SGM Journals
Proteobacteria

Nitratireductor aquibiodomus gen. nov., sp. nov., a novel α-proteobacterium from the marine denitrification system of the Montreal Biodome (Canada)

Normand Labbé, Serge Parent, Richard Villemur

  • 1INRS – Institut Armand-Frappier, 531 Boul. des Prairies, Laval, Canada H7V 1B7
  • 2Biodôme de Montréal, 4777 Pierre-De Coubertin, Montreal, Canada H1V 1B3
  • Correspondence
    Richard Villemur
    richard.villemur{at}inrs-iaf.uquebec.ca

    • International Journal of Systematic and Evolutionary Microbiology 2004; 54(1):269–273 · https://doi.org/10.1099/ijs.0.02793-0

    View at publisher PubMed

    Abstract

    The Montreal Biodome operates a methanol-fed denitrification system that treats the water in its three million litre marine mesocosm. An unknown bacterium, named strain NL21T, was isolated from this system on TSA and R2A agar. The organism is a Gram-negative, rod-shaped (1×3 μm) facultative aerobe. Optimal growth conditions on R2A agar are 30–35 °C, pH 7–7·5 and 1 % (w/w) NaCl. Phylogenetic analysis of the 16S rDNA sequence reveals that strain NL21T forms a novel lineage in the family ‘Phyllobacteriaceae’ within the α2 subgroup of the Proteobacteria. The closest related genera are Aminobacter, Pseudaminobacter, Mesorhizobium and Defluvibacter. Major cellular fatty acids are C18 : 1ω7c (75 %), C19 : 0ω8c cyclopropane (9·4 %) and C18 : 0 (4·2 %). The DNA G+C content of strain NL21T (57 mol%) differs from those of all other described members of the ‘Phyllobacteriaceae’ (60–64 mol%). Strain NL21T reduces nitrate to nitrite, but does not reduce nitrite to nitrogen gas. Only a few sugars and amino acids can serve as carbon sources. Strain NL21T is able to grow without salt and tolerates up to 5 % NaCl. Phylogenetic analysis, as well as physiological and biochemical tests, showed that strain NL21T was different from all other members of the ‘Phyllobacteriaceae’ with validly published names. Strain NL21T therefore represents a novel genus, for which the name Nitratireductor aquibiodomus gen. nov., sp. nov. is proposed, with the type strain NL21T (=DSM 15645T=ATCC BAA-762T).

    • Published online ahead of print on 12 September 2003 as DOI 10.1099/ijs.0.02793-0.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NL21T is AF534573.

    Nitrate is a pollutant that accumulates quickly in closed systems, such as marine aquariums. It is usually removed biologically in a denitrification system, where oxygen is replaced by nitrate as an electron acceptor in bacterial respiration. A large variety of bacterial species are able to reduce nitrate to nitrite or to molecular nitrogen [see Zumft (1997) for an exhaustive list]. Many of these species belong to the family ‘Phyllobacteriaceae’ of the α-Proteobacteria, which comprises the genera Phyllobacterium (Knösel, 1984), Aminobacter (Urakami et al., 1992), Mesorhizobium (Jarvis et al., 1997), Pseudaminobacter (Kämpfer et al., 1999), Defluvibacter (Fritsche et al., 1999) and Aquamicrobium (Bambauer et al., 1998). This family was originally described by Knösel (1984). It may also contain the uncharacterized strain B1G-2, which was isolated by Duncan et al. (2001), and the Mesorhizobium strain WG, which was isolated from a denitrification process by Costa et al. (2000).

    During the characterization of organisms that were isolated from the marine denitrification system of the Montreal Biodome (Labbé et al., 2003), strain NL21T was recovered on R2A and TSA agar. Sequence analysis of the 16S rRNA gene (rDNA) revealed that this bacterium was related to the family ‘Phyllobacteriaceae’. The goal of the present study was to further characterize strain NL21T and to classify it within the family ‘Phyllobacteriaceae’.

    Gram-staining was performed as described by Gerhardt et al. (1994). Cell morphology was observed under a Nikon light microscope at ×1000 with cells that were grown for 3 days at 35 °C. Physiological characteristics were investigated by using API systems: API 50CH strips inoculated with the medium described by Velázquez et al. (2001) were used for acid production and sugar assimilation, API 20NE strips were used for biochemical reactions (nitrate and nitrite reduction, urease and indole formation) and the assimilation of selected carbon sources and API ZYM strips were used to examine extracellular enzyme activity. Strips were incubated at 30 °C for 24 h (API ZYM) or 72 h (API 50CH and API 20NE). Strain NL21T differs from most members of the family ‘Phyllobacteriaceae’ by being positive for citrate assimilation, negative for urease and positive for the presence of N-acetyl-β-glucosamidase (Table 1⇓). Other differences in substrate assimilation between NL21T and other representatives of the family ‘Phyllobacteriaceae’ are shown in Table 1⇓.

    Table 1.

    Physiological characteristics of the family ‘Phyllobacteriaceae’

    Taxa: 1, strain NL21T; 2, Pseudaminobacter salicylatoxidans DSM 6986T; 3, Aminobacter aminovorans DSM 7048T; 4, Phyllobacterium myrsinacearum LMG 2t2T; 5, Mesorhizobium tianshanense DSM 11417T; 6, Defluvibacter lusatiensis DSM 11099T [data from Lechner et al. (1995) and Fritsche et al. (1999)]. All strains are positive for assimilation of d-glucose, d-arabinose, d-fructose, ribose, d-xylose and l-fucose and the presence of alkaline phosphatase, acid phosphatase, leucine arylamidase, cysteine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase and α-glucosidase. All strains are negative for indole formation, gelatinase, assimilation of caprate, phenylacetate, salicin, inulin, starch and erythritol and the presence of α-galactosidase, β-glucuronidase, α-mannosidase, α-fucosidase and lipase C14. +, Positive reaction; (+), weakly positive reaction; −, negative reaction; v, variable; nd, not determined.

    The fatty acid profile was traced with cells that were grown on TSA agar for 3 days at 35 °C. Analysis was carried out in accordance with the standard protocol of the Microbial Identification system (MIDI; Microbial ID). Major cellular fatty acids of strain NL21T are C18 : 1ω7c (75 %), C19 : 0ω8c cyclo (9·4 %) and C18 : 0 (4·2 %). This profile supports the affiliation of strain NL21T to the ‘Phyllobacteriaceae’, in which most species have C18 : 1ω7c and C19 : 0ω8c cyclo as major components. The fatty acid profile of strain NL21T (Table 2⇓) differs from those of all species of the genus Mesorhizobium (Tighe et al., 2000) because of the absence of 11-methyl C18 : 1ω7c. The two genera also have different hydroxy fatty acids. Compared to the genus Pseudaminobacter, strain NL21T has a lower amount of C17 : 0 and C19 : 0ω8c cyclo and a higher amount of C18 : 1ω7c. Compared to the genus Aminobacter, strain NL21T has a higher amount of C18 : 1ω7c, fewer unknown fatty acids and different hydroxylated fatty acids (Table 2⇓). Compared to the genus Phyllobacterium, strain NL21T has 17-carbon fatty acids (C17 : 0, C17 : 1, iso-C17 : 0 and C17 : 0 cyclo) and different hydroxy fatty acids. Finally, it differs from the genus Defluvibacter in that it lacks the fatty acids C16 : 1ω7c, C20 : 1ω7c, C12 : 0 3-OH and 10-methyl C19 : 0. Additional differences in fatty acid profiles are shown in Table 2⇓.

    Table 2.

    Fatty acid profiles of bacteria in the family ‘Phyllobacteriaceae’

    Taxa: 1, Nitratireductor aquibiodomus NL21T; 2, Pseudaminobacter salicylatoxidans DSM 6986T (data from Kämpfer et al., 1999); 3, Aminobacter aminovorans DSM 7048T (data from Kämpfer et al., 1999); 4, Phyllobacterium myrsinacearum LMG 2t2T (data from Mergaert et al., 2002); 5, Mesorhizobium tianshanense (data from Tighe et al., 2000); 6, Defluvibacter lusatiensis DSM 11099T (data from Lechner et al., 1995).

    DNA extraction was performed by dispersing colonies of strain NL21T in 250 μl TEN buffer (50 mM Tris/HCl, pH 8·0; 10 mM EDTA, pH 8·0; and 150 mM NaCl) and then mixing them with 250 mg sterilized 0·4–0·5 mm glass beads (B. Braun Melsungen). The DNA pellet was prepared as described by Labbé et al. (2003) and was then washed with 70 % ethanol and resuspended in 50 μl water.

    The DNA G+C content of strain NL21T was determined as described by Mesbah et al. (1989) by using two 15 cm C18 columns. λ-Phage DNA was used for calibration, whilst salmon sperm served as a control. The DNA G+C content of strain NL21T was lower than those of other characterized members of the family ‘Phyllobacteriaceae’ (57 vs 60–64 mol%). The 16S rDNA sequence of NL21T was determined as reported previously (Labbé et al., 2003) (GenBank accession no. AF534573); both DNA strands of the resulting PCR product were sequenced.

    Sequences were analysed by using BioEdit software, version 5.0.9.1 (). Comparisons of bacterial sequences in gene databases were done with the blastn program (National Center for Biotechnology Information; ). The clustalw 1.4 program (included in the BioEdit software package) was used to align sequences. Phylogenetic analyses were carried out with software applications from the phylip package, version 3.5 (). Bootstrap values (1000 replicates) were derived by using the seqboot program. The dnadist program was used to generate a distance matrix for each bootstrap replicate. Each number in the matrix represented the distance between a pair of sequences. The fitch program was used to calculated a tree according to the Fitch–Margoliash algorithm. Lastly, the consense program was used to derive consensus trees. Strain designations and GenBank accession numbers for 16S rDNA sequences of reference strains are given in Fig. 1⇓.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic analysis of 16S rDNA sequences. Evolutionary distances among 16S rDNA of strain NL21T and all species of the family ‘Phyllobacteriaceae’ with validly published names are illustrated (Hyphomicrobium denitrificans was used as the outgroup species). The tree was inferred from a matrix of pairwise distances by using 1400 nt (dnadist). The fitch program was used to derive the best phylogenetic tree for each replicate. Lastly, the consense program was used to derive the consensus tree. Numbers indicate percentages of 1000 bootstrap resamplings; only values >50 % are shown. Bar, 0·01 nucleotide substitution per site.

    Representative members of the family ‘Phyllobacteriaceae’ showed 16S rDNA sequence similarity values of approximately 95 % with strain NL21T. The closest species with validly published names to strain NL21T were Mesorhizobium tianshanense and Mesorhizobium chacoense (95·1 %). However, phylogenetic analysis showed that strain NL21T is not related closely to any genus within the family ‘Phyllobacteriaceae’ (Fig. 1⇑). Parsimony and neighbour-joining analyses were also done with phylip software (dnapars and NJ). These two methods produced phylogenetic trees that were similar to the consensus tree. Sequence similarity analysis also revealed that strain NL21T shows 99·8 % similarity (only three different nucleotides) to strain TUT1018 (GenBank accession no. AB098586), which was isolated from a biowaste sequence-batch composting system (Hiraishi et al., 2003).

    Description of Nitratireductor gen. nov.

    Nitratireductor (Ni.tra.ti.re.duc′tor. N.L. n. nitras nitrate; L. v. reducere to bring back, reduce; N.L. masc. n. Nitratireductor nitrate-reducing bacterium).

    Gram-negative rods, 1 μm in diameter and 2–3 μm in length. Multiplies by budding. Cells are pleomorphic in rapid growth, motile and oxidase- and catalase-positive. Colonies on R2A agar are white, 2–3 mm in diameter, smooth, circular and convex. Optimum growth conditions are 30–35 °C and pH 7–7·5. No growth occurs at pHs lower than 7. Major fatty acids are C18 : 1ω7c (75 %), C19 : 0ω8c cyclo (9·4 %) and C18 : 0 (4·2 %). DNA G+C content is 57 mol%. Physiological characteristics are shown in Table 1⇑. Phylogenetically, the genus is a member of the α-subclass of the Proteobacteria. The type species is Nitratireductor aquibiodomus.

    Description of Nitratireductor aquibiodomus sp. nov.

    Nitratireductor aquibiodomus (a.qui.bi.o.do′mus. L. fem. n. aqua water; N.L. fem. n. biodomus Biodome; N. L. gen. n. aquibiodomus of the water of the Montreal Biodome).

    Description is the same as that given for the genus. Cells can reduce nitrate to nitrite, but not nitrite to nitrogen gas. NaCl is not required for growth, but 1 % NaCl stimulates growth. Strain NL21T grows at 0–50 g NaCl l−1. Physiological characteristics are shown in Table 1⇑.

    The type strain is NL21T (=DSM 15645T=ATCC BAA-762T). Isolated from the marine denitrification system of the Montreal Biodome, Canada.

    Acknowledgments

    This research was supported by a grant to R. V. from the Natural Sciences and Engineering Research Council of Canada, by funding from the Montreal Biodome and by a scholarship to N. L. from the INRS – Institut Armand-Frappier. We are grateful to Encarna Velázquez for providing Phyllobacterium myrsinacearum and to Serge Messier for fatty acid analysis. Brian Colwill kindly revised the English style and grammar of this paper.

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