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Isolation and Characterization of a New Denitrifying Spirillum. Capable of Anaerobic Degradation of Phenol. YOSHIFUMI SHINODA,1 YASUYOSHI SAKAI,1 ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2000, p. 1286–1291 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 4

Isolation and Characterization of a New Denitrifying Spirillum Capable of Anaerobic Degradation of Phenol ´ ,1 AKIRA HIRAISHI,2 YOSHIFUMI SHINODA,1 YASUYOSHI SAKAI,1 MAKIKO UE

AND

NOBUO KATO1*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,1 and Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441-8580,2 Japan Received 25 October 1999/Accepted 6 January 2000

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under ironlimited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the ␣ subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features. Bacterial degradation of aromatic compounds under anaerobic conditions has been intensely studied, especially in the past decade. These reactions are of great interest, because unlike aerobic degradation by (di)oxygenases, alternative strategies are required, for example, to dearomatize and cleave a chemically stable benzene moiety without the use of molecular oxygen. Various kinds of bacteria or bacterial consortia are now known to degrade aromatic compounds under anaerobic conditions (9). In many cases, the compounds are converted into benzoyl coenzyme A (benzoyl-CoA) as a common central intermediate (peripheral pathways) and then degraded further into acetyl-CoA (benzoyl-CoA pathway) (8). Most denitrifying aromatic compound-degrading bacteria that have been isolated so far are Thauera (1) or Azoarcus (26) species which are members of the ␤ subclass of the class Proteobacteria (9). Bakker (3) and Khoury et al. (13) described spiral bacteria in mixed cultures that degraded phenol under denitrifying conditions but did not characterize these organisms. In this paper, we describe two isolates of denitrifying phenol-degrading bacteria obtained from the same enrichment culture during 3 years of incubation. A rod-shaped bacterium, strain CC-11, belongs to the genus Azoarcus, but the other isolate, which has spiral cells, is phylogenetically most closely related to strains belonging to the genus Magnetospirillum. The latter strain, designated CC-26, is the first example of a denitrifying aromatic compound-degrading bacterium that belongs to the ␣ subclass of the Proteobacteria.

with 0.1 M HCl; and dilution rate, 0.0067 h⫺1. The enrichment medium in the fermentor was inoculated with a suspension of soil obtained from a rice paddy and bubbled gently with pure nitrogen gas (99.999% pure; Sumitomo-seika Chemicals Co., Ltd., Osaka, Japan). When there was a biofilm on the surface of the flask (at intervals of 2 to 6 months), part of the enrichment culture was transferred to fresh medium and enriched in the same way. We attempted to isolate denitrifying phenol-degrading bacteria twice, once after 1 year of enrichment and once after 3 years of enrichment. An aliquot of the 1-year-old culture was diluted in basal medium (24) and then plated onto the enrichment medium containing 2% Noble agar (Difco Laboratories, Detroit, Mich.) and phenol as the sole carbon source. After 2 weeks of anaerobic incubation at 30°C, some of the small colonies that formed on the agar surface could grow on phenol under denitrifying conditions. Strain CC-11 was isolated from one of these cultures by repeated plating on nutrient agar (Difco Laboratories) with aerobic incubation. After 3 years of enrichment, part of the culture was transferred to an anaerobic culture tube (Bellco Glass Inc., Vineland, N.J.) and cultivated batchwise in the enrichment medium. After six transfers, the batch culture was diluted with the basal medium, spread onto a nutrient agar plate, and then incubated aerobically at 30°C. Small colonies which appeared after 10 days of incubation were able to grow on phenol anaerobically. Strain CC-26 was isolated from one of these cultures in the same way that strain CC-11 was isolated. Cultivation and growth medium. Strains CC-11 and CC-26 were maintained aerobically on nutrient agar slants containing KNO3 (5 mM) and were transferred every 1 or 2 weeks. The growth media used were prepared by using mineral base E (7, 18) for strain CC-11 and the enrichment medium described above for strain CC-26, eliminating components which did not affect anaerobic growth of the strains on phenol, and adding ferrous sulfate to the latter medium. The resulting compositions of the media were as follows. The growth medium used for strain CC-11 contained (per liter) 0.5 g of (NH4)2SO4, 1.04 g of K2HPO4, 0.75 g of KH2PO4, 0.033 g of CaCl2 䡠 2H2O, 0.2 g of MgSO4 䡠 7H2O, 15 mg of disodium EDTA, 1.5 mg of FeSO4 䡠 7H2O, 0.48 mg of CoCl2 䡠 6H2O and 0.45 mg of Na2MoO4 䡠 2H2O, and the growth medium used for strain CC-26 contained (per liter) 1.6 g of Na2HPO4, 1.0 g of KH2PO4, 0.5 g of NH4Cl, 0.06 g of K2SO4, 0.025 g of CaCl2 䡠 2H2O, 0.1 g of MgCl2 䡠 6H2O, 15 mg of disodium EDTA, 1.5 mg of FeSO4 䡠 7H2O, 20 ␮g of vitamin B12, and 50 ␮g of paminobenzoate. The substrates used were the same substrates that were used in the enrichment medium described above. The pH values of the media were 7.0 to 7.1. Aerobic cultivation was conducted with free gaseous exchange with the atmosphere. Microaerobic cultivation was conducted in sealed culture vessels under a gas mixture containing N2 (98%) and O2 (2%). The inoculated media were shaken reciprocally under both conditions. An anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, Mich.) was used for anaerobic cultivation. Each medium was equilibrated inside the chamber for 1 to 2 days before use in order to remove dissolved oxygen (2). Ascorbate (5 mM) was used to reduce the media, and reduction was confirmed by complete decolorization of resazurin (2 ␮g/ml) in the media. The inoculated media were cultivated in sealed culture vessels without shaking, and the gaseous phase in the vessels was N2 containing ca. 5% H2. All cultures were incubated at 30°C.

MATERIALS AND METHODS Enrichment and isolation. A bacterial consortium that degraded phenol under denitrifying conditions was enriched in a 2-liter jar fermentor (Mitsuwa Scientific Corp., Osaka, Japan) that was continuously fed with phenol as the sole carbon source. The enrichment medium was the medium of Tschech and Fuchs (24) containing phenol (1 mM), NaHCO3 (5 mM), and KNO3 (5 mM) as substrates. The fermentor was aseptically run under the following conditions: working volume, 1.5 liters; stirring at 500 rpm; temperature, 30°C; pH 7.2 to 7.4, controlled

* Corresponding author. Mailing address: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-753-6385. Fax: 81-75-753-6385. E-mail: [email protected]. 1286

VOL. 66, 2000 Growth of a liquid culture was monitored by measuring the optical density at 610 nm of samples with a spectrophotometer (model 101 with a test tube holder or model 220A; Hitachi Ltd., Tokyo, Japan). Growth substrates. Various carbon compounds were tested to determine whether they supported growth of each strain under aerobic, anaerobic (denitrifying), and microaerobic (only strain CC-26) conditions. The media used were mineral base E for strain CC-11 and the enrichment medium described above for strain CC-26. KNO3 was omitted under aerobic and microaerobic conditions. The concentration of each compound was 0.6 to 3 mM depending on its toxicity and the number of carbon atoms in its molecule. Cells grown anaerobically on phenol to the late exponential phase were used as inocula. All cultivations were performed in duplicate, and the results were judged by determining the optical density at 610 nm of each culture after 10 days. The purity of a culture that exhibited growth was checked microscopically. Magnetotaxis. Strain CC-26 was cultivated under aerobic, microaerobic, and anaerobic conditions with various iron sources added to the enrichment medium at concentrations of 10 and 50 ␮M. Acetate (3 mM) was used as the carbon source. The following iron chelates were tested (5, 16): ferric catechol, ferric chloride (without a chelating agent), ferric citrate, ferric L-3,4-dihydroxyphenylalanine ferric EDTA, ferric L-epinephrine, ferric gallate, ferric malate, ferric protocatechuate, and ferric quinate. The magnetic response of the cells was determined by microscopy during the exponential phase of growth. Magnetic selection (5) was also used to study strain CC-26. This strain was cultivated microaerobically and anaerobically in the growth medium used for Magnetospirillum magnetotacticum MS-1 (5) supplemented with acetate (3 mM) as a carbon source. A small permanent magnet was attached to the culture vessel, and after growth became evident, an aliquot of the culture close to the magnet was transferred to fresh medium. This procedure was repeated several times. Chemical analysis. The concentrations of phenol, nitrate, and nitrite were determined by high-performance liquid chromatography performed with a UV detector (model LC-10A type PIA; Shimadzu Corp. Kyoto, Japan). Phenol was detected at 275 nm on a Wakosil 5C18 HG column (150 by 4.6 mm; Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 30°C. The solvent system used was 0.1% H3PO4–methanol (50:50) at a flow rate of 1.0 ml/min. The nitrate and nitrite concentrations were measured at 210 nm on a TSKgel IC-Anion PW column (50 by 4.6 mm; Tosoh Corp., Tokyo, Japan) at 40°C. The eluent used contained 1.3 mM potassium gluconate, 1.3 mM sodium tetraborate, 30 mM borate, 10% (vol/vol) acetonitrile, and 0.5% (vol/vol) glycerol, and the flow rate was 1.2 ml/min. The culture fluid was centrifuged at 15,000 ⫻ g for 10 min, and 10 ␮l of the diluted supernatant was analyzed. The retention times of phenol, nitrate, and nitrite were 3.7, 5.8, and 3.8 min, respectively. The N2 gas content was measured by gas chromatography with a thermal conductivity detector (TCD) (model GC-7A; Shimadzu Corp.). The headspace of each culture vessel was replaced with He gas prior to cultivation, and samples (100 ␮l) were injected onto a Molecular Sieve 13X 60/80 mesh column (2.1 m by 2.6 mm; GL Sciences Inc., Tokyo, Japan) by using He as the carrier gas (40 ml/min). The column, injector, and detector temperatures were all 50°C. The TCD filament current was 100 mA. The retention time of N2 gas was 1.4 min. Electron micrography. Phenol-grown cells in the late exponential phase were fixed with glutaraldehyde (final concentration, 3%). Washed cell suspensions were stained with 2% (wt/vol) uranyl acetate and photographed with a model JEM-2000ES transmission electron microscope (JEOL Ltd., Tokyo, Japan) at an accelerating potential of 100 kV. Phylogenetic analysis. The 16S ribosomal DNA (rDNA) was selectively amplified by PCR from cell lysates of the strains (10, 11) by using oligodeoxynucleotide primers fD1 and rD1 or rP2, which were designed to anneal to conserved regions of the eubacterial 16S rDNA (25). The purified PCR products were used as templates for direct sequencing of 16S rDNA (10). Six oligodeoxynucleotide primers corresponding to the complements of positions 536 to 518, 821 to 803, 1111 to 1093, 1406 to 1389, 1094 to 1112, and 907 to 926 (Escherichia coli 16S rRNA numbering) were used as primers. Dideoxy sequencing reactions were performed with a SequiTherm Long Read Cycle sequencing kit with 7-deazadGTP (Epicentre Technologies Corp., Madison, Wis.) as specified by the manufacturer. Automated electrophoresis and analysis of DNA sequence reactions were performed with an ALFexpress DNA sequencer (Amersham Pharmacia Biotech AB, Uppsala, Sweden) or an automated fluorescence DNA sequencer (model DSQ-1000L; Shimadzu Corp.). Sequence analyses were performed by using the DNA Data Bank of Japan (DDBJ) website (http://www.ddbj.nig.ac.jp). Related sequences were obtained by using the FASTA 3.0 search program (19). The sequences were aligned and phylogenetic distances were calculated by using the CLUSTAL W algorithm (23), and phylogenetic trees were drawn by using the Tree View program (version 1.5.3; Division of Environmental and Evolutionary Biology, Institute of Biomedical and Life Sciences, University of Glasgow [http://taxonomy.zoology.gla.ac.uk/rod/treeview.html]). Taxonomic studies were also performed by workers at NCIMB Japan Co., Ltd. (Shizuoka, Japan). Nucleotide sequence accession numbers. The 16S rDNA sequences of strains CC-11 and CC-26 have been deposited in the DDBJ database under accession no. AB033745 and AB033746, respectively. The accession numbers of other nucleotide sequences used to determine phylogenetic relationships among the strains are as follows: Rhodospirillum fulvum, M59065; Agrobacterium tumefa-

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FIG. 1. Phylogenetic positions of strains CC-11 and CC-26 in the class Proteobacteria based on 16S rDNA sequence comparisons. Anacystis nidulans was used as an outgroup. Bar ⫽ 1 nucleotide substitution per 1,000 nucleotides.

ciens, M11223; Rhodobacter sphaeroides, X53854; Azoarcus tolulyticus Tol-4, L33694; Azoarcus indigens VB32, L15531; Pseudomonas testosteroni, M11224; E. coli, V00348; Desulfovibrio desulfuricans, M34113; Anacystis nidulans, X03538; M. magnetotacticum MS-1, Y10110; Magnetospirillum gryphiswaldense MSR-1, Y10109; Magnetospirillum sp. strain AMB-1, D17514; Magnetospirillum sp. strain MGT-1, D17515; Magnetospirillum sp. strain MSM-3, Y17389; Magnetospirillum sp. strain MSM-4, Y17390; and Magnetospirillum sp. strain MSM-6, Y17391.

RESULTS Enrichment and isolation. An enrichment culture that degraded phenol under denitrifying conditions was established in a continuous-feed fermentor by using soil from a rice paddy as the source of microorganisms. Rod-shaped bacteria were dominant in this mixed culture, and a phenol-degrading denitrifier, strain CC-11, was isolated after 1 year of enrichment. After 3 years of operation, however, actively motile spirilla were observed, and these organisms constituted about one-quarter to one-third of the total bacterial population. In an anaerobic batch culture of this consortium containing phenol as the sole carbon source these spirilla were dominant. On nutrient agar plates inoculated with this batch culture, many colonies of rod-shaped bacteria were observed during the first 3 days of incubation. Although we expected that strain CC-11 would be reisolated after the procedure described above, none of the colonies grew on phenol anaerobically. Colonies that contained spiral bacteria were less than 1 mm in diameter and appeared after 10 days of incubation. The strain isolated was designated strain CC-26. Phylogenetic analysis and identification. Almost complete sequences of the 16S rDNA of strain CC-11 (1,458 bases) and strain CC-26 (1,452 bases) were determined. The taxonomic positions of the strains inferred from these sequences are shown in Fig. 1, which shows that strains CC-11 and CC-26 belong to the ␤ and ␣ subclasses of the class Proteobacteria, respectively. The 16S rDNA of strain CC-11 exhibited more than 99% homology with the 16S rDNA of Azoarcus sp. strains (22), especially strain T (14) and strains Td-17 and Td-15 (22, 26). Thus, strain CC-11 was classified as a member of this taxon, whereas the nucleotide sequence of strain CC-26 exhibited 94.5 to 98.4% homology with the nucleotide sequences of Magnetospirillum spp. An unrooted phylogenetic tree based on a comparison of the sequences is shown in Fig. 2. Cell morphology and bacteriological properties. Strain CC-11 had rod-shaped cells that were 1 to 2 ␮m long and 0.5 ␮m in diameter, some of which had one polar flagellum. The

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FIG. 2. Phylogenetic relationship of strain CC-26 and previously described strains of Magnetospirillum species based on 16S rDNA sequence comparisons. The numbers are bootstrap values for branches based on 1,000 replicates. Bar ⫽ 1 nucleotide substitution per 10,000 nucleotides.

cells were gram negative, catalase negative, and oxidase positive. The cells of strain CC-26 were helical and were 2 to 5 ␮m long and 0.4 to 0.5 ␮m in diameter (Fig. 3). The helix was clockwise, and each cell had a single flagellum at each end. In

an old culture of the strain, however, coccoid bodies were observed. These morphological features are similar to morphological features of M. magnetotacticum MS-1 (4) and other strains belonging to the genus Magnetospirillum. The strain CC-26 cells were gram negative, catalase negative, and oxidase positive, like Magnetospirillum sp. strain AMB-1 cells (16). Growth on various carbon sources. Table 1 shows the growth of strains CC-11 and CC-26 on various carbon sources under aerobic, denitrifying, and microaerobic (only strain CC26) conditions. Strain CC-11 grew on phenol only under denitrifying conditions, but some of the other aromatic compounds were utilized under both aerobic and denitrifying conditions. Some organic acids, sugars, alcohols, and amino acids also supported the growth of this strain. In the case of strain CC-26, there was no difference in growth on the substrates tested under microaerobic and aerobic conditions. Phenol supported growth only under denitrifying conditions, which was true for most of the other aromatic compounds tested. Organic acids were good substrates, but no sugars, alcohols, or amino acids tested supported growth of this strain under any conditions, like M. magnetotacticum MS-1 growth (5). Growth on phenol under denitrifying conditions. Figure 4 shows the growth curves for and substrate consumption in cultures of strains CC-11 and CC-26 cultivated in growth medium containing phenol as the sole carbon source under denitrifying conditions. The mean doubling times of strains CC-11 and CC-26 in the exponential growth phase were 7.78 and

FIG. 3. Electron micrograph of strain CC-26.

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TABLE 1. Growth of strains CC-11 and CC-26 on various carbon sources under aerobic, denitrifying, and microaerobic conditions Growth ofa: b

Aromatic compounds 2-Aminobenzoate 3-Aminobenzoate 4-Aminobenzoate Benzaldehyde Benzoate Benzyl alcohol p-Cresol Gentisate 2-Hydroxybenzoate 3-Hydroxybenzoate 4-Hydroxybenzoate Phenol Phenylacetate Organic acids Acetate Adipate Butyrate Caproate Cyclohexanecarboxylate Fumarate Glutarate ␤-Hydroxybutyrate Isobutyrate ␣-Ketoglutarate Lactate Malate Mandelate Oxaloacetate Pimelate Propionate Pyruvate Succinate Tartrate Sugars D-Fructose D-Glucose Maltose Sucrose Alcohols Ethanol Methanol Amino acids Glutamate Phenylalanine

Strain CC-26c

Strain CC-11

Substrate

Aerobic conditions

Denitrifying conditions

Aerobic conditions

Microaerobic conditions

Denitrifying conditions

⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹

Weak ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ Weak ⫹ Weak ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹

⫺ ⫺ ⫺ Weak ⫹ Weak NT NT ⫺ ⫺ ⫺ ⫺ NT

⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹

⫹ ⫺ ⫹ NT ⫹ ⫹ ⫹ NT ⫹ NT ⫹ ⫹ NT ⫹ ⫺ ⫹ ⫹ ⫹ ⫺

⫹ ⫺ ⫹ NT ⫹ ⫹ ⫹ NT ⫹ NT ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺

⫹ NT ⫹ NT NT ⫹ NT NT NT ⫹ ⫹ ⫹ NT ⫹ NT ⫹ ⫹ ⫹ NT

⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺

NT NT NT NT

⫺ ⫺ ⫺ ⫺

⫹ ⫺

⫹ ⫺

⫺ ⫺

NT NT

⫺ ⫺

⫹ ⫹

⫹ ⫹

⫺ ⫺

NT NT

⫺ ⫺

a

⫹, growth; ⫺, no growth; NT, not tested. The following substrates were not used by strain CC-11 under any of the conditions tested: alanine, benzene, o- and m-cresols, ethylbenzene, galactose, lysine, mannose, protocatechuate, phthalate, ribose, toluene, and xylenes. c The following substrates were not used by strain CC-26 under any of the conditions tested: aconitate, alanine, arabinose, benzene, butanol, catechol, cellobiose, citrate, o- and m-cresols, cysteine, L-␤-3,4-dihydroxyphenylalanine, dulcitol, L-epinephrine, ethylbenzene, formate, galactose, gluconate, glycerol, inositol, isocitrate, isopropanol, lysine, malonate, mannitol, mannose, melibiose, melizitose, methionine, oxalate, peptone, propanol, phthalate, quinate, raffinose, rhamnose, ribose, sorbitol, sulfanilate, toluene, trehalose, tyrosine, xylenes, xylitol, and xylose. b

8.29 h, respectively. In the strain CC-11 culture, accumulation of nitrite was observed along with reduction of nitrate; these results were similar to results obtained with known aromatic compound-degrading denitrifiers (6, 24). In contrast, strain CC-26 did not accumulate nitrite. Production of N2 was measured during growth of strain CC-26, and we found that the amount of nitrogen released as N2 was equivalent to at least 86% of the nitrate reduced, indicating that this strain assimilates phenol through denitrification. Omitting bicarbonate from the growth medium inhibited the growth of strain CC-26 when phenol was used as the sole

carbon source. 4-Hydroxybenzoate and benzoate supported good growth of this strain. These facts suggest that the degradation pathway for phenol in strain CC-26 is the same as or similar to the pathway proposed for Thauera aromatica K172 (15). Growth under iron-limiting conditions. Enrichment medium containing 0 to 5 ␮M ferrous sulfate was used to cultivate each strain under denitrifying conditions with phenol as the carbon source. Figure 5 shows that both strain CC-11 and strain CC-26 required ferrous ions and that strain CC-26 grew better than strain CC-11 under iron-limiting conditions. Con-

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FIG. 6. Growth of strain CC-26 under aerobic, microaerobic, denitrifying, and denitrifying reduced conditions. Symbols: E, aerobic conditions; 䊐, microaerobic conditions; ‚, denitrifying conditions; ƒ, denitrifying reduced conditions. OD610, optical density at 610 nm. FIG. 4. Degradation of phenol by strains CC-11 (A) and CC-26 (B) under denitrifying conditions. Symbols: F, growth; E, phenol concentration; 䊐, nitrate concentration; ‚, nitrite concentration. OD610, optical density at 610 nm.

centrations greater than 0.5 ␮M did not improve the growth of either strain any further. Growth of strain CC-26 under microaerobic or reduced conditions. Strain CC-26 was cultivated under aerobic, microaerobic, denitrifying, and denitrifying reduced conditions in growth medium containing 5 mM KNO3 and 3 mM sodium acetate as the carbon source. Cells were subcultured four to seven times before growth under each set of conditions became stable, and the results were compared. As shown in Fig. 6, strain CC-26 grew fastest under microaerobic conditions. Strain CC-26 grew as well in reduced medium as in nonreduced medium. As described previously for M. magnetotacticum MS-1 (5), when strain CC-26 was cultivated in semisolid enrichment medium containing 0.01% agar, the cells initially grew as a fine band near the bottom of the culture tube and then migrated upward to the agar-air surface, and the band of cells became more dense. When an anaerobically grown liquid culture was exposed to air, strain CC-26 cells immediately migrated downward to the bottom of the culture vessel. These observations indicate that strain CC-26 is a microaerophile. Magnetotaxis of strain CC-26. A temporary loss of magnetism has been observed for previously described magnetic bacteria due to a lack of an iron source, aerobic growth conditions, an inappropriate nitrogen source, or repeated subculturing; however, magnetism could be restored (5, 16, 21). Strain CC-26 was cultivated with various iron chelates under aerobic, microaerobic, and anaerobic conditions. “Magnetic selection” was also performed with cells that were cultivated microaero-

FIG. 5. Growth of strains CC-11 (A) and CC-26 (B) under iron-limiting conditions. Symbols: E, 0.5 ␮M iron; ‚, 0.3 ␮M iron; 䊐, 0.1 ␮M iron; F, no iron. OD610, optical density at 610 nm.

bically or anaerobically in the growth medium used for M. magnetotacticum MS-1 (5). These procedures failed to induce magnetotaxis in this strain. DISCUSSION A new spirillum, strain CC-26, which degrades phenol and other aromatic compounds through denitrification, was isolated from an enrichment culture from which a phenol-degrading denitrifying rod-shaped bacterium, Azoarcus sp. strain CC11, had been isolated previously. The latter strain was isolated after 1 year of enrichment, but when strain CC-26 was isolated 2 years later, strain CC-11 was not found in the culture. This change in phenol degraders in the enrichment culture may have been the result of a difference in the requirement for ferrous ions between the strains; that is, strain CC-26 grows better than strain CC-11 under iron-limited conditions. As the continuous enrichment culture was incubated, ferrous ions, which originally came from the inoculated soil suspension, were gradually diluted by the iron-free enrichment medium, and thus the growth of strain CC-11 was inhibited. At this point, the number of strain CC-26 cells started to increase, and eventually strain CC-26 occupied the niche that strain CC-11 had occupied in the enrichment culture. In support of this hypothesis, we have isolated several strains of spiral bacteria of this kind from another soil sample by performing enrichment in batch cultures with medium containing a low concentration of iron. This implies that aromatic compound-degrading denitrifying spirilla could be widespread in certain environments and could play a role in the natural degradation of aromatic compounds, especially in an iron-limited environment. Strain CC-26 was most closely related to Magnetospirillum sp. (21). The morphology of this strain, its chemotaxonomic characteristics, most of its growth substrates, and its microaerophilic features are consistent with this classification. However, strain CC-26 has several features which are different from those of previously described strains of Magnetospirillum sp., as described below. (i) Growth on aromatic compounds has not been reported for the previously described strains of this genus. Phenol inhibits the growth of M. magnetotacticum MS-1 (5). (ii) Growth under reduced conditions has not been reported previously. Magnetospirillum sp. strain AMB-1 can grow without O2 (17), but there has been no report of growth under reduced conditions. The most closely related strain, as inferred from the 16S rDNA sequences, M. magnetotacticum MS-1, requires O2 for growth (5). (iii) Strain CC-26 has not exhibited magnetotaxis under any of the conditions examined

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so far. Magnetotaxis is the most important feature, along with 16S rDNA sequences, when the genus Magnetospirillum is distinguished from the genus Aquaspirillum (21). The genus Aquaspirillum is known to be very heterogeneous, and most of the species need to be renamed (20). Recently, the phylogenetic relationships of the helical bacteria in the ␣ subclass of the Proteobacteria, including Aquaspirillum spp. and Magnetospirillum spp., were analyzed based on their 16S rDNA sequences (12). Because of the high level of sequence similarity, Kawasaki et al. (12) proposed that Aquaspirillum polymorphum is a Magnetospirillum species that lost its magnetotactic ability during evolution. Considering the characteristics of strain CC-26 and the taxonomic background, the exact classification of this strain will not be possible until more strains of this kind and other Aquaspirillum sp. and Magnetospirillum sp. strains have been isolated and characterized. Strain CC-26 is the first aromatic compound-degrading denitrifier belonging to the ␣ subclass of the Proteobacteria. Additional biochemical and genetical analyses of this strain should result in a better understanding of the distribution and evolution of anaerobic degradation of aromatic compounds in the microbial world. ACKNOWLEDGMENTS We thank T. Matsunaga, Tokyo University of Agriculture and Technology, for suggestions and K. Takabe, Kyoto University, for advice regarding the use of the electron microscope. This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan, to N.K. REFERENCES 1. Anders, H.-J., A. Kaetzke, P. Ka ¨mpfer, W. Ludwig, and G. Fuchs. 1995. Taxonomic position of aromatic-degrading denitrifying pseudomonad strains K 172 and KB 740 and their description as new members of the genera Thauera, as Thauera aromatica sp. nov., and Azoarcus, as Azoarcus evansii sp. nov., respectively, members of the beta subclass of the Proteobacteria. Int. J. Syst. Bacteriol. 45:327–333. 2. Aranki, A., S. A. Syed, E. B. Kenney, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17:568–576. 3. Bakker, G. 1977. Anaerobic degradation of aromatic compounds in the presence of nitrate. FEMS Lett. 1:103–108. 4. Balkwill, D. L., D. Maratea, and R. P. Blakemore. 1980. Ultrastructure of a magnetotactic spirillum. J. Bacteriol. 141:1399–1408. 5. Blakemore, R. P., D. Maratea, and R. S. Wolfe. 1979. Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium. J. Bacteriol. 140:720–729. 6. Chee-Sanford, J. C., J. W. Frost, M. R. Fries, J. Z. Zhou, and J. M. Tiedje. 1996. Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4. Appl. Environ. Microbiol. 62:964–973. 7. Fries, M. R., J. Zhou, J. Chee-Sanford, and J. M. Tiedje. 1994. Isolation, characterization, and distribution of denitrifying toluene degraders from a

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variety of habitats. Appl. Environ. Microbiol. 60:2802–2810. 8. Harwood, C. S., G. Burchhardt, H. Herrmann, and G. Fuchs. 1999. Anaerobic metabolism of aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol. Rev. 22:439–458. 9. Heider, J., and G. Fuchs. 1997. Microbial anaerobic aromatic metabolism. Anaerobe 3:1–22. 10. Hiraishi, A. 1992. Direct automated sequencing of 16S rDNA amplified by polymerase chain reaction from bacterial cultures without DNA purification. Lett. Appl. Microbiol. 15:210–213. 11. Hiraishi, A., Y. K. Shin, Y. Ueda, and J. Sugiyama. 1994. Automated sequencing of PCR-amplified 16S rDNA on ‘Hydrolink’ gels. J. Microbiol. Methods 19:145–154. 12. Kawasaki, H., K. Yamamoto, and J. Sugiyama. 1997. Phylogenetic relationships of the helical-shaped bacteria in the ␣ Proteobacteria inferred from 16S rDNA sequences. J. Gen. Appl. Microbiol. 43:89–95. 13. Khoury, N., W. Dott, and P. Ka ¨mpfer. 1992. Anaerobic degradation of phenol in batch and continuous cultures by a denitrifying bacterial consortium. Appl. Microbiol. Biotechnol. 37:524–528. 14. Krieger, C. J., H. R. Beller, M. Reinhard, and A. M. Spormann. 1999. Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp. strain T. J. Bacteriol. 181:6403–6410. 15. Lack, A., and G. Fuchs. 1994. Evidence that phenol phosphorylation to phenylphosphate is the first step in anaerobic phenol metabolism in a denitrifying Pseudomonas sp. Arch. Microbiol. 161:132–139. 16. Matsunaga, T., T. Sakaguchi, and F. Tadokoro. 1991. Magnetite formation by a magnetic bacterium capable of growing aerobically. Appl. Microbiol. Biotechnol. 35:651–655. 17. Matsunaga, T., and N. Tsujimura. 1993. Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically. Appl. Microbiol. Biotechnol. 39:368–371. 18. Owens, J. D., and R. M. Keddie. 1969. The nitrogen nutrition of soil and herbage coryneform bacteria. J. Appl. Bacteriol. 32:338–347. 19. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444–2448. 20. Pot, B., M. Gillis, and J. De Ley. 1992. The genus Aquaspirillum, p. 2569– 2582. In A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer, New York, N.Y. 21. Schleifer, K. H., D. Schu ¨ler, S. Spring, M. Weizenegger, R. Amann, W. Ludwig, and M. Ko ¨hler. 1991. The genus Magnetospirillum gen. nov. Description of Magnetospirillum gryphiswaldense sp. nov. and transfer of Aquaspirillum magnetotacticum to Magnetospirillum magnetotacticum comb. nov. Syst. Appl. Microbiol. 14:379–385. 22. Song, B., M. M. Haggblom, J. Z. Zhou, J. M. Tiedje, and N. J. Palleroni. 1999. Taxonomic characterization of denitrifying bacteria that degrade aromatic compounds and description of Azoarcus toluvorans sp. nov. and Azoarcus toluclasticus sp. nov. Int. J. Syst. Bacteriol. 49:1129–1140. 23. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680. 24. Tschech, A., and G. Fuchs. 1987. Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads. Arch. Microbiol. 148: 213–217. 25. Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697– 703. 26. Zhou, J. Z., M. R. Fries, J. C. Chee-Sanford, and J. M. Tiedje. 1995. Phylogenetic analyses of a new group of denitrifiers capable of anaerobic growth on toluene and description of Azoarcus tolulyticus sp. nov. Int. J. Syst. Bacteriol. 45:500–506.