Download Brevifollis gellanilyticus gen. nov., sp. nov., a gellan-gum

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3075–3078
DOI 10.1099/ijs.0.048793-0
Brevifollis gellanilyticus gen. nov., sp. nov., a
gellan-gum-degrading bacterium of the phylum
Verrucomicrobia
Shigeto Otsuka,1 Taku Suenaga,1 Hoan Thi Vu,1 Hiroyuki Ueda,1
Akira Yokota2 and Keishi Senoo1
1
Correspondence
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Shigeto Otsuka
[email protected]
2
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
The taxonomic properties of strain DC2c-G4T, a Gram-staining-negative, ovoid, gellan-gumdegrading bacterial isolate, were examined. Phylogenetic analysis based on 16S rRNA gene
sequences identified this isolate as a member of the phylum Verrucomicrobia and closest to the
genus Prosthecobacter. The 16S rRNA gene sequence similarities between this isolate and any of
the type strains of species of the genus Prosthecobacter were less than 95 %. In addition, the
absence of a single prostheca and the predominant menaquinone MK-7(H2) supported the
differentiation of this isolate from the genus Prosthecobacter. Here, we propose Brevifollis
gellanilyticus gen. nov., sp. nov. to accommodate the isolate. The type strain of the type species is
DC2c-G4T (5NBRC 108608T5CIP 110457T).
Bacteria belonging to the phylum Verrucomicrobia are
recognized as being typically difficult to cultivate. Although
recent advances in cultivation methods and patient efforts
have gradually increased the diversity of cultured isolates and
described species belonging to this phylum (e.g. Janssen et al.,
2002; Sangwan et al., 2005; Yoon et al., 2007a, b, 2008, 2010),
the number of cultured strains is still limited. Improved PCR
techniques and metagenomic analyses are now revealing the
ubiquitous distribution and even, in some cases, the
dominance of members of the phylum Verrucomicrobia in
the environment (Bergmann et al., 2011; Kielak et al., 2010).
However, the potential difficulties in cultivation have kept
the ecological functions of these organisms unknown.
Therefore, it is desirable to obtain more and diverse isolates
of the phylum Verrucomicrobia.
In our previous study, we established consortia of Chlorella
vulgaris NIES-227 (a green microalga) and bacteria
originating from soil (Ueda et al., 2010). The bacterial
culture fluid was collected from one of the consortia,
diluted, and inoculated onto 2 % gellan gum plates
containing live Chlorella prepared as previously reported
(Otsuka et al., 2013). Strain DC2c-G4T was isolated after
two weeks of cultivation at 30 uC, as one of the visible
colonies formed on the medium. Afterward, it turned out
Abbreviations: NJ, neighbour-joining; ML, maximum-likelihood.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of Brevifollis gellanilyticus DC2c-G4T is AB552872.
A supplementary figure is available with the online version of this paper.
048793 G 2013 IUMS
that this strain grows on liquid and agar-gelled R2A
(Seviour et al., 1994) and MMB (Staley & Mandel, 1973)
media, but not on nutrient broth (Difco). Strain DC2c-G4T
lyses gellan gum, and the surface of the gel around the
colonies sagged locally when this strain was cultivated on
gellan-gum-gelled media. The strain is maintained in our
laboratory in glycerol stock at –80 uC.
Gram staining was performed as described by Gerhardt
et al. (1994). Cell morphology was observed by micrographs (Fig. 1) taken by JEOL Datum (Tokyo, Japan) using
transmission electron microscopy with negative staining
using the model 1010 at an acceleration voltage of 100Kv,
with cells grown for 3 days or 1 week at 30 uC, namely at
their middle and late exponential growth phase, on 1.5 %
agar plates of R2A and MMB (separately). The bacterial
cells stain Gram-negative, and are non-motile, non-gliding,
single, unbranched, ovoid without prosthecae (Fig. 1) and
0.8–1.0 mm in width, 1.5–2.0 mm in length after division
and up to 3.5 mm in length shortly before division.
Without DNA extraction, the almost full-length 16S rRNA
gene was amplified by PCR with the primers 27F and 1492R
(Lane, 1991) by the same method and reaction conditions as
described elsewhere (Otsuka et al., 2008), with Biotaq DNA
polymerase (Bioline) and a GeneAmp PCR system 9700
thermal cycler (Applied Biosystems). The amplified products were subjected to sequencing commercially performed
by Takara Bio (Mie, Japan). The determined 16S rRNA gene
sequence of strain DC2c-G4T was a continuous stretch of
1469 bp.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 30 Apr 2017 09:10:06
Printed in Great Britain
3075
S. Otsuka and others
was checked, and distances (according to the Kimura twoparameter model) and clustering with the maximumlikelihood (ML) and neighbour-joining (NJ) methods were
determined by using bootstrap values based on 1000
replications. In the ML and NJ trees (Fig. 2 and Fig. S1
available in IJSEM Online, respectively), strain DC2c-G4T
was located within the cluster including all species of the
genus Prosthecobacter (indicated as ‘Prosthecobacter cluster’
in the trees).
Fig. 1. Transmission electron micrograph of negatively stained
cells of strain DC2c-G4T. Bar, 1 mm.
The close relatives of strain DC2c-G4T among species with
validly published names were searched for as described
elsewhere (Vu et al., 2010). Species of the genus
Prosthecobacter were the closest taxa, with sequence
similarities of 92.9–94.9 % calculated by EMBOSS Matcher
(Huang & Miller, 1991). These values are low enough to
describe a novel species without a DNA–DNA hybridization experiment (Stackebrandt & Goebel, 1994) or even a
new genus (Martinez-Romero & Caballero-Mellado, 1996)
represented by strain DC2c-G4T. The 16S rRNA gene
sequences were aligned based on secondary structure using
the SILVA SINA Webaligner (Pruesse et al., 2007) and
imported into MEGA 5 (Kumar et al., 2008). The alignment
0.05
50/70 84
/87
The respiratory quinone system and cellular fatty acid
composition of strain DC2c-G4T after 3 days of cultivation
at 30 uC on R2A agar medium were analysed according to
the same conditions described by Katsuta et al. (2005), by
using a GC (GC6890; Hewlett Packard) equipped with a
25 m60.2 mm Ultra 2 capillary column (19091B-102E;
Agient). The fatty acids were identified with MIDI Sherlock
Version 4.0 and the TSBA40 database. The G+C content
of the chromosomal DNA was determined, as described by
Mesbah et al. (1989), using reversed-phase HPLC.
Menaquinone MK-7(H2) was detected as the predominant
quinone. The major fatty acids were iso-C14 : 0 (27.1 %),
C16 : 0 (27.0 %), C16 : 1v11c (10.4 %), C16 : 1v5c (8.4 %),
C14 : 0 (6.5 %), anteiso-C15 : 0 (6.1 %) and C15 : 0 (4.9 %). The
DNA G+C content of strain DC2c-G4T was 59.4 mol%.
Growth under anaerobic conditions was tested by cultivating strain DC2c-G4T in an anaerobic chamber with an
O2-absorbing and CO2-generating agent (AnaeroPack;
Mitsubishi Gas Chemical). Salinity and pH ranges for
growth were tested by incubating the strain for a week at
Brevifollis gellanilyticus DC2c-G4T (AB552872)
Prosthecobacter fusiformis FC4T (U60015)
Prosthecobacter dejongeii FC1T (U60012)
99
Prosthecobacter debontii FC3T (U60014)
/100
Prosthecobacter vanneervenii FC2T (U60013)
98/100
Prosthecobacter fluviatilis JCM 14805T (AB305640)
100/100
100/100
‘Prosthecobacter
cluster’
Verrucomicrobium spinosum DSM 4136T (X90515)
96/96
43/–
Roseimicrobium gellanilyticum DC2a-G7T (AB552861)
Akkermansia muciniphila ATCC BAA-835T (AY271254)
91/94
Luteolibacter pohnpeiensis KCTC 22041T (AB331895)
82/85
Haloferula rosea KCTC 22201T (AB372853)
Roseibacillus ishigakijimensis KCTC 12986T (AB331888)
99/99
93/89
97/98
Rubritalea marina DSM 17716T (DQ302104)
Persicirhabdus sediminis KCTC 22039T (AB331886)
Opitutus terrae JCM 15787T (AJ229235)
Chlamydia trachomatis ATCC VR-571BT (D89067)
Fig. 2. ML tree reconstructed based on the nearly full-length 16S rRNA gene sequences of strain DC2c-G4T and the type
strains of phylogenetically related species. Chlamydia trachomatis ATCC VR-571BT was used as the outgroup. Accession
numbers in DDBJ/EMBL/GenBank databases are indicated in parentheses. Bootstrap values based on 1000 replications for
ML and NJ analyses are indicated at the corresponding nodes (ML/NJ). Tree topologies differed between ML and NJ trees at the
node indicated by an open circle. Bar, 0.05 nucleotide substitutions per site.
3076
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 63
IP: 88.99.165.207
On: Sun, 30 Apr 2017 09:10:06
Brevifollis gellanilyticus gen. nov., sp. nov.
30 uC in R2A medium supplemented with 0, 1, 4, 8, 9, or
10 % NaCl (w/v) and in R2A medium adjusted to pH 5 to
10 at 1 pH unit intervals using HCl or NaOH. Tolerance
of antibiotics was examined in three repetitions by Biolog
Gen III MicroPlate during 1 week of incubation at 30 uC.
This period of time was necessary for the positive control
to become clearly positive. Growth on saccharides,
catalase activity, oxidase activity and the temperature
range for growth were determined as previously described
(Otsuka et al., 2013). The results are given in the species
description.
As described above, strain DC2c-G4T can be differentiated
from members of the genus Prosthecobacter by the
menaquinone profile and the absence of prostheca (Table
1), although the strain is located within the ‘Prosthecobacter
cluster’ in the phylogenetic trees (Figs 2 and S1). In order to
describe a novel species of the genus Prosthecobacter
represented by strain DC2c-G4T, we would need to emend
the genus description to remove the distinguishing characteristic of this genus, the presence of a single prostheca,
which may cause confusion. Alternatively, if we describe a
new genus represented by strain DC2c-G4T, we should
admit the presence of a different genus within the
‘Prosthecobacter cluster’ in the phylogenetic trees (Figs 2
and S1). Considering that a new taxon can arise from an
already existing taxon, it is not awkward if the
‘Prosthecobacter cluster’ embeds a different genus as a result
of evolutionary differentiation. Therefore, we propose that
strain DC2c-G4T represents a novel species in a new genus.
Description of Brevifollis gen. nov.
Brevifollis (Bre.vi.fol9lis. L. adj. brevis short, small; L. masc.
n. follis a windball; N.L. masc. n. Brevifollis a short ball,
intended to mean a small ovoid cell).
Cells are ovoid to short fusiform, single, unbranched, non–
motile, non–gliding and stain Gram-negative. Obligately
aerobic. Chemo-organotrophic. The major respiratory
quinone is menaquinone MK-7(H2). The DNA G+C
content is approximately 59–60 mol%. The type species is
Brevifollis gellanilyticus. The differentiation of this genus
from the phylogenetically close genus Prosthecobacter is
possible chiefly by the major menaquinone MK-7(H2) and
the absence of a single polar prostheca.
The type species is Brevifollis gellanilyticus.
Description of Brevifollis gellanilyticus sp. nov.
Brevifollis gellanilyticus [gel.lan.i.ly9ti.cus. N.L. neut. n.
gellanum gellan; N.L. masc. adj. lyticus (from Gr. adj.
lutikon) able to loose, able to dissolve; N.L. masc. adj.
gellanilyticus gellan-dissolving].
Cells stain Gram-negative and are non-motile, non-gliding,
single, unbranched, ovoid to short fusiform and 0.8–162–
3.5 mm in diameter. Forms opaque pale-yellow colonies with
a smooth, convex and circular surface after 3 days to 1 week
of cultivation at 30 uC on R2A agar and MMB agar media.
The predominant respiratory quinone is menaquinone MK7(H2). The major fatty acids are iso-C14 : 0 and C16 : 0. Grows
well at 26 to 30 uC, can grow at 10 to 37 uC, but does not
grow or hardly grows at 4 or 42 uC; grows in media at pH 6
to 8 but not at pH 5 or 9; can tolerate 8 % but not 9 %
salinity. Sensitive to tetrazolium violet and tetrazolium blue.
Insensitive to 1 % sodium lactate, troleandomycin, lincomycin, vancomycin, nalidixic acid, aztreonam, fusidic acid,
D-serine, rifamycin SV, minocycline, guanidine HCl, lithium
chloride, potassium tellurite, sodium butyrate and sodium
bromate. Growth occurs on D-glucose, D-galactose, Dfructose (slow growth), L-rhamnose, D-mannose, L-xylose,
Table 1. Characteristics differentiating strain DC2c-G4T from phylogenetically close genera
Taxa: 1, DC2c-G4T; 2, Prosthecobacter; 3, Verrucomicrobium; 4, Roseimicrobium. Data for Prosthecobacter, Verrucomicrobium and Roseimicrobium
from Hedlund et al. (1997), Hedlund (2010), Otsuka et al. (2013) & Takeda et al. (2008). +, Positive; –, negative; NA, data not available.
Characteristic
Cell shape
Cell length
Cell width
Multiple short prosthecae
Single polar prostheca
Colony colour
Optimal growth temperature (uC)
pH range for growth
Salinity tolerance (% NaCl)
Aerobic growth
Anaerobic growth
Major menaquinones
DNA G+C content (mol%)
1
2
3
Ovoid to short fusiform
2–3.5
0.8–1
–
–
Pale yellow
10–37
6–8
8
+
–
MK-7(H2)
59.4
Fusiform to vibrioid
2–10
0.5–1
–
+
Pale yellow
1–40
6.5–7*
Rod
1–3.8
0.8–1
+
–
Pale yellow
26–33
NA
+
+
MK-6(H2)
54.6–62.9
4
Ovoid to rod
2–4.5
0.9–1.5
–
–
Orangish-red
26–30
NA
6–7.5
1
1
+
+
–
–
MK-9, MK-10, MK-10(H2) MK-9, MK-10, MK-11
58–59
60.1
*pH range for growth of Prosthecobacter is that of Prosthecobacter fluviatilis HAQ-1T (Takeda et al., 2008).
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 30 Apr 2017 09:10:06
3077
S. Otsuka and others
D-sorbitol, D-gluconic acid, sucrose, starch, pullulan, inulin,
curdlan, gellan gum, xanthan gum, galactomannan and
xylan (very slow growth). No growth occurs on Dglucuronic acid, glycerol, pyruvate, cellulose (soluble) or
alginate. Positive for cytochrome oxidase and catalase.
Otsuka, S., Sudiana, I.-M., Komori, A., Isobe, K., Deguchi, S.,
Nishiyama, M., Shimizu, H. & Senoo, K. (2008). Community
The type strain is DC2c-G4T (5NBRC 108608T5CIP
110457T), isolated from an artificial consortium of
Chlorella vulgaris (green alga) and a mixed population of
bacteria originating from soil. The DNA G+C content
of strain DC2c-G4T is 59.4 mol%.
nov., a new member of the class Verrucomicrobiae. Int J Syst Evol
Microbiol 63, 1982–1986.
structure of soil bacteria in a tropical rainforest several years after
fire. Microbes Environ 23, 49–56.
Otsuka, S., Ueda, H., Suenaga, T., Uchino, Y., Hamada, M., Yokota,
A. & Senoo, K. (2013). Roseimicrobium gellanilyticum gen. nov., sp.
Pruesse, E., Quast, C., Knittel, K., Fuchs, B. M., Ludwig, W., Peplies,
J. & Glöckner, F. O. (2007). SILVA: a comprehensive online resource
for quality checked and aligned ribosomal RNA sequence data
compatible with ARB. Nucleic Acids Res 35, 7188–7196.
Sangwan, P., Kovac, S., Davis, K. E. R., Sait, M. & Janssen, P. H.
(2005). Detection and cultivation of soil verrucomicrobia. Appl
Acknowledgements
This work was supported by a research fund from the Institute for
Fermentation, Osaka (IFO), Japan.
Environ Microbiol 71, 8402–8410.
Seviour, E. M., Williams, C., DeGrey, B., Soddell, J. A., Seviour, K. C.
& Lindrea, K. C. (1994). Studies on filamentous bacteria from
Australian activated sludge plants. Water Res 28, 2335–2342.
References
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
Bergmann, G. T., Bates, S. T., Eilers, K. G., Lauber, C. L., Caporaso,
J. G., Walters, W. A., Knight, R. & Fierer, N. (2011). The under-
DNA-DNA reassociation and 16S rRNA sequence analysis in the
present species definition in bacteriology. Int J Syst Bacteriol 44, 846–
849.
recognized dominance of Verrucomicrobia in soil bacterial communities. Soil Biol Biochem 43, 1450–1455.
Staley, J. T. & Mandel, M. (1973). Deoxyribonucleic acid base
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors)
(1994). Methods for General and Molecular Bacteriology. Washington,
DC: American Society for Microbiology.
Hedlund, B. P. (2010). Phylum XXIII. Verrucomicrobia phyl. nov. In
Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 4, pp. 795–
841. Edited by N. R. Krieg, J. T. Staley, D. R. Brown, B. P. Hedlund &
B. J. Paster. New York: Springer.
Hedlund, B. P., Gosink, J. J. & Staley, J. T. (1997). Verrucomicrobia
div. nov., a new division of the bacteria containing three new species
of Prosthecobacter. Antonie van Leeuwenhoek 72, 29–38.
Huang, X. & Miller, W. (1991). A time-efficient, linear-space local
similarity algorithm. Adv Appl Math 12, 337–357.
Janssen, P. H., Yates, P. S., Grinton, B. E., Taylor, P. M. & Sait, M.
(2002). Improved culturability of soil bacteria and isolation in pure
culture of novel members of the divisions Acidobacteria,
Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl Environ
Microbiol 68, 2391–2396.
Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005).
Ferrimonas marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
Kielak, A., Rodrigues, J. L. M., Kuramae, E. E., Chain, P. S. G., van
Veen, J. A. & Kowalchuk, G. A. (2010). Phylogenetic and
metagenomic analysis of Verrucomicrobia in former agricultural
grassland soil. FEMS Microbiol Ecol 71, 23–33.
Kumar, S., Nei, M., Dudley, J. & Tamura, K. (2008). MEGA: a biologist-
centric software for evolutionary analysis of DNA and protein
sequences. Brief Bioinform 9, 299–306.
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid
Techniques in Bacterial Systematics, pp. 115–147. Edited by
E. Stackebrandt & M. Goodfellow. New York: John Wiley & Sons.
Martinez-Romero, E. & Caballero-Mellado, J. (1996). Rhizobium
phylogenies and bacterial genetic diversity. Crit Rev Plant Sci 15, 113–
140.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
measurement of the G+C content of deoxyribonucleic acid by highperformance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
3078
composition of Prosthecomicrobium and Ancalomicrobium strains. Int
J Syst Bacteriol 23, 271–273.
Takeda, M., Yoneya, A., Miyazaki, Y., Kondo, K., Makita, H., Kondoh,
M., Suzuki, I. & Koizumi, J. (2008). Prosthecobacter fluviatilis sp. nov.,
which lacks the bacterial tubulin btubA and btubB genes. Int J Syst
Evol Microbiol 58, 1561–1565.
Ueda, H., Otsuka, S. & Senoo, K. (2010). Bacterial communities
constructed in artificial consortia of bacteria and Chlorella vulgaris.
Microbes Environ 25, 36–40.
Vu, H. T., Otsuka, S., Ueda, H. & Senoo, K. (2010). Cocultivated
bacteria can increase or decrease the culture lifetime of Chlorella
vulgaris. J Gen Appl Microbiol 56, 413–418.
Yoon, J., Yasumoto-Hirose, M., Matsuo, Y., Nozawa, M., Matsuda, S.,
Kasai, H. & Yokota, A. (2007a). Pelagicoccus mobilis gen. nov., sp.
nov., Pelagicoccus albus sp. nov. and Pelagicoccus litoralis sp. nov.,
three novel members of subdivision 4 within the phylum
‘Verrucomicrobia’, isolated from seawater by in situ cultivation. Int J
Syst Evol Microbiol 57, 1377–1385.
Yoon, J., Matsuo, Y., Matsuda, S., Adachi, K., Kasai, H. & Yokota, A.
(2007b). Rubritalea spongiae sp. nov. and Rubritalea tangerina sp.
nov., two carotenoid- and squalene-producing marine bacteria of the
family Verrucomicrobiaceae within the phylum ‘Verrucomicrobia’,
isolated from marine animals. Int J Syst Evol Microbiol 57, 2337–2343.
Yoon, J., Matsuo, Y., Adachi, K., Nozawa, M., Matsuda, S., Kasai, H. &
Yokota, A. (2008). Description of Persicirhabdus sediminis gen. nov.,
sp. nov., Roseibacillus ishigakijimensis gen. nov., sp. nov., Roseibacillus
ponti sp. nov., Roseibacillus persicicus sp. nov., Luteolibacter pohnpeiensis gen. nov., sp. nov. and Luteolibacter algae sp. nov., six marine
members of the phylum ‘Verrucomicrobia’, and emended descriptions
of the class Verrucomicrobiae, the order Verrucomicrobiales and the
family Verrucomicrobiaceae. Int J Syst Evol Microbiol 58, 998–1007.
Yoon, J., Matsuo, Y., Matsuda, S., Kasai, H. & Yokota, A. (2010).
Cerasicoccus maritimus sp. nov. and Cerasicoccus frondis sp. nov., two
peptidoglycan-less marine verrucomicrobial species, and description
of Verrucomicrobia phyl. nov., nom. rev. J Gen Appl Microbiol 56,
213–222.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 63
IP: 88.99.165.207
On: Sun, 30 Apr 2017 09:10:06