International Journal of Systematic and Evolutionary Microbiology (2012), 62, 942–948
DOI 10.1099/ijs.0.030304-0
Erwinia typographi sp. nov., isolated from bark
beetle (Ips typographus) gut
V. Skrodenyte? -Arbačiauskiene? ,1 S. Radžiute? ,1 V. Stunže? nas1 and V. Būda1,2
Correspondence
Vesta Skrodenyte? -Arbačiauskiene?
[email protected]
1
Institute of Ecology, Nature Research Centre, Akademijos str. 2, Vilnius LT-08412, Lithuania
2
Faculty of Natural Sciences, Vilnius University, M.K. Čiurlionio 21/27, Vilnius LT-03101, Lithuania
Gram-negative-staining bacteria that were resistant to monoterpene myrcene (7-methyl-3methylene-1.6-octadiene, C10H16, at concentrations of up to 10 ml ml”1 in TSB) were isolated
from the gut contents of adult bark beetles Ips typographus (Coleoptera, Scolytidae). The beetles
were collected from the bark of Norway spruce (Picea abies) in Lithuania. Bark beetles feed on
conifers, which produce myrcene among many other defensive compounds. It has been
suggested that the micro-organisms present within the beetles’ guts could be involved in their
resistance towards this plant defensive compound. The most resistant bacterial strains were
isolated and characterized by phenotypic assays as well as fatty acid analysis, 16S rRNA gene
sequencing, multilocus sequence analyses (MLSA) based on the rpoB, atpD and infB genes and
DNA–DNA hybridization. Biochemical characterization indicated that the bacteria belonged to the
family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA gene sequences and MLSA of
the novel strains revealed that they belonged to the genus Erwinia, but represented a novel
species. The dominant cellular fatty acids were C16 : 0 and C17 : 0 cyclo. The DNA G+C content
was 49.1 mol%. The results obtained in this study indicated that these bacteria from the bark
beetle gut represented a novel species, for which the name Erwinia typographi sp. nov. is
proposed, with the type strain DSM 22678T (5Y1T5LMG 25347T).
Three novel strains, DSM 22678T (5Y1T), DSM 24222 (5Y4)
and DSM 24223 (5Y7), were isolated from the gut contents of
healthy bark beetles Ips typographus L. (Coleoptera, Scolytidae)
as previously described by Skrodenyte? -Arbačiauskiene? et al.
(2006a). Bark beetles were sampled from infested Norway
spruce (Picea abies) in Lithuania. Phytophagous insects, such
as bark beetles, feed on conifers, which produce large amounts
of terpenes. Terpenes are known to be defensive compounds
and many of them are toxic to both herbivorous insects
and micro-organisms (Phillips & Croteau, 1999; Keeling
& Bohlmann, 2006; Seybold et al., 2006; Gershenzon &
Dudareva, 2007; Zhao et al., 2010). Monoterpene myrcene (7methyl-3-methylene-1.6-octadiene, C10H16) is one of the
defensive compounds produced by conifers. The novel strains
DSM 22678T, DSM 24222 and DSM 24223 were the most
resistant to myrcene and were the only surviving strains at
concentrations of myrcene (~90 % GC, Fluka, Sigma-Aldrich
Chemie) at up to 10 ml ml21 in TSB (Oxoid).
Genomic DNA was extracted from the isolates following
the method reported by Skrodenyte-Arbaciauskiene et al.
Abbreviations: MLSA, multilocus sequence analysis; MP, maximumparsimony; NJ, neighbour-joining.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences of strains DSM 22678T, DSM 24222 and DSM 24223 are
GU166291, HQ540302 and HQ452956, respectively.
942
(2006b). Universal bacterial primers w001 F8 (59AGAGTTTGATCMTGGCTC-39) and w002 R1509 (59GNTACCTTGTTACGACTT-39) (Godon et al., 1997) were
used to amplify 16S rRNA gene sequences. The PCR mixtures
contained 106 PCR buffer with (NH4)2SO4 (Fermentas),
2 mM dNTP Mix (Fermentas), 1.5 mM MgCl2, 20 pmol
each primer, 1 ng DNA in 10 ml, and 1 U of Taq DNA
polymerase (Fermentas) in a total volume of 50 ml. PCRs
were performed using initial denaturation for 3 min at 95 uC
followed by 30 cycles of denaturation for 3 min at 95 uC,
primer annealing for 1 min at 50 uC and primer extension for
2 min at 72 uC. This procedure was followed by a final
extension reaction at 72 uC for 10 min. Bands of 1.5 kb were
excised. The 16S rRNA gene PCR product was extracted from
agarose gel using a Cyclo-pure gel extraction kit (Amresco).
The purified PCR product was cloned into Escherichia coli
DH5a using the GeneJet PCR Cloning kit/TransformAid
Bacterial Transformation kit (Fermentas). Recombinant
plasmid DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen). The cloned 1.5 kb fragments were
sequenced by automated DNA sequencing. Sequence data
were confirmed and sequence ambiguities were resolved,
where possible, by manual scanning of the individual chromatograms. Similarity searches were performed using the
BLAST program (Altschul et al., 1997). In phylogenetic tree
analyses, sequences were aligned by CLUSTAL W 1.6 (with a gap
opening penalty of 15 and a gap extension penalty of 6.66)
Downloaded from www.microbiologyresearch.org by
030304 G 2012 IUMS
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
Printed in Great Britain
Erwinia typographi sp. nov.
using MEGA4 (Tamura et al., 2007). Sequences were analysed
against 16S rRNA gene sequences from GenBank (Stoesser
et al., 2001). In this analysis, insertions and deletions were
treated as a fifth base. Almost complete 16S rRNA gene
sequences of strains DSM 22678T, DSM 24222 and DSM
24223 comprising 1406 nt were determined and used for
phylogenetic analysis. The sequences of the type strains of
Erwinia billingiae and Erwinia toletana were too short for the
Fig. 1. Bayesian phylogenetic tree of 16S rRNA gene sequences showing the relationships of strains DSM 22678T, DSM 24222
and DSM 24223 among genera of the family Enterobacteriaceae. See text for details of tree construction. Plesiomonas shigelloides
and Pectobacterium carotovorum were used as outgroups. Nodal support values indicate posterior clade probabilities; only values
.70 are shown. GenBank accession nos of strains are given in parentheses. Bar, 0.1 substitutions per nucleotide position.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
943
V. Skrodenyte? -Arbačiauskiene? and others
alignment (1218 and 1165 nt long, respectively) with the
novel strains; therefore the sequences of E. toletana CFBP
6644 (GenBank accession no. AF130964) and E. billingiae
Eb661 (FP236843) were used for the alignment (Rojas et al.,
2004; Kube et al., 2010). Bayesian phylogenetic trees derived
from these analyses are shown in Fig. 1. The General Time
Reversible model of evolution including invariable sites and
variation among sites (GTR+I+G) as defined by Modeltest
3.7 (Posada & Crandall, 1998) was used. The tree was constructed with a sample frequency of every 100th generation
over 10 million generations generating 100 000 trees. Before
constructing a majority consensus tree, 25 % of the initial
trees were discarded as burn in periods. Nodal support values
indicated the posterior clade probabilities. The tree showed
the same topology with high confidence values. The
neighbour-joining (NJ) (Saitou & Nei, 1987) and maximum-parsimony (MP) (Fitch, 1971) trees were obtained
and analysed using MEGA4 (bootstrap analyses with 10 000
replicates were conducted; not shown). Their topology was
identical to the Bayesian phylogenetic tree.
The 16S rRNA gene sequences determined for strains DSM
22678T, DSM 24222 and DSM 24223 were 1406 nt long. The
novel strains possessed signature nucleotides identical to the
signatures described by Hauben et al. (1998) for the genus
Erwinia: A408, A594, C598, G639, G646, C839, G847, G987,
G988, C989, G1216, C1217, C1218, C1308 and G1329, using
the E. coli 16S rRNA gene sequence numbering (Brosius et al.,
Fig. 2. Bayesian phylogenetic tree based on the concatenated nucleotide sequences of the rpoB, atpD and infB genes of
strains DSM 22678T, DSM 24222 and DSM 24223 among species of the genera Erwinia, Pantoea and Tatumella. See text for
details of tree construction. Shigella dysenteriae and Citrobacter rodentium were included as outgroups. Nodal support values
indicate posterior clade probabilities, only values .70 are shown. Bar, 0.1 substitutions per nucleotide position.
944
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
Erwinia typographi sp. nov.
1981). A 16S rRNA gene sequence similarity of .97 % was
found with only one species, ‘Pantoea cedenensis’ (97.8 %).
The sequence similarity values were ,97 % with other type
strains of recognized species of the genera Erwinia and
Pantoea such as Pantoea agglomerans DSM 3493T (96.7 %), E.
toletana DSM 18073T (96.5 %), Erwinia tasmaniensis DSM
17950T (96.1 %), Erwinia aphidicola DSM 19347T (96.0 %),
Erwinia oleae DSM 23398T (96.0 %), Pantoea cypripedii DSM
3873T (96.0 %), Erwinia rhapontici DSM 4484T (95.9 %),
Erwinia amylovora DSM 30165T (95.9 %), Erwinia persicina
ATCC 35598T (95.9 %), Pantoea ananatis ATCC 33244T
(95.9 %), Erwinia pyrifoliae DSM 12163T (95.8 %), Erwinia
mallotivora DSM 4565T (95.6 %), Erwinia psidii LMG 7034
(95.5 %), Erwinia tracheiphila LMG 2906T (95.3 %), Pantoea
stewartii subsp. stewartii LMG 2715T (95.2 %), Erwinia
piriflorinigrans CFBP 5888T (95.1 %), Erwinia papayae DSM
16540T (93.7 %) and E. billingiae LMG 2613T (91.6 %).
The phylogenetic tree (Fig. 1) shows the position of the
novel strains DSM 22678T, DSM 24222 and DSM 24223
among the type strains of species of the genera Pantoea and
Erwinia as well as other genera belonging to the family
Enterobacteriaceae. The novel strains formed a robust clade
with ‘P. cedenensis’, a non-validly published species name,
(clade probability value 100 %, sequence similarity 97.8 %)
and the type strain of E. toletana (clade probability value
93 %, sequence similarity 96.5 %). The novel strains also
grouped with other species of the genus Erwinia (E.
billingiae, E. rhapontici, E. persicina, E. tasmaniensis, E.
amylovora and E. pyrifoliae) in a robust clade (clade
probabilities: 82 %). All species of the genus Erwinia
clustered in one well-supported clade that could be clearly
differentiated from the clades of species of the genera
Pantoea and Tatumella (Fig. 1). These results corroborated
those of a previous study (Gardan et al., 2004) that
indicated that ‘P. cedenensis’ clearly belongs to the genus
Erwinia and should be, therefore, be formally described as a
member of this genus. In conclusion, strains DSM 22678T,
DSM 24222 and DSM 24223 represent a novel species of
the genus Erwinia.
The partial housekeeping rpoB (637 bp), atpD (563 bp)
and infB (615 bp) gene sequences for strains DSM 22678T,
DSM 24222, DSM 24223 and ‘P. cedenensis’ CFBP 6627
were determined following Brady et al. (2008). Gene
sequences of the other species needed for phylogenetic
analysis were obtained from genome sequencing databases
(http://www.ncbi.nlm.nih.gov). Sequence analysis and tree
construction were performed as described above, and a
phylogenetic tree based on concatenated sequences of the
three genes is presented in Fig. 2. The branching patterns of
the novel species in NJ and MP trees were identical to
those found in the Bayesian phylogenetic tree. Also, the
phylogenetic position of the novel strains was identical to
that found in Bayesian, NJ and MP amino acid trees of
the housekeeping genes. The Bayesian phylogenetic tree
demonstrated that the novel strains fell into well-supported
clusters within the genus Erwinia. The recognized species of
the genera Pantoea and Tatumella were more distantly
http://ijs.sgmjournals.org
related, with Shigella dysenteriae and Citrobacter rodentium
used as outgroups. The MLSA data suggest that strains
DSM 22678T, DSM 24222 and DSM 24223 represent a
novel species within the genus Erwinia.
For DNA–DNA hybridization studies, cells were disrupted
by using a French pressure cell (Thermo Spectronic) and the
DNA in the crude lysate was purified by chromatography on
hydroxyapatite as described by Cashion et al. (1977). DNA–
DNA hybridization was carried out as described by De Ley
et al. (1970) with the modifications described by Huß et al.
(1983) using a Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 666 multicell
changer and an in situ temperature probe (Varian).
According to Stackebrandt & Goebel (1994), organisms
that have ,97 % 16S rRNA gene sequence similarity will not
show DNA–DNA reassociation values of .60 %. Therefore,
DNA–DNA hybridizations were conducted on strains
exhibiting the highest 16S rRNA gene pairwise similarity
to strain DSM 22678T. ‘P. cedenensis’ CFBP 6627 showed a
relatedness value of 47 % with strain DSM 22678T, making it
the most closely related organism to the novel strain.
E. toletana DSM 18073T and P. agglomerans DSM 3493T
showed DNA–DNA relatedness values of 20 % and 13 %,
respectively, with strain DSM 22678T (Table 1). When the
recommendation of a threshold value of 70 % DNA–DNA
similarity for the definition of bacterial species is considered
(Wayne et al., 1987; Vandamme et al., 1996; Rosselló-Mora
& Amann, 2001), strain DSM 22678T was clearly separate
from these recognized species.
The DNA G+C content of strain DSM 22678T was
determined by HPLC (Cashion et al., 1977; Tamaoka &
Komagata, 1984; Mesbah et al., 1989) and was found to
be 49.1 mol%. This was slightly lower than the range
previously reported for members of the genus Erwinia:
49.8–54.1 mol% (Hauben et al., 1998). This indicated that
strain DSM 22678T differed from other recognized species
of the genus Erwinia.
Physiological and biochemical tests were performed on
strains DSM 22678T, DSM 24222, DSM 24223 and the type
strains of phylogenetically related type species of the genus
Erwinia, as well as ‘P. cedenensis’ CFBP 6627. The API 20E,
Table 1. Levels of DNA–DNA reassociation among Erwinia
typographi sp. nov. strains and strains of ‘P. cedenensis’, P.
agglomerans and E. toletana
Strain
Erwinia typographi sp. nov.
DSM 22678T (5Y1T5LMG
25347T)
DSM 24222 (5Y45LMG 26160)
DSM 24223 (5Y75LMG 26161)
‘Pantoea cedenensis’ CFBP 6627
Erwinia toletana DSM 18073T
Pantoea agglomerans DSM 3493T
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
Relative DNA binding (%)
100
86
82
47
20
13
945
V. Skrodenyte? -Arbačiauskiene? and others
API 50 CHE (bioMérieux) and Tween 80 (Tindall et al.,
2007) tests were used. The novel strains could be differentiated from their closest phylogenetic neighbours by
their ability to utilize mannose, trehalose and mannitol and
their inability to metabolize glycerol, inositol, maltose,
raffinose, sucrose, rhamnose, arabitol, melibiose, salicin or
cellobiose. Other distinguishing characteristics were a
negative result in the Voges–Proskauer test and for gelatin
liquefaction (Table 2). Detailed phenotypic and biochemical properties of strain DSM 22678T are given in the
species description below.
the genus Erwinia as representing a novel species. The
DNA–DNA relatedness between the phylogenetically closest neighbours and strain DSM 22678T showed values
below the 70 % cut-off value used to delineate species.
Therefore, it is suggested that the new strains identified in
this study represent a novel species of the genus Erwinia,
for which the name Erwinia typographi sp. nov. (type strain
DSM 22678T) is proposed.
Description of Erwinia typographi sp. nov.
Erwinia typographi (ty.po9gra.phi. N.L. gen. n. typographi
of typographus referring to the host bark beetle Ips
typographus; the type strain was isolated from the intestinal
tract of the beetles).
Whole-cell fatty acid methyl ester analysis was performed
as described by Kämpfer & Kroppenstedt (1996). Table 3
shows the percentage of characteristic fatty acids (.0.5 %)
found in the strains examined. For strain DSM 22678T, the
most abundant fatty acids were C16 : 0 (35.54 %) and C17 : 0
cyclo (29.27 %), accounting for 65 % of the total fatty
acids. Thus, the composition of fatty acids in strain DSM
22678T differed from that found for the closely related
species ‘P. cedenensis’, E. toletana and E. billingiae.
Cultures grow well on TSA (Oxoid) and TSB (Oxoid)
between 22 uC and 36 uC, but not at 41 uC. Colonies on
TSA are translucent, beige, non-pigmented, round, convex
and smooth with entire margins at 28 uC within 3 days.
Cells are Gram-negative-staining, rod-shaped and vary in
length from 2.0 to 4.0 mm and in diameter from 0.7 to
0.8 mm. Cells occur singly and are motile and non-sporeforming. Facultatively anaerobic, oxidase-negative, catalase-positive and able to ferment D-glucose without gas
formation. b-Galactosidase, phenylalanine deaminase and
aminopeptidase (Cerny) are produced. The Methyl red test
gives a positive result. DNase activity, Voges–Proskauer test
and tests for gelatin liquefaction are negative. Indole, H2S
and urease are not produced. The following carbon sources
are utilized at 28 uC within 3–6 days: L-arabinose, D-ribose,
D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol,
In conclusion, the isolates from the gut contents of
bark beetles showed the general characteristics expected
for species of the genus Erwinia in the family
Enterobacteriaceae (Hauben & Swings, 2005). The novel
strains were Gram-staining negative, motile, facultatively
anaerobic, oxidase-negative, catalase-positive and able to
ferment D-glucose without gas formation. Phylogenetic
analyses of 16S rRNA gene sequences, using three different
methods (Bayesian, NJ and MP), together with MLSA
confirmed the taxonomic position of the novel strains in
Table 2. Differentiating phenotypic characteristics of strains DSM 22678T, DSM 24222, DSM 24223, phylogenetically related type
species of the genus Erwinia and ‘P. cedenensis’ CFBP 6627
Species/strains: 1, DSM 22678T, DSM 24222, DSM 24223; 2, ‘P. cedenensis’ CFBP 6627; 3, E. toletana DSM 18073T; 4, E. amylovora CFBP 1232T; 5,
E. aphidicola CFBP 6829T; 6, E. billingiae CFBP 6830T; 7, E. mallotivora CFBP 2503T; 8, E. rhapontici CFBP 3163T; 9, E. tracheiphila CFBP 2355T; 10,
E. tasmaniensis CFBP 7177T; +, 90–100 % strains positive in 3–6 days; (+), weakly positive; 2, 90–100 % of strains negative. All data were
obtained from this study.
Characteristic
1
2
3
4
5
6
7
8
9
10
Voges–Proskauer test
Gelatin liquefaction
Utilization of:
Arabitol
Cellobiose
Glycerol
Inositol
Maltose
Mannitol
Mannose
Melibiose
Raffinose
Rhamnose
Salicin
Sucrose
Trehalose
2
2
2
2
2
2
+
+
+
2
+
2
+
2
+
2
(+)
2
+
2
2
2
2
2
2
+
+
2
2
2
2
2
+
+
2
(+)
+
2
+
+
(+)
2
+
+
2
+
+
+
2
+
+
+
2
+
2
2
+
2
+
2
2
2
2
2
2
2
2
2
2
2
+
+
2
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
2
+
+
+
+
2
+
+
2
+
2
2
2
2
2
+
+
2
2
2
2
+
+
2
+
+
+
+
+
+
+
+
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
2
2
+
2
(+)
2
2
2
+
+
946
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
Erwinia typographi sp. nov.
Table 3. Cellular fatty acid profiles of strain DSM 22678T and
related species
Taxa: 1, DSM 22678T; 2, ‘P. cedenensis’ CFBP 6627; 3, E. toletana DSM
18073T; 4, E. billingiae Eb661T (data from Geider et al., 2006).
Fatty acid
C12 : 0
C14 : 0
C16 : 0
C17 : 0
C17 : 0 cyclo
C14 : 0 3-OH
C16 : 1v7c
C18 : 1v7c
1
2
3
4
2.35
3.61
35.54
1.42
29.27
5.32
7.67
5.06
3.60
4.79
35.08
0.57
13.72
7.30
18.91
12.84
6.50
0.93
31.88
1.05
10.37
7.72
24.12
14.43
3.68
5.81
31.26
3.18
8.92
10.03
22.15
9.61
N-acetylglucosamine, trehalose and malonate. The following
carbon sources are not utilized at 28 uC within 3–6 days:
glycerol, erythritol, D-arabinose, D-xylose, L-xylose, Dadonitol, methyl b-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, methyl a-D-mannoside, methyl
a-D-glucoside, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, inulin, melezitose,
raffinose, starch, glycogen, xylitol, gentiobiose, turanose, Dlyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol,
gluconate, 2- ketogluconate, 5-ketogluconate and Tween 80.
The major fatty acids are C16 : 0 (35.54 %), C17 : 0 cyclo
(29.27 %) and C16 : 1v7c (7.67 %).
T
T
T
The type strain, DSM 22678 (5Y1 5LMG 25347 ), was
isolated from the gut of bark beetles infesting Norway
spruce (Picea abies) in Lithuania. Strain DSM 22678T is
resistant to myrcene (7-methyl-3-methylene-1.6-octadiene,
C10H16, known as the defensive compound of Norway
spruce), and is capable of surviving at a concentration as
high as 10 ml myrcene ml21 in TSB. The G+C content of
the type strain is 49.1 mol%.
Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. (1981). Gene
organization and primary structure of a ribosomal RNA operon from
Escherichia coli. J Mol Biol 148, 107–127.
Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977).
A rapid method for the base ratio determination of bacterial DNA.
Anal Biochem 81, 461–466.
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative
measurement of DNA hybridization from renaturation rates. Eur J
Biochem 12, 133–142.
Fitch, W. M. (1971). Toward defining the course of evolution:
minimum change for a specific tree topology. Syst Zool 20, 406–416.
Gardan, L., Christen, R., Achouak, W. & Prior, P. (2004). Erwinia
papayae sp. nov., a pathogen of papaya (Carica papaya). Int J Syst Evol
Microbiol 54, 107–113.
Geider, K., Auling, G., Du, Z., Jakovljevic, V., Jock, S. & Völksch, B.
(2006). Erwinia tasmaniensis sp. nov., a non-phytopathogenic
bacterium from apple and pear trees. Int J Syst Evol Microbiol 56,
2937–2943.
Gershenzon, J. & Dudareva, N. (2007). The function of terpene
natural products in the natural world. Nat Chem Biol 3, 408–414.
Godon, J. J., Zumstein, E., Dabert, P., Habouzit, F. & Moletta, R.
(1997). Molecular microbial diversity of an anaerobic digestor as
determined by small-subunit rDNA sequence analysis. Appl Environ
Microbiol 63, 2802–2813.
(2005). Genus XIII. Erwinia Winslow,
Broadhurst, Buchanan, Krumweide, Rogers and Smith 1920, 209AL.
In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2B,
pp. 670–679. Edited by D. J. Brenner, N. R. Krieg, J. R. Staley &
G. M. Garrity. New York: Springer.
Hauben, L. & Swings, J.
Hauben, L., Moore, E. R. B., Vauterin, L., Steenackers, M., Mergaert, J.,
Verdonck, L. & Swings, J. (1998). Phylogenetic position of phyto-
pathogens within the Enterobacteriaceae. Syst Appl Microbiol 21, 384–
397.
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the
spectrophotometric determination of DNA hybridization from
renaturation rates. Syst Appl Microbiol 4, 184–192.
Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of
fatty acid patterns of coryneform bacteria and related taxa. Can J
Microbiol 42, 989–1005.
Keeling, C. I. & Bohlmann, J. (2006). Genes, enzymes and chemicals
of terpenoid diversity in the constitutive and induced defence of
conifers against insects and pathogens. New Phytol 170, 657–675.
Acknowledgements
This work was supported by a grant from the Research Council of
Lithuania (Contract no. PBT-03/2010). We are grateful to Dr Peter
Schumann (DSMZ) for G+C content and fatty acid analyses, Dr
Cathrin Spröer (DSMZ) for DNA–DNA hybridization and Dr
Nomeda Kuisiene? (Department of Microbiology and Biotechnology,
Vilnius University) for comments on the manuscript.
Kube, M., Migdoll, A. M., Gehring, I., Heitmann, K., Mayer, Y., Kuhl, H.,
Knaust, F., Geider, K. & Reinhardt, R. (2010). Genome comparison of
References
Phillips, M. A. & Croteau, R. B. (1999). Resin-based defenses in
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new
Posada, D. & Crandall, K. A. (1998).
the epiphytic bacteria Erwinia billingiae and E. tasmaniensis with the
pear pathogen E. pyrifoliae. BMC Genomics 11, 393.
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 Evol Microbiol 39,
159–167.
conifers. Trends Plant Sci 4, 184–190.
generation of protein database search programs. Nucleic Acids Res 25,
3389–3402.
Brady, C. L., Cleenwerck, I., Venter, S. N., Vancanneyt, M., Swings, J.
& Coutinho, T. A. (2008). Phylogeny and identification of Pantoea
species associated with plants, humans and the natural environment
based on multilocus sequence analysis (MLSA). Syst Appl Microbiol
31, 447–460.
http://ijs.sgmjournals.org
MODELTEST:
testing the model of
DNA substitution. Bioinformatics 14, 817–818.
Rojas, A. M., de Los Rios, J. E. G., Fischer-Le Saux, M., Jimenez, P.,
Reche, P., Bonneau, S., Sutra, L., Mathieu-Daudé, F. & McClelland, M.
(2004). Erwinia toletana sp. nov., associated with Pseudomonas
savastanoi-induced tree knots. Int J Syst Evol Microbiol 54, 2217–2222.
Rosselló-Mora, R. & Amann, R. (2001). The species concept for
prokaryotes. FEMS Microbiol Rev 25, 39–67.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48
947
V. Skrodenyte? -Arbačiauskiene? and others
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA 4: molecular
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol
Evol 24, 1596–1599.
Seybold, S. J., Huber, D. P. W., Lee, J. C., Graves, A. D. & Bohlmann, J.
(2006). Pine monoterpenes and pine bark beetles: a marriage of
convenience for defense and chemical communication. Phytochem Rev
5, 143–178.
Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007).
phagous insect Ips typographus. Ekologija 4, 1–6.
Phenotypic characterization and the principles of comparative
systematics. In Methods for General Molecular Microbiology, 3rd edn,
pp. 330–393. Edited by T. J. Beveridge, J. A. Breznak, G. A. Marzluf,
T. M. Schmidt & L. R. Synder. Washington: American Society for
Microbiology.
Skrodenyte-Arbaciauskiene, V., Sruoga, A. & Butkauskas, D.
(2006b). Assessment of microbial diversity in the river trout Salmo
Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K. & Swings, J.
(1996). Polyphasic taxonomy, a consensus approach to bacterial
trutta fario L. intestinal tract identified by partial 16S rRNA gene
sequence analysis. Fish Sci 72, 597–602.
systematics. Microbiol Rev 60, 407–438.
Skrodenyte? -Arbačiauskiene? , V., Būda, V., Radžiute? , S. & Stunže? nas, V.
(2006a). Myrcene-resistant bacteria isolated from the gut of phyto-
Stackebrandt, E. & Goebel, 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.
Stoesser, G., Baker, W., van den Broek, A., Camon, E., GarciaPastor, M., Kanz, C., Kulikova, T., Lombard, V., Lopez, R. & other
authors (2001). The EMBL nucleotide sequence database. Nucleic
Acids Res 29, 17–21.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
948
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.
Zhao, T., Krokene, P., Björklund, N., Långström, B., Solheim, H.,
Christiansen, E. & Borg-Karlson, A.-K. (2010). The influence of
Ceratocystis polonica inoculation and methyl jasmonate application on
terpene chemistry of Norway spruce, Picea abies. Phytochemistry 71,
1332–1341.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 22:13:48