-cult to isolate in pure cultures. This, together with usuallylow numbers (less than 0.1% of direct counts), hamperedour knowledge on the distribution and relative abundanceof these bacteria in nature until molecular approaches be-came available.

2.4.1. Immuno£uorescence approachesAOB were counted directly by application of £uorescent

polyclonal antibodies (FA). The production of FA de-pends on the availability of pure cultures. A considerableserological diversity was noticed within the isolates fromthe same sample of soil [77]. FA developed for selectivecounting of several Nitrosomonas spp. allowed the species-speci¢c counting of attached and suspended bacteria [78].On the other hand, FA produced against several marineammonia or nitrite oxidizers reacted with a broad spec-trum of isolates of the same physiological type [79]. About70% of 30 AOB isolates and eight of nine nitrite oxidizersreacted with at least one of the FA raised against ammo-



nia or nitrite oxidizers, respectively. These bacteria repre-sented only 0.1^0.8% of the total bacterial counts in oce-anic waters. As a consequence, extensive counting timesare needed to get statistically signi¢cant numbers. Anotherdrawback is the variability of the clonal composition ofthe antibodies as discussed in Section 3.2. Nevertheless,compared to the most-probable-number (MPN) techniquethe use of FA provides results within hours and has ahigher resolution and sensitivity.

For a long time, Nitrosomonas spp. were believed to bethe dominant [80], or at least the most common, [81] am-monia oxidizers in aquatic environments, whereas Nitro-sospira spp. were most frequent in soil [82]. Nitrosomonasspp. were the dominant nitri¢ers in the lower part of theriver Elbe, as determined from high MPN dilutions [83],by immuno£uorescence microscopy [84], as well as DNAhybridization and partial sequence analysis of 16S rRNApartial gene sequences [83]. However, more recent inves-tigations of other locations have changed this view (seebelow).

2.4.2. Phylogenetic approachesThe fact that nitrifying bacteria (ammonia and nitrite

oxidizers) seem to have only one operon for 16S rRNAgenes [85,86] greatly facilitates community analyses by mo-lecular approaches. The knowledge of these sequences al-lows di¡erent approaches for studying the communitycomposition of nitrifying bacteria. Most of the work hasbeen done for the monophyletic group of the AOB withinthe L-Proteobacteria.

2.4.2.1. Fluorescent in situ hybridization (FISH). Sev-eral oligonucleotide probes for detecting AOB of the L-Proteobacteria have been described [87^89], but there areonly few applications of FISH for estimating the abun-dance of nitrifying bacteria in natural environments. Theprobe NEU [87] was designed to be speci¢c for Nitroso-monas spp. At present the probes NSO190 and NSO1225have the broadest speci¢city and are therefore the mostsuitable for this approach. According to a recent BLASTsearch, both probes match sequences of 16S rRNA inAOB and a few unidenti¢ed clones. They could be appliedsimultaneously (or separately as controls for one another)because their speci¢city is slightly di¡erent.

Voytek et al. [90] reported the detection and quanti¢ca-tion of L subclass Proteobacteria using FISH in samplescollected from several permanently ice-covered lakes in theTaylor Valley of Antarctica. Abundances derived fromFISH compared well with those estimated by immuno£uo-rescence (maximum of about 2000 cells ml31) and slightapparent di¡erences in cell distribution were attributed tothe slightly di¡erent speci¢cities of the antibodies versusthe DNA probes. In samples from the Baltic Sea, FISHyielded cell numbers near the detection limit, when a con-trol with a nonsense probe was used (S. Bauer and G.Jost, unpublished). Therefore, the application of more spe-

ci¢c probes that distinguish di¡erent groups of AOB mightbe feasible only in habitats with high AOB abundances,such as sewage treatment plants. So far, no probes forAOB of the Q-Proteobacteria have been described, whichwould be especially useful for enumerating AOB in marinesamples.

2.4.2.2. PCR-based techniques. From the 16S rRNAgene sequences known up to 1997, 30 oligonucleotideswith known speci¢city for AOB of the L-Proteobacteriawere suggested (for review see [91]) for PCR or hybrid-izations with extracted DNA/RNA or whole cells. Nearlyall knowledge about the diversity of AOB communities innatural environments has been obtained using these oligo-nucleotides. Oligonucleotide primers for N. oceani werereported [92] but have not been widely applied [93].

PCR with primer combinations of known speci¢city (forall AOB of the L-Proteobacteria or subgroups thereof) wasused to amplify 16S rDNA of the ammonia-oxidizingcommunity from natural samples. Either direct PCR[94^97] or nested PCR (¢rst PCR with primers for eubac-teria) [98^101] was conducted. In some cases, nested PCRrevealed positive results from samples where direct PCRfailed, which was mainly attributed to low abundances ofAOB [102,103].

During investigations of di¡erent environmental sam-ples (water and soil, nutrient-poor and enriched) it becameevident that Nitrosospira-like AOB are nearly ubiquitous[98,99,101,104^106]. From di¡erent soils, a variety of se-quences were isolated that grouped into four clusters re-lated to Nitrosospira, and three clusters related to Nitro-somonas [107]. Clones from marine and soil samplesgrouped into di¡erent clusters [94,96,108].

Nitrosospira-like AOB seemed to be ubiquitouslypresent and also the dominant AOB in most natural envi-ronments. Nevertheless, Nitrosomonas-like AOB couldalso be detected in many environmental samples. N. euro-paea-type 16S rDNA was present in di¡erent depths ofthree lakes in northern Germany [103]. Cloned 16SrDNA sequences formed a strong monophyletic clustertogether with the sequences from N. ureae [95]. Signi¢cantdi¡erences in the community structure between free-livingand attached AOB have been found in marine samples.Whereas the majority of the partial sequences obtainedfrom planktonic samples were related to cluster 1 withinthe Nitrosospira spp. nearly all sequences from particle-associated material were related to cluster 7 within theN. eutropha/europaea lineage [100]. It has long been knownthat at least some, if not all, of the AOB tend to adhere tosurfaces [2]. Due to their ability to produce extracellularpolymers [84], they can form aggregates, attach to soil,sediments, suspended particles in water, or surfaces ofculture vessels. As a survival strategy, exopolymers mayhelp recovery after desiccation stress in soil [109], survivalin starvation conditions [29,34] or facilitate nitri¢cation atlow pH [110]. However, strong adsorption to particles may



in£uence the extraction of cells from soil [111] as well asthe permeability of the cells for oligonucleotide probes inFISH, or the quantitative recovery of DNA for PCR.

The probability of detection of members of the N. eu-tropha/europaea lineage rises with increasing nutrientconcentrations [97,100,101,104]. Sometimes, even if Nitro-somonas-like sequences could not be detected in environ-mental DNA samples, enrichment cultures prepared fromthe same sampling site revealed Nitrosomonas spp.[98,99,112]. Whitby et al. [101] provided evidence thateven within di¡erent sediment types (littoral and profun-dal sediments) distinct groups related to either N. europaeaor N. eutropha occurred. 16S rRNA sequences most sim-ilar to those from N. europaea and N. eutropha wererecovered from the highly saline waters of Mono Lake,California [93].

Interestingly, within a steadily increasing number of en-vironmental studies sequences belonging to similar clustersof the AOB were retrieved from very di¡erent locations(water and sediment samples from freshwater and an es-tuary) with di¡erent composition of their `total' microbialcommunities. On the other hand, the data give the impres-sion of great diversity within a group for which only a fewstrains are in culture and are not physiologically well char-acterized.

In fertilized soil a drastic increase in nitri¢cation rate,but not in population size, was estimated by competitivePCR based on 16S rRNA and amoA genes. This suggestedphenotypic changes within the AOB community, while thepopulation size increased 6 weeks later [106]. In a studyusing 16S rDNA fragments to characterize the communityof AOB in the root zone and bare sediment of a shallowlake in the Netherlands, no evidence was found that anyparticular taxonomic/phylogenetic group was speci¢c forthese periodically anoxic environments [113]. It would beinteresting to see if such adaptations could be monitoredwhen targeting mRNAs of key enzymes (AMO, HAO) ofammonia oxidation.

2.4.3. Comparison of the community analysis by either16S rRNA or functional genes

As mentioned above, AOB of the L subclass of theProteobacteria form a distinct group within this subclass.Their 16S rRNA sequence has high similarity with that ofthe phototrophic Rhodocyclus purpureus and the iron-oxi-dizing bacterium Gallionella ferruginea. Therefore, a slightlack of speci¢city in the PCR reaction with `speci¢c' prim-ers may shift the spectrum of sequences that is obtainedtowards phylogenetically related but physiologically andecologically di¡erent organisms. As none of the primercombinations described for ampli¢cation of L-proteobac-terial AOB seems to be absolutely speci¢c [91,95,96,107],there is a risk of erroneous results. Our own experiences(J. Ho«ppner and K.-P. Witzel, unpublished) showed thatthe proportion of non-nitri¢er sequences in cloned PCRproducts from freshwater samples increased dramatically

when the PCR was not performed under high stringency.Many of these unspeci¢c clones were identi¢ed as Gallio-nella or Rhodocyclus spp.

Important physiological di¡erences may exist withinbacteria of a monophyletic 16S rDNA group with highsequence identity [95]. It is questionable whether 16SrDNA sequences are optimally suited for the speci¢c de-tection of AOB even if the sequence homology is high[46,48,86]. The highly variable, selectively neutral, inter-genic spacer regions between the 5S and 16S or 16S and23S rRNA genes [86,114] might provide better targets. Atpresent, there is not enough information available on thissubject for comparative studies.

Under functional or ecological aspects, genes encodingkey enzymes may be a better target for ¢ne-scale resolu-tion because they are under high selection pressure [48].For a marine isolate of AOB, belonging to the Q subclass,the 16S rDNA sequence was nearly identical (only one andtwo bases out of 976 were di¡erent) to the 16S rDNAfrom two strains of N. oceani. In contrast, a comparisonof all three amo genes of these bacteria revealed a muchlower identity of around 88^90% [42]. AMO and HAO,which are crucial for the existence of these bacteria, musthave been optimized during evolution for the prevailingconditions in the natural habitat. Therefore, it may bespeculated that di¡erent ecotypes of these enzymes musthave evolved for optimal function in di¡erent habitats.

For community structure analyses, it seems rather un-likely that other parts of the bacterial genome could fullyreplace the 16S rDNA gene as target sites, not only due toinsu¤cient sequence information for comparisons, butalso because AOB have only one copy of the rRNA oper-on. More important than an enhanced resolution withinthe AOB is information about ecological requirements andphysiological capacities in relation to environmental fac-tors of the organisms to which the retrieved sequencesbelong. The next step is probably a comparison betweenmere presence and activity, for example, by comparingamoA gene sequences with the related mRNA analyses.

3. Denitri¢cation

3.1. Introduction

Denitri¢cation (dissimilatory nitrate reduction, nitraterespiration) is characterized by consecutive steps startingfrom nitrate via nitrite, nitric oxide (NO), nitrous oxide(N2O) to the dinitrogen gas (N2). The ability to denitrify iswidespread among bacteria of unrelated systematic a¤li-ations, most likely due to lateral gene transfer in evolution[115,116]. Denitri¢cation has been described for some Ar-chaea [7,116] and even for the mitochondria of some fungi[117]. While the NO reductase from the fungus Fusariumoxysporum has been crystallized and its structure described[118], molecular details and the ecological signi¢cance of



Tab

le1

Enz

ymes

ofni

tri¢

cati

onan

dde

nitr

i¢ca

tion

a

Enz

yme

EC

num

ber

Rea

ctio

npe

rfor

med

Gen

esP

rost

heti

cgr

oup

Fun

ctio

nof

gene

prod

uct

Gen

esi

ze(b

p)B

acte

ria

from

whi

chge

nes

wer

ese

quen

cedc

NO

3 3re

duct

aseb

1.7.

99.4

NO

3 3+

2e3

+2H� C

NO

3 2+

H2O

narG

[4F

e^4S

],m

olyb

dopt

erin

cata

lyti

cce

nter

3588

^378

61,

2,3,

5,6,

7;

12ot

hers

part

ly

narH

3[4

Fe^

4S]+

[3F

e^4S

]co

nfer

se3

from

Nar

Ito

Nar

G14

64^1

677

1,2,

3,4,

5,6,

7,pa

rtly

8

narJ

func

tion

unkn

own

516^

741

1,2,

3,4,

5,6,

7na

rI2

cyt

bm

embr

ane

anch

or,

QH

2ox

idat

ion

672^

741

1,2,

3,4,

5,6,

7

Hem

eN

O3 2

redu

ctas

e1.

7.2.

1N

O3 2

+e3

+2H� C

NO

+H

2O

nirS

cyt

cd1/s

ubun

it(K

2)

cata

lyti

cce

nter

1665

^179

14,

9,10

;fo

urot

hers

part

lyC

uN

O3 2

redu

ctas

eN

O3 2

+e3

+2H� C

NO

+H

2O

nirK

Cu(

I),

Cu(

II)

(K2)

cata

lyti

cce

nter

1092

^114

011

,12

,13

,14

,15

,16

,17

;cr

ysta

lst

ruct

ure

wit

h2.

3Aî

reso

luti

onav

aila

ble

NO

redu

ctas

e1.

7.99

.72N

O+

2e3

+2H�C

N2O

+H

2O

norB

cyt

bca

taly

tic

cent

er13

41^1

542

13,

18,

4,19

,5,

10,

15,

17,

part

ly20

norC

cyt

cca

taly

tic

cent

er44

1^45

313

,2U

4,19

,5,

10,

2U17

,15

N2O

redu

ctas

e1.

7.99

.6N

2O

+2e

3+

2H�C

N2+

H2O

nosZ

4C

u/su

buni

t(K

2)

cata

lyti

cce

nter

1905

^195

911

,13

,2U

4,5,

10,

9,21

;43

othe

rspa

rtly

Am

mon

iam

onoo

xyge

nase

1.13

.12

NH

3+

2[H

]+O

2C

NH

2O

H+

H2O

amoA

acti

vesi

te74

4^82

5(1

530)

22,

23,

24,

25,

26,

27,

28;

69ot

hers

part

ly

amoB

invo

lved

inac

tivi

ty12

50^1

262

23,

24,

26;

two

othe

rspa

rtly

amoC

func

tion

unkn

own

813^

880

26,

29,

30;

two

othe

rspa

rtly

Hyd

roxy

lam

ine

oxid

ored

ucta

se1.

7.3.

4N

H2O

H+

H2OC

HN

O2+

4[H

]ha

o7

cyt

c,he

me

P46

0

(K3)

cata

lyti

cce

nter

1713

26,

31;

one

part

ly

aT

heta

ble

was

com

pile

dby

C.

Ro «s

ch(C

olog

ne)

from

data

bank

info

rmat

ion

avai

labl

ein

Mar

ch20

00.

bE

.co

liha

sa

seco

ndm

embr

ane-

boun

dni

trat

ere

duct

ase

(enc

oded

byna

rZY

WV

),an

da

furt

her,

peri

plas

mic

diss

imila

tory

enzy

me

(nap

FD

AG

HB

C).

c The

num

bers

repr

esen

tth

efo

llow

ing

bact

eria

:(1

)B

acill

ussu

btili

s,(2

)E

sche

rich

iaco

li,(3

)M

ycob

acte

rium

tube

rcul

osis

,(4

)P

arac

occu

sde

nitr

i¢ca

ns,

(5)

Pse

udom

onas

aeru

gino

sa,

(6)

Sta

phyl

ococ

cus

carn

o-su

s,(7

)T

herm

usth

erm

ophi

lus,

(8)

Pse

udom

onas

£uor

esce

ns,

(9)

Ral

ston

iaeu

trop

ha,

(10)

Pse

udom

onas

stut

zeri

,(1

1)`A

chro

mob

acte

rcy

cloc

last

es',

(12)

Alc

alig

enes

faec

alis

,(1

3)B

rady

rhiz

obiu

mja

poni

cum

,(1

4)P

seud

omon

asch

loro

raph

is,

(15)

Pse

udom

onas

sp.,

(16)

Rhi

zobi

umhe

dysa

ri,

(17)

Rho

doba

cter

spha

eroi

des,

(18)

Nit

roba

cter

ham

burg

ensi

s,(1

9)P

arac

occu

sha

lode

nitr

i¢ca

ns,

(20)

Syn

echo

cyst

issp

.,(2

1)S

i-no

rhiz

obiu

mm

elilo

ti,

(22)

Pse

udom

onas

puti

da,

(23)

Nit

roso

cocc

usoc

eani

,(2

4)N

itro

soco

ccus

sp.

C-1

13,

(25)

Nit

roso

spir

am

ulti

form

is,

(26)

Nit

roso

mon

aseu

ropa

ea,

(27)

Nit

roso

spir

abr

iens

is,

(28)

Nit

roso

spir

ate

nuis

,(2

9)N

itro

sosp

ira

sp.

NpA

V,

(30)

Nit

roso

mon

assp

.T

K79

4,(3

1)N

itro

som

onas

sp.

EN

I-11

.



denitri¢cation in these organisms still need to be charac-terized in more detail. In nitrate respiration, some bacteriareduce nitrate only to nitrite or N2O. In addition, specieshave been described which grow anaerobically with nitriteor N2O as the sole respiratory electron acceptor. The ex-pression of denitri¢cation genes is subject to complex reg-ulation. Anaerobic conditions and NO are major factorsfor the expression of denitri¢cation genes which are undercontrol of the transcription factor FNR, and also by aphosphorylated NarL protein in dissimilatory nitrate re-duction of Escherichia coli [119]. For details on this sub-ject, the reader is referred to recent reviews [7,8,13,120,121].

All molecular studies on the ecology of denitrifying bac-teria are based on functional genes and their products.Genes involved in denitri¢cation (nar, nor, nirS, nirKand nos, see Table 1) contain highly conserved DNA re-gions which can be successfully exploited for developinggene probes. From a mechanistic point of view, NO re-ductase is a very interesting enzyme, because it catalyzesthe formation of the dinitrogen bond in N2O. Little isknown about this process. The conversion of nitrite tonitric oxide (or nitrous oxide) is the crucial step in thereaction sequence because it leads to gas formation. There-fore, most of the ecological work has been done withprobes for both types of nitrite reductases [122,123]. De-nitrifying bacteria possess either a cytochrome cd1 (cd1

NIR) encoded by nirS, or a Cu-containing enzyme fornitrite reduction (Cu NIR) encoded by nirK. The nirKgene encoding Cu NIR from Pseudomonas aureofacienscan be transferred and functionally expressed in a muta-tionally cd1 NIR-free background of P. stutzeri [124]. It isbelieved that nirS is more widespread, but less conservedamong bacteria than is nirK. Therefore, it is more di¤cultto design broad-range primers or probes for nirS than fornirK [125,126]. The nirK gene occurs in bacteria of totallyunrelated systematic a¤liation but is apparently reason-ably conserved throughout the bacterial world. A generalprobe recognizing both nirK and nirS cannot be devel-oped, since these structurally di¡erent enzymes are en-coded by genes which do not share sequence homology.Recently, the distribution and diversity of denitri¢ers inmarine sediment has been investigated by the use ofN2O reductase (NosZ) gene-speci¢c primers and sequenceanalysis [127^129].

Denitri¢cation is related to nitrate ammoni¢cation andto the newly discovered anaerobic ammonia oxidation(anammox) reaction. Nitrate ammoni¢cation is typicalfor the Enterobacteriaceae, and e.g. Wolinella succinogenesand Sulforospirillum deleyianum. The ¢rst step, the reduc-tion of nitrate to nitrite, is catalyzed by a nitrate reductasewith properties similar to that enzyme in denitrifying bac-teria. E. coli contains at least two di¡erent membrane-bound nitrate reductases (encoded by narGHJI and nar-ZYWV) and a periplasmic nitrate reductase (napF-DAGHBC). Recent work with a mutant defective in

both membrane-bound enzymes showed that nitrate respi-ration solely via the periplasmic enzyme is possible [130].Nitrite is then directly reduced to ammonia by a cyto-chrome c-dependent nitrite reductase of which two formshave been isolated [131]. The enzyme from S. deleyianumhas been crystallized [132], and at least ¢ve di¡erent se-quences for the napC/nirT gene family (see Table 1) havebeen published. Thus, it should now be possible to developspeci¢c probes for this enzyme to assess its distribution inbacteria of di¡erent ecological habitats. Remarkably, E.coli can even convert N2O to N2 [133].

Recently, an anammox reaction in bio¢lms has beendescribed for bacteria whose closest relative is a plancto-mycete [120,134]. Ammonia and nitrite are comproportio-nated mainly to dinitrogen gas. However, nitrate is alsoformed to some extent in this reaction. Such bacteria maybe ubiquitous in nature and were claimed to account for asubstantial proportion of the bacterial population of thewastewater bioreactor in this study [134].

3.2. Serological approaches to study denitri¢cation

Immunological techniques have been used mainly tostudy the distribution of the cd1 NIR and Cu NIRamongst di¡erent bacteria. Ko«rner et al. [135] raised apolyclonal antiserum against the nitrous oxide reductaseand cd1 NIR from Pseudomonas perfectomarina (renamedP. stutzeri) and screened di¡erent pseudomonads and oth-er selected denitrifying bacteria for these enzymes usingOuchterlony double immunodi¡usion tests. While almostall tested Pseudomonas strains showed reactivity with theanti-N2O reductase antiserum, cross-reactivity with theanti-cd1 NIR antiserum was limited to one strain of P.stutzeri. The same antiserum was used to investigate theonset and cessation of denitri¢cation enzyme productionby P. stutzeri in continuous cultures at de¢ned concentra-tions of dissolved O2 covering the full range of transitionfrom air saturation to complete anaerobiosis [136]. A poly-clonal antiserum against the cd1 NIR of the same P. stut-zeri strain was raised by Ward et al. [137]. Using Westernblot analysis, the serum reaction was almost strain-specif-ic. These results clearly demonstrated that the antigeniccharacter of cd1 NIR is variable even among closely re-lated strains. Therefore, antisera which show cross-reactiv-ity with a broader range of bacteria containing cd1 NIRare di¤cult to produce. A DNA probe encoding cd1 NIRshowed a broader range of reactivity with di¡erent cd1

NIR-containing bacteria than did the serological results[137]. Michalski and Nicholas [138] used highly speci¢cpolyclonal antisera against the nitrate, nitrite, and nitrousoxide reductases of a photosynthetic, denitrifying bacte-rium (Rhodobacter sphaeroides sp.f. denitri¢cans) to inves-tigate the distribution of bacterial denitrifying enzymes.ELISA and Western blot analysis revealed that the mo-lybdenum-containing nitrate reductase and the multi-cop-per nitrous oxide reductase appear to be common proteins



among denitrifying bacteria. The pattern of immunologi-cal cross-reactivity for nitrite reductase, however, con-¢rmed that there are two di¡erent kinds of the enzyme,namely cd1 NIR and Cu NIR, commonly distributedamongst bacteria [138]. The Cu NIR type from R. sphae-roides was also detected in Alcaligenes denitri¢cans, twoPseudomonas spp. and Achromobacter cycloclastes. Coyneet al. [139] raised two di¡erent polyclonal antisera to iden-tify cd1 NIR and Cu NIR in 100 isolates of denitrifyingbacteria from diverse environments, mainly agriculturalsoils. Using Western blotting, the Cu NIR type was iden-ti¢ed in the species Alcaligenes (renamed Ralstonia) eutro-phus, Bacillus azotoformans and Corynebacterium nephridiiexclusively, while Aquaspirillum itersonii, Flavobacteriumspp. and Pseudomonas £uorescens contained the cd1 NIRtype. Coyne et al. [139] concluded from their results thatCu NIR can be found in taxonomically more unrelatedstrains.

Until now immunological studies concerning denitri¢ca-tion have only been performed with bacterial isolates.However, the ex situ measurement of the functions ofselected microorganisms provides only a measure of po-tential activity. Recently, an immunological test system forin situ detection of actually denitrifying bacterial commun-ities in di¡erent habitats was described on the basis of thein situ distribution of the Cu NIR [140], which is widelydistributed among di¡erent bacterial taxa (see above).

Polyclonal antisera, which were used in pure culturestudies, have three main limitations for in situ use:(1) they are mixtures of antibodies with di¡erent speci¢c-ities and it is therefore di¤cult to validate the exact cross-reactivities in complex ecosystems; (2) the availableamount is limited by the blood volume of the animalused for immunization; and (3) the exact composition ofantibodies cannot be reproduced by immunizing a secondanimal [141]. Monoclonal antibodies (mAbs) [142], as used

Table 2Characterization of polyclonal antisera (1^4) and monoclonal antibodies (5,6) raised against Cu NIR and cd1 NIR

Bacterial strain NIR type Antibodya

1 2 3 4 5 6

Achromobacter cycloclastes ATCC 15466 Copper positive n.d. n.d. n.d. n.d. n.d.ATCC 21921 Copper n.d. positive n.d. n.d. n.d. n.d.

Achromobacter xylosoxidans NCIB 11015 Copper n.d. positive n.d. n.d. n.d. n.d.DSM 30026 Copper n.d. n.d. n.d. n.d. positive negative

Agrobacterium tumefaciens DSM 30205 Copper n.d. n.d. n.d. n.d. positive negativeAlcaligenes denitri¢cans ATCC 27061 Copper positive n.d. n.d. n.d. n.d. n.d.Alcaligenes faecalis ATCC 8750 Copper n.d. n.d. n.d. negative n.d. n.d.

DSM 30030 Copper n.d. n.d. n.d. n.d. positive negativeAquaspirillum itersonii ATCC 11331 cd1 n.d. n.d. negative n.d. n.d. n.d.Azospirillum lipoferum ATCC 29707 cd1 n.d. negative n.d. n.d. n.d. n.d.Bacillus azotoformans ATCC 29788 Copper n.d. positive n.d. n.d. n.d. n.d.Corynebacterium nephridii ATCC 11425 Copper n.d. positive n.d. n.d. n.d. n.d.Flavobacterium sp. ATCC 33514 cd1 n.d. n.d. negative n.d. n.d. n.d.Ochrobactrum anthropi LMG 18952 Copper n.d. n.d. n.d. n.d. positive positiveOchrobactrum tritici LMG 18957 Copper n.d. n.d. n.d. n.d. positive negativeOchrobactrum grignonense LMG 18954 Copper n.d. n.d. n.d. n.d. positive negativeParacoccus denitri¢cans ATCC 19367 cd1 n.d. negative n.d. n.d. n.d. n.d.Pseudomonas aeruginosa DSM 50071 cd1 n.d. n.d. negative n.d. n.d. n.d.

ATCC 10145 cd1 n.d. negative n.d. n.d. n.d. n.d.DSM 10 cd1 n.d. n.d. n.d. n.d. negative negative

Pseudomonas alcaligenes ATCC 14909 Copper positive n.d. n.d. n.d. n.d. n.d.Pseudomonas aureofaciens ATCC 13985 Copper n.d. positive n.d. negative n.d. n.d.Pseudomonas denitri¢cans ATCC 13543 Copper positive n.d. n.d. n.d. n.d. n.d.

ATCC 13867 Copper n.d. positive n.d. negative n.d. n.d.Pseudomonas £uorescens ICPB 2708-401 cd1 n.d. n.d. negative n.d. n.d. n.d.

DSM 50415 cd1 n.d. n.d. negative n.d. n.d. n.d.ATCC 17802 cd1 n.d. negative n.d. n.d. n.d. n.d.ATCC 33512 cd1 n.d. n.d. n.d. negative n.d. n.d.

Pseudomonas mendocina DSM 50017 cd1 n.d. n.d. negative n.d. n.d. n.d.Pseudomonas nautica DSM 50418 cd1 n.d. n.d. negative n.d. n.d. n.d.Pseudomonas putida ATCC 12633 cd1 n.d. n.d. n.d. negative n.d. n.d.Pseudomonas stutzeri ATCC 17588 cd1 n.d. n.d. positive n.d. n.d. n.d.

ATCC 14405 cd1 n.d. n.d. positive positive n.d. n.d.ATCC 11607 cd1 n.d. n.d. n.d. positive n.d. n.d.ATCC 17588 cd1 n.d. n.d. n.d. positive n.d. n.d.

Rhodobacter sphaeroides ATCC 17023 Copper positive n.d. n.d. n.d. n.d. n.d.

a(1) Michalski and Nicholas [138], (2) Coyne et al. [139], (3) Ko«rner et al. [135], (4) Ward et al. [137], (5) Metz and Schloter [140], (6) Metz andSchloter [140].



by the authors, do not show these limitations. To obtainthe Cu NIR protein in high amounts and purity suitablefor immunization, two di¡erent ways were chosen. (1) CuNIR from Ochrobactrum anthropi, a typical denitrifyingsoil bacterium [143], was puri¢ed using a classical proteinisolation and puri¢cation approach [144]. (2) The wholeCu NIR gene from Alcaligenes faecalis S6, which showsvery high homology to the Cu NIR gene from O. anthropi[145], was ampli¢ed by PCR, cloned and expressed in E.coli IM105. The recombinant enzyme, which had a 6UHisa¤nity tag at the N-terminal end, was puri¢ed using im-mobilized metal a¤nity chromatography.

The hybridoma cell lines produced mAbs with di¡erentspeci¢city. While the mAb from procedure (1) was veryspeci¢c and showed only cross-reactivity with the Cu NIRfrom the O. anthropi strain used for immunization, mostof the mAbs obtained from procedure (2) showed a broad-er range of cross-reactivities with the copper-containingnitrite reductase from bacteria of di¡erent taxa, e.g. Ach-romobacter xylooxidans, Alcaligenes faecalis, Agrobacte-rium tumefaciens, Ochrobactrum spp. These distinct reac-tivities of the monoclonal antibodies might be explainedby the di¡erent epitopes which are recognized. The mAbraised via procedure (1) could bind to £anking region of

the Cu NIR protein, which show high variabilities, where-as mAbs raised via procedure (2) could react with theactive part of the enzyme which is highly conserved. Thedata on cross-reactivities of both mAb types are listed inTable 2. The mAbs could detect 10 ng puri¢ed Cu NIRprotein in ELISA procedures.

The mAbs raised via procedure (2) were used for in situdetection of denitrifying bacteria in di¡erent environ-ments. Fig. 1 shows one example with FITC-labeled anti-bodies applied to bacteria which express the Cu NIR phe-notype. The bacteria were detected in bio¢lms from anaerated nitri¢cation^denitri¢cation basin of an industrialwastewater treatment plant, which is known for its highloads of nitrate and its high number of denitrifying bac-teria [146].

In future, double labeling with mAbs raised via proce-dure (2) could be combined with taxonomic oligonucleo-tide probes in order to identify the bacteria which showthe Cu NIR phenotype in situ [147]. Together with theenrichment by £ow cytometry of cells labeled with a £uo-rescent mAb [148] these techniques might have great po-tential to detect and enrich denitrifying bacteria from com-plex ecosystems and to analyze directly their genotypewithout further cultivation.

Fig. 1. Phase contrast (a) and epi£uorescence (b) microscopic pictures of a bio¢lm with denitrifying bacteria from an aerated nitri¢cation^denitri¢cationbasin of industrial wastewater treatment plant. The denitrifying bacteria were labeled with a monoclonal antibody against cd1 NIR, labeled withFLUOS0. The green £uorescence was excited at 488 nm.



3.3. Detection of denitrifying bacteria in soils

Any investigation of soil is immediately faced with theproblem that this substrate is highly variable on smallscales, both horizontally and vertically in the soil layer.In the case of higher plants, it is impossible to explainand predict why one plant species occupies one soil spotbut not the next one on a given soil type, because factorsdetermining the competitiveness of a plant are many andcomplex. The same might also apply to bacterial commun-ities in soils. Due to our inability to cultivate most soilbacteria and due to the impossibility of detecting individ-ual genes present in single copy or low copy numbers inintact bacterial cells [149], recent attempts to analyze soilbacteria have concentrated mainly on characterizing soilDNA and the relative abundance of genes encoding deni-tri¢cation and nitri¢cation functions.

Such an approach requires the isolation of soil DNA inhigh yields and of high purity. Several protocols have beenpublished [150^153], and each investigator seems to haveoptimized his/her own recipe for a particular application.The potential for widespread use of uniform techniques isnot clear. DNA recoveries are determined by seeding ex-periments with culturable bacteria. The e¤ciency and re-producibility of the DNA extraction method can be eval-uated by competitive PCR [154]. It is not known whetherindigenous bacteria stick more tightly to soil particles orcan as readily be retrieved from soils as the culturablemicroorganisms stirred into a soil suspension. Soils con-tain substances such as tannins, polyphenols and polysac-charides in variable amounts, and these interfere withDNA isolation and detection ([151], and references there-in). In extractions of DNA directly from soils, factors suchas the e¤ciency of bacterial cell wall lysis and the non-speci¢c adsorption of DNA to clay strongly in£uence theyield [155]. In contaminated DNA preparations, restric-tion enzyme digestion and Taq DNA polymerase activitymay be a¡ected and the e¤ciency of DNA^DNA hybrid-izations may also be decreased [156]. Preparations oftencontain a large amount of sheared DNA and are often notamenable to restriction analysis. Commercially producedkits for rapid preparation of soil DNA of such quality arenow available (e.g. Mo Bio Laboratories Inc, SolanaBeach, CA, USA), but the details of the kit methods arenot published for obvious reasons.

Instead of analyzing total DNA and its relative contentit would be even more rewarding to perform transcriptanalysis of the respective genes. However, compared toDNA, mRNA is even more unstable. Transcript analysisof genes involved in denitri¢cation has not yet been per-formed in soil samples.

When the experiments [157] started some 10 years ago,only a few sequences for denitri¢cation genes were avail-able, and the gene probes used at that time also containedother sequences not encoding denitri¢cation genes. Theseprobes, however, recognized denitri¢cation genes in a

large number of bacteria [157]. These results of DNA^DNA hybridization experiments (dot-blot analyses) withthese gene probes were interpreted as indicating that cul-turable denitrifying bacteria are enriched in the upper soillayer and in the vicinity of plant roots. Using a similarhybridization approach [137,158], high abundances of pu-tative denitrifying bacteria were detected in marine watercolumns and sediment environments. Since these initialreports, many further sequences of denitri¢cation geneshave been published (Table 1). Thus, it is not di¤cult tosynthesize by PCR speci¢c probes which recognize thetarget genes in a wide range of bacteria. With probes of0.3^1.0 kb in size, the distribution of denitri¢cation geneswere assessed in di¡erent Hyphomicrobium species orstrains occurring in a sewage plant and its incoming water[159,160]. Experiments with these gene probes also con-¢rmed that the concentration of culturable denitrifyingbacteria is highest in the upper 5 cm and steadily decreaseswith the depth of the soils investigated [161].

The two molecular approaches which have been appliedto date to study the ecology of denitri¢cation are PCRampli¢cation using 15^25-oligomer primers and DNA^DNA hybridizations with 0.4^1.0-kb probes targetedagainst nitrite reductase genes. The use of a set of oligo-nucleotide primers makes it possible to amplify segmentsof nirS or nirK, and the products generally have the cor-rect molecular mass and DNA sequence. However, thePCR approach is often not successful, particularly whenthe DNA template is not entirely free of contaminants. Insome cases, such as two Pseudomonas denitri¢cans and oneAlcaligenes (renamed Ralstonia) eutrophus strains [126],PCR products could not be obtained for nirS, althoughactivity measurements and biochemical data did show thepresence of this enzyme in these strains. To obtain quan-titative data on the relative distribution of denitri¢cationgenes in bacterial genomes, quantitative PCR would berequired. Despite the advertisements of companies sellinginstruments for quantitative PCR, this technique is seem-ingly still in its infancy and has not yet successfully beenemployed with soil DNA. Competitive PCR, or even com-petitive RT-PCR, o¡ers a possible alternative.

Hybridization of extracted DNA with 0.4^1.0-kb probesfor genes involved in denitri¢cation gives positive scoresmore often than in the PCR approach using enrichmentcultures. Distinct hybridization bands are often obtained,and the data from the DNA^DNA hybridization are usu-ally in accordance with the results from activity measure-ments. However, Azospirillum lipoferum Sp59 has beendescribed as expressing a cd1 NIR and produces N2Ofrom nitrite (in the presence of acetylene and under anoxicconditions), but hybridizations with a nirS probe were al-ways negative with DNA from this bacterium under allstringencies employed (A. Mergel and H. Bothe, unpub-lished). Faint signals are often obtained in DNA^DNAhybridization, even at high stringencies. This is the majorproblem when the occurrence of denitri¢cation genes in a



bacterial isolate from a soil is to be assessed. In our studies[162,163], the nir probes used (for nirS and nirK) did hy-bridize with DNA isolated from the soils, in contrast topreparations used in other studies [164]. However, for thispurpose, DNA extracted from soils must be highly puri-¢ed (close to OD260=280nm = 1.8). Experiments with cruderpreparations yielded erratic data (A. Mergel and H. Bothe,unpublished).

In principle, probing soil DNA provides informationabout the relative abundance of the target gene in a soillayer or habitat. The intensity of the signal is, however,di¡erent from bacterium to bacterium, mainly dependingon the sequence alterations with each bacterium. There-fore, signal intensities cannot be transformed to cell num-bers, as has also been pointed out for rRNA dot-blothybridizations [165]. Apart from this, signal intensities ofthe bands can normally be compared only on a givenmembrane. Many factors determine the signal intensityin DNA^DNA hybridizations (the hybridization strin-gency; the quality of the membrane, which is often notuniform; the labeling intensity of the probe with any labelbeing variable from preparation to preparation, etc.).Thus, it is advisable to use the same membrane ¢rst forthe DNA probe developed for a functional gene, then tostrip the membrane and to rehybridize it with a generalprobe recognizing all bacterial DNA (e.g. a general 16SrRNA probe). Signal intensities can be quanti¢ed densito-metrically.

This approach was used to study the distribution ofdenitrifying bacteria in acid soil (humisol type) under anoak^hornbeam forest in the vicinity of Cologne [162]. Thehybridization data indicated that most bacteria of the totalpopulation lived in the upper (5 cm) soil layer. Hybrid-izations with all denitri¢cation probes (with the exceptionof nirK) also showed that denitrifying bacteria are en-riched in the vicinity of plant roots compared to thebulk, root-free soil. Unexpectedly, denitrifying bacteriawere not enriched in the deeper soil layer (V25 cm depth)where the concentration of nitrate was still not growthlimiting and where the O2 partial pressure might be low.Altogether, it was estimated that less than 5% of the totalbacterial population possess the denitri¢cation genes. Withall soil samples assayed, denitri¢cation activity (N2O for-mation) was only detected when the assays were supple-mented with high amounts of nitrate.

Bacteria living in such soils may not gain a selectiveadvantage from possessing the denitri¢cation genes. Sucha soil is rarely waterlogged, so that the concentration ofO2 in the upper 5 cm may always be high enough tosuppress the expression of denitri¢cation genes. It remainsto be shown whether any aerobic denitri¢cation occurs insuch habitats. The ability to respire with nitrate as anelectron acceptor in the presence of oxygen is seeminglywidespread among bacteria [166]. It has been estimatedthat 104^107 bacteria per g of soil or sediment are ableto respire with nitrate under oxic conditions [167]. In ad-

dition, anoxic microsites may also occur in such soils, andsome evidence for denitri¢cation in such microsites hasbeen published [168^170]. Exact oxygen concentrationsin densely packed soils cannot be determined by micro-electrodes. Measurements have been reported for soil ag-gregates and ¢r litter [168,171]. As in the soil of the oak^hornbeam forest near Cologne, enrichment of denitrifyingbacteria was observed in the upper layer of a Norwayspruce forest in southern Germany [163] where the N in-put to the soil from the air was marginal due to low in-dustrial activity in this area. In this study [163], the num-ber of culturable bacteria, determined by the MPNmethod, and of denitrifying bacteria showed large seasonal£uctuations during the investigation period December1994^August 1998. Seasonal £uctuations in the bacterialpopulation hybridizing with the nitrogenase probe nifHwere also observed at a Douglas ¢r forest site in the Or-egon Cascade mountain range [172]. Such £uctuationsmay be responsible for the peak activities in N2O forma-tion often observed in spring in ¢eld studies [173,174].Fertilization of one area of the Norway spruce stand insouthern Germany with a high load of ammonium nitratedid not cause a shift in the population of denitrifyingbacteria [163]. The nitrate content and the presence ofplants in habitats may, however, determine the composi-tion of the nitrate-respiring bacterial community at otherlocations [175].

In the long run, an analysis of the complete populationof denitrifying and nitrifying bacteria in soils is required iftheir potential denitri¢cation activities (NOx £uxes) are tobe predicted. Quanti¢cation of speci¢c genes and geneproducts in complex microbial communities by in situPCR has been employed [149,176]. Such techniques havenot been used very widely so far, probably because theysu¡er from non-speci¢c ampli¢cation of non-target DNAand from non-speci¢c binding of £uorescence probes toDNA, particularly when the preparation is not entirelyfree of contaminants. A laborious and tedious, yet reward-ing avenue is to construct clone libraries of PCR productsfrom soil DNA using primers for bacterial 16S rRNA genesequences [177]. The use of separation methods such asDGGE [113,178] or TGGE [179] largely helps to separatethe products obtained. However, only a limited number,perhaps approx. 100 clones, which represent only a smallpercentage of the roughly 104 ribotypes per g soil, can besequenced in one study. The ¢rst investigations indicatedthat sequences of a large number of uncultured bacteriawith unknown phylogenetic a¤liations are obtained bythis approach [177].

3.4. Denitri¢cation in aquatic habitats

Several molecular studies on denitri¢ers in aquatic en-vironments have been published and only a few can bementioned here. Experiments in lakes, particularly in bio-¢lms, are facilitated by the fact that microelectrodes are



available to monitor the changes in the concentrations ofoxygen, nitrate, ammonia, and other compounds. Speci¢campli¢cation by PCR indicated the occurrence of Alcali-genes xylosoxidans in freshwater lakes [145]. Hybridizationwith the nir speci¢c probe developed from P. stutzeri in-dicated that 0.1-1% of the total bacterial population pos-sess this gene in seawater samples [137]. In sediments of aDanish Fjord estuary denitri¢cation activity determinedby 15N isotope measurements and by the acetylene inhibi-tion technique showed that denitri¢cation capacity washighest in the upper 1 cm of the sediment and declinedwith depth. Denitri¢cation showed peak activities inspring and autumn, whereas nitrate ammoni¢cation rateswere highest in summer [180]. To our knowledge, a studycombining electrode measurements and 15N isotope tech-niques with new molecular approaches for determiningdenitri¢cation in aquatic habitats has not yet been thor-oughly performed. Results for marine sediments, in whichnitrate supplementation was required to detect potentialdenitri¢cation activity as assayed by the acetylene inhibi-tion technique [158], are consistent with observations insoils (above). High abundances of nirS were detected byDNA hybridization in these sediments, but individualstrains of denitrifying bacteria, enumerated by immuno-£uorescence, were present at very low abundance.

4. Conclusions and future developments

Molecular approaches have made the study of unknownmicroorganisms and their behavior in diverse environ-ments much more accessible. However, we are far fromunderstanding the complexity of bacterial life in any nat-ural habitat. As mentioned above, about 104 di¡erent ge-nomes per gram occur in soils, and the number may bevery high in aquatic habitats as well. Target DNA (se-quences encoding 16S rRNA or functional genes) can beampli¢ed by PCR, cloned and sequenced. Techniques suchas DGGE, TGGE or T-RFLP might help to screen largesample sets for a comparison of local and seasonal varia-tions. However, only a limited number (102^103, theamount depending on the sequence length and the enthu-siasm of the group involved) can be cloned and sequenced.From the functional genes, e.g. amoA for nitri¢cation ornirS for denitri¢cation, only few sequences have been de-posited. Thus most sequences obtained for functionalgenes will be new. This situation is not so dramatic inthe case of the 16S rRNA sequences, but clearly the char-acterization of any new habitat will add new sequenceinformation to the richness of the 16S rRNA data banks.When the 16S rRNA sequence is new, it does not provideinformation on the physiological capabilities of the organ-ism. Due to the monophyletic origin of most AOB knownso far, it can at least be guessed whether a detected 16SrRNA gene sequence belongs to a nitri¢er. This does notapply at all to denitri¢ers or heterotrophic nitri¢ers. This

means that sequences of each of the desired target genes,like 16S rDNA, nirK, amoA etc., have to be obtained fromthe same bacterial population. New techniques, like micro-arrays (DNA/RNA chips) with diverse copies of all nitri-¢cation and denitri¢cation genes, will likely facilitatescreening of natural communities for their speci¢c meta-bolic potential in the N cycle. When a large percentage ofthe genomes in soil or water have been examined for theirnitri¢cation and denitri¢cation traits, it may help to obtaina better understanding of factors which regulate NOx

£uxes. Such guesses are necessary for any prediction ofthe impact of global NOx emissions on climate changescaused by nitrifying and denitrifying bacteria. Informationis needed not only about the microbial community struc-ture, but also about their activities. As a ¢rst step, thisrequires the development of protocols to isolate and iden-tify stable transcripts (mRNA) for the key enzymes of theprocesses. Antibodies may help to quantify enzyme con-centrations.

Besides all the excitement about the molecular ap-proaches that enable us to study microorganisms directlyin their natural habitats, we need more attempts to culti-vate and characterize those organisms that are still un-known.

Acknowledgements

This work was in part ¢nanced by the Deutsche For-schungsgemeinschaft within the priority research program`Structure/function analysis of natural microbial commun-ities'. The authors are indebted to two anonymous re-viewers for valuable comments.

References

[1] Tiedje, J.M. (1988) Ecology of denitri¢cation and dissimilatory ni-trate reduction to ammonium. In: Biology of Anaerobic Microorgan-isms (Zehnder, A.J.B., Ed.), pp. 179^244. Wiley, New York.

[2] Prosser, J.I. (1989) Autotrophic nitri¢cation in bacteria. Adv. Mi-crob. Physiol. 30, 125^181.

[3] Revsbech, N.P. and SÖrensen, J. (Eds.) (1990) Denitri¢cation in Soiland Sediment. Plenum Press, New York.

[4] Ferguson, S.J. (1994) Denitri¢cation and its control. Antonie vanLeeuwenhoek 66, 89^110.

[5] Ward, B.B. (1996) Nitri¢cation and denitri¢cation: probing the nitro-gen cycle in aquatic environments. Microb. Ecol. 32, 247^261.

[6] Hooper, A.B., Vannelli, T., Bergmann, D.J. and Arciero, D.M.(1997) Enzymology of the oxidation of ammonia to nitrite by bac-teria. Antonie van Leeuwenhoek 71, 59^67.

[7] Zumft, W.G. (1997) Cell biology and molecular basis of denitri¢ca-tion. Microbiol. Mol. Biol. Rev. 61, 533^616.

[8] Ferguson, S.J. (1998) Nitrogen cycle enzymology. Curr. Opin. Chem.Biol. 2, 182^193.

[9] Hagopian, D.S. and Riley, J.G. (1998) A closer look at the bacteriaof nitri¢cation. Aquacult. Eng. 18, 223^244.

[10] Jetten, M.S.M., Strous, M., van de Pas-Schoonen, K.T., Schalk, J.,van Dongen, U.G.J.M., van de Graaf, A.A., Logemann, S., Muyzer,



G., van Loosdrecht, M.C.M. and Kuenen, J.G. (1999) The anaerobicoxidation of ammonium. FEMS Microbiol. Rev. 22, 421^437.

[11] Herbert, R.A. (1999) Nitrogen cycling in coastal marine ecosystems.FEMS Microbiol. Rev. 23, 563^590.

[12] Philippot, L. and Hojberg, O. (1999) Dissimilatory nitrate reductasesin bacteria. Biochim. Biophys. Acta 1446, 1^23.

[13] Richardson, D.J. and Watmough, N.J. (1999) Inorganic nitrogenmetabolism in bacteria. Curr. Opin. Chem. Biol. 3, 207^219.

[14] Torsvik, V., Goksoyr, J. and Daae, F.L. (1990) High diversity ofDNA of soil bacteria. Appl. Environ. Microbiol. 56, 782^787.

[15] Klug, M.J. and Tiedje, J.M. (1993) Response of microbial commun-ities to changing environmental conditions: chemical and physiolog-ical approaches. In: Trends in Microbial Ecology (Guerrero, R. andPedros-Alio, C., Eds.), pp. 371^374. Spanish Society for Microbiol-ogy, Barcelona.

[16] Alexander, M. (Ed.) (1977) Introduction to Soil Microbiology. Wiley,New York.

[17] Amann, R.I., Ludwig, W. and Schleifer, K.H. (1995) Phylogeneticidenti¢cation and in situ detection of individual microbial cells with-out cultivation. Microbiol. Rev. 59, 143^169.

[18] Meincke, M., Krieg, E. and Bock, E. (1989) Nitrosovibrio spp., thedominant ammonia-oxidizing bacteria in building sandstone. Appl.Environ. Microbiol. 55, 2108^2110.

[19] Sorokin, D.Y. (1998) On the possibility of nitri¢cation in extremelyalkaline soda biotopes. Microbiology 67, 335^339.

[20] Arrigo, K.R., Dieckmann, G., Gosselin, M., Robinson, D.H., Frit-sen, C.H. and Sullivan, C.W. (1995) High resolution study of theplatelet ice ecosystem in McMurdo Sound, Antarctica: biomass, nu-trient, and production pro¢les within a dense microalgal bloom. Mar.Ecol. Prog. Ser. 127, 1^3.

[21] Golovatcheva, R.S. (1976) Thermophilic nitrifying bacteria from hotsprings. Microbiology 45, 329^331.

[22] Diaz, M.C. and Ward, B.B. (1997) Sponge-mediated nitri¢cation intropical benthic communities. Mar. Ecol. Prog. Ser. 156, 97^107.

[23] Koops, H.-P. and Mo«ller, U.C. (1992) The lithotrophic ammonia-oxidizing bacteria. In: The Prokaryotes (Balows, A., Tru«per, H.G.,Dworkin, M., Harder, W. and Schleifer, K.H., Eds.), pp. 2625^2637.Springer Verlag, New York.

[24] Head, I.M., Hiorns, W.D., Embley, T.M., McCarthy, A.J. andSaunders, J.R. (1993) The phylogeny of autotrophic ammonia-oxidiz-ing bacteria as determined by analysis of 16S ribosomal RNA genesequences. J. Gen. Microbiol. 139, 1147^1153.

[25] Teske, A., Alm, E., Regan, J.M., Toze, S., Rittmann, B.E. and Stahl,D.A. (1994) Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. J. Bacteriol. 176, 6623^6630.

[26] Focht, D.D. and Verstraete, W. (1977) Biochemical ecology of nitri-¢cation and denitri¢cation. In: Advances in Microbial Ecology, Vol.1 (Alexander, M., Ed.), pp. 135^214. Plenum Press, New York.

[27] Robertson, L.A., van Niel, E.W.J., Torremans, R.A.M. and Kuenen,J.G. (1988) Simultaneous nitri¢cation and denitri¢cation in aerobicchemostat cultures of Thiosphaera pantotropha. Appl. Environ. Mi-crobiol. 54, 2812^2818.

[28] Bock, E., Schmidt, I., Stu«ven, R. and Zart, D. (1995) Nitrogen losscaused by denitrifying Nitrosomonas cells using ammonium or hydro-gen as electron donors and nitrite as electron acceptor. Arch. Micro-biol. 163, 16^20.

[29] Abeliovich, A. (1987) Nitrifying bacteria in wastewater reservoirs.Appl. Environ. Microbiol. 53, 754^760.

[30] Abeliovich, A. and Vonshak, A. (1992) Anaerobic metabolism ofNitrosomonas europaea. Arch. Microbiol. 158, 267^270.

[31] Schmidt, I. and Bock, E. (1997) Anaerobic ammonia oxidation withnitrogen dioxide by Nitrosomonas eutropha. Arch. Microbiol. 167,106^111.

[32] Lusby, F.E., Gibbs, M.M., Cooper, A.B. and Thompson, K. (1998)The fate of groundwater ammonium in a lake edge wetland. J. En-viron. Qual. 27, 459^466.

[33] Smorczewski, W.T. and Schmidt, E.L. (1991) Numbers, activities and

diversity of autotrophic ammonia-oxidizing bacteria in a freshwater,eutrophic lake sediment. Can. J. Microbiol. 37, 828^833.

[34] Diab, S. and Shilo, M. (1988) E¡ect of adhesion to particles on thesurvival and activity of Nitrosomonas sp. and Nitrobacter sp.. Arch.Microbiol. 150, 377^393.

[35] Bodelier, P.L.E., Libochant, J.A., Blom, C.W.P.M. and Laanbroek,H.J. (1996) Dynamics of nitri¢cation and denitri¢cation in root-oxy-genated sediments and adaptation of ammonia-oxidizing bacteria tolow-oxygen or anoxic habitats. Appl. Environ. Microbiol. 62, 4100^4107.

[36] Arciero, D.M., Balny, C. and Hooper, A.B. (1991) Spectroscopic andrapid kinetic studies of reduction of cytochrome c554 by hydroxyl-amine oxidoreductase from Nitrosomonas europaea. Biochemistry-US30, 11466^11472.

[37] Miller, D.J. and Nicholas, D.J.D. (1985) Characterization of a solu-ble cytochrome oxidase/nitrite reductase from Nitrosomonas euro-paea. J. Gen. Microbiol. 131, 2851^2854.

[38] DiSpirito, A.A., Lipscomb, J.D. and Hooper, A.B. (1986) A three-subunit cytochrome aa3 from Nitrosomonas europaea. J. Biol. Chem.261, 17048^17056.

[39] Moir, J.W.B., Crossman, L.C., Spiro, S. and Richardson, D.J. (1996)The puri¢cation of ammonia monooxygenase from Paracoccus deni-tri¢cans. FEBS Lett. 387, 71^74.

[40] Daum, M., Zimmer, W., Papen, H., Kloos, K., Nawrath, K. andBothe, H. (1998) Physiological and molecular biological character-ization of ammonia oxidation of the heterotrophic nitri¢er Pseudo-monas putida. Curr. Microbiol. 37, 281^288.

[41] Sayavedra-Soto, L.A., Hommes, N.G., Alzerreca, J.J., Arp, D.J.,Norton, J.M. and Klotz, M.G. (1998) Transcription of the amoC,amoA and amoB genes in Nitrosomonas europaea and Nitrosospirasp. NpAV. FEMS Microbiol. Lett. 167, 81^88.

[42] Alzerreca, J.J., Norton, J.M. and Klotz, M.G. (1999) The amo oper-on in marine, ammonia-oxidizing gamma-proteobacteria. FEMS Mi-crobiol. Lett. 180, 21^29.

[43] McTavish, H., Fuchs, J.A. and Hooper, A.B. (1993) Sequence of thegene coding for ammonia monooxygenase in Nitrosomonas europaea.J. Bacteriol. 175, 2436^2444.

[44] McTavish, H., LaQuier, F., Arciero, D., Logan, M., Mundfrom, G.,Fuchs, J.A. and Hooper, A.B. (1993) Multiple copies of genes codingfor electron transport proteins in the bacterium Nitrosomonas euro-paea. J. Bacteriol. 175, 2445^2447.

[45] Bergmann, D.J. and Hooper, A.B. (1994) Sequence of the gene,amoB, for the 43-kDa polypeptide of ammonia monooxygenase ofNitrosomonas europaea. Biochem. Biophys. Res. Commun. 204, 759^762.

[46] Rotthauwe, J.-H., Deboer, W. and Liesack, W. (1995) Comparativeanalysis of gene sequences encoding ammonia monooxygenase ofNitrosospira sp. AHB1 and Nitrosolobus multiformis C-71. FEMSMicrobiol. Lett. 133, 131^135.

[47] Klotz, M.G., Alzerreca, J. and Norton, J.M. (1997) A gene encodinga membrane protein exists upstream of the amoA/amoB genes inammonia oxidizing bacteria: a third member of the amo operon?FEMS Microbiol. Lett. 150, 65^73.

[48] Rotthauwe, J.-H., Witzel, K.-P. and Liesack, W. (1997) The ammo-nia monooxygenase structural gene amoA as a functional marker:molecular ¢ne-scale analysis of natural ammonia-oxidizing popula-tions. Appl. Environ. Microbiol. 63, 4704^4712.

[49] Horz, H.-P., Rotthauwe, J.-H., Lukow, T. and Liesack, W. (2000)Identi¢cation of major subgroups of ammonia-oxidizing bacteria inenvironmental samples by T-RFLP analysis of amoA PCR products.J. Microbiol. Methods 39, 197^204.

[50] Limburg, P., Horz, H.-P. and Witzel, K.-P. (2000) Detection of am-monia-oxidizing bacteria by denaturing gradient gel electrophoresis(DGGE) of amoA-fragments. (submitted).

[51] Bedard, C. and Knowles, R. (1989) Physiology, biochemistry, andspeci¢c inhibitors of CH4, NH�4 , and CO oxidation by methano-trophs and nitri¢ers. Microbiol. Rev. 53, 68^84.



[52] Ensign, S.A., Hyman, M.R. and Arp, D.J. (1993) In vitro activationof ammonia monooxygenase from Nitrosomonas europaea by copper.J. Bacteriol. 175, 1971^1980.

[53] Holmes, A.J., Costello, A., Lidstrom, M.E. and Murrell, J.C. (1995)Evidence that particulate methane monooxygenase and ammoniamonooxygenase may be evolutionarily related. FEMS Microbiol.Lett. 132, 203^208.

[54] Semrau, J.D., Chistoserdov, A., Lebron, J., Costello, A., Davagnino,J., Kenna, E., Holmes, A.J., Finch, R., Murrell, J.C. and Lidstrom,M.E. (1995) Particulate methane monooxygenase genes in methano-trophs. J. Bacteriol. 177, 3071^3079.

[55] Zahn, J.A., Duncan, C. and DiSpirito, A.A. (1994) Oxidation ofhydroxylamine by cytochrome P-460 of the obligate methylotrophMethylococcus capsulatus Bath. J. Bacteriol. 176, 5879^5887.

[56] Bergmann, D.J., Zahn, J.A. and DiSpirito, A.A. (2000) Primarystructure of cytochrome cP of Methylococcus capsulatus Bath: evi-dence of a phylogenetic link between P460 and cP-type cytochromes.Arch. Microbiol. 173, 29^34.

[57] Bergmann, D.J. and Hooper, A.B. (1994) The primary structure ofcytochrome P460 of Nitrosomonas europaea : presence of a c-hemebinding motif. FEBS Lett. 353, 324^326.

[58] Arciero, D.M. and Hooper, A.B. (1998) Consideration of a phlorinstructure for haem P-460 of hydroxylamine oxidoreductase and itsimplications regarding reaction mechanism. Biochem. Soc. Trans. 26,385^389.

[59] Arciero, D.M. and Hooper, A.B. (1993) Hydroxylamine oxidoreduc-tase from Nitrosomonas europaea is a multimer of an octa-heme sub-unit. J. Biol. Chem. 268, 14645^14654.

[60] Hoppert, M., Mahony, T.J., Mayer, F. and Miller, D.J. (1995) Qua-ternary structure of the hydroxylamine oxidoreductase from Nitro-somonas europaea. Arch. Microbiol. 163, 300^306.

[61] Igarashi, N., Moriyama, H., Fujiwara, T., Fukumori, Y. and Tana-ka, N. (1997) The 2.8 Aî structure of hydroxylamine oxidoreductasefrom a nitrifying chemoautotrophic bacterium, Nitrosomonas euro-paea. Nature Struct. Biol. 4, 276^284.

[62] Moir, J.W., Wehrfritz, J.M., Spiro, S. and Richardson, D.J. (1996)The biochemical characterization of a novel non-haem iron hydroxyl-amine oxidase from Paracoccus denitri¢cans GB17. Biochem. J. 319,823^827.

[63] Jetten, M.S., de Bruijn, P. and Kuenen, J.G. (1997) Hydroxylaminemetabolism in Pseudomonas PB16: involvement of a novel hydroxyl-amine oxidoreductase. Antonie van Leeuwenhoek 71, 69^74.

[64] Bergmann, D.J., Arciero, D.M. and Hooper, A.B. (1994) Organiza-tion of the hao gene cluster of Nitrosomonas europaea : genes for twotetraheme c cytochromes. J. Bacteriol. 176, 3148^3153.

[65] Norton, J.M., Low, J.M. and Martin, G. (1996) The gene encodingammonia monooxygenase subunit A exists in three nearly identicalcopies in Nitrosospira sp. NpAV. FEMS Microbiol. Lett. 139, 181^188.

[66] Hommes, N.G., Sayavedra-Soto, L.A. and Arp, D.J. (1998) Muta-genesis and expression of amo, which codes for ammonia mono-oxygenase in Nitrosomonas europaea. J. Bacteriol. 180, 3353^3359.

[67] Klotz, M.G. and Norton, J.M. (1998) Multiple copies of ammoniamonooxygenase (amo) operons have evolved under biased AT/GCmutational pressure in ammonia-oxidizing autotrophic bacteria.FEMS Microbiol. Lett. 168, 303^311.

[68] Hommes, N.G., Sayavedra-Soto, L.A. and Arp, D.J. (1994) Sequenceof hcy, a gene encoding cytochrome c-554 from Nitrosomonas euro-paea. Gene 146, 87^89.

[69] Sayavedra-Soto, L.A., Hommes, N.G. and Arp, D.J. (1994) Charac-terization of the gene encoding hydroxylamine oxidoreductase in Ni-trosomonas europaea. J. Bacteriol. 176, 504^510.

[70] Sayavedra-Soto, L.A., Hommes, N.G., Russell, S.A. and Arp, D.J.(1996) Induction of ammonia monooxygenase and hydroxylamineoxidoreductase mRNAs by ammonium in Nitrosomonas europaea.Mol. Microbiol. 20, 541^548.

[71] Hyman, M.R. and Arp, D.J. (1995) E¡ects of ammonia on the de

novo synthesis of polypeptides in cells of Nitrosomonas europaea de-nied ammonia as an energy source. J. Bacteriol. 177, 4974^4979.

[72] Stein, L.Y., Arp, D.J. and Hyman, M.R. (1997) Regulation of thesynthesis and activity of ammonia monooxygenase in Nitrosomonaseuropaea by altering pH to a¡ect NH3 availability. Appl. Environ.Microbiol. 63, 4588^4592.

[73] Nejidat, A., Shmuely, H. and Abeliovich, A. (1997) E¡ect of ammo-nia starvation on hydroxylamine oxidoreductase activity of Nitroso-monas europaea. J. Biochem. Tokyo 121, 957^960.

[74] Stein, L.Y. and Arp, D.J. (1998) Ammonium limitation results in theloss of ammonia-oxidizing activity in Nitrosomonas europaea. Appl.Environ. Microbiol. 64, 1514^1521.

[75] Stein, L.Y. and Arp, D.J. (1998) Loss of ammonia monooxygenaseactivity in Nitrosomonas europaea upon exposure to nitrite. Appl.Environ. Microbiol. 64, 4098^4102.

[76] Wilhelm, R., Abeliovich, A. and Nejidat, A. (1998) E¡ect of long-term ammonia starvation on the oxidation of ammonia and hydrox-ylamine by Nitrosomonas europaea. J. Biochem. Tokyo 124, 811^815.

[77] Belser, L.W. and Schmidt, E.L. (1978) Serological diversity within aterrestrial ammonia-oxidizing population. Appl. Environ. Microbiol.36, 589^593.

[78] Scha«fer, B. and Harms, H. (1995) A case study on the oxygen budgetin the freshwater part of the Elbe estuary. 5. Distribution of di¡erentammonia-oxidizing bacteria in the river Elbe downstream of Ham-burg at low and normal oxygen concentrationsTITLEs . Arch. Hy-drobiol. Suppl. Unters. Elbe Aestuars 110, 77^82.

[79] Ward, B.B. and Carlucci, A.F. (1985) Marine ammonia- and nitrite-oxidizing bacteria: serological diversity determined by immuno£uo-rescence in culture and in the environment. Appl. Environ. Micro-biol. 50, 194^201.

[80] Hall, G.H. (1986) Nitri¢cation in lakes. In: Nitri¢cation (Prosser,J.I., Ed.), pp. 127^156. IRL Press, Oxford.

[81] Pommerening-Ro«ser, A., Rath, G. and Koops, H.P. (1996) Phyloge-netic diversity within the genus Nitrosomonas. Syst. Appl. Microbiol.19, 344^351.

[82] Belser, L.W. (1979) Population ecology of nitrifying bacteria. Annu.Rev. Microbiol. 33, 309^333.

[83] Stehr, G., Bo«ttcher, B., Dittberner, P., Rath, G. and Koops, H.P.(1995) The ammonia-oxidizing nitrifying population of the RiverElbe estuary. FEMS Microbiol. Ecol. 17, 177^186.

[84] Stehr, G., Zo«rner, S., Bo«ttcher, B. and Koops, H.P. (1995) Exopol-ymers: an ecological characteristic of a £oc-attached, ammonia-oxi-dizing bacterium. Microb. Ecol. 30, 115^126.

[85] Navarro, E., Fernandez, M.P., Grimont, F., Clays-Josserand, A. andBardin, R. (1992) Genomic heterogeneity of the genus Nitrobacter.Int. J. Syst. Bacteriol. 42, 554^560.

[86] Aakra, Aî ., Uta®ker, J.B. and Nes, I.F. (1999) RFLP of rRNA genesand sequencing of the 16S-23S rDNA intergenic spacer region ofammonia-oxidizing bacteria: a phylogenetic approach. Int. J. Syst.Bacteriol. 49, 123^130.

[87] Wagner, M., Rath, G., Amann, R., Koops, H.P. and Schleifer, K.H.(1995) In situ identi¢cation of ammonia-oxidizing bacteria. Syst.Appl. Microbiol. 18, 251^264.

[88] Mobarry, B.K., Wagner, M., Urbain, V., Rittmann, B.E. and Stahl,D.A. (1996) Phylogenetic probes for analyzing abundance and spatialorganization of nitrifying bacteria. Appl. Environ. Microbiol. 62,2156^2162.

[89] Schramm, A., de Beer, D., Wagner, M. and Amann, R. (1998) Iden-ti¢cation and activities in situ of Nitrosospira and Nitrospira spp. asdominant populations in a nitrifying £uidized bed reactor. Appl.Environ. Microbiol. 64, 3480^3485.

[90] Voytek, M.A., Priscu, J.C. and Ward, B.B. (1999) The distributionand relative abundance of ammonia-oxidizing bacteria in lakes of theMcMurdo Dry Valley, Antarctica. Hydrobiologia 401, 113^130.

[91] Uta®ker, J.B. and Nes, I.F. (1998) A qualitative evaluation of thepublished oligonucleotides speci¢c for the 16S rRNA gene sequencesof the ammonia-oxidizing bacteria. Syst. Appl. Microbiol. 21, 72^88.



[92] Voytek, M.A. (1996) Relative abundance and species diversity ofautotrophic amonia-oxidizing bacteria in aquatic systems. Biology240.

[93] Ward, B.B., Martino, D.P., Diaz, M.C. and Joye, S.B. (2000) Anal-ysis of ammonia-oxidizing bacteria from hypersaline Mono Lake,California, on the basis of 16S rRNA sequences. Appl. Environ.Microbiol. 66, 2873^2881.

[94] Kowalchuk, G.A., Stephen, J.R., De Boer, W., Prosser, J.I., Emb-ley, T.M. and Woldendorp, J.W. (1997) Analysis of ammonia-oxi-dizing bacteria of the L subdivision of the class Proteobacteria incoastal sand dunes by denaturing gradient gel electrophoresis andsequencing of PCR-ampli¢ed 16S ribosomal DNA fragments. Appl.Environ. Microbiol. 63, 1489^1497.

[95] Speksnijder, A.G., Kowalchuk, G.A., Roest, K. and Laanbroek,H.J. (1998) Recovery of a Nitrosomonas-like 16S rDNA sequencegroup from freshwater habitats. Syst. Appl. Microbiol. 21, 321^330.

[96] Stephen, J.R., Kowalchuk, G.A., Bruns, M.A.V., McCaig, A.E.,Phillips, C.J., Embley, T.M. and Prosser, J.I. (1998) Analysis ofbeta-subgroup proteobacterial ammonia oxidizer populations insoil by denaturing gradient gel electrophoresis analysis and hierarchi-cal phylogenetic probing. Appl. Environ. Microbiol. 64, 2958^2965.

[97] McCaig, A.E., Phillips, C.J., Stephen, J.R., Kowalchuk, G.A., Har-vey, S.M., Herbert, R.A., Embley, T.M. and Prosser, J.I. (1999)Nitrogen cycling and community structure of proteobacterial L-sub-group ammonia-oxidizing bacteria within polluted marine ¢sh farmsediments. Appl. Environ. Microbiol. 65, 213^220.

[98] Hiorns, W.D., Hastings, R.C., Head, I.M., McCarthy, A.J., Saun-ders, J.R., Pickup, R.W. and Hall, G.H. (1995) Ampli¢cation of 16Sribosomal RNA genes of autotrophic ammonia-oxidizing bacteriademonstrates the ubiquity of Nitrosospiras in the environment. Mi-crobiology 141, 2793^2800.

[99] Hastings, R.C., Saunders, J.R., Hall, G.H., Pickup, R.W. and Mc-Carthy, A.J. (1998) Application of molecular biological techniquesto a seasonal study of ammonia oxidation in a eutrophic freshwaterlake. Appl. Environ. Microbiol. 64, 3674^3682.

[100] Phillips, C.J., Smith, Z., Embley, T.M. and Prosser, J.I. (1999) Phy-logenetic di¡erences between particle-associated and planktonic am-monia-oxidizing bacteria of the beta subdivision of the class Proteo-bacteria in the northwestern Mediterranean Sea. Appl. Environ.Microbiol. 65, 779^786.

[101] Whitby, C.B., Saunders, J.R., Rodriguez, J., Pickup, R.W. and Mc-Carthy, A. (1999) Phylogenetic di¡erentiation of two closely relatedNitrosomonas spp. that inhabit di¡erent sediment environments inan oligotrophic freshwater lake. Appl. Environ. Microbiol. 65,4855^4862.

[102] Voytek, M.A. and Ward, B.B. (1995) Detection of ammonium-ox-idizing bacteria of the beta-subclass of the class Proteobacteria inaquatic samples with the PCR. Appl. Environ. Microbiol. 61, 1444^1450.

[103] Ward, B.B., Voytek, M.A. and Witzel, K.-P. (1997) Phylogeneticdiversity of natural populations of ammonia oxidizers investigatedby speci¢c PCR ampli¢cation. Microb. Ecol. 33, 87^96.

[104] Hastings, R.C., Ceccherini, M.T., Miclaus, N., Saunders, J.R., Baz-zicalupo, M. and McCarthy, A.J. (1997) Direct molecular biologicalanalysis of ammonia oxidising bacteria populations in cultivated soilplots treated with swine manure. FEMS Microbiol. Ecol. 23, 45^54.

[105] Bruns, M.A., Stephen, J.R., Kowalchuk, G.A., Prosser, J.I. andPaul, E.A. (1999) Comparative diversity of ammonia oxidizer 16SrRNA gene sequences in native, tilled, and successional soils. Appl.Environ. Microbiol. 65, 2994^3000.

[106] Mendum, T.A., Sockett, R.E. and Hirsch, P.R. (1999) Use of mo-lecular and isotopic techniques to monitor the response of autotro-phic ammonia-oxidizing populations of the beta subdivision of theclass Proteobacteria in arable soils to nitrogen fertilizer. Appl. En-viron. Microbiol. 65, 4155^4162.

[107] Stephen, J.R., McCaig, A.E., Smith, Z., Prosser, J.I. and Embley,T.M. (1996) Molecular diversity of soil and marine 16S rRNA gene

sequences related to beta-subgroup ammonia-oxidizing bacteria.Appl. Environ. Microbiol. 62, 4147^4154.

[108] Bano, N. and Hollibaugh, J.T. (2000) Diversity and distribution ofDNA sequences with a¤nity to ammonia-oxidizing bacteria of thebeta subdivision of the class Proteobacteria in the Arctic Ocean.Appl. Environ. Microbiol. 66, 1960^1969.

[109] Allison, S.M. and Prosser, J.I. (1991) Survival of ammonia oxidisingbacteria in air-dried soil. FEMS Microbiol. Lett. 79, 65^68.

[110] Allison, S.M. and Prosser, J.I. (1993) Ammonia oxidation at low pHby attached populations of nitrifying bacteria. Soil Biol. Biochem.25, 935^941.

[111] Aakra, Aî ., Hesselsoe, M. and Bakken, L.R. (2000) Surface attach-ment of ammonia-oxidizing bacteria in soil. Microb. Ecol. 39, 222^235.

[112] McCaig, A.E., Embley, T.M. and Prosser, J.I. (1994) Molecularanalysis of enrichment cultures of marine ammonia oxidisers.FEMS Microbiol. Lett. 120, 363^368.

[113] Kowalchuk, G.A., Bodelier, P.L.E., Heilig, G.H.J., Stephen, J.R.and Laanbroek, H.J. (1998) Community analysis of ammonia-oxi-dising bacteria, in relation to oxygen availability in soils and root-oxygenated sediments, using PCR, DGGE and oligonucleotideprobe hybridisation. FEMS Microbiol. Ecol. 27, 339^350.

[114] Fisher, M.M. and Triplett, E.W. (1999) Automated approach forribosomal intergenic spacer analysis of microbial diversity and itsapplication to freshwater bacterial communities. Appl. Environ. Mi-crobiol. 65, 4630^4636.

[115] Stewart, V. (1988) Nitrate respiration in relation to facultative me-tabolism in Enterobacteria. Microbiol. Rev. 52, 190^232.

[116] Zumft, W.G. (1992) The denitrifying prokaryotes. In: The Prokary-otes (Balows, A., Tru«per, H.G., Dworkin, M., Harder, W. andSchleifer, K.H., Eds.), pp. 554^582. Springer Verlag, New York.

[117] Kobayashi, M., Matsuo, Y., Takimoto, A., Suzuki, S., Maruo, F.and Shoun, H. (1996) Denitri¢cation, a novel type of respiratorymetabolism in fungal mitochondrion. J. Biol. Chem. 271, 16263^16267.

[118] Park, S.Y., Shimizu, H., Adachi, S., Nakagawa, A., Tanaka, I.,Nakahara, K., Shoun, H., Obayashi, E., Nakamura, H., Iizuka,T. and Shiro, Y. (1997) Crystal structure of nitric oxide reductasefrom denitrifying fungus Fusarium oxysporum. Nature Struct. Biol.4, 827^832.

[119] Stewart, V. (1993) Nitrate regulation of anaerobic respiratory geneexpression in Escherichia coli. Mol. Microbiol. 9, 425^434.

[120] Jetten, M.S.M., Logemann, S., Muyzer, G., Robertson, L.A., Dev-ries, S., Vanloosdrecht, M.C.M. and Kuenen, J.G. (1997) Novelprinciples in the microbial conversion of nitrogen compounds. An-tonie van Leeuwenhoek 71, 75^93.

[121] Stouthamer, A.H., de Boer, A.P., van der Oost, J. and van Span-ning, R.J. (1997) Emerging principles of inorganic nitrogen metab-olism in Paracoccus denitri¢cans and related bacteria. Antonie vanLeeuwenhoek 71, 33^41.

[122] Braker, G., Zhou, J., Wu, L., Devol, A.H. and Tiedje, J.M. (2000)Nitrite reductase genes (nirK and nirS) as functional markers toinvestigate diversity of denitrifying bacteria in paci¢c northwest ma-rine sediment communities. Appl. Environ. Microbiol. 66, 2096^2104.

[123] Michotey, V., Mejean, V. and Bonin, P. (2000) Comparison ofmethods for quanti¢cation of cytochrome cd(1)-denitrifying bacteriain environmental marine samples. Appl. Environ. Microbiol. 66,1564^1571.

[124] Glockner, A.B., Jungst, A. and Zumft, W.G. (1993) Copper-con-taining nitrite reductase from Pseudomonas aureofaciens is function-al in a mutationally cytochrome cd1-free background (NirS3) ofPseudomonas stutzeri. Arch. Microbiol. 160, 18^26.

[125] Ward, B.B. (1995) Diversity of culturable denitrifying bacteria. Lim-its of rDNA RFLP analysis and probes for the functional gene,nitrite reductase. Arch. Microbiol. 163, 167^175.

[126] Hallin, S. and Lindgren, P.E. (1999) PCR detection of genes encod-



ing nitrite reductase in denitrifying bacteria. Appl. Environ. Micro-biol. 65, 1652^1657.

[127] Scala, D.J. and Kerkhof, L.J. (1998) Nitrous oxide reductase (nosZ)gene-speci¢c PCR primers for detection of denitri¢ers and threenosZ genes from marine sediments. FEMS Microbiol. Lett. 162,61^68.

[128] Scala, D.J. and Kerkhof, L.J. (1999) Diversity of nitrous oxidereductase (nosZ) genes in continental shelf sediments. Appl. Envi-ron. Microbiol. 65, 1681^1687.

[129] Scala, D.J. and Kerkhof, L.J. (2000) Horizontal heterogeneity ofdenitrifying bacterial communities in marine sediments by terminalrestriction fragment length polymorphism analysis. Appl. Environ.Microbiol. 66, 1980^1986.

[130] Potter, L.C., Millington, P.D., Thomas, G.H., Rothery, R.A., Gior-dano, G. and Cole, J.A. (2000) Novel growth characteristics andhigh rates of nitrate reduction of an Escherichia coli strain,LCB2048, that expresses only a periplasmic nitrate reductase.FEMS Microbiol. Lett. 185, 51^57.

[131] Simon, J., Gross, R., Einsle, O., Kroneck, P.M.H., Kro«ger, A. andKlimmek, O. (2000) A NapC/NirT-type cytochrome c (NrfH) is themediator between the quinone pool and the cytochrome c nitritereductase of Wolinella succinogenes. Mol. Microbiol. 35, 686^696.

[132] Einsle, O., Messerschmidt, A., Stach, P., Bourenkov, G.P., Bartu-nik, H.D., Huber, R. and Kroneck, P.M.H. (1999) Structure ofcytochrome c nitrite reductase. Nature 400, 476^480.

[133] Kaldorf, M., Linne von Berg, K.-H., Meier, U., Servos, U. andBothe, H. (1993) The reduction of nitrous oxide to dinitrogen byEscherichia coli. Arch. Microbiol. 160, 432^439.

[134] Strous, M., Fuerst, J.A., Kramer, E.H.M., Logemann, S., Muyzer,G., van de Pas-Schoonen, K.T., Webb, R., Kuenen, J.G. and Jetten,M.S.M. (1999) Missing lithotroph identi¢ed as new planctomycete.Nature 400, 446^449.

[135] Ko«rner, H., Frunzke, K., Do«hler, K. and Zumft, W.G. (1987) Im-munological patterns of distribution of nitrous oxide reductase andnitrite reductase (cytochrome cd1) among denitrifying pseudomo-nads. Arch. Microbiol. 148, 20^24.

[136] Ko«rner, H. and Zumft, W.G. (1989) Expression of denitri¢cationenzymes in response to the dissolved oxygen level and respiratorysubstrate in continuous culture of Pseudomonas stutzeri. Appl. En-viron. Microbiol. 55, 1670^1676.

[137] Ward, B.B., Cockcroft, A.R. and Kilpatrick, K.A. (1993) Antibodyand DNA probes for detection of nitrite reductase in seawater.J. Gen. Microbiol. 139, 2285^2293.

[138] Michalski, W.P. and Nicholas, D.J.D. (1988) Immunological pat-terns of distribution of bacterial denitrifying enzymes. Phytochem-istry 27, 2451^2456.

[139] Coyne, M.S., Arunakumari, A., Averill, B.A. and Tiedje, J.M.(1989) Immunological identi¢cation and distribution of dissimila-tory heme cd1 and nonheme copper nitrite reductases in denitrifyingbacteria. Appl. Environ. Microbiol. 55, 2924^2931.

[140] Metz, S., Schloter, M. and Hartmann, A. (2000) Characterization ofmonoclonal antibodies for the in situ detection of denitrifying bac-teria. Hybridoma (submitted).

[141] Schloter, M., AMmus, B. and Hartmann, A. (1995) The use of im-munological methods to detect and identify bacteria in the environ-ment. Biotechnol. Adv. 13, 75^90.

[142] Ko«hler, G. and Milstein, C. (1975) Continous cultures of fused cellssecreting antibodies of prede¢ned speci¢ty. Nature 256, 495^497.

[143] Lebuhn, M., Achouak, W., Schloter, M., Berge, O., Meier, H.,Hartmann, A. and Heulin, T. (2000) Taxonomic characterisationof Ochrobactrum sp. isolates from soil samples and wheat roots,and description of Ochrobactrum tritici sp. nov. and Ochrobactrumgrinonense sp. nov. J. Sys. Bacteriol. (in press).

[144] Michalski, W.P. and Nicholas, D.J.D. (1985) Molecular character-ization of a copper-containing nitrite reductase from Rhodopseudo-monas sphaeroides forma sp. denitri¢cans. Biochim. Biophys. Acta828, 130^137.

[145] Braker, G., Fesefeldt, A. and Witzel, K.-P. (1998) Development ofPCR primer systems for ampli¢cation of nitrite reductase genes(nirK and nirS) to detect denitrifying bacteria in environmental sam-ples. Appl. Environ. Microbiol. 64, 3769^3775.

[146] Juretschko, S., Timmermann, G., Schmid, M., Schleifer, K.H., Pom-merening-Ro«ser, A., Koops, H.P. and Wagner, M. (1998) Combinedmolecular and conventional analyses of nitrifying bacterium diver-sity in activated sludge: Nitrosococcus mobilis and Nitrospira-likebacteria as dominant populations. Appl. Environ. Microbiol. 64,3042^3051.

[147] AMmus, B., Schloter, M., Kirchhof, G., Hutzler, P. and Hartmann,A. (1997) Improved in situ tracking of rhizosphere bacteria usingdual staining with £uorescence-labeled antibodies and rRNA-tar-geted oligonucleotides. Microb. Ecol. 33, 32^40.

[148] Wallner, G., Fuchs, B., Spring, S., Beisker, W. and Amann, R.(1997) Flow sorting of microorganisms for molecular analysis.Appl. Environ. Microbiol. 63, 4223^4231.

[149] Hodson, R.E., Dustman, W.A., Garg, R.P. and Moran, M.A.(1995) In situ PCR for visualization of microscale distribution ofspeci¢c genes and gene products in prokaryotic communities. Appl.Environ. Microbiol. 61, 4074^4082.

[150] Tsai, Y.L. and Olson, B.H. (1991) Rapid method for direct extrac-tion of DNA from soil and sediments. Appl. Environ. Microbiol. 57,1070^1074.

[151] Tebbe, C.C. and Vahjen, W. (1993) Interference of humic acids andDNA extracted directly from soil in detection and transformation ofrecombinant DNA from bacteria and a yeast. Appl. Environ. Mi-crobiol. 59, 2657^2665.

[152] Porteous, L.A., Armstrong, J.L., Seidler, R.J. and Watrud, L.S.(1994) An e¡ective method to extract DNA from environmentalsamples for polymerase chain reaction ampli¢cation and DNA ¢n-gerprint analysis. Curr. Microbiol. 29, 301^307.

[153] Schwieger, F. and Tebbe, C.C. (1997) E¤cient and accurate PCRampli¢cation and detection of a recombinant gene in DNA directlyextracted from soil using the Expand1 High Fidelity PCR systemand T4 gene 32 protein. Boehringer Mannheim Biochem. Inf., pp.73^75.

[154] Wikstro«m, P., Wiklund, A., Andersson, A.C. and Forsman, M.(1996) DNA recovery and PCR quanti¢cation of catechol 2,3-dioxy-genase genes from di¡erent soil types. J. Biotechnol. 52, 107^120.

[155] Frostegard, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D.,Le Gall, F., Jeannin, P., Nesme, X. and Simonet, P. (1999) Quanti-¢cation of bias related to the extraction of DNA directly from soils.Appl. Environ. Microbiol. 65, 5409^5420.

[156] Ste¡an, R.J. and Atlas, R.M. (1988) DNA ampli¢cation to enhancedetection of genetically engineered bacteria in environmental sam-ples. Appl. Environ. Microbiol. 54, 2185^2191.

[157] Linne von Berg, K.-H. and Bothe, H. (1992) The distribution ofdenitrifying bacteria in soils monitored by DNA-probing. FEMSMicrobiol. Ecol. 86, 331^340.

[158] Golet, D.S. and Ward, B.B. (2000) Vertical distribution of denitri-¢cation potential, denitrifying bacteria and benzoate utilization inintertidal microbial mat communities. Microb. Ecol. (in press).

[159] Kloos, K., Fesefeldt, A., Gliesche, C.G. and Bothe, H. (1995) DNA-probing indicates the occurrence of denitri¢cation and nitrogen ¢x-ation genes in Hyphomicrobium. Distribution of denitrifying andnitrogen ¢xing isolates of Hyphomicrobium in a sewage treatmentplant. FEMS Microbiol. Ecol. 18, 205^213.

[160] Fesefeldt, A., Kloos, K., Bothe, H., Lemmer, H. and Gliesche, C.G.(1998) Distribution of denitri¢cation and nitrogen ¢xation genes inHyphomicrobium spp. and other budding bacteria. Can. J. Micro-biol. 44, 181^185.

[161] Kloos, K., Hu«sgen, U.M. and Bothe, H. (1998) DNA-probing forgenes coding for denitri¢cation, N2-¢xation and nitri¢cation in bac-teria isolated from di¡erent soils. Z. Naturforsch. C 53, 69^81.

[162] Mergel, A., Schmitz, O., Mallmann, T., Ouziad, F., Nawrath, K.and Bothe, H. (2000) The distribution of dinitrogen ¢xing and de-



nitrifying bacteria and of mycorrhizal fungi in a forest soil. (sub-mitted).

[163] Mergel, A., Kloos, K. and Bothe, H. (2000) Seasonal £uctuations inthe population of denitrifying and N2-¢xing bacteria in an acid soilof a Norway spruce forest. (submitted).

[164] Smith, G.B. and Tiedje, J.M. (1992) Isolation and characterizationof a nitrite reductase gene and its use as a probe for denitrifyingbacteria. Appl. Environ. Microbiol. 58, 376^384.

[165] Amann, R., Glo«ckner, F.-O. and Neef, A. (1997) Modern methodsin subsurface microbiology: in situ identi¢cation of microorganismswith nucleic acid probes. FEMS Microbiol. Rev. 20, 191^200.

[166] McDevitt, C., Burrell, P., Blackall, L.L. and McEwan, A.G. (2000)Aerobic nitrate respiration in a nitrite-oxidising bioreactor. FEMSMicrobiol. Lett. 184, 113^118.

[167] Carter, J.P., Hsaio, Y.H., Spiro, S. and Richardson, D.J. (1995) Soiland sediment bacteria capable of aerobic nitrate respiration. Appl.Environ. Microbiol. 61, 2852^2858.

[168] Hojberg, O., Revsbech, N.P. and Tiedje, J.M. (1994) Denitri¢cationin soil aggregates analyzed with microsensors for nitrous oxide andoxygen. Soil Sci. Soc. Am. J. 58, 1691^1698.

[169] Horn, R., Stepniewski, W., Wlodarzyk, T., Walenzik, G. andEckhardt, F.E.W. (1994) Denitri¢cation and microbial distribu-tion within homogenous model soil aggregates. Int. Agrophys. 8,65^74.

[170] Philippot, L., Renault, R., Sierra, J., Clays-Josserand, A., Chenu,C., Chaussod, R. and Lensi, R. (1996) Dissimilatory nitrite reduc-tase provides a competitive advantage to Pseudomonas sp. RTC01 tocolonise the centre of soil aggregates. FEMS Microbiol. Ecol. 21,175^185.

[171] Sexstone, A.J., Revsbech, N.P., Parkin, T.B. and Tiedje, J.M. (1985)Direct measurement of oxygen pro¢les and denitri¢cation rates insoil aggregates. Soil Sci. Soc. Am. J. 49, 645^651.

[172] Widmer, F., Sha¡er, B.T., Porteous, L.A. and Seidler, R.J. (1999)Analysis of nifH gene pool complexity in soil and litter at a Douglas¢r forest site in the Oregon cascade mountain range. Appl. Environ.Microbiol. 65, 374^380.

[173] Bremner, J.M., Robbins, S.G. and Blackmer, A.M. (1980) Seasonalvariability in emission of nitrous oxide from soil. Geophys. Res.Lett. 7, 641^644.

[174] Sommerfeld, R.A., Mosier, A.R. and Musselman, R.C. (1993) CO2,CH4 and N2O £ux through a Wyoming snowpack and implicationsfor global budgets. Nature 361, 140^141.

[175] Nijburg, J.W. and Laanbroek, H.J. (1997) The in£uence of Glyceriamaxima and nitrate input on the composition and nitrate metabo-lism of the dissimilatory nitrate-reducing bacterial community.FEMS Microbiol. Ecol. 22, 57^63.

[176] Tani, K., Kurokawa, K. and Nasu, M. (1998) Development of adirect in situ PCR method for detection of speci¢c bacteria in nat-ural environments. Appl. Environ. Microbiol. 64, 1536^1540.

[177] Kuske, C.R., Barns, S.M. and Busch, J.D. (1997) Diverse unculti-vated bacterial groups from soils of the arid southwestern UnitedStates that are present in many geographic regions. Appl. Environ.Microbiol. 63, 3614^3621.

[178] Muyzer, G., de Waal, E.C. and Uitterlinden, A.G. (1993) Pro¢lingof complex microbial populations by denaturing gradient gel elec-trophoresis analysis of polymerase chain reaction-ampli¢ed genescoding for 16S rRNA. Appl. Environ. Microbiol. 59, 695^700.

[179] Felske, A., Vancanneyt, M., Kersters, K. and Akkermans, A.D.(1999) Application of temperature-gradient gel electrophoresis intaxonomy of coryneform bacteria. Int. J. Syst. Bacteriol. 49, 113^121.

[180] Joergensen, K.S. (1989) Annual pattern of denitri¢cation and nitrateammoni¢cation in estuarine sediment. Appl. Environ. Microbiol. 55,1841^1847.



molecular analysis of ammonia oxidation and ... · nia oxidation [10]; nitrogen cycling in coastal...

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