The Lithoautotrophic Nitrite-Oxidizing

Eva Spieck and Eberhard Bock

The lithotrophic nitrite oxidizers are Gram-negative eubacteria that are able to use nitrite as a sole source of energy and CO2 as the main source of carbon. Some strains are able to grow mixotrophically. These organisms are obligate lithoautotrophs with the exception of , which can grow heterotrophi- cally. Nitrobacter has been shown to grow anaerobically by dissi- milatory nitrate reduction (Freitag et al., 1987).

MORPHOLOGICAL CHARACTERISTICS The nitrite oxidizers are a diverse group of rods, cocci, and spi- rilla. Historically, the classification of genera was founded pri- marily on cell shape and arrangement of intracytoplasmic mem- branes, and taxonomic categorization was based on the work of Sergei and Helene Winogradsky (Winogradsky, 1892). Four mor- phologically distinct genera (Nitrobacter, Nitrococcus, Nitrospina, and Nitrospira) have been described (Watson et al., 1989; Bock and Koops, 1992). Cells of Nitrobacter are pleomorphic short rods FIGURE 1. Spiral-shaped cell of Nitrospira marina grown lithoautotroph- .nm 500 ס containing a polar cap of intracytoplasmic membranes. Nitrococ- ically. Negative staining with uranylacetate. Bar cus occurs in form of coccoid cells with tubular intracytoplasmic membranes. Cells of Nitrospina appear as long rods, intracyto- plasmic membranes in the form of flattened vesicles or tubes are but much higher phylogenetical diversity may be present in this missing. The genus Nitrospira is characterized by a spiral shape genus (Schramm et al., 1999). and the absence of intracytoplasmic membranes. Some strains are motile by means of a single polar or subpolar flagellum. ECOLOGY AND DISTRIBUTION The best-investigated nitrite-oxidizing bacterium is Nitrobacter, PHYLOGENY which was believed to dominate in most natural environments Unlike the ammonia oxidizers, which are restricted to two line- except marine ones. This picture has changed significantly over ages within the , the nitrite oxidizers are more scat- the last few years, and current investigations, especially molecular tered phylogenetically (Fig. 1 of the chapter “Nitrifying Bacte- ones, are focused on the occurrence of Nitrospira. Members of ria”). The genus Nitrobacter belongs to the the genera Nitrococcus and Nitrospina have only been found in (Woese et al., 1984a; Stackebrandt et al., 1988), whereas Nitro- marine habitats to date. coccus is affiliated with the Gammaproteobacteria (Woese et al., Nitrobacter is a soil and freshwater organism that is tolerant of 1985). The genus Nitrospina seems to be a member of the Del- changing environmental conditions. Members of this genus also taproteobacteria (Teske et al., 1994), although this assignment re- occur in sewage and marine environments. Other isolates were mains preliminary. Nitrospira was thought to be related to Nitro- originally obtained from extreme environments such as concrete spina, but Nitrospira was later shown not to be a member of the and natural stones, desert soils and sulfidic ore mines. One acid- Proteobacteria. Ehrich et al. (1995) demonstrated that this nitrite- ophilic strain with a pH optimum of 5.5 was isolated from an oxidizing bacterium occupies a phylogenetically isolated position acidic forest soil (Hankinson and Schmidt, 1988). Facultatively and represents a new phylum, Nitrospirae, of the domain Bacteria. alkalophilic strains of Nitrobacter were recently be isolated from Nitrospira was described in Volume I of the Manual; some data soda lakes in Siberia and Kenya and described as a new species, are also presented here to facilitate comparison. N. alkalicus (Sorokin et al., 1998). The genus Nitrobacter is comprised of four described species Although nitrite oxidizers do not form endospores, they can (Bock and Koops, 1992; Sorokin et al., 1998), whereas one species survive long periods of starvation and dryness. One survival strat- is known for each of the genera Nitrococcus and Nitrospina (Watson egy used by these organisms may be the formation and accu- and Waterbury, 1971). Two species of Nitrospira have been de- mulation of extracellular compatible solutes. Nitrobacter was scribed in the literature (Watson et al., 1986; Ehrich et al., 1995), found to produce trehalose and was able to accumulate glycine

149 150 THE LITHOAUTOTROPHIC NITRITE-OXIDIZING BACTERIA betaine and sucrose from the medium. An increase in the treatment plants was postulated to be “unculturable,” microcol- amounts of compatible solutes was reproducibly found in cul- onies of Nitrospira from wastewater samples from Dradenau in tures exposed to salt stress and dryness. Nitrobacter vulgaris can Hamburg were highly enriched in laboratory cultures (Fig. 2). survive a period of 24 months without water (L. Lin, personal Nitrospira can be regularly enriched using adapted cultivation communication). Diab and Shilo (1988) found that adhesion to techniques that include the avoidance of turbulence, and cul- particles had a positive effect on both the activity and the survival tures originating from a wide range of habitats such as permafrost of Nitrobacter cells. soil (Bartosch et al., 2002), caves, and hot springs are being When the habitat-specific distribution of three species of Ni- investigated. trobacter was examined using automated pattern matching of pro- So far, phylogenetic analysis of Nitrospira has revealed four teins, it was found that Nitrobacter vulgaris was the dominant spe- sublineages based on environmental sequences from various cies in building stone (T. Krause-Kupsch, personal communica- aquatic environments (summarized by Daims et al., 2001). Two tion). Nitrobacter hamburgensis was only found in soil, whereas of the sublineages include the described species N. moscoviensis Nitrobacter winogradskyi occurred in various habitats such as soils, and N. marina. A third species, originating from a Moscow heat- fresh water, sewage and concrete. According to Both et al. (1992) ing system, will be described in the future (Lebedeva, personal Nitrobacter winogradskyi out-competes Nitrobacter hamburgensis in communication). A thermophilic culture derived from a hot well-aerated soils under nitrite-limiting conditions, since the for- spring at Lake Baikal differed from known Nitrospira isolates mer has a lower Km for nitrite under autotrophic as well as mix- based on DGGE (Alawi and Lebedeva, personal communication). otrophic conditions. However, the activity of Nitrobacter hambur- It is likely that the phylogenetic tree of Nitrospira will become gensis increases when oxygen tension decreases. more complex as new representatives are isolated and sequence Immunological and molecular investigations of Nitrobacter analysis of environmental samples is carried out. populations demonstrated that several strains of this genus can coexist (Stanley and Schmidt, 1981; Degrange et al., 1997). Na- GROWTH CHARACTERISTICS varro et al. (1992) characterized natural populations of Nitrobacter Lithotrophic growth of nitrite oxidizers is slow. The generation by PCR/RFLP (restriction fragment length polymorphism). time varies from 8 h to several days. Growth rates are controlled These authors differentiated several coexisting strains in various by substrate concentration, temperature, pH, light, and oxygen soils and a lake; the coexistence of several strains may reflect the concentration. Most nitrite oxidizers grow best at nitrite concen- existence of local niches. Genetic distances obtained by amplified trations of 2–30 mM at a pH of 7.5–8.0 and at temperatures of ribosomal DNA restriction analysis (ADRA) of the 16S-23S rRNA 25–30ЊC. Some strains are able to grow mixotrophically; the cell intergenic spacer regions and partial sequences of the 23S rRNA yield from mixotrophically grown cultures can be ten-fold greater gene enable comparison of Nitrobacter species in soil (Grund- than that from lithotrophically grown cultures. mann and Normand, 2000). Two 16S rRNA-targeted oligonucle- otide probes specific for Nitrobacter have been developed for the BIOCHEMISTRY in situ analysis of nitrite oxidizers (Wagner et al., 1996). Although Most biochemical investigations have been performed on the this genus has been regarded as the most abundant nitrite oxi- genus Nitrobacter. Initial biochemical studies of Nitrospira revealed dizer in various environments, it could not be detected in acti- several significant differences between Nitrospira and Nitrobacter vated sludge samples and reactor biofilms. The authors suggested (Watson et al., 1986). Little is known about the biochemistry of that still unknown organisms might be responsible for nitrifi- Nitrococcus and Nitrospina. The genera Nitrobacter and Nitrococcus cation in these habitats. This hypothesis was confirmed recently are similar in cytochrome content and in the location and mo- when several groups reported that Nitrospira-like bacteria seem lecular masses of the nitrite-oxidizing enzymes, whereas the gen- to be the dominant nitrite oxidizers in freshwater aquaria, bio- era Nitrospina and Nitrospira differ from Nitrobacter and Nitrococcus films and activated sludge (Burrell et al., 1998; Hovanec et al., but are similar to each other with respect to these characteristics. 1998; Juretschko et al., 1998). Schramm et al. (1998) found in Nitrite, the substrate for aerobic nitrification, is thought to a nitrifying reactor organisms that formed two phylogenetically be transported into the bacteria by a nitrite/nitrate antiport sys- distinct groups affiliated with Nitrospira moscoviensis. The novel tem (Wood, 1986). Nitrate, the electron acceptor for the reverse genus Nitrospira marina was first isolated by Watson et al. (1986) reaction, is assumed to be transferred by the same transporter. from the Gulf of Maine (Fig. 1). Nitrospira moscoviensis was first The key enzyme of nitrite oxidation has been studied in Ni- isolated from a heating system in Moscow (Ehrich et al., 1995). trobacter and Nitrospira; this enzyme is called the nitrite oxido- Similar nitrite-oxidizing organisms have been enriched from soil reductase (NOR) in the genus Nitrobacter and nitrite-oxidizing samples, sediments, beach sands, and salt marshes (Watson et system (NOS) in the genera Nitrococcus, Nitrospina and Nitrospira. al., 1989). It seems that although the genus Nitrospira is ubiq- The occurrence of membrane-bound particles containing the uitous, it is outcompeted by Nitrobacter when standard isolation enzyme is a general characteristic of all members of nitrite-oxi- procedures are used ( Johnson and Sieburth, 1976). In studies dizing bacteria; these particles are densely packed on the surface using monoclonal antibodies that recognize the nitrite oxido- of the cytoplasmic and intracytoplasmic membranes. The loca- reductase (NOR) enzyme of Nitrobacter, Bartosch et al. (1999) tion of the particles is coincident with immunolabeling of the demonstrated that different genera of nitrite oxidizers were en- NOR and NOS enzymes (Spieck et al., 1996a). In Nitrobacter and riched from activated sludge depending on the substrate con- Nitrococcus the key enzyme is located on the inner side of the centration of the media. When enrichments were made in ac- cytoplasmic and intracytoplasmic membranes (Watson and cordance with the instructions of Watson et al. (1989), Nitrospira Waterbury, 1971; Sundermeyer and Bock, 1981a). In cells of was the most abundant nitrite oxidizer in enrichment cultures Nitrospina and Nitrospira, which do not possess intracytoplasmic grown in mixotrophic medium containing 0.2 g NaNO2 per liter. membranes, the nitrite-oxidizing system is found in the peri- In contrast, cells of Nitrobacter dominated when the medium con- plasmic space and is associated with the outer surface of the cell tained 2 g NaNO2 per liter. Although Nitrospira from wastewater membrane in Nitrospira (Spieck et al., 1998). This location of the THE LITHOAUTOTROPHIC NITRITE-OXIDIZING BACTERIA 151

A B FIGURE 2. Microcolonies of Nitrospira-like bacteria in activated sludge from waste water treatment plant in Dradenau, Hamburg. A) Ultrathin section of activated sludge. Cells are similar in ultrastructure to those of the genus Nitrospira with respect to the extended perimplasmic space and lack of nm. B) Fluorescence in situ hybridization (FISH) of a nitrite-oxidizing enrichment culture with oligonucleotide 250 ס intracytoplasmic membranes. Bar probe S-*Ntspa-1026-a-A-18 specific for N. moscoviensis ( Juretschko et al., 1998). Cells were grown in mixotrophic medium containing 0.2 g NaNO2/l. Picture courtesy of S. Bartosch

enzyme may explain the higher sensitivity of Nitrospina and Ni- respiratory chain (Cobley, 1976; Aleem and Sewell, 1981). The trospira to nitrite in comparison to Nitrobacter and Nitrococcus. The reduction of cytochrome c is a thermodynamically unfavorable מ מ molecular masses of the b-NOR of Nitrobacter and the b-NOS of step because the NO2 /NO3 couple has a redox potential of mV. Nitrite-oxidizing cells of Nitrobacter winogradskyi 420 ם ס Nitrococcus are identical (65 KDa), whereas the b-NOSs of Nitro- E0Ј spina (48 KDa) and Nitrospira (46 KDa) differ (Bartosch et al., have a very low energy charge of 0.37 during the logarithmic 1999). Images showing the location and arrangement of the NOR growth phase (Eigener, 1975). The inefficiency of energy gen- can be found in the chapters describing the genera Nitrobacter, eration in Nitrobacter may be compensated for by high levels of Nitrococcus, and Nitrospina. For Nitrospira, see Volume 1. NOR, which may comprise 10–30% of total protein (Bock et al., The NOR of Nitrobacter forms a periodic arrangement in 1991). The primary energy product is NADH (Sundermeyer and paired rows. Tsien and Laudelout (1968) provided the first evi- Bock, 1981b), which is used for ATP synthesis (Freitag and Bock, dence that a minimum of four particles had to remain associated 1990). It is not clear how energy conservation occurs because in order to retain enzymatic activity. The integrity of a structure the postulated reverse electron flow for the generation of NADH extending between neighboring particles was assumed to be nec- has not yet been demonstrated. The nitrite oxidase system of essary for conservation of activity. The molecular weight of a Nitrobacter winogradskyi was reconstituted in proteoliposomes with single particle was 186 KDa; this result suggests that each particle isolated nitrite oxidoreductase, cytochrome c oxidase and the is an ␣b-heterodimer (Spieck et al., 1996b). subtrate nitrite. In this system oxygen was consumed in the pres-

The biochemistry of Nitrobacter has been reviewed by several ence of membrane-bound cytochrome c550 (Nomoto et al., 1993). authors (Wood, 1986; Yamanaka and Fukumori, 1988; Hooper, A purified ATPase from N. winogradskyi has been characterized 1989; Bock et al. 1991, 1992; Bock and Wagner, 2001.). Two by Hara et al. (1991). electrons are released during the oxidation of nitrite to nitrate Cells of Nitrobacter hamburgensis seem to utilize different ter- as shown in the following equation. The third oxygen atom in minal oxidases in response to different growth conditions. Dur- the nitrate molecule is derived from water (Aleem et al., 1965). ing nitrite oxidation, cytochrome aa3 is active, whereas a b-type מ ם מ מ 2e cytochrome is used as a terminal oxidase for heterotrophic ם 2H ם H2OrNO3 ם NO2 מ ם O2rH2O growth (Kirstein et al., 1986). Nitrobacter and Nitrococcus are rich 0.5 ם 2e ם 2H מ מ O2rNO3 in cytochromes c and a; dense cell suspensions exhibit a typical 0.5ם NO2 The electron flux from nitrite to oxygen is thought to flow red to brownish color. Characteristic peaks occur at 420, 440, through the following electron carriers. 550, 587 and 600 nm in oxidized/dithionite-reduced difference nitritermolybdopterinriron-sulfur-clustersrcytochrome spectra. The other two genera of nitrite-oxidizing bacteria, Nitro- a1rcytochrome crcytochrome aa3rO2 spina and Nitrospira, apparently lack type a cytochromes (Watson The electron transfer from nitrite to cytochrome a1 is cata- et al., 1989). lyzed by the enzyme nitrite oxidoreductase (NOR) which con- Lithoautotrophic nitrite oxidizers fix carbon dioxide via the tains molybdopterin and iron-sulfur clusters. Cytochrome a1 is Calvin cycle. About 80% of the energy generated by nitrite ox- necessary to channel electrons from nitrite to cytochrome c (Ya- idation is used for CO2 fixation. In Nitrobacter ribulose-1,5-bis- manaka and Fukumori, 1988), where the electrons enter the phosphate carboxylase/oxygenase (RubisCO) is responsible for 152 THE LITHOAUTOTROPHIC NITRITE-OXIDIZING BACTERIA this reaction. In Nitrobacter (Shively et al., 1977) and Nitrococcus, multiple passages in liquid medium of colonies from nitrite agar the enzyme may be soluble as well as carboxysome-bound; car- (Sorokin et al., 1998). Nitrite oxidizers like Nitrospira can be boxysomes are found in most but not all species of Nitrobacter. separated from heterotrophic contaminants by Percoll gradient In Nitrobacter winogradskyi the soluble form of RubisCO has a centrifugation and subsequent serial dilution (Ehrich et al., molecular mass of 480 KDa. In Nitrobacter hamburgensis X14 the 1995). enzyme occurs in two forms with different molecular masses— 480 KDa and 520 KDa; both forms have an L8S8 quaternary MAINTENANCE PROCEDURES FOR STOCK CULTURES structure. In this species two different genes and gene products Nitrifying organisms can survive starvation for more than one for the large subunit of RubisCO have been identified; one is year when kept at 17ЊC in liquid media. Nevertheless, cells should located on the chromosome and the other on a plasmid (Harris be transferred to fresh media every four months. In Table 1 three et al., 1988). The Calvin cycle genes are located in two separate different growth media for nitrite oxidizers are listed. Freezing clusters on the chromosome in Nitrobacter vulgaris (Strecker et in liquid nitrogen is a suitable technique for maintenance of al., 1994). stock cultures that are suspended in a cryoprotective buffer con- taining sucrose and histidine. When freeze-dried on lavalite or ENRICHMENT AND ISOLATION PROCEDURES polyurethane, about 0.5% of Nitrobacter cells survive for one year (L. Lin, personal communication). Another possibility for the Nitrite oxidizers can be isolated using a mineral medium con- storage of Nitrobacter for several years is cultivation in 1l-bottles taining nitrite; the compositions of media for lithotrophic, mix- filled to the top with complex medium and closed by a screw otrophic, and heterotrophic growth are given in Table 1. Serial top. Glycerol should be used instead of pyruvate to keep the pH dilutions of enrichment cultures must be incubated for one to stable for a long period. Since the bacteria are able to oxidize several months in the dark. Since nitrite oxidizers are sensitive nitrite to nitrate aerobically and subsequently able to reduce the to high partial pressures of oxygen, cell growth on agar surfaces nitrate anaerobically, a high cell yield can be obtained using this is limited. Pure cultures of Nitrobacter alkalicus were obtained by method (Freitag et al., 1987).

DIFFERENTIATION OF THE FOUR GENERA OF NITRITE-OXIDIZING TABLE 1. Three different media for lithoautotrophic (medium A for BACTERIA terrestrial strains; medium B for marine strains), mixotrophic (medium C), and heterotrophic (medium C without NaNO2) growth Morphological, genotypic, and chemotaxonomic characteristics of nitrite oxidizers that can be used to differentiate the four genera of nitrite-oxi- Culture medium dizing bacteria are given in Tables 2, 3, and 4. Ingredient Aa, Bb Cc, d Specific reaction patterns of a set of three monoclonal anti- Distilled water (ml) 1000 300 1000 bodies (MAbs) that recognize the nitrite-oxidizing system (Aa- Seawater (ml) 700 mand et al., 1996) were shown to be useful for taxonomic in- NaNO2 (mg) 200–2000 69 200–2000 vestigations of pure and enrichment cultures by western blot MgSO4•7H2O (mg) 50 100 50 analysis and immunofluorescent labelling (Bartosch et al., 1999). CaCl2•2H2O (mg) 6 The three different MAbs have different degrees of specificity CaCO (mg) 3 3 3 that permit classification to the genus level. MAb Hyb 153-2 rec- KH2PO4 (mg) 150 1.7 150 FeSO4•7H2O (mg) 0.15 0.15 ognizes the ␣-NOR of the described species of Nitrobacter. MAb Chelated iron (13%, Geigy) (mg) 1 Hyb 153-1 recognizes the b-NOS of Nitrobacter and Nitrococcus, Na2MoO4•2H2O (lg) 30 whereas MAb Hyb 153-3 reacts with the b-NOS of all known (NH4)2Mo7O24•4H2O (lg) 50 50 MnCl •6H O (lg) 66 nitrite-oxidizing bacteria. The differing molecular masses of the 2 2 b CoCl2•6H2O (lg) 0.6 -NOSs enable differentiation of the four genera. CuSO4•5H2O (lg) 6 The results summarized in Table 2 indicate that the epitope ZnSO4•7H2O (lg) 30 of the b-subunit recognized by MAb Hyb 153-3 is highly con- NaCl (mg) 500 500 served. The finding of such conserved regions in the key enzyme Sodium pyruvate (mg) 550 Yeast extract (Difco) (mg) 1,500 of nitrite oxidation does not support the hypothesis of Teske et Peptone (Difco) (mg) 1,500 al. (1994) that the nitrifiers arose independently multiple times, pH adjusted toe 8.6 6 7.4 possibly from different photosynthetic ancestors. The specific re- aFor terrestrial strains from Bock et al. (1983). actions of the MAbs suggest a close correlation between phylog- bFor marine strains modified from Watson and Waterbury (1971). eny and function and underscore the utility of investigation of cFor terrestrial strains from Bock et al. (1983). the comparative biochemistry of proteins involved in energy me- d For heterotrophic growth medium C without NaNO2 is used. tabolism as an approach to the study of bacterial evolution eAfter sterilization pH should be 7.4–7.8. (Brock, 1989). THE LITHOAUTOTROPHIC NITRITE-OXIDIZING BACTERIA 153

TABLE 2. Differentiation of the four genera of nitrite-oxidizing bacteria Characteristic Nitrobacter Nitrococcus Nitrospina Nitrospira Phylogenetic position Alphaproteobacteria Gammaproteobacteria Deltaproteobacteria Phylum Nitrospirae (preliminary) Morphology Pleomorphic short rods Coccoid cells Straight rods Curved rods to spirals Intracytoplasmic Polar cap Tubular Lacking Lacking membranes 2.2–0.9 ן 0.4–0.2 6.6–1.7 ן 0.5–0.3 1.8–1.5 2.0–1.0 ן Size (lm) 0.5–0.9 מ מ ם ם Motility Reproduction: Budding or binary fission Binary fission Binary fission Binary fission Main cytochrome typesa a, c a, c c b, c Location of the nitrite Cytoplasmic Cytoplasmic Periplasmic Periplasmic oxidizing system on membranes MAb-labeled subunits 130 and 65 65 48 46 (KDa)b Crystalline structure of Rows of particle dimers Particles in rows Hexagonal pattern Hexagonal pattern membrane-bound particles aLithoautotrophic growth. bMAbs, monoclonal antibodies.

TABLE 3. Properties of the nitrite-oxidizing bacteria Nitrobacter Nitrobacter Nitrobacter Nitrobacter Nitrococcus Nitrospina Nitrospira Nitrospira Characteristic winogradskyi alkalicus hamburgensis vulgaris mobilis gracilis marina moscoviensis C of the DNA 61.7 62 61.6 59.4 61.2 57.7 50 56.9 ם Mol% G מ מ מ ם ם ם מ ם Carboxysomes Habitat: ם ם Fresh water ם ם Waste water ם Brackish water ם ם ם ם Oceans ם Soda lakes ם ם ם Soil ם Soda soil ם ם Stones ם Heating system

TABLE 4. Primary fatty acids of the described species of nitrite-oxidizing bacteriaa,b Nitrobacter Nitrobacter Nitrobacter Nitrobacter Nitrococcus Nitrospina Nitrospira Nitrospira Fatty acid winogradskyi Engel alkalicus AN4 hamburgensis X14 vulgaris Z mobilis 231 gracilis 3 marina 295 moscoviensis M1

ם C14:1cis9 ם ם םםם ם ם ם C14:0 םם םםם C16:1cis7 םםם םםם ם ם ם ם C16:1cis9 םםם םםם C16:1cis11 ם ם C16:03OH םםם םםם םם םםם םם םם םם םם C16:0 םםם ם C16:0 11methyl ם ם ם ם C18:1cis9 ם ם ם םםם םםםם םםםם םםםם םםםם C18:1cis11 ם םם ם ם ם ם ם C18:0 ם ם ם ם ם C19:0cyclo11-12 .Ͼ60% ,םםםם ;60%–16 ,םםם ;15%–6 ,םם ;Ͻ5% ,ם :aSymbols bStirred cultures were grown autotrophically at 28ЊC (Nitrospira moscoviensis at 37ЊC) and collected at the end of exponential growth. Modified from Lipski et al., (2001).