International Journal of Systematic and Evolutionary Microbiology (2015), 65, 2919–2924
DOI 10.1099/ijs.0.000356
Sphingobium barthaii sp. nov., a high molecular
weight polycyclic aromatic hydrocarbon-degrading
bacterium isolated from cattle pasture soil
Allyn H. Maeda,1 Marie Kunihiro,1 Yasuhiro Ozeki,1 Yuichi Nogi2 and
Robert A. Kanaly1
Correspondence
Robert A. Kanaly
1
Department of Life and Environmental System Science, Graduate School of Nanobiosciences,
Yokohama City University, 22-2 Seto, Kanazawa, Kanagawa, Yokohama 236-0027, Japan
[email protected]
2
Institute of Biogeosciences (Biogeos), Japan Agency for Marine-Earth Science and Technology
(JAMSTEC), 2–15 Natsushima-cho, Yokosuka 237-0061, Japan
Received 31 March 2015
Accepted 21 May 2015
A Gram-stain-negative, yellow, rod-shaped bacterium, designated strain KK22T, was isolated
from a microbial consortium that grew on diesel fuel originally recovered from cattle pasture soil.
Strain KK22T has been studied for its ability to biotransform high molecular weight polycyclic
aromatic hydrocarbons. On the basis of 16S rRNA gene sequence phylogeny, strain KK22T
was affiliated with the genus Sphingobium in the phylum Proteobacteria and was most closely
related to Sphingobium fuliginis TKPT (99.8 %) and less closely related to Sphingobium
quisquiliarum P25T (97.5 %). Results of DNA–DNA hybridization (DDH) revealed relatedness
values between strain KK22T and strain TKPT and between strain KK22T and strain P25T of
21¡4 % (reciprocal hybridization, 27¡2 %) and 15¡2 % (reciprocal hybridization, 17¡1 %),
respectively. Chemotaxonomic analyses of strain KK22T showed that the major respiratory
quinone was ubiquinone Q-10, that the polar lipid profile consisted of phosphatidylglycerol,
phosphatidylethanolamine, phosphatidylcholine, phosphatidyl-N-methylethylethanolamine and
sphingoglycolipid, and that C18 : 1v7c and C14 : 0 2-OH were the main fatty acid and
hydroxylated fatty acids, respectively. This strain was unable to reduce nitrate and the genomic
DNA G+C content was 64.7 mol%. Based upon the results of the DDH analyses, the fact that
strain KK22T was motile, and its biochemical and physiological characteristics, strain KK22T
could be separated from recognized species of the genus Sphingobium. We conclude that
strain KK22T represents a novel species of this genus for which the name Sphingobium barthaii
sp. nov. is proposed; the type strain is KK22T (5DSM 29313T5JCM 30309T).
The genus Sphingobium, which was proposed by Takeuchi
et al. (2001), is composed of aerobic, Gram-staining-negative,
rod-shaped, chemo-organotrophic bacteria characterized
chemotaxonomically by possessing sphingoglycolipids in
their outer membranes, fatty acid profiles consisting of
C18 : 1v7c as the major fatty acid and C14 : 0 2-OH as the
main hydroxylated fatty acid, and which contain ubiquinone Q-10 as the main respiratory quinone (Young et al.,
2007; Stolz, 2009). At the time of writing, there are 37
members of the genus Sphingobium with validly published
Abbreviations: DDH, DNA–DNA hybridization; HMW, high
molecular weight; PAH, polycyclic aromatic hydrocarbon.
The GenBank/EMBL/DDBJ accession number for the 16S
rRNA gene sequence of strain KK22T is HQ830159.
Two supplementary figures and one supplementary table are
available with the online Supplementary Material.
000356 G 2015 IUMS
names and 23 of these species have been proposed in the
last five years. This genus includes members that are
known for their abilities to biodegrade environmental pollutants and some that biodegrade polycyclic aromatic
hydrocarbons (PAHs) (Kanaly & Harayama, 2000, 2010;
Stolz, 2009). PAHs comprised of four or more aromatic
rings are considered to be more resistant to biodegradation
by bacteria and are often referred to as high molecular
weight (HMW) PAHs. The greater molecular stability of
HMW PAHs is a contributing factor to their environmental persistence. Many are suspected carcinogens, displaying
genotoxicity, immunotoxicity and causing oxidative cell
damage, and so they are considered to be a threat to
human health (Cerniglia, 1992; Kanaly & Harayama, 2010).
Strain KK22T was isolated by enrichment on phenanthrene
from a bacterial consortium that was studied for its ability
to biodegrade diesel fuel and mineralize benzo[a ]pyrene
and which was originally recovered from cattle pasture
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39
Printed in Great Britain
2919
A. H. Maeda and others
soil in the Gulf region of Texas, USA, circa 1994 (Kanaly
et al., 1997, 2000; Kanaly & Bartha, 1999). This strain was studied for its abilities to biotransform the HMW PAHs benz[a ]anthracene, fluoranthene and benzo[k ]flouranthene
(Kunihiro et al., 2013; Maeda et al., 2014). Strain KK22T
was maintained on phenanthrene (300 mg l21) as the sole
source of carbon and energy in Stanier’s Basal Medium
(SBM; Atlas, 1993) at 30 8C by rotary shaking at 175 r.p.m.
in the dark. On Noble agar (Becton Dickinson) supplemented with phenanthrene crystals, strain KK22T produced brownish-yellow, round, convex and shiny colonies
of approximately 2–3 mm in diameter after 3 to 4 days.
This strain also grew and produced yellow colonies on trypticase soy agar (TSA), LB agar and Noble agar (all Becton Dickinson) supplemented with 1 % (w/v) glucose.
16S rRNA gene PCR and sequence determination were conducted by colony PCR following the growth of strain KK22T
on SBM Noble agar plates with crystalline phenanthrene. 16S
rRNA gene fragments were amplified and sequenced using
primers, as described previously (Kunihiro et al., 2013).
The 16S rRNA gene sequences of members of the genus
Sphingobium with validly published names were retrieved
from the NCBI database and were aligned using CLUSTAL W
version 1.8 software and alignments were refined by visual
inspection (Thompson et al., 1994). Phylogenetic trees
based upon comparison of 1481 bases were reconstructed
by the neighbour-joining and maximum-likelihood algorithms with 1000 bootstrap analysis using MEGA 5.0 software
(Saitou & Nei, 1987; Tamura et al., 2011). The results showed
that strain KK22T was related to members of the genus Sphingobium and was most closely related to Sphingobium fuliginis
TKPT (Prakash & Lal, 2006) and less closely related to
members of other genera of the family Sphingomonadaceae:
Sphingomonas, Sphingopyxis, Sphingosinicella and Novosphingobium (Fig. 1). The 16S rRNA gene sequence of
strain KK22T showed 99.8 % similarity to Sphingobium fuliginis TKPT, 97.5 % similarity to Sphingobium quisquiliarum
P25T (Bala et al., 2010) and was less than 97.0 % similar to
the next closest strains, Sphingobium herbicidovorans MBIC
3166T (Zipper et al., 1996; Takeuchi et al., 2001) and Sphingobium rhizovicinum CCM 7491T (Young et al., 2008).
Sequence divergence values of 3 % or greater are considered
to be strong evidence that organisms are not related at the
species level (Stackebrandt & Goebel, 1994). At the same
time, DNA–DNA hybridization (DDH) is necessary to clarify the taxonomic relationships of strains when they have
greater than 97.0 % (Tindall et al., 2010), or greater than
98.5 % (Stackebrandt & Ebers, 2006; Stackebrandt, 2011),
16S rRNA gene sequence similarity. Therefore, DDH was
conducted at the Japan Agency for Marine-Earth Science
and Technology (JAMSTEC) according to the methods of
Ezaki et al. (1989). Hybridizations were conducted in triplicate and the means of the resultant values determined. Based
upon the recommendations of Tindall et al. (2010), reciprocal analyses were also performed between all strains. DDH
revealed low DNA relatedness between strain KK22T and
strain TKPT (21¡4 %; reciprocal, 27¡2 %); average and
2920
standard deviation and between strain KK22T and strain
P25T (15¡2 %; reciprocal, 17¡1 %) (Table S1, available
in the online Supplementary Material). These levels of
DNA–DNA relatedness were less than the threshold value
of 70 % suggested for the delineation of bacterial species
according to Wayne et al. (1987), and so it was concluded
that strain KK22T should be separated and considered as a
representative of a novel species of the genus Sphingobium.
The cell morphology of strain KK22T was investigated.
It was grown on 300 mg phenanthrene l21 for 3 days and
streaked onto and cultivated on Noble agar plus crystalline
phenanthrene. The morphology and dimensions were then
determined from photomicrographs using transmission
electron microscopy (TEM) and phase-contrast microscopy.
For TEM analyses, samples were prepared by negative staining with 1.0 % phosphotungstic acid (pH 7.6), 0.4 %
sucrose and 0.01 % sodium azide solution (all w/v) on a
Formvar-coated copper grid, dried and analysed using a
transmission electron microscope (JEOL model JEM-1210)
equipped with an Olympus camera (model SIS Megaview
III CCD) at JAMSTEC. Gram staining was conducted by
using a Becton Dickinson Gram stain kit. Strain KK22T
was Gram-stain-negative, motile and rod-shaped or short
rod-shaped. TEM revealed that this organism possessed
one polar flagellum of up to approximately 5–6 mm in
length (Fig. S1). Cell size was approximately 0.8–1.3 mm in
length and 0.5–0.6 mm in width. The detection of the monotrichous flagellum differentiated strain KK22T from Sphingobium fuliginis TKPT and Sphingobium quisquiliarum P25T,
which are non-motile organisms (Prakash & Lal, 2006;
Bala et al., 2010).
Oxidase and catalase tests were conducted according to standard procedures (Smibert & Krieg, 1994). Hydrolysis of 1.0 %
Tween 20 and Tween 80 (w/v) was indicated by a zone of opacity around the areas of growth according to the method of
Arden Jones et al. (1979). Casein hydrolysis was determined
by the method of Gordon & Mihm (1962). For physiological
and biochemical characterizations, API 20NE (bioMérieux)
and API 50 CHB/E Medium (bioMérieux) test kits were
used, according to the manufacturer’s instructions. DNase
activity was assayed by using a DNase test agar (Becton Dickinson). Growth of strain KK22T at 4, 10, 15, 20, 28, 30, 37, 40,
45 and 50 8C and at a pH of 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 (pH
was adjusted with 6 M HCl or KOH) was evaluated on TSA
and Noble agar supplemented with 1 % (w/v) glucose. Motility was evaluated by the test tube method with nutrient broth
and 0.4 % (w/v) agar prepared in 10 ml tubes and inoculated
by the stab technique. Salinity tolerance was evaluated on TSA
supplemented with NaCl at concentrations of 0, 0.5, 1.5, 2.5,
3.0, 3.5, 4.5 and 5.5 % (w/v) (Becton Dickinson). Growth
on phenanthrene and HMW PAH biotransformation assays
were conducted as described previously and the detection of
ring-cleavage products was used as an indicator of HMW
PAH biotransformation (Kunihiro et al. 2013; Maeda et al.
2014). Strain KK22T cells were oxidase and catalase-positive.
Strong growth was observed between 20 and 40 8C and optimal growth occurred at 30 and 37 8C. Weak growth was
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39
Characterization of Sphingobium barthaii sp. nov.
Sphingobium abikonense IAM 12404T (AB021416)
87
Sphingobium lactosutens DS20T (EU675846)
Sphingobium olei IMMIB HF-1T (AM489507)
Sphingobium xenophagum DSM 6383T (X94098)
79
100 Sphingobium czechense LL01T (JN646865)
88
Sphingobium cupriresistens CU4T (JQ046313)
Sphingobium rhizovicinum CCM 7491T (EF465534)
Sphingobium baderi LL03T (JN695620)
81
91
Sphingobium wenxiniae JZ-1T (FJ686047)
89
Sphingobium faniae JZ-2T (FJ373058)
Sphingobium ummariense RL-3T (EF207155)
68
Sphingobium cloacae S-3T (AB040739)
100 Sphingobium barthaii KK22T (HQ830159)
Sphingobium fuliginis TKPT (DQ092757)
Sphingobium quisquiliarum P25T (EU781657)
54
Sphingobium herbicidovorans MBIC 3166T (AB022428)
Sphingobium mellinum WI4T (KF437546)
Sphingobium lucknowense F2T (EF534725)
95
Sphingobium japonicum UT26T (AF039168)
Sphingobium indicum B90AT (AY519129)
74 88
Sphingobium francense Sp+T (AY519130)
67
50
Sphingobium chinhatense IP26T (EF190507)
83
Sphingobium chungbukense DJ77T (AF159257)
Sphingomonas chlorophenolicum ATCC 33790T (X87161)
Sphingobium vermicomposti CCUG 55809T (AM998824)
Sphingobium scionense WP01T (EU009209)
99
Sphingobium yanoikuyae GIFU 9882T (D13728)
73
Sphingobium fontiphilum Chen16-4T (HQ667767)
Sphingobium amiense YTT (AB047364)
60
Sphingobium limneticum 301T (JN591313)
Sphingobium qiguonii X23T (EU095328)
100
Sphingobium jiangsuense BA-3T (HM748834)
100
Sphingobium vulgare HU1-GD12T (FJ177535)
Sphingobium xanthum NL9T (KF437579)
Sphingobium aromaticiconvertens RW16T (AM181012)
Sphingobium sufflavum HL-25T (JQ060960)
Sphingobium boeckii 469T (JN591315)
Sphingobium suberifaciens Ca1T (D13737)
Sphingopyxis witflariensis W-50T (AJ416410)
65
Novosphingobium aromaticivorans F199T (U20756)
99
Novosphingobium rosa IAM 14222T (D13945)
Sphingomonas oligophenolica S213T (AB018439)
Sphingomonas adhaesiva GIFU 11458T (D16146)
93
Sphingomonas paucimobilis GIFU 2395T (D16144)
Zymomonas mobilis DSM 18599T ( FR749909)
99
0.01
69
89
Fig. 1. Neighbour-joining phylogenetic dendrogram, based on 16S rRNA gene sequence comparisons, showing the position
of strain K22T and all type strains of species from the genera Sphingobium with validly published names on 18 June 2014
plus related genera within the family Sphingomonadaceae. The sequence of Zymomonas mobilis DSM 18599T was used as
the out-group. Accession numbers of sequences are given in parentheses. Bootstrap values are indicated at branch nodes
(per 100 resamplings). Bar, 0.01 substitutions per nucleotide position.
observed at 10 and 15 8C and strain KK22T did not grow at 4,
45, or 50 8C. Strain KK22T grew at pH 5.0 to 9.0 but not at
pH 4.0. This strain grew with NaCl up to a concentration of
http://ijs.sgmjournals.org
3.0 % (w/v) but not 3.5 % (w/v) or greater. It hydrolysed
Tween 20, but not Tween 80 and not casein. It was
DNase-negative. As shown in Table 1, comparisons of the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39
2921
A. H. Maeda and others
Table 1. Differential phenotypic and biochemical characteristics of strain KK22T and phylogenetically closely related
members of the genus Sphingobium
Strains: 1, KK22T; 2, Sphingobium fuliginis TKPT; 3, Sphingobium quisquiliarum P25T; 4, Sphingobium herbicidovorans MBIC 3166T; 5,
Sphingobium rhizovicinum CCM 7491T. All data are from this study.
All strains were negative for nitrate reduction, indole production, glucose fermentation, urease, DNase, hydrolysis of Tween 80, and assimilation of D -mannitol, D -sorbitol, myo-inositol, N-acetylglucosamine,
adipic acid, lactose, sucrose, D -ribose, L -xylose and casein. All strains
were positive for b-glucosidase, oxidase, and assimilation of D -glucose,
D -galactose and L -arabinose. +, Positive; 2, negative.
Characteristic
1
2
3
4
5
Motility
Production of catalase
b-Galactosidase
Hydrolysis of:
Tween 20
Gelatin
Starch
Assimilation of:
D -Fructose
Maltose
D -Xylose
Cellobiose
Raffinose
Trehalose
D -Arabinose
D -Mannose
Gentiobiose
Citrate
Phenanthrene
Biotransformation of:
Fluoranthene
Benz[a ]anthracene
Benzo[k ]fluoranthene
+
+
2
2
+
2
2
+
2
+
+
2
+
2
+
+
2
+
+
2
2
+
2
2
2
2
2
+
+
2
+
+
+
+
2
+
+
2
+
2
+
+
+
+
+
2
+
+
2
+
2
+
2
+
+
+
+
+
+
2
+
2
+
2
2
2
2
2
2
2
2
2
2
2
+
+
+
+
2
+
+
+
2
+
+
+
+
+
+
+
+
2
+
2
2
2
2
2
2
2
phenotypic properties of strain KK22T with closely related
type strains revealed differences which further supported the
conclusion that it was distinct from species of the genus Sphingobium with validly published names.
To conduct chemotaxonomic analyses, strains KK22T,
TKPT, P25T, MBIC 3166T and CCM 7491T were harvested
from liquid cultures at the late exponential phase of growth
on trypticase soy broth at 30 8C (with rotary shaking at
175 r.p.m. in the dark) (Becton Diskinson). The composition
of cellular fatty acids was analysed at the Technosuraga Laboratory (Shizuoka, Japan) by using the Sherlock Microbial
Identification (MIDI) System version 6.0 (MIDI). The
extraction and analysis of polar lipids by two-dimensional
TLC was performed according to standard procedures (Minnikin et al., 1979). Quinones were extracted and analysed by
using a Waters 600 Series HPLC System in-line with a phosphodiode array detector (Nishijima et al., 1997). The fatty
acids present in strain KK22T supported its assignment to
2922
the genus Sphingobium; fatty acids belonging to summed feature 8 that corresponded to the fatty acids C18 : 1v6c and/or
C18 : 1v7c were the major fatty acid(s) detected (64.3 %) and
C14 : 0 2-OH was the major hydroxylated fatty acid detected
(9.2 %). Only two other fatty acids were detected at levels
exceeding 4 % of the total: C16 : 0 (11.3 %) and 11-methyl
C18 : 1v7c (4.2 %). A difference of greater than 4 % was
observed in C16 : 0 when the composition was compared
with that of strain TKPT Table 2). When compared with
the other four most closely related strains, strain KK22T possessed the highest amount of fatty acids from summed feature 8, C14 : 0 and C14 : 0 2-OH. Comparisons of the
cellular fatty acid profiles of strain KK22T and the most closely related species of the genus Sphingobium are summarized in Table 2. The results of polar lipid analyses of strain
KK22T showed that it contained phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylN-methylethylethanolamine and sphingoglycolipid plus
three unidentified polar lipids and two unidentified phospholipids (Fig. S2). The polar lipids identified are those commonly reported for members of the genus Sphingobium. The
major respiratory quinone was ubiquinone Q-10 and other
respiratory quinones were not detected. Ubquinone Q-10
is found in all members of the phylum Proteobacteria and
it is an important feature of sphingomonads (Dadhwal
et al., 2009). All members of the genus Sphingobium contain
ubquinone Q-10 (Busse et al., 1999). The genomic DNA
G+C content of strain KK22T was 64.7 mol% (Maeda
et al., 2013), which fell within the range of 62–67 mol%
observed for other members of the genus Sphingobium
(Takeuchi et al., 2001). Based upon the results of DDH
and the phenotypic and chemotaxonomic analyses, strain
Table 2. Cellular fatty acid profiles of strain KK22T and the
type strains of related species of the genus Sphingobium
Strains: 1, KK22T; 2, Sphingobium fuliginis TKPT; 3, Sphingobium quisquiliarum P25T; 4, Sphingobium herbicidovorans MBIC 3166T; 5,
Sphingobium rhizovicinum CCM 7491T. Values are percentages of
the total fatty acids. 2, Not detected. All data are from this study.
Fatty acid
C14 : 0
C16 : 0
C18 : 0
C16 : 1v5c
C17 : 1v6c
C18 : 1v5c
C14 : 0 2-OH
C16 : 0 2-OH
11-Methyl C18 : 1v7c
Summed features*
3
8
1
2
3
4
5
0.6
11.3
0.5
1.1
–
1.2
9.2
0.5
4.2
0.5
15.5
0.6
0.8
–
0.9
9.0
0.4
4.8
0.4
14.8
0.5
1.0
1.0
2.1
8.5
0.4
7.9
0.2
6.0
0.4
1.5
1.5
3.9
7.5
0.7
11.4
0.1
16.3
0.4
1.2
–
1.8
7.0
0.8
0.9
7.1
64.3
6.6
61.1
7.2
54.1
10.6
53.2
14.8
56.8
*Summed feature 3 corresponds to C16 : 1v7c and/or C16 : 1v6c and
summed feature 8 corresponds to C18 : 1v6c and/or C18 : 1v7c.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39
Characterization of Sphingobium barthaii sp. nov.
KK22T is proposed as a representative of a novel species of the
genus Sphingobium, for which the name Sphingobium barthaii
sp. nov. is proposed.
strains from soil bearing the specific epithet rectivirgula. J Gen
Microbiol 115, 343–354.
Atlas, R. M. (1993). Handbook of Microbiological Media. Boca Raton,
FL: CRC Press.
Description of Sphingobium barthaii sp. nov.
Sphingobium barthaii (bar.tha9i.i. N.L. gen. n. barthaii of
Bartha in honour of Dr Richard Bartha, Professor Emeritus
of the Department of Biochemistry and Microbiology, Cook
College, Rutgers University, NJ, USA for his outstanding
contributions to the fields of microbial ecology and environmental microbiology including the biodegradation of aromatic hydrocarbon pollutants).
Cells are aerobic, Gram-stain-negative, polar flagellated rods
(0.5|1.3 mm). Colonies are shiny, opaque, convex, circular
and yellow–light brown with smooth edges that become
rough after extended incubation on TSA. Growth occurs at
up to 40 uC, but not at 45 uC. Luxuriant growth occurs at up
to 37 uC. Profuse growth occurs at pH 5.0–8.0 with weaker
growth at pH 9.0. Catalase- and oxidase-positive. Nitrate is
not reduced to nitrite. Hydrolyses starch and Tween 20, but
not Tween 80, gelatin or casein. b-Galactosidase activity is
not detected. Indole production is negative. Acid is produced
from D -glucose, D -xylose, D -fructose, D -galactose, cellobiose,
trehalose, D -arabinose, maltose and potassium gluconate,
but not from sucrose, D -mannitol, raffinose, D -sorbitol, lactose, D -mannose, myo-inositol, N-acetylglucosamine, capric
acid, adipic acid, malic acid or citrate. Whole-cell fatty acid
profiles consist mostly of C18 : 1v7c and C16 : 0 and the main
hydroxylated fatty acid is C14 : 0 2-OH. Ubiquinone Q-10
is the major respiratory quinone. Contains phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine,
phosphatidyl-N-methylethylethanolamine and sphingoglycolipid plus three unidentified polar lipids and two unidentified phospholipids. Grows on phenanthrene as a sole source
of carbon and energy up to concentrations as high as
500 mg l21 and biotransforms the following HMW PAHs to
downstream ring-fission products: fluoranthene, benz[a ]anthracene and benzo[k ]fluoranthene.
The type strain, KK22T (5DSM 29313T5JCM 30309T),
was isolated from a diesel-fuel-degrading bacterial consortium that was originally recovered from cattle pasture soil
in the Gulf region of Texas, USA. The DNA G+C content
of the type strain is 64.7 mol%.
Acknowledgements
This work was supported in part by Yokohama City University
Strategic Research Grant, K20002 and the Japan Society for the
Promotion of Science (JSPS) KAKENHI Grant no. 26505010 to R. A. K.
Bala, K., Sharma, P. & Lal, R. (2010). Sphingobium quisquiliarum
sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium isolated from an HCH-contaminated soil. Int J Syst Evol Microbiol 60,
429–433.
Busse, H.-J., Kämpfer, P. & Denner, E. B. M. (1999). Chemotaxonomic
characterisation of Sphingomonas. J Ind Microbiol Biotechnol 23,
242–251.
Cerniglia, C. E. (1992). Biodegradation of polycyclic aromatic
hydrocarbons. Biodegradation 3, 351–368.
Dadhwal, M., Jit, S., Kumari, H. & Lal, R. (2009). Sphingobium
chinhatense sp. nov., a hexachlorocyclohexane (HCH)-degrading
bacterium isolated from an HCH dumpsite. Int J Syst Evol
Microbiol 59, 3140–3144.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in
which radioisotopes are used to determine genetic relatedness among
bacterial strains. Int J Syst Bacteriol 39, 224–229.
Gordon, R. E. & Mihm, J. M. (1962). Identification of Norcadia caviae
(erikson) nov. comb. Annals NY Acad Sci 98, 628–636.
Kanaly, R. A. & Bartha, R. (1999). Cometabolic mineralization of
benzo[a]pyrene caused by hydrocarbon additions to soil Environ
Toxicol Chem 18, 2186–2190.
Kanaly, R. A. & Harayama, S. (2000). Biodegradation of high-
molecular-weight polycyclic aromatic hydrocarbons by bacteria.
J Bacteriol 182, 2059–2067.
Kanaly, R. A. & Harayama, S. (2010). Advances in the field of high-
molecular-weight polycyclic aromatic hydrocarbon biodegradation
by bacteria. Microb Biotechnol 3, 136–164.
Kanaly, R., Bartha, R., Fogel, S. & Findlay, M. (1997). Biodegradation
of [(sup14)C]benzo[a]pyrene added in crude oil to uncontaminated
soil. Appl Environ Microbiol 63, 4511–4515.
Kanaly, R. A., Bartha, R., Watanabe, K. & Harayama, S. (2000). Rapid
mineralization of benzo[a]pyrene by a microbial consortium growing
on diesel fuel. Appl Environ Microbiol 66, 4205–4211.
Kunihiro, M., Ozeki, Y., Nogi, Y., Hamamura, N. & Kanaly, R. A.
(2013). Benz[a]anthracene biotransformation and production of
ring fission products by Sphingobium sp. strain KK22. Appl Environ
Microbiol 79, 4410–4420.
Maeda, A. H., Nishi, S., Ozeki, Y., Ohta, Y., Hatada, Y. & Kanaly, R. A.
(2013). Draft genome sequence of Sphingobium sp. strain KK22, a
high-molecular-weight polycyclic aromatic hydrocarbon-degrading
bacterium isolated from cattle pasture soil. Genome Announc 1,
e00911–e00913.
Maeda, A. H., Nishi, S., Hatada, Y., Ozeki, Y. & Kanaly, R. A. (2014).
Biotransformation of the high-molecular weight polycyclic aromatic
hydrocarbon (PAH) benzo[k ]fluoranthene by Sphingobium sp.
strain KK22 and identification of new products of non-alternant
PAH biodegradation by liquid chromatography electrospray
ionization tandem mass spectrometry. Microb Biotechnol 7, 114–129.
Minnikin, D. E., Collins, M. D. & Goodfellow, M. (1979). Fatty acid and
polar lipid composition in the classification of Cellulomonas,
Oerskovia and related taxa. J Appl Bacteriol 47, 87–95.
References
Nishijima, M., Araki-Sakai, M. & Sano, H. (1997). Identification
Arden Jones, M. P., McCarthy, A. J. & Cross, T. (1979). Taxonomic
of isoprenoid quinones by frit-FAB liquid chromatographymass spectrometry for the chemotaxonomy of microorganisms.
J Microbiol Methods 28, 113–122.
and serologic studies on Micropolyspora faeni and Micropolyspora
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39
2923
A. H. Maeda and others
Prakash, O. & Lal, R. (2006). Description of Sphingobium fuliginis
sp. nov., a phenanthrene-degrading bacterium from a fly ash dumping site, and reclassification of Sphingomonas cloacae as Sphingobium
cloacae comb. nov. Int J Syst Evol Microbiol 56, 2147–2152.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol Biol Evol 4,
406–425.
Smibert, R. M. & Krieg, N. R. (1994). Pehnotypic characterization.
In Methods for General and Molecular Bacteriology, pp. 607–655.
Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood &
N. R. Krieg. Washington, DC: American Society for Microbiology.
Stackebrandt, E. (2011). Molecular taxonomic parameters. Microbiol
Aust 32, 59–61.
Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited:
tarnished gold standards. Microbiol Today 33, 152–155.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
DNA-DNA reassociation and 16S rRNA sequence analysis in the
present species definition in bacteriology. Int J Syst Evol Microbiol
44, 846–849.
Stolz, A. (2009). Molecular characteristics of xenobiotic-degrading
sphingomonads. Appl Microbiol Biotechnol 81, 793–811.
Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus
Sphingomonas sensu stricto and three new genera, Sphingobium,
Novosphingobium and Sphingopyxis, on the basis of phylogenetic
and chemotaxonomic analyses. Int J Syst Evol Microbiol 51,
1405–1417.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar,
S. (2011). MEGA 5: molecular evolutionary genetics analysis using
2924
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
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.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Tindall, B. J., Rosselló-Móra, R., Busse, H.-J., Ludwig, W. & Kämpfer,
P. (2010). Notes on the characterization of prokaryote strains for
taxonomic purposes. Int J Syst Evol Microbiol 60, 249–266.
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler,
O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. &
other authors (1987). International Committee on Systematic
Bacteriology. Report of the ad hoc committee on reconciliation of
approaches to bacterial systematics. Int J Syst Evol Microbiol 37,
463–464.
Young, C. C., Ho, M.-J., Arun, A. B., Chen, W.-M., Lai, W.-A., Shen,
F.-T., Rekha, P. D. & Yassin, A. F. (2007). Sphingobium olei sp. nov.,
isolated from oil-contaminated soil. Int J Syst Evol Microbiol 57,
2613–2617.
Young, C. C., Arun, A. B., Kämpfer, P., Busse, H.-J., Lai, W.-A., Chen,
W.-M., Shen, F.-T. & Rekha, P. D. (2008). Sphingobium rhizovicinum
sp. nov., isolated from rhizosphere soil of Fortunella hindsii
(Champ. ex Benth.) Swingle. Int J Syst Evol Microbiol 58, 1801–1806.
Zipper, C., Nickel, K., Angst, W. & Kohler, H.-P. E. (1996). Complete
microbial degradation of both enantiomers of the chiral herbicide
mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in
an enantioselective manner by Sphingomonas herbicidovorans
sp. nov. Appl Environ Microbiol 62, 4318–4322.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 65
IP: 88.99.165.207
On: Sun, 18 Jun 2017 02:57:39