International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2690–2699
DOI 10.1099/ijs.0.047787-0
Methylobacterium trifolii sp. nov. and
Methylobacterium thuringiense sp. nov., methanolutilizing, pink-pigmented bacteria isolated from leaf
surfaces
S. Wellner, N. Lodders, S. P. Glaeser and P. Kämpfer
Correspondence
Peter Kämpfer
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen,
Germany
peter.kaempfer@umwelt.
uni-giessen.de
Three pink-pigmented, aerobic, Gram-stain-negative, rod-shaped and facultatively methylotrophic
strains were isolated from the phyllosphere of Trifolium repens and Cerastium holosteoides. 16S
rRNA gene sequence analysis support the affiliation of all strains to the genus Methylobacterium.
The closest relatives of strains C34T and T5 were Methylobacterium gnaphalii 23eT (98.0 and
98.5 % sequence similarity, respectively) and Methylobacterium organophilum JCM 2833T (97.0
and 97.2 %, respectively). Strain TA73T showed the highest sequence similarities to
Methylobacterium marchantiae JT1T and Methylobacterium bullatum F3.2T (both 97.9 %),
followed by Methylobacterium phyllosphaerae CBMB27T and Methylobacterium brachiatum
DSM 19569T (both 97.8 %), Methylobacterium cerastii C15T and Methylobacterium
radiotolerans JCM 2831T (both 97.7 %). The major components in the fatty acid profiles were
C18 : 1v7c, C16 : 0 and one unknown fatty acid for strain TA73T and C18 : 1v7c, C16 : 1v7c/isoC15 : 0 2-OH, C18 : 0 and C16 : 0 for strains C34T and T5. Physiological and biochemical analysis,
including DNA–DNA hybridization, revealed clear differences between the investigated strains
and their closest phylogenetic neighbours. DNA–DNA hybridization studies also showed high
similarities between strains C34T and T5 (59.6–100 %). Therefore, the isolates represent two
novel species within the genus Methylobacterium, for which the names Methylobacterium trifolii
sp. nov. (type strain TA73T5LMG 25778T5CCM 7786T) and Methylobacterium thuringiense sp.
nov. (type strain C34T5LMG 25777T5CCM 7787T) are proposed.
Strains of the genus Methylobacterium are typically strictly
aerobic, Gram-negative, rod-shaped and facultatively
methylotrophic bacteria. They are able to grow on onecarbon compounds such as formate, formaldehyde or
methanol as the sole source of carbon and energy, as well as
on a wide range of multicarbon substrates (Green, 2006).
Most Methylobacterium strains, except Methylobacterium
nodulans ORS 2060T and Methylobacterium jeotgali S2R039T, show the characteristic pink pigmentation caused by
carotenoids (Urakami et al., 1993; Konovalova et al., 2007).
The genus Methylobacterium, with the Methylobacterium
organophilum as the type species, belongs to the class
Alphaproteobacteria. It was initially proposed by Patt et al.
(1976) and, at the time of writing, contained 42 species
with validly published names (http://www.bacterio.cict.fr/
Abbreviations: pNA, para-nitroanilide; pNP, para-nitrophenyl.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA/
MxaF gene sequences sequence of strains T5, C34T and TA73T are
FR847846/FR847847, FR847848/FR847843 and FR847844/
FR847845, respectively.
2690
m/methylobacterium.html). However, based on high DNA–
DNA similarity values, Kato et al. (2005) stated that Methylobacterium chloromethanicum (McDonald et al., 2001) and
Methylobacterium dichloromethanicum (Doronina et al., 2000)
should be regarded as synonyms of Methylobacterium
extorquens (Bousfield & Green, 1985) and Methylobacterium
lusitanum (Doronina et al., 2002) should be regarded as a
synonym of Methylobacterium rhodesianum (Green et al.,
1988). Additionally, the names Methylobacterium dankookense
(Lee et al., 2009), M. bullatum (Hoppe et al., 2011), M. cerastii
(Wellner et al., 2012), M. longum (Knief et al., 2012), M.
oxalidis (Tani et al., 2012a), M. soli (Cao et al., 2011) and
M. gnaphalii (Tani et al., 2012b) have been validly published
recently.
Although methylobacteria can be found in such diverse
habitats as soil, freshwater, sewage, in the human mouth
and on feet (Doronina et al., 2002; Kato et al., 2008; Anesti
et al., 2004; Anesti et al., 2005), they are particularly known
as phyllosphere inhabitants (Corpe & Rheem, 1989;
Holland & Polacco, 1994; Trotsenko et al., 2001). A recent
meta-proteomic study confirmed that Methylobacterium is
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Two novel species of the genus Methylobacterium
a predominant genus in the phyllosphere (Delmotte et al.,
2009). Methylobacteria have been detected on leaves of
many agricultural plants like maize, cotton, sunflower,
soybean, red clover, winter wheat and rice (Balachandar
et al., 2007; Delmotte et al., 2009; Knief et al., 2010;
Madhaiyan et al., 2007; Madhaiyan et al., 2009; Omer et al.,
2004; Raja et al., 2008). Additionally, they have been found
on leaves of trees (Doronina et al., 2004; Kang et al., 2007;
Van Aken et al., 2004), mosses and herbs like Cerastium
holosteoides, Bellis perennis and Taraxacum officinale (Knief
et al., 2008; Wellner et al., 2011). Besides leaves,
Methylobacterium strains have also been isolated from
stem tissue (Madhaiyan et al., 2007) and root nodules
(Jourand et al., 2004). Methylobacteria are able to utilize
methanol emitted from plants and, in turn, produce plantgrowth promoting substances such as indole-3-acetic acid
(IAA), cytokinins or vitamins (Ivanova et al., 2000, 2001,
2006; Koenig et al., 2002; Trotsenko et al., 2001).
All strains were isolated from the phyllosphere of leaves
collected in the framework of a comprehensive biodiversity
program (Fischer et al., 2010). Strain TA73T was isolated
from the leaf surface of Trifolium repens collected in 2009
in the region Schwäbische Alb, Germany. Strain T5 was
isolated from the phyllosphere of Trifolium repens and
strain C34T was from Cerastium holosteoides, both collected
in 2008 in the Hainich-Dün region, Germany.
Potassium phosphate buffer (6.75 g KH2PO4, 8.75 g
K2HPO4 per litre) and mechanical treatment (Stomacher
80 Biomaster; Seward Laboratory Systems) were used to
remove bacteria from the leaf surface. Serial dilutions were
plated on mineral salt medium supplemented with 0.5 %
methanol (M125, according to the DSMZ, Germany) and
incubated at 25 uC for 14 days. Pink-pigmented colonies
with different morphologies were isolated.
Exponentially grown M125-broth cultures were used to
determine cellular morphology and motility with a
fluorescence microscope (Axiophot2; ZEISS). Gram-staining was performed as described by Gerhardt et al. (1994).
All strains are aerobic, Gram-stain-negative, rod-shaped,
non-motile and formed pink- to red-pigmented colonies.
According to Gerhardt et al. (1994), 3 % H2O2 and 1 %
tetramethyl-p-phenylenediamine dihydrochloride, respectively, were used to test the production of catalase and
oxidase. All strains produced these enzymes.
Growth on the following media was monitored over a
period of 21 days at 28 uC: CASO agar (Carl Roth),
medium M85 (according to DSMZ), K7 (1 g yeast extract,
1 g peptone, 1 g glucose, 15 g agar per litre, pH 7.2), Luria
broth (LB; Sigma-Aldrich Chemie), medium M65 (according to DSMZ), marine agar 2216 (Becton Dickinson),
nutrient agar (NA; Becton Dickinson), nutrient agar
(Oxoid), PCA (5 g casein peptone, 2.5 g yeast extract, 1 g
glucose, 9 g agar per litre, pH 7.0), PYE (3 g yeast extract,
3 g peptone, 15 g agar per litre, pH 7.2), PYG (20 g
glucose, 10 g yeast extract, 10 g peptone, 15 g agar per
http://ijs.sgmjournals.org
litre), R2A (Oxoid), medium M1207 (according to DSMZ)
and tryptic soy agar (TSA; Becton Dickinson).
Growth in M125 broth at various temperatures (4, 10, 16,
20, 25, 28, 36 and 45 uC), different initial methanol
concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 1.5 and 2.0 %) and
different initial pH-values (4.0, 5.0, 6.0, 7.0, 7.2, 8.0 and
10.0) was documented after 3 and 14 days, respectively.
Salt (NaCl) tolerance was assessed in M125 broth
supplemented with 0, 0.5, 1.0, 2.0, 3.0 and 4.0 % (w/v)
NaCl over a period of 14 days. Bacteria grown on modified
Bennett agar (Williams et al., 1989) were covered with
iodine solution to test for hydrolysis of starch. Indole
formation, hydrolysis of adonitol, citrate, glucose, inositol,
malonate, n-nitrophenyl galactopyranoside, rhamnose,
sucrose and xylose as well as the production of the
enzymes arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease
were determined with the micronaut E test pate (Merlin
Diagnostika) following the manufacturer’s instructions.
Substrate assimilation was tested either in M125 broth or
in microtitre plates according to Kämpfer et al. (1991).
M125 broth was supplemented with 0.5 % acetate, Larabinose, citrate, ethanol, D-fructose, D-glucose, L-glutamate, tartrate and D-xylose, respectively, instead of
methanol. To examine growth on different C1 compounds,
0.1 % formaldehyde, formate, formamide and methylamine, respectively, were added to M125 broth instead of
methanol. Assimilation of methane was carried out in
M125 broth where methane was provided as headspace gas
in the proportions 50 : 50 methane : air according to Green
(2006). Like other members of the genus, all strains grew
on methanol. However, they failed to grow on formaldehyde, formamide, formate and methylamine. None of the
strains grew on methane, not even M. organophilum LMG
6083T, which was reported to utilize methane in some cases
(Patt et al., 1974, 1976). However, the possibility that M.
organophilum can lose the ability to utilize methane was
observed previously by Green & Bousfield (1983). In
contrast to M. organophilum LMG 6083T, strains C34T and
T5 did not utilize L-aspartate, glutarate, itaconate, trehalose
or L-tryptophan as sole carbon sources. Strains C34T and
T5 also showed differences in substrate utilization to each
other, for example for L-glutamate, DL-lactate, pyruvate or
T
D-ribose. Strain TA73 differed from M. phyllosphaerae
T
CBMB27 , M. fujisawaense DSM 5686T and M. oryzae
CBMB20T for example in its ability to utilize L-malate,
mesaconate, 2-oxoglutarate and pyruvate and its inability
to utilize adipate, azelate and suberate. Other physiological
features of the tested strains and differences to their closest
relatives are summarized in the species description and
Table 1.
The extraction of pigments was performed as described
by Wellner et al. (2011). The resulting supernatant was
measured at wavelengths from 300 to 900 nm in an Infinite
200 Pro microplate reader (Tecan Deutschland) against
80 % methanol as reference. Pigments of all isolates showed
absorption peaks at 490 and 520 nm, respectively, and a
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2691
S. Wellner and others
Table 1. Differential phenotypic characteristics of the isolates and members of other related species of the genus Methylobacterium
Taxa: 1, C34T; 2, T5; 3, TA73T; 4, M. organophilum LMG 6083T; 5, M. gnaphalii 23eT; 6, M. phyllosphaerae DSM 19779T; 7, M. oryzae DSM 18207T;
8, M. fujisawaense DSM 5686T; 9, M. marchantiae JT1T. All data were obtained comparatively in this study. +, Positive; 2, negative; (+), weak, V,
variable results; ND, not determined. Results in square brackets are data from the following: 4, Patt et al. (1976, 1974); 5, Tani et al. (2012b); 6,
Madhaiyan et al. (2009); 7, Madhaiyan et al. (2007); 8, Green et al. (1988); 9, Schauer et al. (2011). ‘‘/’’: Different test systems were applied, the tests
according to Kämpfer et al. (1991) or alternative test systems listed below the table. The test systems applied for each tested substance are given
behind the respective name. If different results were obtained in the tests, the results according to Kämpfer et al. (1991) are given in first place. All
strains were positive for hydrolysis of L-alanine-pNA*|| and acid production from rhamnoseD. pNP, para-nitrophenyl; pNA, para-nitroanilide.
Characteristic
Acid produced from:
D-Glucose*D
L-Arabinose*
D-Xylose*D
D-Mannose*
Assimilation as sole carbon source:
Methaned
Formamide§
Formaldehyde§
Methylamine§
Formate§
L-Glutamate||
Tartrate||
Ethanol||
N-Acetyl-D-Galactosamine*
N-Acetyl-D-Glucosamine*
L-Arabinose*||
D-Fructose*||
D-Galactose*
D-Glucose*||
D-Ribose*
Sucrose*
Trehalose*
D-Xylose*||
Maltitol
D-Mannitol
D-Sorbitol*
Putrescine*
Acetate (sodium salt) *||
Propionate*
Adipate*
Azelate*
Citrate*||
Fumarate*
Glutarate*
DL-3-Hydroxybutyrate*
Itaconate*
DL-Lactate*
L-Malate*
Mesaconate*
Oxo-glutarate*
Pyruvate*
Suberate*
L-Alanine*
b-Alanine*
L-Aspartate*
L-Phenylalanine*
L-Tryptophan*
3-Hydroxy-benzoate*
2692
1
2
3
4
5
6
7
8
9
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
2/2
+
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
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
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/+ [(+)]
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/+ [2]
2
+
+
2/2 [2]
2
2
+
2
2
2
2
2
2
+
2
2
2
2
2
2
ND
ND
ND
[2]
[+]
[+]
ND
ND
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 [V]
2 [+]
(+) [V]
+
2
2
2
2
+/+ [+] 2/2 [2]
+/+ [V] +/+ [+]
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
2
2
+
2
2
2
2
2
2
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International Journal of Systematic and Evolutionary Microbiology 63
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Two novel species of the genus Methylobacterium
Table 1. cont.
Characteristic
1
2
3
4
5
6
7
8
9
Hydrolysis of:
pNP-b-D-Xylopyranoside
pNP-Phenyl-phosphonate*
Bis-pNP-phosphate*
pNP-Phosphate-disodium salt
2
+
+
+
2
2
+
+
+
(+)
(+)
+
2
2
+
+
2
2
(+)
+
2
+
+
+
2
+
+
+
2
+
+
+
2
+
+
2
*Result from the method according to Kämpfer et al. (1991).
DResult from Micronaut E.
dGrowth in M125 where methane was provided as head space gas in the proportions 50 : 50 methane : air.
§Growth in M125 supplemented with 0.1 % substrate.
||Growth in M125 supplemented with 0.5 % substrate.
weak peak at 460 nm. These absorption maxima have been
reported for previously described strains of the genus
Methylobacterium (Schauer et al., 2011; Urakami et al.,
1993) indicating that they contain the same carotenoids.
Furthermore, a peak at 360 nm was detected for all strains,
which was also present in the non-pigmented control M.
jeotgali S2R03-9T. Strains C34T and T5 showed a weak
absorption maximum at 770 nm indicating that the same
carotenoids are present in these strains.
The commercially available GenElute Plant Genomic DNA
Miniprep kit (Sigma-Aldrich) was used to extract genomic
DNA following the manufacturer’s instructions. For
PCR amplification of nearly full-length 16S rRNA gene
sequences (1348/1350 nt), universal eubacterial 16S rRNA
gene primers 27F and 1492R (Lane, 1991) were used with
the following cycle conditions: an initial denaturation of
95 uC for 3 min, 33 cycles of 94 uC for 45 s, 57.3 uC for
45 s, 72 uC for 2 min, and a final elongation step of 72 uC
for 15 min. The PCR mixture contained 32 ml RNase/
DNase-free water, 5 ml 106 Taq Buffer (containing KCl),
4 ml MgCl2 (25 nM), 5 ml dNTPs (2 mM), 0.9 ml of each
primer (10 mM), 0.2 ml Taq DNA polymerase (5 U ml21)
and 1 ml of genomic DNA in a total volume of 50 ml.
A partial sequence of the mxaF gene was amplified using
primers 1003f and 1561r (McDonald et al., 1995) and the
following PCR conditions: an initial denaturation of 95 uC
for 3 min, 30 cycles of 94 uC for 60 s, 55 uC for 60 s, 72 uC
for 60 s, and a final elongation step of 72 uC for 5 min.
PCR amplification was performed in a total volume of
50 ml. The reaction mixture contained 31.8 ml RNase/
DNase-free water, 5 ml 106 Taq Buffer (containing KCl),
6 ml MgCl2 (25 nM), 4 ml dNTPs (2 mM), 1 ml of each
primer (10 mM), 0.2 ml Taq DNA polymerase (5 U ml21)
and 1 ml of genomic DNA. PCR products were purified
with the QIAquick PCR purification system (Qiagen) and
sequenced with the primers listed above.
Phylogenetic analyses of the 16S rRNA gene sequences were
performed in ARB release 5.2 (Ludwig et al., 2004) using the
‘All-Species Living Tree’ Project (LTP; Yarza et al., 2008)
database release LTPs106 (August 2011). Sequences not
http://ijs.sgmjournals.org
included in the LTP database were aligned with SINA
(v1.2.9) according to the SILVA seed alignment (http://
www.arb-silva.de; Pruesse et al., 2007) and implemented in
the ARB database. The alignment was controlled manually
based on secondary structure information. Sequence
similarities were calculated in ARB without the use of an
evolutionary substitution model. Phylogenetic trees were
constructed with the maximum-likelihood method using
RAxML v7.04 (Stamatakis, 2006) with GTR-GAMMA and
rapid bootstrap analysis, the neighbour-joining method
with the Jukes–Cantor correction (Jukes & Cantor, 1969),
and the maximum-parsimony method using DNAPARS v 3.6
(Felsenstein, 2005). All phylogenetic trees were calculated
with 100 resamplings (bootstrap analysis; Felsenstein,
1985) and based on 16S rRNA gene positions 85 to
1432 (numbering according to Brosius et al., 1978).
Comparative 16S rRNA gene sequence analysis clearly
allocated the novel strains to the genus Methylobacterium
(Fig. 1). Sequence similarity calculations revealed that
strain C34T and T5 shared 99.3 % sequence similarity and
were most closely related to M. gnaphalii 23eT (98.0 and
98.5 %, respectively) and M. organophilum JCM 2833T
(97.0 and 97.2 %, respectively). Strain TA73T shared only
96.4 % sequence similarity with C34T and T5 and was
closely related to M. marchantiae JT1T and M. bullatum
F3.2T (both 97.9 %), M. phyllosphaerae CBMB27T and M.
brachiatum DSM 19569T (both 97.8 %), M. radiotolerans
JCM 2831T and M. cerastii C15T (both 97.7 %), and M.
fujisawaense DSM 5686T, M. oryzae CBMB20T, M. longum
440T and M. tardum RB677T, and M. mesophilicum DSM
1708T (all 97.6 %). The construction of phylogenetic trees
showed that strains C37T and T5 formed a distinct cluster
with the type strains of M. gnaphalii (supported by high
bootstrap values) and M. organophilum. In contrast, TA73T
was only loosely linked to the type strains with high 16S
rRNA gene sequence similarities (.97 %) (Fig. 1).
MxaF sequences were aligned according to respective
amino acid sequences using the software package MEGA5
version 5.05 (Tamura et al., 2011). The full-length mxaF
sequence of the genome sequenced M. extorquens strain
AM1 (Chistoserdova et al., 2003) was used to determine
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2693
S. Wellner and others
M. podarium FM4T (AF514774)
M. rhodesianum DSM 5687T (AB175642)
75 M. populi BJ001T (AY251818)
M. thiocyanatum ALL/SCN-PT (U58018)
M. zatmanii DSM 5688T (AB175647)
M. aminovorans JCM 8240T (AB175629)
M. extorquens JCM 2802T (D32224)
M. suomiense NCIMB 13778T (AB175645)
92
M. salsuginis MRT (EF015478)
96 *
M. rhodinum DSM 2163T (AB175644)
81
M. aquaticum GR16T (AJ635303)
*
97 *
M. platani PMB02T (EF426729)
98 *
M. variabile GR3T (AJ851087)
* 100 M. isbiliense AR24T (AJ888239)
77
M. nodulans ORS 2060T (AF220763)
*
98
M. soli YIM 48816T (EU860984)
M. oxalidis 35aT (AB607860)
*
100 M. gregans 002-074T (AB252200)
M. hispanicum GP34T (AJ635304)
M. longum 440T (FN868949)
M. tardum RB677T (AB252208)
82 M. aerolatum 5413S-11T (EF174498)
98 *
M. persicinum 002-165T (AB252202)
M. komagatae 002-079T (AB252201)
M. radiotolerans JCM 2831T (D32227)
M. fujisawaense DSM 5686T (AJ250801)
M. oryzae CBMB20T (AY683045)
81 M. phyllosphaerae CBMB27T (EF126746)
M. brachiatum B0021T (AB175649)
100 M. mesophilicum DSM 1708T (AB175636)
M. dankookense SW08-7T (FJ155589)
Methylobacterium trifolii TA73T (FR847848)
M. cerastii C15T (FR733885)
Methylobacterium thuringiense C34T (FR847847)
90 * Methylobacterium thuringiense T5 (FR847846)
M. gnaphalii 23eT (AB627071)
M. organophilum ATCC 27886T (AB175638)
M. jeotgali S2R03-9T (DQ471331)
M. bullatum F3.2T (FJ268657)
M. marchantiae JT1T (FJ157976)
M. geosingense iEII3T (AY364020)
M. gossipiicola Gh-105T (EU912445)
M. adhaesivum AR27T (AM040156)
M. iners 5317S-33T (EF174497)
R. palustris ATCC 17001T (AB498815)
0.10
Fig. 1. Maximum-likelihood tree showing the phylogenetic position of strains C34T, T5 and TA73T among all type strains of the
genus Methylobacterium. The tree was generated in ARB using RAxML (GTR-GAMMA, Rapid Bootstrap analysis, 100
bootstraps) and based on 16S rRNA gene sequences between positions 85 to 1432 [E. coli numbering, Brosius et al. (1978)].
GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values .70 % (100
replicates). Asterisks represent nodes that are supported by high bootstrap values in neighbour-joining and maximumparsimony trees. Rhodopseudomonas palustris ATCC 17001T was used as an outgroup. Bar, 0.10 substitutions per site.
the correct open reading frame. Phylogenetic trees were
constructed based on DNA and amino acid sequences
using the neighbour-joining (Saitou & Nei, 1987) and
maximum-likelihood methods. Kimura’s two-parameter
(Kimura, 1980) and Jones–Thornton–Taylor (JTT; Jones
et al., 1992) evolutionary models were used for DNA- and
amino acid-based analyses, respectively. Bootstrap confidence analyses were performed on 100 replicates to determine the reliability of the tree topology obtained (Felsenstein,
1985). The phylogenetic relationships obtained by mxaF and
MxaF analyses confirmed 16S rRNA gene based analysis.
Strains C34T and T5 clustered with M. gnaphalii 23eT but
were still different to this strain even based on amino acid
sequence-based analysis. In contrast strain M. gnaphalii 23eT
2694
shared identical amino acid sequences with the type strain of
M. brachiatum. Strain TA73T was distinct from all other
Methylobacterium species based on mxaF analysis but
clustered close to type strains to which strain TA73T also
shared high 16S rRNA gene sequence similarity including M.
marchantiae and M. cerastii if amino acid sequences were
analysed (Fig. 2a, b).
For differentiation of closely related strains three genomic
fingerprinting methods were used, two repetitive element
primed (rep)-PCRs, including BOX- and (GTG)5-PCR
and a random amplification polymorphic DNA (RAPD)
analysis. All PCRs were performed in a total volume of
15 ml including 60 ng genomic DNA, 16 DreamTaq
Buffer, 0.2 mM of each dNTP, 1.0 mM of each primer,
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Two novel species of the genus Methylobacterium
(a)
(b)
T
87 M. fujisawaense DSM 5686 T (FJ157951)
* M. phyllosphaerae CBMB27 (EF562496)
M. oryzae CBMB20T (EF562478)
94 M. brachiatum NBRC 103629T (HQ588891)
* M. mesophilicum A47T (FJ157953)
M. radiotolerans DSM 1819T (FJ157954)
Methylobacterium thuringiense T5 (FR847843)
Methylobacterium thuringiense C34T (FR847844)
M. gnaphalii 23eT (JX683688)
M. jeotgali S2R03-9T (FJ157952)
T
* M. iners KACC 11765 (EU912497)
100 M. platani KCTC 12901T (EU912500)
M. gregans NBRC 103626T (HQ588893)
*
97 M. hispanicum DSM 16372T (EF562468)
M. organophilum ATCC 27886T (M22629)
M. thiocyanatum DSM 11490T (EF562475)
M. aminovorans DSM 8832T (EU194911)
M. zatmanii DSM 5688T (EF031553)
M. salsuginis MRT (EF030550)
M. suomiense KCTC 12963T (EF562474)
97 M. lusitanum KCTC 12964T (EF562469)
* M. rhodesianum DSM 5687T (EF562473)
95 M. aerolatum KACC 11766T (EU912496)
* M. podarium FM4T (AY468366)
M. dichloromethanicum DSM 6343T (AJ878068)
T
99* M. extorquens DSM 1337 T(EF562466)
89* M. rhodinum ATCC 14821 (U70527)
Methylobacterium trifolii TA73T (FR847845)
M. cerastii C15T (FR847841)
M. goesingense iEII3T (FJ157955)
92 M. adhaesivum DSM 17169T (FJ157950)
M. gossipiicola Gh-105T (HQ588892)
*
M. bullatum F3.2T (GU353343)
M. marchantiae JT1T (FJ157956)
74
M. aquaticum DSM 16371T (EF562464)
*
M. nodulans ORS 2060T (AF220764)
83
R. palustris BisB18 (CP000301)
0.05
M. extorquens DSM 1337T (EF562466)
M. dichloromethanicum DSM 6343T (AJ878068)
M. rhodinum ATCC 14821T (U70527)
M. podarium FM4T (AY468366)
M. aerolatum KACC 11766T (EU912496)
M. organophilum ATCC 27886T (M22629)
M. suomiense KCTC 12963T (EF562474)
M. rhodesianum DSM 5687T (EF562473)
M. aminovorans DSM 8832T (EU194911)
M. lusitanum KCTC 12964T (EF562469)
M. zatmanii DSM 5688T (EF031553)
M. salsuginis MRT (EF030550)
M. jeotgali S2R03-9T (FJ157952)
M. thiocyanatum DSM 11490T (EF562475)
M. phyllosphaerae CBMB27T (EF562496)
88 M. radiotolerans DSM 1819T (FJ157954)
* M. oryzae CBMB20T (EF562478)
M. fujisawaense DSM 5686T (FJ157951)
Methylobacterium thuringiense T5 (FR847843)
Methylobacterium thuringiense C34T (FR847844)
80 M. iners KACC 11765T (EU912497)
M. platani KCTC 12901T (EU912500)
*
T
78 M. gnaphalii 23e (JX683688) T
M. brachiatum NBRC 103629 (HQ588891)
M. mesophilicum A47T (FJ157953)
Methylobacterium trifolii TA73T (FR847845)
M. adhaesivum DSM 17169T (FJ157950)
M. cerastii C15T (FR847841)
M. gossipiicola Gh-105T (HQ588892)
M. bullatum F3.2T (GU353343)
M. goesingense iEII3T (FJ157955)
76 M. marchantiae JT1T (FJ157956)
M. hispanicum DSM 16372T (EF562468)
M. gregans NBRC 103626T (HQ588893)
M. aquaticum DSM 16371T (EF562464)
M. nodulans ORS 2060T (AF220764)
R. palustris BisB18 (CP000301)
0.05
94
Fig. 2. Phylogenetic relationship of strains C34T, T5 and TA73T to species of the genus Methylobacterium based on partial mxaF
(a) and MxaF (b) sequences. The phylogenetic trees were calculated in MEGA 5 with the maximum-likelihood method and based on
455 nt or 151 amino acids, respectively. GenBank accession numbers are given in parentheses. Numbers at branch nodes refer
to bootstrap values .70 % (100 replicates). Asterisks represent nodes that are supported by high bootstrap values in respective
neighbour-joining trees. Rhodopseudomonas palustris BisB18 was used as outgroup. Bars, 0.05 substitutions per side.
and 0.8 U DreamTaq DNA Polymerase (Fermentas).
Primer BOXA1R (59-CTACGGCAAGGCGACGCTGACG39) and (GTG)5 (59-GTGGTGGTGGTGGTG-39) were used
for BOX-PCR and (GTG)5-PCR (Versalovic et al., 1994),
respectively. RAPD-PCR was performed with primer
(GTG)5-PCR
A (59-CTGGCGGCTTG-39) (Ziemke et al., 1997). PCRconditions were as followed: 95 uC for 3 min, followed by
30 cycles of 94 uC for 30 s, 53 uC for 1 min, and 70 uC for
8 min (BOX-PCR) or 3 min [(GTG)5-PCR], and finally
70 uC for 16 min. RAPD-PCR conditions were: 95 uC for
RAPD-PCR
BOX-PCR
M 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 M M 1 2 3 4 5 6 7 8 9 10 M
Fig. 3. Genomic fingerprint pattern generated with (GTG)5-, RAPD-, and BOX-PCR. Lanes: 1, C34T; 2, T5; 3, M.
organophilum LMG 6083T; 4, M. gnaphalii 23eT, 5, TA73T; 6, M. phyllosphaerae DSM 19779T; 7, M. fujisawaense DSM
5686T; 8, M. oryzae DSM 18207T, 9, M. marchantiae JT1T; 10, M. brachiatum DSM 19569T; M, GeneRuler 100 bp Plus DNA
Ladder (Fermentas). Ethidium Bromide stained fingerprint pattern separated on an agarose gel (1.5 %).
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2695
S. Wellner and others
Table 2. Major fatty acid compositions (%) of type strains and members of the genus Methylobacterium that are closely related to the
investigated strains
Taxa: 1, C34T; 2, T5; 3, TA73T: 4, M. organophilum LMG 6083T; 5, M. gnaphalii 23eT; 6, M. phyllosphaerae DSM 19779T; 7, M. oryzae DSM 18207T;
8, M. fujisawaense DSM 5686T; 9, M. marchantiae JT1T; 10, M. cerastii C15T. All data were obtained comparatively in this study. 2, Not detected.
Fatty acid
anteiso-C15 : 0
C16 : 0
C18 : 0
C18 : 1v7c*
Summed feature: D
3
2
Unknownd
14.263
14.959
1
2
3
4
5
6
7
8
9
10
2
2.4
5.4
74.3
2
3.5
4.3
65.4
2
5.5
2
87.6
2
3.2
4.6
89.5
2
10.4
2
89.6
2
5.0
3.2
87.2
2
5.0
5.4
85.1
2
4.8
2.7
86.5
2
6.2
1.5
74.8
1.7
3.9
0.9
81.3
14.8
1.6
23.2
1.9
2
2
2
2.8
2
2
2.4
2
2
2
2.7
2
15.1
2.5
8.4
1.8
2
1.5
2
1.7
6.9
2
2
2
2
2
2.2
2
4.6
2
3.3
2
2
2
2
2.1
*For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (v) end of the carbon chain.
DSummed features are groups of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 2 contained one
or more of the fatty acids iso-C16 : 1 I and C14 : 0 3-OH. Summed feature 3 contained one or more of the fatty acids C16 : 1v7c and iso-C15 : 0 2-OH.
dThe unknown fatty acids have no name listed in the peak library file of the MIDI system and therefore cannot be identified.
3 min, followed by 45 cycles of 95 uC for 15 s, 34 uC for
1 min, and 72 uC for 2 min, and finally 72 uC for 10 min.
All PCRs were performed in a MyCycler (Bio-Rad). PCR
products were separated on a 1.5 % agarose gels in 16 TBE
buffer for 2.5 h at 5.1 V cm21, strained with ethidium
bromide, and documented using a Fluor-S MultiImager
(Bio-Rad). All investigated strains exhibit clearly different
DNA fingerprint pattern to the closely related Methylobacterium species (Fig. 3).
Genomic DNA for hybridization experiments was isolated
according to Pitcher et al. (1989). DNA–DNA hybridization studies were carried out according to Ziemke et al.
(1998) with a hybridization temperature of 73.4 uC. These
experiments resulted in DNA–DNA relatedness values of
47.6 % (reciprocal 41.9 %) and 59.7 % (reciprocal 20.8 %)
for pairing of M. gnaphalii 23eT and 14.9 % (reciprocal
25.9 %) and 11.2 % (reciprocal 24.4 %) in pairing of M.
organophilum JCM 2833T with C34T and T5, respectively.
Pairing of C34T and T5 showed DNA–DNA relatedness
values of 100 % (reciprocal 59.6 %). Based on genomic
fingerprint and DNA–DNA hybridization results, strains
C34T and T5 represent two different strains of the same
species. The DNA–DNA similarity values between TA73T
and M. phyllosphaerae CBMB27T, M. fujisawaense DSM
5686T, M. oryzae CBMB20T and M. brachiatum DSM
19569T were 37.5 % (reciprocal 31.9 %), 19.4 % (reciprocal
22.3 %), 39.1 % (reciprocal 41.8 %) and 16.8 % (reciprocal
45.6 %), respectively.
Extraction and analysis of whole-cell fatty acids was
performed using the MIDI protocol as previously described
by Kämpfer & Kroppenstedt (1996); cells were grown on
M125 at 25 uC prior to fatty acid extraction. The samples were
investigated using a model 5898A microbial identification
2696
system (Microbiol ID) and the Microbial Identification
System Standard Software (Microbial ID, USA, Version
TSBA 4.1). Major fatty acids of strain TA73T were C18 : 1v7c,
C16 : 0 and one unknown fatty acid. Strains C34T and
T5 contained mainly C18 : 1v7c, C16 : 1v7c/iso-C15 : 0 2-OH
(summed feature 3), C18 : 0 and C16 : 0. The fatty acid profiles
of the investigated strains and closely related members of the
genus Methylobacterium were very similar (Table 2).
On the basis of phylogenetic, chemotaxonomic and
phenotypic analysis, we propose two novel Methylobacterium species. Strain TA73T should be the type strain
of the proposed species Methylobacterium trifolii sp. nov.
and strain C34T should be the type strain of the proposed
species Methylobacterium thuringiense sp. nov. Based on
high DNA–DNA relatedness values strain T5 should be
considered as strain of M. thuringiense sp. nov.
Description of Methylobacterium trifolii sp. nov.
Methylobacterium trifolii [tri.fo9li.i. L. n. trifolium threeleaved grass, trefoil and also a scientific genus name
(Trifolium); L. gen. n. trifolii of trefoil, isolated from
Trifolium repens].
Cells are aerobic, Gram-stain-negative, rod-shaped, approx.
0.8–0.962.6–3.2 mm in size and immotile. Slow-growing,
with a growth rate (m) of approximately 0.03–0.06 h21.
Grows well on M125 supplemented with 0.1–2.0 % (v/v)
methanol but growth on R2A, PYE, PCA, K7 and NA is weak.
Colonies are pale red, convex, circular, with entire margins
and smaller than 1 mm in diameter on M125 agar after
7 days of incubation at 25 uC. Pigments have absorption
maxima in 80 % (v/v) methanol at 360, 490 and 520,
respectively and a weak absorption maximum at 460 nm.
Produces the enzymes catalase and oxidase. Grows at
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Two novel species of the genus Methylobacterium
10–28 uC (optimum 20 uC) and pH values of 4.0–8.0
(optimum 5.0–7.2). Does not grow in medium supplemented with 0.5 % NaCl or higher salt concentrations. Does not
hydrolyse starch, but is able to hydrolyse para-nitrophenyl
(pNP)-b-D-xylopyranoside, bis-pNP-phosphate, pNP-phenyl-phosphonate sodium salt, 2-deoxythymidine-5-pNPphosphate and L-alanine- para-nitroanilide (pNA). Indole
test is negative and Voges–Proskauer test is positive. Urease is
produced but H2S, arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, tryptophan deaminase are
not. The following compounds serve as sole carbon source: Lglutamate, D-ribose, citrate, itaconate, DL-lactate, L-malate,
mesaconate, 2-oxoglutarate and pyruvate. Results for citrate
are variable: shows a positive reaction in the physiological
test panel (Kämpfer et al., 1991) but a negative reaction in
M125 supplemented with this substrate. Does not grow on
acetate, tartrate, D-fructose, D-glucose, D-xylose, L-arabinose
or ethanol. Produces acid from D-xylose and rhamnose but
not from D-glucose or D-mannose. Major fatty acids are
C18 : 1v7c and C16 : 0 and one unknown fatty acid.
The type strain is TA73T (5LMG 25778T5CCM 7786T),
isolated from the leaf surface of Trifolium repens collected
in 2009 in the region Schwäbische Alb, Germany.
Description of Methylobacterium thuringiense
sp. nov.
Methylobacterium thuringiense (thu.rin.gi.en9se. N.L. neut.
adj. thuringiense referring to Thuringia, the Latin name of
the region Thüringen in Germany from where the type
strain was isolated).
Both strains cells are aerobic, Gram-stain-negative, rodshaped and immotile. Cells are approx. 0.6–1.062.0–
5.5 mm in size. Slow-growing with a growth rate (m) of
approximately 0.03–0.05 h21. Grows on R2A, NA, PYG,
PYE, CASO, PCA, K7, M1207 and on M125 supplemented
with 0.1–2.0 % (v/v) methanol. Colonies are pink, shiny,
smooth, circular, with entire margins and a diameter of
1.0–2.0 mm on M125 agar after 7 days of incubation at
25 uC. Pigments have absorption maxima in 80 % (v/v)
methanol at 360, 490, 520 and 770, respectively, and a weak
maximum at 460 nm. Produces catalase and oxidase. Grow
at 10–28 uC (optimum 16–25 uC) and pH values of 4.0–
10.0 (optimum 5.0–7.2). Does not grow in medium
supplemented with 1.0 % NaCl or higher salt concentrations. Does not hydrolyse starch but bis-pNP-phosphate,
2-deoxythymidine-5-pNP-phosphate, L-alanine-pNA are
hydrolysed; hydrolyses of pNP-phenylphosphonate is variable. Indole test is negative and Voges–Proskauer test is
positive. Urease is produced but H2S, arginine dihydrolase,
ornithine decarboxylase, lysine decarboxylase and tryptophan
deaminase are not. Differential utilization of and acid
production from several carbon sources by the isolates and
their closest relatives is summarized in Table 1. Assimilation
of ethanol and N-acetyl-D-glucosamine as sole carbon
substrates; variable for the assimilation of D-ribose, maltitol,
D-mannitol, D-sorbitol, putrescine, propionate, fumarate,
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DL-3-hydroxybutyrate, DL-lactate, L-malate,
oxo-glutarate,
pyruvate, L-phenylalanine and 3-hydroxy-benzoate and Lglutamate (test system: physiological test panel according to
Kämpfer et al., 1991). Acid production from rhamnose and
variable for L-arabinose. For D-xylose, results were different
dependent on the test system, acid production was variable in
the micronaut E test plate but not observed in the
physiological test panel according to Kämpfer et al. (1991).
Major fatty acids are C18 : 1v7c, C16 : 1v7c/iso-C15 : 0 2-OH
(summed feature 3), C18 : 0 and C16 : 0.
The type strain is C34T (5LMG 25777T5CCM 7787T),
isolated from the phyllosphere of Cerastium holosteoides
collected in 2008 in the Hainich-Dün region, Germany.
As a result of recent work, including DNA–DNA
hybridization data and genomic fingerprint studies, we
propose that strain T5 (5DSM 236315CCM 7790), which
was isolated from the phyllosphere of Trifolium repens, is a
second strain of M. thuringiense.
Acknowledgements
We are grateful to Gundula Will, Maria Sowinsky, and Jan
Rodrigues-Fonseca for excellent technical assistance. We thank
Dr Jean Euzéby for his nomenclatural advice. The studies were
supported by the DFG Priority Program 1374 ‘InfrastructureBiodiversity-Exploratories’, grant number Ka 875/6-1 to P. K., which
is gratefully acknowledged.
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