The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis

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Systematic and Applied Microbiology 30 (2007) 297–308 www.elsevier.de/syapm

The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis Bram Vanparysa,, Eva Spieckb, Kim Heylena, Lieven Wittebollec, Joke Geetsc, Nico Boonc, Paul De Vosa a

Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium b Biozentrum Klein Flottbek, Abteilung Mikrobiologie, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany c Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium Received 11 October 2006

Abstract Strains of Nitrobacter mediate the second step in the nitrification process by oxidizing nitrite to nitrate. The phylogenetic diversity of the genus is currently not well investigated. In this study, a rep-PCR profile and the nearly complete 16S rRNA gene sequence of 30 strains, comprising a wide physiological as well as ecological diversity and encompassing representatives of the four species, were determined. The sequence diversity of the 16S rRNA gene between different species was low, indicating the need for additional phylogenetic markers. Therefore, primers were developed for amplifying the complete nxrX gene and a 380 bp fragment of the nxrB1 gene, which are both genes involved in the nitrite oxidation process. These genes confirmed the division into phylogenetic groups revealed by the 16S rRNA gene but showed a better discriminatory power. They can be a valuable additional tool for phylogenetic analysis within the genus Nitrobacter and can assist in the identification of new Nitrobacter isolates. r 2006 Elsevier GmbH. All rights reserved. Keywords: Nitrobacter; Nitrite oxidoreductase; nxrA; nxrB; nxrX; 16S rRNA; Rep-PCR; Phylogenetic diversity

Introduction Nitrification, the microbiological conversion of ammonia to nitrate, is a key process in the nitrogen cycle. The process consists of two subsequent steps: (i) the oxidation of ammonia to nitrite and (ii) the oxidation of nitrite to nitrate. The latter reaction is performed by the nitrite oxidizing bacteria (NOB), which belong to four phylogenetically unrelated genera: Nitrobacter (a-Proteobacteria), Nitrococcus (g-Proteobacteria), Nitrospina Corresponding author. Tel.: +32 9 264 51 01; fax: +32 9 264 53 46.

E-mail address: [email protected] (B. Vanparys). 0723-2020/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2006.11.006

(preliminarily assigned to the d-Proteobacteria) and Nitrospira (separate phylum; [11,40]). Although Nitrospira is currently regarded as more important than Nitrobacter in, for example, wastewater treatment plants and aquaria, Nitrobacter has been shown to coexist with Nitrospira in plants with temporally or spatially elevated nitrite concentrations [9]. Furthermore, Nitrobacter is still considered an important genus in fertilized as well as unfertilized soils [14], which are regarded as the privileged habitats of Nitrobacter [10,15]. Nitrobacter is also associated with landfill leachate treatment plants [20] and rivers [8], and is important in the erosion of rocks [23] and the deterioration of historic monuments [24].

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Nitrobacter is a phylogenetically young group [30,35] and the genome is conserved within this genus [15]. The genus currently consists of four valid species, Nitrobacter winogradskyi [44,48], Nitrobacter hamburgensis [6], Nitrobacter vulgaris [4] and Nitrobacter alkalicus [36]. Previous studies indicated that the 16S rRNA gene has limited discriminatory power within Nitrobacter [30,35] and might not be a suitable phylogenetic tool [2], although more data are necessary to confirm this observation. Sequence analysis of functional genes could overcome this constraint, since protein coding genes show more sequence diversity than the 16S rRNA gene. Nitrite oxidation in Nitrobacter is mediated by the nitrite oxidoreductase enzyme (previously abbreviated as NOR, but now called NXR [38]). The catalytically active NXR enzyme of N. hamburgensis X14T consists of two subunits [25], encoded by nxrA and nxrB [21]. Kirstein and Bock [21] showed that a nxrX gene, assumed to encode a peptidyl-prolyl cis–trans isomerase that may assist in the folding of the nitrite oxidoreductase enzyme [38], was located between nxrA and nxrB (further denominated as the nxrAXB gene cluster; Fig. 1). The NXR enzymes of the different Nitrobacter species have serological homology [1,2] and recently the nxrAXB gene cluster was also shown to be present in representatives of the other Nitrobacter species [38,42]. A complete genome sequence analysis showed that N. winogradskyi Nb-255T contains, besides the nxrA (nxrA1) and nxrB (nxrB1) genes located in the nxrAXB cluster, additional nxrA (nxrA2) and nxrB (nxrB2) genes segregated at distant points in the genome. The two copies of nxrA (94%) and nxrB (97%) were only moderately related [38]. Currently, only a limited number of nxr gene sequences are publicly available and no primers amplifying these genes have been described in the literature. The value of the nxr genes in the phylogenetic analysis of Nitrobacter has hitherto not been evaluated. In this study, a set of 30 Nitrobacter strains, comprising a wide physiological as well as ecological diversity and encompassing representatives of the four valid species of the genus, was analysed. The nearly complete 16S rRNA gene sequence, a rep-PCR profile and, using newly developed primers, the complete nxrX

and a 380 bp fragment of the nxrB1 gene sequence of all strains were determined.

Materials and methods Strains and culture conditions All strains used in this study are listed in Table 1. A frozen cell suspension of N. alkalicus AN2 was obtained from D. Sorokin (Delft University of Technology, The Netherlands). An active liquid culture of N. vulgaris DSM 10236T was obtained from DSMZ. Active cultures of N. winogradskyi ATCC 25391T and ATCC 14123 and N. hamburgensis X14T were obtained from P. Bodelier (Netherlands Institute of Ecology, The Netherlands). An active culture of Nitrospira moscoviensis NCIMB 13793T was obtained from Zena Smith (NCIMB Ltd., UK). The other strains originated from the Institut fu¨r Allgemeine Botanik, Abteilung Microbiology, Universita¨t Hamburg (Germany), that provided strains 339, 329, 219, 311 and 263 isolated by S.T. Watson. Strains Engel, 219, Nato I, F83KO, 339, GT Oman, AB1, K48, K55 and 263 were cultivated in a basal mineral salt medium [11], supplemented with 2 g/l NaNO2. Strains 5F/3, LIP, 4111/1, BS5/19, AB3, R1.30, 329, Termite 2, Yukatan, BB3, C2, D6/13 and Y were cultivated in a basal mineral salt medium [11] containing 0.2 g/l NaNO2. Strain Io Acid was cultivated in an acidic medium [34] at a pH of 5.5 and strain 311 in a marine medium [45]. Cultures were grown at 28 1C and periodically assayed for nitrite consumption. To obtain a sufficient amount of cells, cultures with 0.2 g/l NaNO2 were supplemented with nitrite up to eight times. Strains were checked for contamination analogous to Steinmu¨ller and Bock [39]. Cells were harvested by centrifugation, suspended in 0.9% NaCl and stored at –20 1C upon DNA extraction.

DNA extraction DNA was extracted by alkalic lyses. Cell suspensions were centrifuged for 5 min at 16,000g, whereupon

NxrA1

NxrX

bp 1

3645 NxrA-1F NxrX-1F

1

NxrB1

702 1

1542

NxrB-1F

NxrB-1R

NxrX-1R

Fig. 1. Organization of the Nitrobacter nxrAXB gene cluster. Each arrow represents one gene. Primers used in this study are indicated.

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Table 1.

299

Overview of the strains used in this study

Strain Nitrobacter N. alkalicus AN2 N. hamburgensis X14T Y N. vulgaris ZT ¼ DSM 10236T 329 339 AB1 BB3 C2 K48 K55 N. winogradskyi ATCC 25391T ¼ Nb-255T ATCC 14123 ¼ AG ¼ 106 219 311 AB3 Engel F83KO Nato I R 1.30 Nitrobacter sp. 263 ¼ ATCC 25393 LIP 4111/1 BS 5/19 Gt Oman Termite 2 Yukatan D6/13 Io Acid 5 F/3 Nitrospira N. moscoviensis NCIMB 13793T

Origin

Reference

Sediment soil, Siberia (Russia)

[36]

Soil from botanical garden, Hamburg (Germany) Soil from Uxmal, Yucatan (Mexico)

[6] [6]

Sand filter from waterworks, Hamburg (Germany) Soil, Yalta (Russia) Soil, Rhodos (Greece) Sewage system, Hamburg (Germany) Brackish water from river Elbe, Hamburg (Germany) Salt water from river Elbe, Hamburg (Germany) Sandstone from cathedral, Cologne (Germany) Sandstone from cathedral, Cologne (Germany)

[4] [4] [4] [4] [4] [4] [4] [4]

Soil Soil Paramaribo river (Surinam) Atlantic near coast of Africa Sewage system, Hamburg (Germany) Soil from botanical garden, Hamburg (Germany) Concrete from cooling tower, Cologne (Germany) Activated carbon filter, Hannover (Germany) Stone from cathedral, Regensburg (Germany)

[45] [45] Watson, unpublished Watson, unpublished [7] [4] [4] [4] [4]

Soil, Galapagos Islands (Ecuador) Concrete, Lipetsk (Russia) Concrete, Frankfurt (Germany) Sulfidic ore mine, Baia Sprie (Romania) Oasis (Oman) Termite heap, Macheke (Zimbabwe) Soil from Uxmal, Yucatan (Mexico) Ore mine, Dubova (Romania) Hoosier national forest (USA) Permafrost soil, Siberia (Russia)

[22] [22] [22] [22] [22] [22] [22] [22] [16] [3]

Iron pipe, Moscow (Russia)

[11]

supernatant was removed, 20 ml of lysis buffer (0.5 ml 10% SDS; 1 ml 1 M NaOH; 18.5 ml MilliQ water) was added and the tube was placed at 95 1C for 15 min. Subsequently, 180 ml MilliQ water was added, the tube was centrifuged for 5 min at 16,000g and the supernatant was transferred to a new tube. DNA extracts were stored at –20 1C until use.

16S rRNA gene sequencing Amplification of both strands of the nearly complete (1495 bp; positions 27–1522 in the Escherichia coli numbering system) 16S rRNA gene sequence was performed as described previously [18]. The PCR-amplified 16S rRNA

gene products were purified using the Nucleofasts 96 PCR clean up membrane system (Machery-Nagel, Germany). For each sequence reaction, a mixture was made using 1 ml purified product, 1 ml of BigDyeTM Termination RR mix version 3.1 (Perkin Elmer), 1.5 ml of BigDyeTM buffer (5  ), 1.5 ml MilliQ and 3 ml (20 ng ml1) of one of the eight sequencing primers used (forward primer, position 339–358, 50 -tcctacgggaggcagcact-30 ; forward, 519–536, 50 -cagcagccgcggtaatac-30 ; forward, 908–926, 50 -aactcaaaggaattgacgg-30 ; forward, 1093–1112, 50 -agtcccgcaacgagcgcaac-30 ; reverse, 358–339, 50 -actgctgcctcccgtaggag-30 ; reverse, 536–519, 50 -gtattaccgcggctgctg-30 ; reverse, 1112–1093, 50 gttgcgctcgttgcgggact-30 and reverse, 1241–1222, 50 -gctacacacgtgctacaatg-30 ). The thermal programme consisted of 30 cycles (15 s at 96 1C, 1 sec at 351C and 4 min at 60 1C).

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Sequencing products were purified and sequenced as described previously [26]. Sequence assembly was performed with BioNumerics version 4.5 (Applied Maths, Belgium). A phylogenetic tree was constructed based on the neighborjoining method.

Nxr primer design and gene sequencing The organization of the nxrAXB cluster with indication of the primers used in this study is represented in Fig. 1. Primers were designed using the four nxrA (AF344872AF344875) and thirteen nxrB gene sequences (AY508477AY508483; L76185-L76190; X66067) available in the EMBL database at the time using the software Kodon version 2.0 (Applied-Maths, Belgium). PCR amplification of the complete nxrX gene and the 50 -end of the nxrB1 gene was performed using the primers nxrA-1F and nxrB-1R. NxrA-1F (50 -gcatggatccggtgtggatca-30 ) is situated at position 3242–3262 of the nxrA sequence of N. hamburgensis X14T (AF344872). NxrB-1R (50 -ccgtgctgttgayctcgttga-30 [y ¼ c or t]) is situated at position 470–490 of the nxrB sequence of N. hamburgensis X14T (L76186). Amplification was performed using a 25 ml PCR mixture containing 2.5 ml PCR buffer (10  ), 2.5 ml dNTP’s (2 mM each), 0.5 ml forward primer (5 mM), 0.5 ml reversed primer (5 mM), 0.5 ml AmpliTaq DNA polymerase (1 U ml1), 17.5 ml sterile MilliQ water and 1.0 ml DNA extract (50 mg ml1). The thermal profile consisted of 10 min at 95 1C, 30 cycles of 1 min at 95 1C, 1 min at 55 1C and 2 min at 72 1C, and a final elongation step of 12 min at 72 1C. The PCR products were purified using the Nucleofasts 96 PCR clean up membrane system. Sequencing reactions were performed analogous to the 16S rRNA gene sequencing reactions, using the same mixture composition and thermal profile. The complete nxrX gene was sequenced using the primers nxrX-1F (50 cgtcgtgcgcaagatggaga-30 ; position 3573–3592 of the nxrA sequence of N. hamburgensis X14T AF344872; [42]) and nxrX-1R (50 -cccggcttggtctccacgt-30 ; position 80–98 of the nxrB sequence of N. hamburgensis X14T L76186; [42]). In a few cases, the nxrX-1F primer generated sequences with ambiguous positions at the 50 -end. For these stains, the amplification primer nxrA-1F was additionally used as a sequence primer. A 380 bp fragment of the nxrB1 gene was sequenced using the primers nxrB-1F (50 -acgtggagaccaagccggg-30 ; reverse complement of nxrX-1R) and nxrB1R. Sequence assembly was performed with BioNumerics version 4.5. Phylogenetic trees were constructed based on the neighbor-joining method.

Rep-PCR Rep-PCR genomic fingerprinting was performed with the REP-, (GTG)5- and BOX-primers [43] using the PCR conditions described previously [33]. Electrophoresis was performed as described by Heyrman et al. [17].

The patterns were digitalized, UPGMA clustering of the Pearson correlation similarity values of the resulting band patterns was performed and cophenetic correlation values were calculated using BioNumerics v. 4.5. Cophenetic correlation is a parameter to express the consistency of a cluster, which is similar to the bootstrap method for sequence clusters, with values above 80 representing reliable clusters. The method calculates the correlation between the dendrogram-derived similarities and the matrix similarities.

Nucleotide sequence accession numbers The 16S rRNA gene sequences have been deposited in the GenBank/EMBL/DDBJ under accession numbers AM114522 and AM286374–AM286398. The nxrX gene sequences have been deposited in the GenBank/EMBL/ DDBJ under accession numbers AM114516–114520 and AM286349–AM286373. The nxrB1 gene sequences have been deposited in the GenBank/EMBL/DDBJ under accession numbers AM286319–AM286348.

Results 16S rRNA gene sequence analysis In total, 30 Nitrobacter strains were analysed in this study. The strain list (Table 1) comprised members of the four valid species and strains unidentified at the species level or with a preliminary taxonomic position. They were obtained from a wide range of habitats and geographical locations. A nearly complete 16S rRNA gene sequence from all strains was determined, and a phylogenetic clustering of these sequences together with reference sequences from the GenBank database is represented in Fig. 2. A 100% similarity was shown between the 16S rRNA gene sequence of N. winogradskyi ATCC 25391T ¼ Nb-255T and N. hamburgensis X14T determined in this study and those from the complete genome sequences of the respective strains ([38]; Copeland et al., unpublished). All sequences were highly (498.8%) related to each other. Based on pairwise sequence similarities and by visual analysis of the dendrogram, five groups were delineated. Group 1 contained six strains with 100% sequence similarity and included the type strain of N. winogradskyi (ATCC 25391T) and other strains of this species. Group 2 showed 99.9% sequence similarity to group 1 and contained three sequences with 100% sequence similarity, including N. winogradskyi ATCC 14123 (former type strain of ‘‘N. agilis’’). Group 3 showed 99.9% sequence similarity to groups 1 and 2, and contained the three sequenced members of N. alkalicus and strain Termite 2, which shared 99.9–100% sequence

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301

1% N. alkalicus AN2 (AF069957)* N. alkalicus AN4 (AF069958)* 62

N. alkalicus AN1T (AF069956)*

3

Nitrobacter sp. Termite2 (AM286391) N. winogradskyi ATCC 25391T (CP000115)* N. winogradskyi Engel (AM286374) 63 N.

winogradskyi F83KO (AM286377)

1

Nitrobacter sp. LIP (AM286385) N. winogradskyi R1.30 (AM286389) N. winogradskyi 311 (AM286397) N. winogradskyi ATCC 14123 (L35506)* 84

N. winogradskyi Nato I (AM286376)

2

N. winogradskyi AB3 (AM286388)

66

N. vulgaris K48 (AM286381) 71 37

N. vulgaris K55 (AM286382) N. vulgaris 329 (AM286390) N. vulgaris 339 (AM286378)

32

N. vulgaris DSM10236T (AM114522)

59

Nitrobacter sp. GT Oman (AM286379) N. vulgaris AB1 (AM286380)

60

Nitrobacter sp. 5F/3 (AM286384)

31

Nitrobacter sp. 4111/1 (AM286386)

4

Nitrobacter sp. Yukatan (AM286392) N. vulgaris C2 (AM286393) Nitrobacter sp. D6/13 (AM286394) 87

Nitrobacter sp. Io Acid (AM286395) N. vulgaris BB3 (AM286398) N. hamburgensis Y (AM286396) Nitrobacter sp. 263 (AM286383)

100 64

N. winogradskyi 219 (AM286375) 76

5

Nitrobacter sp. BS5/19 (AM286387)

N. hamburgensis X14T (NC_007964)* Rhodopseudomonas palustris DSM123T(AB175650)*

Fig. 2. Phylogenetic dendrogram based on neighbour-joining clustering after multiple alignment (1372 bp) of the 16S rRNA gene sequences of the isolates analysed in this study and reference strains from GenBank (indicated with *). Bootstrap values (expressed as percentages of 1000 replications) are shown at the branch points. Accession numbers are shown in parentheses. Rhodopseudomonas palustris DSM123T was used as an outgroup. Bar, % nucleotide substitution.

similarity to each other. Group 4 contained eleven strains sharing 99.9–100% sequence similarity and included the type strain of N. vulgaris (DSM 10236T)

and several other strains identified as belonging to this species by Bock et al. [4]. Group 4 showed 99.4–99.6% sequence similarity to groups 1–3. K48, K55 and 329,

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also belonging to N. vulgaris according to Bock et al. [4], fell outside this group. Group 5 contained three strains with intragroup sequence similarities lower than in the other groups (99.5–99.7%). Sequence similarities with strains of other groups were between 98.9% and 99.6%. The two strains of N. hamburgensis shared only 98.8% sequence similarity and were interspersed with the members of group 5.

nxrX and nxrB1 gene sequence analysis Unambiguous sequences for the complete nxrX gene (684–705 bp, depending on the strain) and a 380 bp fragment of the nxrB1 gene were obtained for all 30 Nitrobacter strains, while no amplicon was obtained for the representative of Nitrospira. Since 100% similarity was found between the nxrX and nxrB1 gene sequences determined in this study for N. winogradskyi ATCC 25391T and those obtained by Starkenburg et al. [38], our amplification and sequence analysis protocol was considered valid. As is common for protein coding genes, the nucleotide sequence similarity between strains was lower for the nxrX (ranging from 79.9% to 100%) and nxrB1 (88.5–100%) genes than for the 16S rRNA gene (95.8–100%). The phylogenetic tree for the nxrX gene (Fig. 3A) was in agreement with the tree based on the corresponding amino acid sequences (data not shown), although the relationship between the representatives of N. hamburgensis and those of group 5 showed some difference. All groups delineated in the 16S rRNA gene tree were retrieved in the nxrX dendrograms. However, due to the lower sequence similarities and higher bootstrap values, the grouping was considered to be more reliable. Alignment of the nxrX amino acid sequences showed multiple strain- or groupdependent indels in the region from amino acid position 7 to 19 (with N. winogradskyi ATCC 14123 used as a reference). For example, a sequence of six amino acids was present in N. hamburgensis Y, while this sequence was absent in N. hamburgensis X14T. At amino acid position 16, a histidine was present in all members of group 2 but was absent in the members of group 1. Furthermore, at position 216, an insert of one amino acid was present in the representatives of N. hamburgensis (threonine) and group 5 (lysine for strain 263 and valine for strains BS5/19 and 219), but it was absent in all other strains. Although some minor variability was observed in the relatedness between the different groups, the phylogenetic tree for the nxrB1 gene (Fig. 3B) was generally in agreement with the nxrX trees and the five groups could be separated from each other. On the contrary, amino acid sequences of all strains were nearly identical to each other, and therefore did not allow delineation of the different groups. The alignment of the nxrB1 amino acid sequences showed no indels.

Rep-PCR In this study, a banding profile of rep-PCR products amplified with REP-, BOX- and (GTG)5-primers was determined for all 30 strains. Banding profiles using REP-primers showed too few bands to allow reliable grouping (data not shown). An UPGMA clustering of Pearson’s correlation similarity coefficients for the combined normalized BOX- and (GTG)5 banding patterns is represented in Fig. 4. The reproducibility of the technique was analysed by replicate analysis of Nitrobacter sp. 263 (i.e. two independent PCR amplification reactions and electrophoresis on different gels). A 96% Pearson correlation similarity was shown between the replicates. All strains of group 1 were located in a cluster that could be reliably (cophenetic correlation value of 88%) separated from all other strains. The strains of group 2 grouped together, although N. winogradskyi ATCC 14123 was more distantly located. However, no good banding profile could be obtained for this strain, even after repeated attempts. Both strains of group 3 clustered together, since they showed nearly identical banding patterns. All strains of group 4 were located in a cluster that could be reliably (cophenetic correlation of 95%) separated from all other strains. As in the 16S rRNA and nxr gene analyses, strains K48, K55 and 329 fell outside group 4. The members of N. hamburgensis did not cluster separately from other species. Furthermore, the three strains of group 5 were located at distantly related positions in the dendrogram but showed low similarities compared with other strains.

Discussion Although generally a good target for studying phylogenetic relationships [41], the 16S rRNA gene lacks discriminatory power in recently evolved taxa [13]. Previous studies [30,35] concluded that the 16S rRNA gene showed a low diversity within the genus Nitrobacter. These studies were, however, based on only three sequences and even to date the public databases contain very few 16S rRNA sequences of identified Nitrobacter strains. Here, the nearly complete 16S rRNA gene sequence of a set of 30 strains comprising a wide physiological as well as ecological diversity and encompassing representatives of the four species was determined. The results showed that the 16S rRNA gene was indeed conserved within Nitrobacter. One misidentified basepair during sequencing could hence result in a completely different position of the strain in the 16S rRNA gene dendrogram. Although the species could be separated from each other, there is a need to complement the 16S rRNA gene sequence data with that of other genes to obtain more reliable phylogenetic information. Therefore, primers were developed that

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targeted the nitrite oxidoreductase genes of Nitrobacter. Unambiguous sequences were obtained for the complete nxrX gene and a 380 bp fragment of the nxrB1 gene from all strains. The nxrX and nxrB1 dendrograms were in agreement with the 16S rRNA gene dendrogram but showed a higher discriminatory power. Both nxrX and nxrB1 dendrograms were able to differentiate clearly the Nitrobacter species from each other. Thus, sequencing of the nxrX and/or nxrB1 gene can be a valuable additional tool for phylogenetic analysis within Nitrobacter and can assist in the identification of new Nitrobacter isolates. Previously, amplified ribosomal DNA restriction analysis [15] and restriction fragment length polymorphism analysis [27,28] has been applied to Nitrobacter, but without representatives of N. alkalicus [15] or N. alkalicus and N. vulgaris [27,28]. In this study, a repPCR profile was obtained for all 30 strains. The results confirmed the observation of Navarro et al. [27] that strains from different geographical areas and/or ecological niches can be genotypically very similar. For example, N. vulgaris C2, isolated from German salt water, and Nitrobacter sp. Yukatan, isolated from Mexican soil, showed 99.4% Pearson correlation similarity while the 16S rRNA and nxr genes of both strains were identical. Strains with very similar banding patterns showed identical 16S rRNA and nxr gene sequences and the rep-PCR technique can hence be applied as a fast initial method for dereplication of sets of Nitrobacter isolates. A combination of the data from the rep-PCR banding patterns and the 16S rRNA, nxrX and nxrB1 gene sequence analysis, and data from the literature provided insights into the phylogenetic relationships between different Nitrobacter strains and species, which led to the following conclusions. (1) N. winogradskyi ATCC 14123 was originally described as the only strain of a separate species, ‘‘N. agilis’’ [29]. Later, it was stated that both species could not be phenotypically separated from each other [19,31,46] and that ‘‘N. agilis’’ should hence be a subjective synonym of N. winogradskyi [44]. Although further studies showed that ‘‘N. agilis’’ could be distinguished from N. winogradskyi by immunofluorescence [12], different growth characteristics in the presence of organic matter [39] and different rRNA gene restriction patterns [27], ‘‘N. agilis’’ is currently considered as an invalid name. Based on DNA-DNA hybridization data, Navarro et al. [27] suggested that ‘‘N. agilis’’ might be a subspecies of N. winogradskyi. This current study showed that N. winogradskyi ATCC 25391T and N. winogradskyi ATCC 14123 have a 99.9% 16S rRNA gene sequence similarity and belong to different groups. In the nxrX and nxrB1 dendrogram, N. winogradskyi ATCC 14123 clustered into group 2 and could be reliably separated (bootstrap values of 100%

303

and 68%, respectively) from group 1 containing N. winogradskyi ATCC 25391T. Although phenotypic analysis is necessary to determine whether both groups can be considered as different species, 16S rRNA gene and nxr sequence analyses indicated additional genomic differences between N. winogradskyi ATCC 25391T and N. winogradskyi ATCC 14123 suggesting a separate taxonomic position at the subspecies level. (2) N. vulgaris was originally described as a new species containing 17 strains. DNA–DNA hybridization studies, however, showed DNA relatedness values between 55% and 97% [4], while 70% is considered the cut-off value for species delineation [47]. Eight strains belonging to N. vulgaris were included in this study. K48 and K55 had very similar rep-PCR patterns, and 16S rRNA and nxr gene sequences. Both strains formed a cluster reliably separated from the cluster containing the N. vulgaris type strain and from other Nitrobacter species. These data were in agreement with the DNA–DNA hybridization values of Bock et al. [4] and indicated that K48 and K55 might belong to a hitherto undescribed Nitrobacter species or N. vulgaris subspecies, although phenotypic data distinguishing them from the other N. vulgaris strains are lacking at the moment. Both strains were isolated from natural stones of historical buildings and it was previously shown that this habitat is a good niche for the isolation of new lineages of nitrifiers [5,32]. N. vulgaris 329 showed DNA relatedness values at the threshold value for species delineation compared with other strains of N. vulgaris. Also, in rep-PCR, 16S rRNA and nxr gene sequence analyses, 329 clustered outside the N. vulgaris strains and could hence belong to a separate species or a N. vulgaris subspecies. (3) N. hamburgensis was described based on two strains [6], both of which were included in this study. While DNA-DNA hybridizations showed 100% DNA relatedness [6], some dissimilarity between both strains was found with the rep-PCR profiles (87.7% similarity) as well as with nxrX (94.1%) and nxrB1 (87.0%) gene sequence analysis. Further study is necessary to elucidate the phylogenetic relationship between both strains. (4) Besides a selection of strains previously assigned to the four currently known species of Nitrobacter, this study also included strains unidentified at the species level or with a preliminary taxonomic position deduced from protein pattern analysis [22]. The combination of rep-PCR, 16S rRNA and nxr gene sequences allowed presumptive allocation at the species level. Strain LIP was highly related to members of N. winogradskyi, while strain Termite 2 most probably belongs to N. alkalicus. Stains 5F/3, Gt Oman, Io Acid, D6/13, 4111/1 and Yukatan were provisionally assigned to N. vulgaris. Our results counter the conclusion of Seewaldt et al. [35] that strain Yukatan can be treated as belonging to N. winogradskyi and were generally in agreement with

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Krause-Kupsch [22], except for strains Termite2 and 4111/1. The phylogenetic position of strain 311 differs between the 16S rRNA and the nxr dendrograms and further studies are necessary to elucidate its exact

taxonomic position at the species level. Of particular interest for further study might be group 5, since 16S rRNA and nxr gene sequence analysis showed that strains 263, BS5/19 and 219 are distantly related to the

10% N. winogradskyi ATCC 25391T (AM114520) N. winogradskyi Engel (AM286349) N. winogradskyi F83KO (AM286352) 100

1

Nitrobacter sp. LIP (AM286360) N. winogradskyi R1.30 (AM286364)

100

N. winogradskyi Nato I (AM286351) 100

N. winogradskyi ATCC 14123 (AM114519)

100 100

2

N. winogradskyi AB3 (AM286363)

N. alkalicus AN2 (AM114516)

3

100

Nitrobacter sp. Termite2 (AM286366) N. winogradskyi 311 (AM286372)

72

N. vulgaris 329 (AM286365) 88

N. vulgaris K55 (AM286357) 58 100

N. vulgaris K48 (AM286356)

Nitrobacter sp. 5F/3 (AM286359)

77

Nitrobacter sp. Io Acid (AM286370) 76 100

Nitrobacter sp. D6/13 (AM286369)

21 54

Nitrobacter sp. GT Oman (AM286354)

49

N. vulgaris 339 (AM286353)

37

N. vulgaris AB1 (AM286355)

100

4

N. vulgaris DSM10236T(AM114518) Nitrobacter sp. 4111/1 (AM286361) Nitrobacter sp. Yukatan (AM286367)

100

N. vulgaris C2 (AM286368) N. vulgaris BB3 (AM286373) N. hamburgensis X14T (AM114517) 27

N. hamburgensis Y (AM286371) Nitrobacter sp. 263 (AM286358) 91

Nitrobacter sp. BS5/19 (AM286362) 99

5

N. winogradskyi 219 (AM286350)

T

Nitrococcus mobilis Nb-231 (ZP_01125622)

Fig. 3. Phylogenetic dendrogram based on neighbour-joining clustering after multiple alignment of the (A; this page) nxrX (702 bp) and (B; next page) nxrB1 (380 bp) gene sequences of the isolates analysed in this study. Bootstrap values (expressed as percentages of 1000 replications) are shown at the branch points. Accession numbers are shown in parentheses. Trees were rooted using (A) a Nitrococcus mobilis Nb-231T nxrX homologue or (B) a Nitrococcus mobilis Nb-231T nxrB homologue. Bar, % nucleotide substitution.

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305

10% N. winogradskyi ATCC 25391T (AM286323) N. winogradskyi Engel (AM286324) N. winogradskyi F83KO (AM286327) 100 N.

1

winogradskyi R1.30 (AM286339)

Nitrobacter sp. LIP (AM286335) 68

N. winogradskyi Nato I (AM286326) 100

N. winogradskyi ATCC 14123 (AM286322)

48 97

2

N. winogradskyi AB3 (AM286338)

N. winogradskyi 311 (AM286347) 39

Nitrobacter sp. Termite2 (AM286341) 100 51

3

N. vulgaris 329 (AM286340)

86

N. vulgaris K48 (AM286331)

100

81

N. alkalicus AN2 (AM286319)

N. vulgaris K55 (AM286332) Nitrobacter sp. 5F/3 (AM286334)

84

Nitrobacter sp. GT Oman (AM286329) Nitrobacter sp. Io Acid (AM286345) 88

85

N. vulgaris 339 (AM286328) 73

Nitrobacter sp. D6/13 (AM286344)

43

78

100

N. vulgaris DSM10236T(AM286321)

4

N. vulgaris AB1 (AM286330) 88

Nitrobacter sp. 4111/1 (AM286336)

100 Nitrobacter

sp. Yukatan (AM286342)

N. vulgaris C2 (AM286343) 100

N. vulgaris BB3 (AM286348) N. hamburgensis X14T (AM286320) 72

N. hamburgensis Y (AM286346) Nitrobacter sp. 263 (AM286333)

48

N. winogradskyi 219 (AM286325) 61

5

Nitrobacter sp. BS5/19 (AM286337)

Nitrococcus mobilis Nb-231T(ZP_01125873)

Fig. 3. (Continued)

strains of all currently known Nitrobacter species, and they might hence belong to another species. In contrast to Watson (unpublished), our results indicate that 219 does not belong to N. winogradskyi. Immunological analysis showed that the nitrite oxidizing system (NOS) from Nitrospira and Nitrospina differs from the NXR enzyme of Nitrobacter [2,37]. No sequence information for the NOS of Nitrospira and

Nitrospina is currently available. The nxr primers were hence solely based on Nitrobacter sequences and no amplicon was obtained for Nitrospira moscoviensis NCIMB 13793T. During the course of this research, a complete genome sequence of Nitrococcus mobilis Nb231T was deposited in GenBank. The genome did not show the nxrAXB gene cluster found in Nitrobacter and the nxrA and nxrB genes of N. mobilis Nb-231 and N.

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N. . vulgaris K48

95 100

N. . vulgaris K55

97

N. . hamburgensis X14T

89

. hamburgensis Y N.

100

89

Box

100

(GTG)5

90

85

80

% Pearson correlation

. Nitrobacter sp. 311

87

. vulgaris 329 N. . Nitrobacter sp. 219 100 92 97

83

. vulgaris BB3 N. . vulgaris C2 N. . Nitrobacter sp. 4111/1

99

. vulgaris AB1 N.

98

. vulgaris DSM 10236T N. 96

4

. vulgaris 339 N.

100

83 95

5

. Nitrobacter sp. Yukatan

. Nitrobacter sp. D6/13

99

. Nitrobacter sp. Io Acid 95

. Nitrobacter sp. GT Oman . Nitrobacter sp. 5F/3

76

100

. Nitrobacter sp. 263

5

. winogradskyi Nato I N.

2

. Nitrobacter sp. AB3

85 100

90

. Nitrobacter sp. Termite2 . alkalicus AN2 N. . winogradskyi ATCC 14123 N.

77

88

100 69 92 82

3 2

. winogradskyi Engel N. . winogradskyi F83KO N. . winogradskyi R1.30 N.

1

. Nitrobacter sp. LIP . winogradskyi ATCC25391T N. Nitrobacter sp. BS5/19 .

5

Fig. 4. Grouping of combined normalized BOX- and (GTG)5-patterns of the isolates in a dendrogram based on UPGMA clustering of Pearson’s correlation similarity coefficients. Cophenetic correlation (i.e. a parameter to express the consistency of a cluster) values are shown at the branch points.

winogradsky Nb-255T showed, respectively, a relatedness of 69.3% and 69.0%. When more complete genome sequences of NOB become available, it will be clearer whether primers amplifying the NOS of all currently known genera of NOB can be developed.

Conclusions In conclusion, the data in this study gave insights into the phylogenetic diversity and relationships in Nitrobacter and indicated that taxonomic re-evaluations

might be necessary. At the moment, the phenotypic differences between different Nitrobacter species and strains are not well investigated. A thorough knowledge of the phylogenetic relatedness between strains may lead to a well-founded choice of representative isolates for comparative phenotypic studies of this environmentally important group. Furthermore, the newly developed nxr primers were able to amplify successfully a large variety of Nitrobacter strains. Nxr gene-based dendrograms were largely in agreement with the 16S rRNA gene tree, but showed a better discriminatory power. These primers could potentially be suitable tools for the

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culture independent monitoring of Nitrobacter in environmental samples.

Acknowledgements This work was supported by Project Grant G.O.A. 1205073 (2003-2008) from the ‘Ministerie van de Vlaamse Gemeenschap, Bestuur Wetenschappelijk Onderzoek’ (Belgium), the FWO project G20156.02 and a Ph.D. Grant (no. 41428) from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). We thank Dr. D. Sorokin (Delft University of Technology, The Netherlands), Dr. P. Bodelier (Netherlands Institute of Ecology, The Netherlands) and Zena Smith (NCIMB Ltd., UK) for supply of biological material.

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