Nocardioides aromaticivorans sp. nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments

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Systematic and Applied Microbiology 28 (2005) 165–174 www.elsevier.de/syapm

Nocardioides aromaticivorans sp. nov., a dibenzofuran-degrading bacterium isolated from dioxin-polluted environments$ Masahiro Kubotaa,b,1, Kazuyoshi Kawaharaa,b,c, Kachiko Sekiyad, Tetsuya Uchidae, Yasuko Hattorie, Hiroyuki Futamatae, Akira Hiraishie, a

Graduate School of Fundamental Life Science, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan The Kitasato Institute, Minato-ku, Tokyo 108-8642, Japan c Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, Yokohama, Kanagawa 236-8501, Japan d School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108-8641, Japan e Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441-8580, Japan b

Received 24 September 2004

Abstract Seven strains of dibenzofuran (DF)-degrading bacteria isolated from dioxin-polluted environments were characterized. These isolates were able to grow with dibenzofuran as the sole carbon and energy source. During the growth with dibenzofuran, they produced a soluble yellow metabolite that exhibited a unique pH-dependent shift of absorption maxima. Dibenzo-p-dioxin and biphenyl were also degraded with pigment production. The isolates were strictly aerobic and chemoorganotrophic and had Gram-positive, nonmotile, rod-shaped cells. Chemotaxonomic analyses showed that cells contained L,L-diaminopimeric acid in the peptidoglycan, branched-chain fatty acids as major fatty acids, and menaquinone MK-8(H4) as the sole respiratory quinone. The G+C content of the DNA of the isolates ranged from 72.0 to 72.4 mol%. The 16S rRNA gene sequences of the isolates were very similar to each other (X99.8%). The phylogenetic analysis showed that the isolates formed a cluster with species of the genus Nocardioides with Nocardioides simplex and Nocardioides nitrophenolicus as their nearest neighbors. DNA–DNA hybridization studies showed that the isolates showed a hybridization level of less than 55% to any tested species of the genus Nocardioides. Based on these data, Nocardioides aromaticivorans sp. nov. is proposed for the new DF-degrading isolates. The type strain is strain H-1 (IAM 14992, JCM 11674, DSM 15131). r 2004 Elsevier GmbH. All rights reserved. Keywords: Nocardioides aromaticivorans; Dibenzofuran degradation; Dioxin pollution

Introduction $

The 16S rDNA sequences of the isolates presented in this study have been deposited under DDBJ accession numbers AB087221–AB087725. Corresponding author. Tel.: +81 532 446913; fax: +81 532 446929. E-mail address: [email protected] (A. Hiraishi). 1 Present address: Kibun Food Chemifa Co., Ltd., Chuo-ku, Tokyo 104-8553, Japan. 0723-2020/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2004.10.002

Pollution of sediment and soil with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), which are known as carcinogens and endocrine disruptors, is an environmental issue of major concern. Much effort has been made to develop physicochemical

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and biological methods for detoxification and removal of these compounds. From ecological and economical points of view, bioremediation techniques using particular microorganisms or microbial consortia may be more effective and cost beneficial than physicochemical methods to be applied for relatively wide and low contaminated areas. In this connection, the diversity of dibenzo-p-dioxin- and dibenzofuran (DF)-degrading microorganisms and the enzymes and genes involved has been reviewed [2,10,28,41]. Representative DFdegrading bacteria well characterized are Burkholderia xenovorans strain LB400T [9,33], Pseudomonas resinovorans strain CA10 [26], Rhodococcus sp. strain YK2 [17], Sphingobium yanoikuyae strain B1 [8,20,47], Sphingomonas wittichii strain RW1T [1,40,42,43], and some Terrabacter strains [16,18,27,32] now classified as members of the genus Janibacter [22]. Previously, we studied the distribution of DFdegrading bacteria in soils and sediments polluted with different levels of PCDD/Fs [7,12], and found that Gram-positive bacteria belonging to the phylum Actinobacteria are the most abundant as culturable DF degraders in the polluted environments [7]. Some isolates of the DF-degrading Gram-positive bacteria, classified as a member of the genus Nocardioides, produced a soluble yellow metabolite during the degradation of DF that showed a unique pH-dependent change of the absorption spectrum. Further attempts to isolate similar Nocardioides strains from PCDD/Fcontaminated aquatic environments gave positive results. In the present paper, therefore, we report the physiological, chemotaxonomic, phylogenetic characteristics of these DF-degrading Nocardioides isolates. A creation of a new species with the name Nocardioides aromaticivorans is proposed for them.

Materials and methods Samples and isolation Surface water and sediment samples were taken from the Hikichi river (Kanagawa, Japan) and the Ayase river (Tokyo, Japan). The sediments of Hikichi and Ayase rivers at which the samples were taken were contaminated with PCDD/Fs at a concentration of 450 and 320 pg-TEQ (toxic equivalent) g
1 dry wt, respectively. The samples were taken in polyethylene bottles and used for testing immediately upon return to the laboratory. For isolation of DF-degrading bacteria, mineral medium RM2 [11] supplemented with vitamin solution PV1 [7,15], designated BSV medium, was used. The river water or sediment samples (3 ml each) were introduced into screw-capped test tubes containing 3 ml of BSV medium and 0.5 ml of filter-sterilized 2% DF solution in

2,20 ,4,40 ,6,8,80 -heptamethylnonane. The test tubes were incubated at 30 1C on a reciprocal shaker. After 3 days of incubation, aliquots (0.5 ml) were transferred to fresh DF-BSV medium and further incubated in the same condition. After 1–4 weeks of incubation, some of the enrichment cultures showed significant growth with yellow-orange pigment production. Then, the cultures were spread on BSV agar medium coated with DF precipitates, which was prepared by spreading 2 ml of 2% DF solution in diethyl ether onto agar and drying the surface of the agar in a clean bench [7]. Inoculated plates were incubated at 30 1C for 7 days. Colonies showing soluble yellow pigment production on the agar plates were picked up and subjected to the standard purification procedure by streaking on peptone-beef extract-yeast extract (PBY) agar medium [7]. The isolates thus purified were maintained on PBY agar slants.

Bacterial strains and culture media Seven strains of the new DF-degrading bacteria were studied. Two of these isolates, designated strains H-1T and H-2, were isolated from the Hikichi river (Kanagawa, Japan). Three of the isolates, designated strains A-1, A-2, and A-3, were isolated from the Ayase river (Tokyo, Japan). The remaining DF-degrading isolates, strains NSA1-1 and NSA1-2, were isolated previously from PCDD/F-polluted soil [7]. The reference organisms used for taxonomic studies were the respective type strains of nine Nocardioides species including N. albus strain JCM 3185T, N. aquaticus strain DSM 11439T, N. fulvus strain JCM 3335T, N. jensenii strain JCM 1364T, N. luteus strain JCM 3358T, N. nitrophenolicus strain JCM 10703T, N. plantarum strain JCM 9626T, N. pyridinolyticus strain JCM 10369T, and N. simplex strain JCM 1363T. The strains with JCM and DSM numbers were obtained from the Japan Collection of Microorganisms, RIKEN (Wako, Japan) and the DSMZDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively. In addition, Rhodococcus erythropolis strain TUT581, which was isolated as a DF degrader from PCDD/Fpolluted river sediment [7], was used in DF degradation tests for comparison. Unless otherwise specified, the test organisms were grown with shaking in either PBY medium or Trypticase soy broth (BBL, Nippon Becton Dickinson Co., Tokyo, Japan) supplemented with 0.75% glucose. N. plantarum JCM 9626T was aerobically grown in the medium containing 0.4% Yeast extract (Difco Laboratories, Detroit, Mich., USA), 1% Malt extract (Difco), and 0.4% glucose. N. aquaticus DSM 11439T was aerobically grown in the medium containing minerals, vitamins, and sea water in addition to Bacto-Peptone (Difco), yeast extract, and glucose at 25 1C as described previously [23].

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Dibenzofuran degradation tests

DNA base composition and DNA–DNA relatedness

Precultures were prepared by growing cells in PBY medium for 36–48 h on a reciprocal shaker. Screwcapped test tubes (30-ml capacity) containing 5.5 ml of DF-BSV medium (6 ml of BSV and 0.5 ml of 0.2% DF solution in heptamethylnonane) were inoculated with 100 ml of the preculture. The test tubes were incubated at 30 1C on a reciprocal shaker, and growth was monitored by measuring the optical density at 660 nm. At appropriate intervals of incubation, the cultures were taken and centrifuged for 10 min at 12,600  g to collect the lipid phase and the aqueous supernatant. Then, DF remaining in the collected sample was extracted with ethyl acetate and measured by reverse-phase HPLC as reported previously [15]. The absorption spectrum of the yellow-colored supernatant was measured with a Shimadzu Biospec-1600 spectrophotometer at 200–600 nm.

Genomic DNA was extracted and purified by the method of Marmur [24]. DNA base composition was determined by the HPLC method of Mesbah et al. [25]. DNA–DNA hybridization studies were performed by the dot-blot hybridization method with alkaline phosphatase labeling and chemiluminescence detection using an Amersham-Pharmacia AlkalPhos kit. Labelling, hybridization, and detection were performed according to the manufacturer’s instructions. Hybridization signals were detected with an Amersham-Pharmacia ECL mini-camera, and their intensity was measured by the NIH image program available at the web site http:// rsb.info.nih.gov/nih-image/.

Morphological, physiological, and biochemical tests General cell morphology and motility were observed under an Olympus phase-contrast microscope, and cells negatively stained with 0.5% phosphotungstic acid were observed under a JEOL model JEM-1010 transmission electron microscope. Catalase activity was determined by detecting bubble formation with 3% H2O2 solution. Oxidase activity was determined using paper discs with tetramethyl-p-phenylenediamine. Urease activity was determined by detecting ammonia produced from urea as described previously [21]. Hydrolysis of elastin and casein was determined by detecting cleared zones formed around colonies on PBY agar containing the test substance. Tween 80 hydrolysis was determined as reported by Sierra [34]. Utilization of carbohydrates was determined using the API 50 CH system (bioMerieux Japan, Tokyo, Japan). All test media were incubated at 30 1C unless otherwise noted.

16S rDNA sequencing and phylogenetic analysis Fragments of 16S rRNA genes corresponding to positions 8-1525 of the Escherichia coli rRNA numbering system [4] were amplified by PCR with a pair set of primers [39] as described previously [13]. PCR products were purified by the polyethylene glycol precipitation method, sequenced directly with a SequiTherm Long Read cycle sequencing kit (Epicentre Technologies, Madison, USA), and analyzed with a Pharmacia ALFexpress DNA sequencer. Sequence data were compiled using the GENETYX-MAC program (Software Developing Co., Tokyo, Japan). Multiple alignment of sequences, calculation of evolutionary distance by Kimura’s two parameter model [19], and construction of a neighbor-joining [30] phylogenetic tree were performed using the CLUSTAL W program [37]. The topology of the trees was evaluated by bootstrapping with 1000 resamplings [6]. Alignment positions with gaps were excluded from the calculations.

Results Chemotaxonomic analyses Degradation of dibenzofuran and related compounds The peptidoglycan was prepared and hydrolyzed with 6 M HCl at 120 1C for 15 min as described previously [31]. The resultant liberated diamino acids were analyzed by thin-layer chromatography (TLC) using Merck silica gel 60 plates and a developing solvent mixture of methanol/water/6 M HCl/pyridine (364:72:19:46, v/v). The acyl type of the peptidoglycan was determined colorimetrically as reported previously [38]. Fatty acid methyl esters were extracted by mixing dry cells with 5% HCl/CH3OH and heating at 100 1C for 3 h and analyzed by gas-liquid chromatography as described [36]. Quinones were extracted from dry cells with a chloroformmethanol mixture, purified by TLC, and analyzed by reverse-phase HPLC as described previously [14].

All of the 7 DF-degrading isolates as well as the reference organism Rhodococcus erythropolis strain TUT581 formed colonies on DF-BSV agar with cleared halo formation and soluble yellow pigment production. When growing in DF-BSV liquid medium, all of the isolates degraded DF completely within 96 h of incubation. An example of this experiment is shown in Fig. 1. In this case, the yellow metabolite was produced before the logarithmic phase of growth and disappeared with time of prolonged incubation (not shown). This profile of pigment production during the degradation of DF is similar to that observed for some previously reported DF-degrading bacteria, such as Porphyrobacter

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1.4 1.2 1.0 0.8

0.1 0.6 0.4 0.2 0.01 0

20

40 60 Time (h)

80

DF concentration (mmol ml-1)

Growth (optical density at 660 nm)

1.0

0 100

Fig. 1. Growth of and dibenzofuran degradation by strain H-1T in DF-BSV medium. Open circles, growth (optical density at 660 nm); closed squares, concentration of DF remaining in the culture.

sanguineus IAM 12620T [15], Ralstonia (Wautersia) sp. strain SBUG 290 [3], Rhodococcus erythropolis strain SBUG 271 [35], and Sphingobium yanoikuyae strain B1 [15,20], which produce 2-hydroxy-4-[3’-oxo-3’H-benzofuran-2’-yliden] but-2-enoic acid (HOBB) as the yellowcolored metabolite from DF. It has been shown that HOBB exhibits a pHdependent shift (pH 3 to 7–10) in the absorption maximum from 400 to 465 nm, which is known as a keto–enol tautomerism of the meta cleavage product. Interestingly, the yellow metabolite from the isolates showed a unique pH-dependent change in absorption maxima. Namely, while the yellow pigment from the R.. erythropolis culture showed an absorption change typical of HOBB at different pHs (Fig. 2a), the yellow pigment from the new isolates had the absorption maximum at 400, 466, and 507 nm at pH 3, 7, and 10, respectively (Fig. 2b). The isolates also showed growth with the production of yellow-orange pigment in the medium containing biphenyl as a sole carbon source. The increase of turbidity in this case was slower than that observed in the DF-containing medium, but significant growth was found within 140 h of incubation. When dibenzo-pdioxin was used as a carbon source instead of DF, all of the isolates showed much weaker growth.

Fig. 2. Absorption spectra of the yellow pigment produced by Rhodococcus erythropolis TUT 581 (a) and strain H-1T (b) during degradation of DF.

Fig. 3. Cell morphology of strain H-1T observed by transmission electron microscopy with negative-staining.

Morphological and cultural characteristics All of the isolates had Gram-positive rod-shaped cells measuring 0.5–0.7 mm in width and 1.0–2.0 mm in length. Neither endospore formation nor motility was observed. Transmission electron microscopy with

negatively stained cells demonstrated the absence of flagella (Fig. 3) and the presence of septa in the middle of some rods (not shown). When grown on PBY agar, the isolates formed smooth, round, and milky white colonies.

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Chemotaxonomic characteristics The diamino acid in the peptidoglycan of the 7 isolates was identified as L,L-diaminopimelic acid (L,L-DAP). The acyl group of muramyl residue in the peptidoglycan was acetyl. The cellular fatty acid profiles of the isolates are shown in Table 1. They contained various branchedchain fatty acids with carbon chain length of 14–19. The major component was iso-C16:0. Significant amounts of iso-C17:0 and C18:1 were also detected. The major respiratory quinone of the isolates was identified as tetrahydrogenated menaquinone with eight isoprane units, MK-8(H4).

Phylogenetic characteristics The nearly complete 16S rDNA sequences of the isolates (1525 bases) were determined. The sequences of the isolates were very similar to each other (X99.8% similarity) and were found to be close to those of species of the genus Nocardioides. A neighbor-joining phylogenetic tree based on 16S rDNA sequences showed that the isolates were clustered with Nocardioides simplex and Nocardioides nitrophenolicus as their nearest phylogenetic neighbors (Fig. 4). The similarity levels of 16S rDNA sequences between the isolates and N. simplex or N. nitrophenolicus were 98.5–98.9%. As shown in Table 2, the levels of DNA–DNA hybridization among the isolates were more than 78%, indicating that they form a genetic coherent group as a single species. The isolates showed a DNA–DNA hybridization level of less than 55% to the type strains of any test species of Nocardioides. Although two Nocardioides species recently described, N. aestuarii [45] and N. ganghwensis [44], were not included in this test, our isolates were clearly distinguishable from these species on the phylogenetic tree (Fig. 4). These results strongly suggested that the DF-degrading isolates represent a distinct species of the genus Nocardioides. The G+C content of the genomic DNA of the isolates ranged from 72.0% to 72.4%. These values fall into the range of G+C contents of the genus Nocardioides species [5,29,36,44–46] and are most similar to those recorded for N. simplex.

Other phenotypic characteristics The isolates shared many other physiological and biochemical characteristics. They were strictly aerobic and chemoorganotrophic. Growth occurred in a pH range of 5–8 with the optimum at pH 7. The temperature range for growth was 22–40 1C with the optimum at 30 1C. Catalase and urease were produced. Oxidase was negative. Casein but not starch or Tween

169

Table 1. Cellular fatty acid profiles of the dibenzofurandegrading isolates Component

C14:0 C15:0 C16:0 C17:0 C18:0 C19:0 C17:1 C18:1 iso-C14:0 iso-C15:0 iso-C16:0 iso-C17:0 iso-C18:0 iso-C19:0 anteiso-C17:0 antesio-C19:0

Composition (%) 7 strains

Strain H-1T

0.1–0.3 0.1–1.2 1.4–3.9 2.0–5.0 0.6–1.8 o0.1–0.2 3.0–5.6 11.8–22.1 1.1–1.5 1.4–1.7 32.8–49.0 11.9–13.5 4.9–7.0 2.6–3.7 2.4–4.2 1.2–1.9

0.1 1.1 1.6 2.6 0.8 0.2 4.8 12.2 1.5 1.6 47.8 12.0 6.9 2.8 2.6 1.8

80 was hydrolyzed. Simple organic compounds, in particular sugars, were good carbon and energy sources for growth. As shown in Table 3, the isolates were distinguishable from their phylogenetic neighbors N. simplex and N. nitrophenolicus in the phenotypic characteristics noted above as well as in DF and biphenyl degradation. Other characteristics of the isolates are given below.

Discussion Although large numbers of dibenzo-p-dioxin- and DF-degrading bacteria have so far been isolated from different environments [10,41], the available information about the relationship between the occurrence of these bacteria and pollution levels of environments has been scanty. Our previous study showed that a higher isolation frequency for DF degraders was obtained with soil samples contaminated with higher concentrations of PCDD/Fs and that a member of the genus Nocardioides described herein and some other actinobacteria constitute the major populations of culturable DF-degrading bacteria in the polluted environments [7]. In this study, the DF-degrading strains assigned to the member of the genus Nocardioides noted above were also isolated from the dioxin-polluted rivers. These findings suggest that this Nocardioides group is a common member of the DF-degrading bacterial population in dioxin-polluted environments. An interesting observation reported herein and previously [7] is that the Nocardioides isolates produce

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Strain H-1

T

Strain NSA1-2 0.01 1000

Strain H-2 DF-degrading isolates

Strain A-3 436 1000

Strains A-1 and A-2 Strain NSA1-1 T

Nocardioides nitrophenolicus NSP41 (AF005024) 446

T

Nocardioides simplex KCTC 9106 (AF005009) T

Nocardioides ganghwensis JC2055 (AY423718) T

462

Nocardioides aestuarii JC2056 (AY423719) 491

T

Nocardioides aquaticus EL-17K (X94145)

314

T

Nocardioides plantarum NCIMB 12834 (AF005008) 989

347

T

1000

Nocardioides pyridinolyticus OS4 (U61298) T

Nocardioides aquiterrae GW-9 (AF529063)

979

T

1000

Nocardioides luteus KCTC 9575 (AF005007) T

Nocardioides albus KCTC 9186 (AF004988) T

Nocardioides jensenii DSM 20641 (Z78210) T

Nocardioides fastidiosus NCIB 12713 (X53189) T

Friedmannella antarctica DSM 11053 (Z78206) Fig. 4. Phylogenetic tree based on 16S rDNA sequences showing relationships between the dibenzofuran-degrading isolates and the Nocardioides species. The sequence of Friedmannella antarctica was used as an outgroup to root the tree. The accession numbers for the sequences used for comparison are given in parentheses. Scale=1% nucleotide substitution rate (Knuc). Bootstrap values with 1000 resamplings (4700) are shown at branching points.

a characteristic soluble yellow pigment during the degradation of DF. The production of soluble yelloworange metabolites has been reported in several Grampositive and Gram-negative bacteria that degrade DF via the lateral dioxygenation pathway [3,15,20,35]. In this pathway of DF degradation, the yellow pigment HOBB is formed as the meta-cleavage product that exhibits a pH-dependent shift (pH 3 to 7–10) in the absorption maximum from 400 to 465 nm. On the other hand, the yellow metabolite of our isolates showed a unique pH-dependent shift in the absorption maximum from 400 to 507 nm. This suggests that the degradation pathway of DF in the isolates is different from the typical lateral dioxygenation pathway. Our concurrent study has shown that one of the isolates, strain NSA1-2, contains at least three different genes coding for aromatic hydrocarbon dioxygenases. Details of DF degradation and the genes involved in this organism will be reported elsewhere. The phylogenetic assignment of the isolates to the genus Nocardioides based on 16S rDNA comparisons is

supported unequivocally by the chemotaxonomic profiles, i.e., the presence of L,L-DAP and the N-acetyl group of muramyl residue in the peptidoglycan, isoC16:0. as the major cellular fatty acid component, and MK-8(H4) as the major respiratory quinone. The isolates were similar to each other in 16S rDNA sequences (X99.8%) and DNA–DNA relatedness (X78%), indicating their genetic uniformity at the species level, but exhibited low hybridization levels of less than 55% to all test species of Nocardioides. The nearest phylogenetic neighbors of the isolates, N. simplex and N. nitrophenolicus, were found to be unable to degrade biphenyl, dibenzo-p-dioxin, or DF, and to differ from our isolates in many other phenotypic characteristics. By comparing the previously reported data [5,23,29,44–46] and ours, it is clear that our isolates are phenotypically distinguishable from all other established species of Nocardioides. These results strongly suggest that the isolates represent a distinct species in the genus Nocardioides. Thus, Nocardioides aromaticivorans sp. nov. is proposed for the isolates with strain

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171

Table 2. Genomic DNA relatedness between the dibenzofuran-degrading isolates and the type strains of previously established Nocardioides species Target organism

Isolates H-1T H-2 A-1 A-2 A-3 NSA1-1 NSA1-2 N. albus JCM 3185T N. jensenii JCM 1364T N. luteus JCM 3358T N. nitrophenolicus JCM 10703T N. planatrum JCM 9626T N. simplex JCM 1363T

Mol%

% Hybridization with labeled probe from:a

G+C

H-1T

NSA1-2

JCM 1363T

72.2 72.0 72.2 72.2 72.4 72.3 72.2 68.6b 68.8b 67.5c 71.4d 69.0e 73.0b

100 91 83 84 83 78 79 37 38 36 49 25 55

80 80 93 91 90 89 100 32 37 40 48 28 55

50 nt 51 nt nt nt 48 29 33 31 49 28 100

a

Data show the average values of three different determinations. Cited from Suzuki and Komagata [36]. c Cited from Prauser [29]. d Cited from Yoon et al. [46]. e Cited from Collins et al. [5]. b

Table 3.

Differential phenotypic characteristics of the dibenzofuran-degrading isolates and related organismsa

Characteristic

Isolates

N. simplex

N. nitrophenolicus

7 strains

Strain H-1T

JCM 1363T

JCM 10703T

Oxidase Urease


+


+

+


+ +

Hydrolysis of: Starch Casein Tween 80


+



+


+
+

+ + +

Carbon source: Glycerol L-Arabinose D-Ribose D-Xylose D-Fructose D-Mannose L-Rhamnose D-Mannitol Cellobiose Maltose Biphenyl Dibenzofuran

w + + + + w w +/w +/w +/w + +

w + + + + w w + + + + +

















+ + + w +






a

All of the organisms listed were examined under the same conditions (see Materials and Methods). Symbols and abbreviations: +, reactions positive;
, reactions negative; w, weakly positive; +/w, positive or weakly positive (the first sign indicates the most frequent result).

H-1 as its type strain, which has been deposited with the IAM Culture Collection (Tokyo, Japan) as IAM 14992, the Japan Collection of Microorganisms (Wako, Japan)

as JCM 11674, and the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig) as DSM 15131.

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Description of Nocardioides aromaticivorans sp. nov. Nocardioides aromaticivorans (a.ro.ma.ti.ci.vo’rans. N.L. adj. aromaticus, aromatic; L. part. adj. vorans, devouring; N.L. part. adj. aromaticivorans, devouring aromatic (compounds)). Cells are Gram-positive, asporogenous, nonmotile rods measruing 0.5–0.7 mm in width and 1.0–2.0 mm in length. Colonies are smooth, round and milky-white on complex agar media. Strictly aerobic and chemoorganotrophic. Growth occurs between pH 5 and 8 (optimum, pH 7) and between 22 and 40 1C (optimum, 30 1C). Catalase and urease positive. Oxidase negative. Casein and esculin but not starch and Tween 80 are hydrolyzed. Dibenzo-p-dioxin, DF, and biphenyl are degraded with yellow-orange pigment formation. Good carbon and energy sources are L-arabinose, D-ribose, D-xylose, fructose, glucose, peptone, and yeast extract. Moderate and weak growth occurs with glycerol, L-rhamnose, maltose, sucrose, trehalose, mannitol, and arubutine. Not utilized are erythritol, D-arabinose, L-sorbose, Lxylose, galactose, mannose, lactose, melibiose, melezitose, raffinose, fucose, lyxose, tagatose, arabitol, adonitol, dulcitol, inocitol, sorbitol, xylitol, glycogen, inuline, amygdaline, a-methyl-D-glucoside, a-methyl-D-mannoside, N-acetyl-gulcosamine, citrate, gluconate, 2-ketogluconate, and 5-keto-gluconate. The cell wall peptidoglycan contains L,L-diaminopimelic acid as a diamino acid, and N-acetyl group in the muramyl residue. The major cellular fatty acids are iso-C16:0, iso-C17:0, and C18:1. Menaquinone MK-8(H4) is present. The G+C content of the DNA is 72.0–72.4 mol%. Isolated from polluted river water, sediment, and soil. The type strain is strain H-1 (= IAM 14992, JCM 11674, DSM 15131).

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

Acknowledgments We are grateful to H. Kuraishi for useful advice and encouragement during this study. This work was carried out as a part of ‘‘The Project for Development of Technologies for Analyzing and Controlling the Mechanism of Biodegrading and Processing’’ which was entrusted by the New Energy and Industrial Technology Development Organization (NEDO), and as a part of the 21st Century COE Program ‘‘Ecological Engineering and Homeostatic Human Activities’’ founded by the Ministry of Education, Sports, Culture, Science and Technology, Japan. This study was also supported in part by grants K1433 and K1522 from the Ministry of Environment, Japan.

[12]

[13]

[14]

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