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Acinetobacter nectaris sp. nov. and Acinetobacter boissieri sp. nov., isolated from floral nectar of wild Mediterranean insect-pollinated plants

01 Apr 2013-International Journal of Systematic and Evolutionary Microbiology (Microbiology Society)-Vol. 63, Iss: 4, pp 1532-1539

TL;DR: The taxonomic status of 14 strains of members of the genus Acinetobacter isolated from floral nectar of wild Mediterranean insect-pollinated plants, which did not belong to any previously described species within this genus, was investigated following a polyphasic approach and confirmed that these strains formed two separate lineages.

AbstractThe taxonomic status of 14 strains of members of the genus Acinetobacter isolated from floral nectar of wild Mediterranean insect-pollinated plants, which did not belong to any previously described species within this genus, was investigated following a polyphasic approach. Confirmation that these strains formed two separate lineages within the genus Acinetobacter was obtained from comparative analysis of the partial sequences of the 16S rRNA gene and the gene encoding the β-subunit of RNA polymerase (rpoB), DNA-DNA reassociation data, determination of the DNA G+C content and physiological tests. The names Acinetobacter nectaris sp. nov. and Acinetobacter boissieri sp. nov. are proposed. The type strain of A. nectaris sp. nov. is SAP 763.2(T) ( = LMG 26958(T) = CECT 8127(T)) and that of A. boissieri sp. nov. is SAP 284.1(T) ( = LMG 26959(T) = CECT 8128(T)).

Topics: Acinetobacter (52%)

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Acinetobacter nectaris sp. nov. and Acinetobacter
boissieri sp. nov., isolated from floral nectar of wild
Mediterranean insect-pollinated plants
Sergio A
´
lvarez-Pe
´
rez,
1
3 Bart Lievens,
2,3
3 Hans Jacquemyn
4
and Carlos M. Herrera
1
Correspondence
S. A
´
lvarez-Pe
´
rez
sealperez@gmail.com
B. Lievens
bli@scientiaterrae.org
1
Estacio
´
n Biolo
´
gica de Don
˜
ana, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Avda.
Ame
´
rico Vespucio, E-41092 Sevilla, Spain
2
Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Lessius
University College, De Nayer Campus, Consortium for Industrial Microbiology and Biotechnology
(CIMB), Department of Microbial and Molecular Systems (M
2
S), KU Leuven Association, B-2860
Sint-Katelijne-Waver, Belgium
3
Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium
4
Division of Plant Ecology and Systematics, Biology Department, KU Leuven, B-3001 Heverlee,
Belgium
The taxonomic status of 14 strains of members of the genus Acinetobacter isolated from floral
nectar of wild Mediterranean insect-pollinated plants, which did not belong to any previously
described species within this genus, was investigated following a polyphasic approach.
Confirmation that these strains formed two separate lineages within the genus Acinetobacter was
obtained from comparative analysis of the partial sequences of the 16S rRNA gene and the gene
encoding the b-subunit of RNA polymerase ( rpoB), DNA–DNA reassociation data, determination
of the DNA G +C content and physiological tests. The names Acinetobacter nectaris sp. nov. and
Acinetobacter boissieri sp. nov. are proposed. The type strain of A. nectaris sp. nov. is SAP
763.2
T
(5LMG 26958
T
5CECT 8127
T
) and that of A. boissieri sp. nov. is SAP 284.1
T
(5LMG 26959
T
5CECT 8128
T
).
Members of the genus Acinetobacter are generally regarded
as common, free-living saprophytes that show extensive
metabolic versatility and potential to adapt to different
human-associated and natural environments (Towner,
2006; Doughari et al. , 2011; Sand et al., 2011). Apart from
the well-known human and animal-pathogenic species of
the genus Acinetobacter , several novel species within this
genus have been described during recent years to
accommodate isolates from agricultural soils (Kang et al. ,
2011), activated sludge (Carr et al. , 2003), raw wastewater
(Vaz-Moreira et al., 2011), a hexachlorocyclohexane
dumpsite (Malhotra et al. , 2012) and diverse natural
environmental sources, such as forest soils (Kim et al.,
2008), seawater (Vaneechoutte et al., 2009) and wetlands
(Anandham et al. , 2010). Nevertheless, except for those
species with clinical importance, the distribution and
ecological role(s) of the ‘acinetobacters’ in most environ-
ments are largely unknown (Carr et al., 2003; Towner,
2006). In particular, the possible associations of members
of this bacterial group with plant hosts remain to be
addressed.
Floral nectar is the key component in the mutualism
between angiosperms and their animal pollinators, which
take this sugary solution as a reward for their pollination
services (Brandenburg et al., 2009; Heil, 2011). While
foraging on flowers, pollinators can contaminate floral
nectar with different prokaryotic and eukaryotic micro-
organisms, some of which are particularly well-adapted to
thrive in this ephemeral habitat characterized by high
osmotic pressure and the presence of plant secondary
metabolites with defensive functions (Herrera et al. , 2010;
Pozo et al. , 2012). Nectar micro-organisms can alter
pollinators’ foraging behaviour in different ways, for
example by reducing the nutritional value of floral nectar
3These authors contributed equally to this work.
Abbreviations:
BI, Bayesian inference; ML, maximum-likelihood; NJ,
neighbour-joining; OTU, operational taxonomical unit; PM, Phenotype
MicroArray.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences determined in this study are JQ771129JQ771142 and
those for the partial rpoB gene sequences are JQ771143JQ771156.
A supplementary figure and four supplementary tables are available with
the online version of this paper.

and/or changing other physico-chemical conditions within
the floral microenvironment, and thus potentially interfere
with plant sexual reproduction, as has been recently
suggested for nectar yeasts (Canto et al. , 2008; Herrera
et al., 2008; de Vega et al. , 2009; Herrera & Pozo, 2010;
Peay et al., 2012).
Recently, species of the genus Acinetobacter have been
identified as representing the main bacterial genus
inhabiting the floral nectar of some cultivated plant species
from Northern Israel (Fridman et al. , 2012) and phylo-
genetically diverse wild Mediterranean plants from
Southern Spain (A
´
lvarez-Pe
´
rez & Herrera, 2013). In this
latter study, nectar isolates of members of the genus
Acinetobacter grouped into a single operational taxonom-
ical unit (OTU) defined on the basis of a 3 % dissimilarity
cut-off in the 16S rRNA gene sequence, but into two
different OTUs when this threshold was lowered to 1 %
(A
´
lvarez-Pe
´
rez & Herrera, 2013). Such differentiation was
also supported by some differences in colony morphology
and growth rate in plate cultures, but no additional tests
were conducted to further characterize those isolates. In
this study we explore the taxonomic status of these two
nectar groups of acinetobacters associated with wild
Mediterranean plants.
The 14 strains investigated in this study are listed in Table
S1, available in IJSEM Online. These strains were isolated
on different dates from nectar samples of several plant
species collected at different places within the surroundings
of Don
˜
ana’s Natural Park (Huelva province, southwest
Spain), using the procedure described by A
´
lvarez-Pe
´
rez
et al. (2012). Additionally, for comparative taxonomic
analysis, Acinetobacter calcoaceticus DSM 30006
T
,
Acinetobacter baylyi DSM 14961
T
, Acinetobacter gerneri
DSM 14967
T
and Acinetobacter radioresistens DSM 6976
T
were included in some phenotypic and genotypic assays.
The inclusion of Acinetobacter calcoaceticus in those tests
was justified by its status as the type species for the genus
Acinetobacter, while the other three taxa were identified as
the most closely related species to our nectar strains on the
basis of 16S rRNA gene sequence data (see below).
Colonies of all the nectar strains grown on trypticase soy
agar (TSA; Panreac) were circular, convex to umbilicate,
smooth and slightly opaque with entire margins. After
5 days of incubation at 25
u
C, colonies of these strains were
variable in diameter, ranging from 0.5 to 2.5 mm, although
smaller colonies were also observed for some strains. On
microscope images, cells appeared as non-motile coccoba-
cilli and commonly occurred in pairs, but also alone or in
short chains. No spores were observed. The main
phenotypic properties of the type strains of all studied
taxa are summarized in Table 1, and those of all studied
nectar isolates are shown in Table S2. All tests were carried
out at 25
u
C unless otherwise indicated. Catalase activity
was determined by evaluating bubble production with a
3 % (v/v) hydrogen peroxide solution (Cappuccino &
Sherman, 2002). Oxidase activity was tested using oxidase
test strips (MB 0266 A; Oxoid,). All strains were aerobic,
catalase-positive and oxidase-negative. Whereas no growth
was observed on TSA in an anaerobic jar, all nectar strains
were able to grow at decreased oxygen levels, as assessed by
visual inspection of bacterial cultures grown on TSA in a
candle jar for 5 days at 25
u
C. Growth at 4, 25, 30, 37 and
41
u
C, haemolysis of sheep blood, gelatin hydrolysis and
production of acid from glucose and sucrose in Hugh–
Leifson medium were examined as described previously
(Hugh & Leifson, 1953; Skerman, 1959; Bouvet & Grimont,
1986; Carr et al., 2003). All nectar strains grew at 25 and
30
u
C, but not at 37 or 41
u
C. Strains SAP219.2, SAP239.2,
SAP240.2, SAP241.2, SAP242.2, SAP284.1
T
and SAP320.1
were able to grow at 4
u
C but strains SAP220.2, SAP249.1,
SAP 305.1, SAP763.2
T
, SAP 956.2, SAP970.1 and SAP971.1
could not. All strains were non-haemolytic on Columbia
agar supplemented with sheep blood. Strains SAP 305.1
and SAP 970.1 were found to grow poorly on this medium.
None of the tested isolates were able to hydrolyse gelatin.
All nectar strains produced acid from sucrose and glucose.
Carbon source oxidation was determined by Phenotype
MicroArray (PM) technology (Biolog) using PM plate 1.
Using this technology, kinetic profiles are generated by
continuously monitoring the metabolic activity during
incubation (Bochner et al., 2001). Plates were incubated in
the OmniLog automated incubator-reader (Biolog) for
5 days at 25
u
C and were read every 15 min. Interpretation
of results was performed using OmniLog PM software
according to the manufacturer’s instructions. Clear differ-
ences were observed between the two groups of nectar
strains and between these nectar strains and the type strains
of A. baylyi , A. calcoaceticus , A. gerneri and A. radioresistens .
In contrast to the type strains of the related species of the
genus Acinetobacter , all nectar strains were able to oxidize
sucrose and
D-fructose as the only carbon source. D-
mannose, on the other hand, was only oxidized by the
group which included strain SAP763.2
T
. In addition, D-
glucose was only oxidized by some strains of that group. In
addition to sucrose and
D-fructose, L-malic acid was the
only carbon source that could be oxidized by the majority
of the strains of the group containing strain SAP284.1
T
. For
the group including strain SAP763.2
T
, all strains were
found to oxidize
L-aspartic acid, bromosuccinic acid,
fumaric acid,
L-glutamic acid, L-malic acid, DL-malic acid,
succinic acid,
L-asparagine and L-proline; and some of the
seven strains in this group were able to oxidize
D-xylose, D-
gluconic acid,
a-ketoglutaric acid, mono-methylsuccinate
or
L-alanine (Table S2). In order to compare the results
obtained by PM fingerprinting with more conventional
assimilation tests commonly used for classification of
members of the genus Acinetobacter , some key biochemical
features were also assessed using the phenotypic system
described by Bouvet & Grimont (1986) and adapted by
Nemec et al. (2009). More specifically, tests were
performed for sucrose,
D-glucose, succinic acid and
phenylacetate. Briefly, assimilation tests were performed
using the basal mineral medium of Cruze et al. (1979)

supplemented with 0.1 % (w/v) of the tested carbon source.
The basal medium consisted of the following (l
21
): 10.0 g
KH
2
PO
4
,5.0gNa
2
HPO
4
, 2.0 g (NH
4
)
2
SO
4
, 0.2 g
MgSO
4
.7H
2
O, 0.001 g CaCl
2
.2H
2
O and 0.001 g
FeSO
4
.7H
2
O (pH 7.0). 5 ml of this medium was dispensed
into tubes, inoculated with washed bacterial cells and
incubated at 25
u
C under agitation. Growth on the different
carbon sources was evaluated after 2, 4, 6 and 10 days by
means of visual comparison between inoculated tubes
containing carbon sources and control tubes containing
only inoculated basal medium. In general, results obtained
by these assays confirmed the results obtained by the PM
technology assays (data not shown). From the two groups of
nectar isolates, all isolates were again able to assimilate
sucrose, whereas
D-glucose and succinic acid were only
assimilated by (some of) the isolates of the group which
included SAP763.2
T
. In contrast to other known species of
the genus Acinetobacter , none of the nectar isolates were able
to assimilate acetate.
Sucrose tolerance was determined by culturing the studied
strains in transparent plastic vials containing Luria–Bertani
(LB) broth (Difco) supplemented with 0 (positive control),
10, 20, 30, 40 or 50 % sucrose (w/v, Sigma–Aldrich). All
these liquid media were filter-sterilized and kept at 4
u
C
until use. The range of sugar concentrations tested closely
Table 1. Differential phenotypic characteristics of the type
strains of all studied species of the genus Acinetobacter
Strains: 1, A. nectaris sp. nov. SAP 763.2
T
;2,A. boissieri sp. nov. SAP
284.1
T
;3,A. calcoaceticus DSM 30006
T
;4,A. baylyi DSM 14961
T
;5,A.
gerneri DSM 14967
T
;6,A. radioresistens DSM 6976
T
. A. nectaris sp.
nov. and A. boissieri sp. nov. can be separated from the type strains of
A. calcoaceticus , A. baylyi , A. gerneri and A. radioresistens by some
basic phenotypic characteristics, such as their ability to oxidize
fructose and sucrose, their tolerance to sucrose concentrations above
30 % (w/v) and their inability to grow at ¢37
u
C and assimilate
acetate. + , Positive reaction; 2, negative reaction;
W, weak growth;
ND, no data available.
Characteristic 1 2 3 4 5 6
Growth on TSA at:
4
u
C 2 +
WW2 +
25
u
C ++ + + + +
30
u
C ++ + + + +
37
u
C 22 ++++
41
u
C 22 2
W ++
Anaerobic growth 222222
Growth at decreased oxygen levels ++
ND ND ND ND
Haemolysis on Columbia blood agar 222222
Gelatin hydrolysis 222222
Catalase activity ++ + + + +
Oxidase activity 222222
Growth on LB broth plus sucrose at:
10 % (w/v) ++ + + + +
20 % (w/v) ++ + + + +
30 % (w/v) ++ + + + 2
40 % (w/v) ++ 2222
50 % (w/v) +
W 2222
Acid production from glucose ++ + + + 2
Acid production from sucrose ++ + + + 2
Oxidation of: *
D-Glucose 22 2 + 22
D-Fructose ++ 2222
D-Mannose + 22222
Sucrose ++ 2222
D-Xylose 222222
Acetic acid 22 ++++
D-Aspartic acid 22 2 + 22
L-Aspartic acid + 22 + 22
Bromosuccinic acid + 22 ++2
Citric acid 22 2 ++2
Fumaric acid + 22 ++2
D-Galacturonic acid 22 + 222
D-Gluconic acid + 22 + 22
L-Glutamic acid + 22 ++2
a-Hydroxybutyric acid 22 2 ++2
m-Hydroxyphenyl acetic acid 22 2 2 + 2
p-Hydroxyphenyl acetic acid
22 2 2
+
2
a-Ketobutyric acid 22 2 ++2
a-Ketoglutaric acid 22 2 ++2
L-Lactic acid 22 2 ++2
D-Malic acid
222222
L-Malic acid ++ 2 ++2
DL-Malic acid + 22 ++2
Table 1. cont.
Characteristic 1 2 3 4 5 6
Mucic acid 22 2 + 22
Propionic acid 22 2 ++2
Pyruvic acid 22 2 ++2
D-Saccharic acid 22 2 + 22
Succinic acid + 2 +++2
Tricarballylic acid 22 2 + 22
Methylpyruvate 22 +++2
Mono-methylsuccinate 22 2 ++2
a-Hydroxyglutaric acid-g-Lactone 22 2 2 + 2
2-Aminoethanol 222222
Dulcitol
22
+
222
D-Alanine 22 +++2
L-Alanine 22 2 ++2
L-Asparagine + 22 + 22
L-Glutamine + 22 ++2
Gly–Pro 22 2 2 + 2
Phenylethylamine 22 2 2 + 2
L-Proline + 22 ++2
L-Threonine 22 + 222
Tween 20 22 +++2
Tween 40 22 +++2
Tween 80 22 ++++
*Oxidation of carbon sources was determined by Phenotype
MicroArray (PM) technology (Biolog) using PM plate 1. Further
details on the procedure and the results obtained for all the novel
nectar isolates characterized in this work are provided in Table S2.

matched the range of naturally occurring variation in floral
nectars of the wild Mediterranean plant communities from
which our nectar strains were recovered (S. A
´
lvarez-Pe
´
rez
& C. M. Herrera, unpublished results). Single colonies
picked from 5-day cultures on TSA medium were used to
inoculate the tubes and these were incubated at 25
u
C for
up to 10 days. The turbidity of the cultures with respect to
negative controls (i.e. tubes containing no inoculated
media) was recorded as a positive result. At the end of the
experiment, an aliquot of each test tube was plated on
TSA medium to check for possible contaminations.
Furthermore, acid production by bacterial strains when
growing at different sucrose concentrations was tested by
adding 40
ml methyl red (Panreac) to each tube. All nectar
strains were able to grow at sucrose concentrations ranging
from 10 to 50 % (w/v), although the growth of strains of
the group containing SAP284.1
T
at 40 % and 50 % sucrose
was very weak. Acidification of the culture media was
observed in all tubes containing sucrose, but not in the
positive control (no sucrose). In contrast, A. baylyi DSM
14961
T
, A. calcoaceticus DSM 30006
T
and A. gerneri DSM
14967
T
only grew at sucrose concentrations up to 30 %,
and A. radioresistens DSM 6976
T
only tolerated 10 % and
20 % sucrose. Furthermore, these four reference strains did
not acidify either sucrose-containing culture broths or the
positive control.
Methods for genotypic characterization of the studied
strains included comparative sequence analysis of the 16S
rRNA and the
b-subunit of RNA polymerase (rpoB)-
encoding genes, assessment of overall genomic relatedness
by DNA–DNA hybridizations and determination of the
DNA G+C content.
An almost complete fragment of the 16S rRNA gene
was amplified and subsequently sequenced as described
by A
´
lvarez-Pe
´
rez et al. (2012). Preliminary sequence
comparisons with the 16S rRNA gene sequences stored in
GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and the
Ribosomal Database Project (RDP, http://rdp.cme.msu.
edu/) databases showed that the nectar strains belonged to
the family Moraxellaceae in the Gammaproteobacteria
subdivision, and the best hits for all sequences were the
putative isolates of members of the genus Acinetobacter
recovered by Fridman et al. (2012) from floral nectar of
cultivated plants (99 % and 97–98 % overall similarity to
strains SAP763.2
T
and SAP284.1
T
, respectively). The
SimTable tool available at the EzTaxon server v. 2.1
(http://www.eztaxon.org/, last accessed 10 December 2011;
Chun et al. , 2007) was used to search for neighbours
among species of the genus Acinetobacter with validly
published names on the basis of 16S rRNA gene sequences,
identifying A. baylyi B2
T
, A. gerneri 9A01
T
and A.
radioresistens DSM 6976
T
as the species most closely
related to the novel nectar strains, but with a sequence
similarity value ¡ 96.3 % in all cases (Table S3). The
sequence similarity value between strains SAP 763.2
T
and
SAP 284.1
T
, as determined through the EzTaxon server,
was 97.7 %.
The 16S rRNA gene sequences of the novel nectar strains
and reference strains of members of the genus Acinetobacter
and the family Moraxellaceae were included in a multiple
alignment generated by
CLUSTAL W (Chenna et al., 2003).
The resulting alignment was trimmed with BioEdit v.
7.0.9.0 (Hall, 1999) to ensure that all sequences had the
same start and end point, and analysed with Gblocks
(Castresana, 2000) to eliminate ambiguously aligned
regions, using ‘allow gap positions5with half’, ‘minimum
length of a block
5
5
9
and default settings for all other
options. Following these procedures, 1320 nt positions
(98 % of the original alignment) remained for subsequent
phylogenetic analysis using the neighbour-joining (NJ)
method as implemented in the
MEGA 5 software package
(Tamura et al. , 2011). Pairwise evolutionary distances were
computed by the Jukes–Cantor method, and reliability of
nodes in the NJ phylogram was assessed by running 1000
bootstrap replicates. In the NJ phylogram based on 16S
rRNA gene sequences (Fig. 1) the novel nectar strains
clustered with other members of the genus Acinetobacter ,
but stood apart from the recognised species of this genus
by forming a consistently differentiated group with 99 %
bootstrap support. Furthermore, all strains of the group
which included strain SAP 763.2
T
clustered together with a
100 % bootstrap support, as did all those strains corres-
ponding to the group which included strain SAP 284.1
T
(Fig. 1).
Comparative sequence analysis of two variable regions
zone 1 (approximately 397 bp) and zone 2 (approximately
544 bp) of the rpoB gene was used to confirm both the
within-species relatedness of the two groups of novel
strains, and their separation from each other and from
previously described species of the genus Acinetobacter .
Primer sequences and PCR conditions were as previously
described (Khamis et al. , 2004; La Scola et al., 2006), with
some minor modifications: 250
mM of each dNTP (Sigma–
Aldrich), 0.4
mM of each of the corresponding forward and
reverse primers (Sigma–Aldrich) and 5610
22
U Taq
polymerase
ml
21
(Bioline) were used in reaction mixtures
and 52
u
C was the temperature for primer annealing in
PCR cycles. As with the 16S rRNA gene sequence,
concatenated sequences of rpoB zones 1 and 2 of the
nectar strains and type strains of other species of the genus
Acinetobacter were included in a multiple alignment and
analysed with Gblocks, which resulted in the selection of
843 nucleotide positions (99 % of the original alignment).
A phylogenetic tree was inferred by the NJ method using
the Jukes–Cantor method, and again, the two groups of
nectar strains formed two different clusters with bootstrap
values supporting their distinctness from each other and
from the other acinetobacters (Fig. S1).
The genomic DNA–DNA relatedness between the strains
SAP 763.2
T
and SAP 284.1
T
and between these and the type
strains of A. calcoaceticus , A. baylyi and A. gerneri was
evaluated by DNA–DNA hybridizations. High-molecular-
mass total genomic DNA was extracted by the method of
Wilson (1987). DNA–DNA hybridizations were carried out

0.005
Acinetobacter beijerinckii
LUH 4759
T
(AJ626712)
Acinetobacter haemolyticus
ATCC 17906
T
(Z93437)
Acinetobacter bouvetii
CCM 7196
T
(HQ180181)
Acinetobacter johnsonii ATCC 17909
T
(Z93440)
Acinetobacter gyllenbergii
RUH 442
T
(AJ293694)
Acinetobacter tjemberguae
CCM 7200
T
(HQ180190)
Acinetobacter schindleri LUH 5832
T
(AJ278311)
.
Acinetobacter Parvus LUH 4616
T
(NR 025425)
Acinetobacter tandoii CCM 7199
T
(HQ180189)
Acinetobacter Iwoffii DSM 2403
T
(NR 026209)
Acinetobacter brisouii 5YN5-8
T
(DQ832256)
Acinetobacter nosocomialis
RUH 2376
T
(HQ180192)
Acinetobacter calcoaceticus
DSM 30006
T
(AJ633632)
Acinetobacter pittii LMG 1035
T
(HQ180184)
Acinetobacter ursingii LUH 3792
T
(AJ275038)
Acinetobacter bereziniae ATCC 17924
T
(Z93443)
Acinetobacter guillouiae DSM 590
T
(X81659)
Acinetobacter baumannii ATCC 19606
T
(Z93435)
Acinetobacter junii ATCC 17908
T
(Z93435)
Acinetobacter venetianus ATCC 31012
T
(AJ295007)
Acinetobacter towneri CCM 7201
T
(HQ180191)
Acinetobacter baylyi CCM 7195
T
(AM410709)
Acinetobacter rudis G30
T
(EF204258)
SAP 970.1 (JQ771134)
SAP 971.1 (JQ771135)
SAP 220.2 (JQ771129)
SAP 763.2
T
(JQ771132)
SAP 249.1
(JQ771130)
SAP 240.2 (JQ771138)
SAP 239.2 (JQ771137)
SAP 284.1
T
(JQ771141)
Acinetobacter nectaris
SAP 242.2 (JQ771140)
Acinetobacter boissieri
SAP 3 20.1 (JQ771142)
SAP 219.2 (JQ771136)
SAP 241.2 (JQ771139)
Alkanindiges illinoisensis MVAB Hex1
T
(AF513979)
Perlucidibaca piscinae lMCC1704
T
(DQ664237)
Moraxella lawnala ATCC 17967
T
(AF005160)
Ps
y
chrobacter immobilis ATCC 43116
T
(U39399)
SAP 956.2 (JQ771133)
SAP 305.1 (JQ771131)
99
100
98
Acinetobacter radioresistens M 17694
T
(Z93445)
Acinetobacter indicus A648
T
(HM047743)
Acinetobacter soli B1
T
(EU290155)
100
100
100
Acinetobacter gemeri CCM 7197
T
(HQ180188)
100
99
Fig. 1. Neighbour-joining tree, based on 16S rRNA gene sequences, showing the relationships of nectar strains of A. nectaris
sp. nov. and
A. boissieri
sp. nov. with respect to other members of the genus
Acinetobacter
and representatives of closely
related genera within the family Moraxellaceae. Evolutionary distances were computed using the Jukes–Cantor method and are
in the units of the number of base substitutions per site. There were a total of 1320 positions in the final dataset. All positions

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Journal ArticleDOI
TL;DR: The data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection, and the outstanding diversification of the species occurred largely by horizontal transfer at specific hotspots preferentially located close to the replication terminus.
Abstract: Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species We inferred a robust global phylogeny and delimited several new putative species The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics Our data suggest that A baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens

182 citations


Cites background from "Acinetobacter nectaris sp. nov. and..."

  • ...In the last decade, a small number of complete genome sequences and a large number of draft sequences of Acinetobacter spp. have become available (Barbe et al. 2004; Fournier et al. 2006; Antunes et al. 2013)....

    [...]

  • ...…the species and its large pangenome have led to suggestions that A. baumannnii might have endured one wave of population expansion during the diversification of the species and another very recently after the introduction of antibiotics at the hospital (Diancourt et al. 2010; Antunes et al. 2013)....

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Journal ArticleDOI
16 Apr 2014-PLOS ONE
TL;DR: The crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation, concluding that the crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment.
Abstract: The honey bee is a key pollinator species in decline worldwide. As part of a commercial operation, bee colonies are exposed to a variety of agricultural ecosystems throughout the year and a multitude of environmental variables that may affect the microbial balance of individuals and the hive. While many recent studies support the idea of a core microbiota in guts of younger in-hive bees, it is unknown whether this core is present in forager bees or the pollen they carry back to the hive. Additionally, several studies hypothesize that the foregut (crop), a key interface between the pollination environment and hive food stores, contains a set of 13 lactic acid bacteria (LAB) that inoculate collected pollen and act in synergy to preserve pollen stores. Here, we used a combination of 454 based 16S rRNA gene sequencing of the microbial communities of forager guts, crops, and corbicular pollen and crop plate counts to show that (1) despite a very different diet, forager guts contain a core microbiota similar to that found in younger bees, (2) corbicular pollen contains a diverse community dominated by hive-specific, environmental or phyllosphere bacteria that are not prevalent in the gut or crop, and (3) the 13 LAB found in culture-based studies are not specific to the crop but are a small subset of midgut or hindgut specific bacteria identified in many recent 454 amplicon-based studies. The crop is dominated by Lactobacillus kunkeei, and Alpha 2.2 (Acetobacteraceae), highly osmotolerant and acid resistant bacteria found in stored pollen and honey. Crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment. We conclude that the crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation.

176 citations


Journal ArticleDOI
TL;DR: The microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, are reviewed, which act to impact microbial community assembly and constrain the ecosystem.
Abstract: Microbial habitats that contain an excess of carbohydrate in the form of sugar are widespread in the microbial biosphere. Depending on the type of sugar, prevailing water activity and other substances present, sugar-rich environments can be highly dynamic or relatively stable, osmotically stressful, and/or destabilizing for macromolecular systems, and can thereby strongly impact the microbial ecology. Here, we review the microbiology of different high-sugar habitats, including their microbial diversity and physicochemical parameters, which act to impact microbial community assembly and constrain the ecosystem. Saturated sugar beet juice and floral nectar are used as case studies to explore the differences between the microbial ecologies of low and higher water-activity habitats respectively. Nectar is a paradigm of an open, dynamic and biodiverse habitat populated by many microbial taxa, often yeasts and bacteria such as, amongst many others, Metschnikowia spp. and Acinetobacter spp., respectively. By contrast, thick juice is a relatively stable, species-poor habitat and is typically dominated by a single, xerotolerant bacterium (Tetragenococcus halophilus). A number of high-sugar habitats contain chaotropic solutes (e.g. ethyl acetate, phenols, ethanol, fructose and glycerol) and hydrophobic stressors (e.g. ethyl octanoate, hexane, octanol and isoamyl acetate), all of which can induce chaotropicity-mediated stresses that inhibit or prevent multiplication of microbes. Additionally, temperature, pH, nutrition, microbial dispersion and habitat history can determine or constrain the microbiology of high-sugar milieux. Findings are discussed in relation to a number of unanswered scientific questions.

120 citations


Journal ArticleDOI
TL;DR: The present review summarizes the recent data about the natural reservoir of non-baumannii Acinetobacter including the novel species that have been described for the first time from environmental sources and reported during the last years.
Abstract: Acinetobacter spp. are ubiquitous gram negative and non-fermenting coccobacilli that have the ability to occupy several ecological niches including environment, animals and human. Among the different species, Acinetobacter baumannii has evolved as global pathogen causing wide range of infection. Since the implementation of molecular techniques, the habitat and the role of non-baumannii Acinetobacter in human infection have been elucidated. In addition, several new species have been described. In the present review, we summarize the recent data about the natural reservoir of non-baumannii Acinetobacter including the novel species that have been described for the first time from environmental sources and reported during the last years.

111 citations


Cites background from "Acinetobacter nectaris sp. nov. and..."

  • ...…rpoB, gyrB, 16S-rRNA Krizova et al., 2014 A. boissieri Floral nectar Spain Phenotypic, G+C, fatty acids, 16S-rRNA, rpoB, DNA-DNA hybridization Álvarez-Pérez et al., 2013 A. bouvetii Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. brisouii Wetland (Peat) Korea…...

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  • ...…Korea G+C, 16S-RNA, DNA-DNA hybridization Yoon et al., 2007 A. nectaris Floral nectar Spain Phenotypic, G+C, fatty acids, 16S-rRNA, rpoB, DNA-DNA hybridization Álvarez-Pérez et al., 2013 A. nosocomialis Sewage Denmark 16S-rRNA Geiger et al., 2009 Life environment surface Korea 16S-rRNA rpoB Choi…...

    [...]

  • ...Besides, Acinetobacter boissieri and Acinetobacter nectaris were two novel species that were isolated from nectar samples of plants in Spain (Álvarez-Pérez et al., 2013)....

    [...]

  • ...Acinetobacter species Origin of isolation Country of isolation Identification method References A. albensis Water, soil Czech Republic Phenotypic, 16S-RNA, gyrB, rpoB, gltA, pyrG, recA, Maldi-TOF Krizova et al., 2015a A. anitratus Animal France Phenotypic, 16S-rRNA La Scola et al., 2001 A. antiviralis Plant roots Korea % G+C, fatty acid analysis, 16S-RNA, DNA-DNA hybridization Lee et al., 2009 A. apis Animal Korea DNA-DNA hybridization, 16S rRNA gene and rpoB sequence analysis, % G+C, and fatty acid analysis Kim et al., 2014 A. baylyi Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. beijerinckii Animal Lebanon rpoB Rafei et al., 2015 A. bereziniae Sewage Denmark 16S-rRNA Geiger et al., 2009 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Vegetables Hong Kong UK ARDRA Berlau et al., 1999a; Houang et al., 2001 Meat Lebanon rpoB Rafei et al., 2015 Human skin Germany Hong Kong Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD Seifert et al., 1997; Chu et al., 1999 Animal Lebanon rpoB Rafei et al., 2015 A. bohemicus Soil Czech Republic rpoB, gyrB, 16S-rRNA Krizova et al., 2014 Water Czech Republic rpoB, gyrB, 16S-rRNA Krizova et al., 2014 A. boissieri Floral nectar Spain Phenotypic, G+C, fatty acids, 16S-rRNA, rpoB, DNA-DNA hybridization Álvarez-Pérez et al., 2013 A. bouvetii Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. brisouii Wetland (Peat) Korea Phenotypic, G+C, fatty acids, 16S-rRNA, DNA-DNA hybridization Anandham et al., 2010 A. calcoaceticus Sewage, water Denmark, Croitia 16S-rRNA Geiger et al., 2009; Maravić et al., 2015 Soil Hong Kong Korea Lebanon China ARDRA 16S-rRNA rpoB Houang et al., 2001; Choi et al., 2012; Rafei et al., 2015; Wang and Sun, 2015 Vegetables Lebanon UK rpoB ARDRA Berlau et al., 1999a; Rafei et al., 2015; Al Atrouni et al., 2016 Animal Lebanon rpoB Rafei et al., 2015 Human skin Hong Kong India Phenotypic, ARDRA, RAPD Chu et al., 1999; Patil and Chopade, 2001 A. gandensis Water Croitia – Maravić et al., 2015 Animal – Phenotypic, DNA-DNA hybridization, 16S rRNA rpoB, % G+C, fatty acid, MALDI-TOF MS Smet et al., 2014 A. gerneri Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 Animal Lebanon rpoB Rafei et al., 2015 (Continued) Frontiers in Microbiology | www.frontiersin.org 3 February 2016 | Volume 7 | Article 49 TABLE 1 | Continued Acinetobacter species Origin of isolation Country of isolation Identification method References A. grimontii, Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. guangdongensis lead-zinc ore mine site China Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Feng et al., 2014b A. guillouiae Water Denmark 16S-rRNA Geiger et al., 2009 Vegetables UK ARDRA Berlau et al., 1999a Human skin Hong Kong UK, Netherland Phenotypic, ARDRA, RAPD, AFLP Chu et al., 1999; Dijkshoorn et al., 2005 A. haemolyticus Water Croitia – Maravić et al., 2015 Human skin India Phenotypic Patil and Chopade, 2001 A. harbinensis Water China Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Li et al., 2014b A. indicus Dump site India Phenotypic, G+C, fatty acids, 16S-rRNA, rpoB, DNA-DNA hybridization Malhotra et al., 2012 A. johnsonii Activated sludge Germany Pcr fingerprinting Wiedmann-al-Ahmad et al., 1994 Sewage, water, sea food Denmark, Croitia, China 16S-rRNA Geiger et al., 2009; Zong and Zhang, 2013; Maravić et al., 2015 Animal Lebanon rpoB Rafei et al., 2015 Human skin Germany Hong Kong UK, Netherland Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD, AFLP Seifert et al., 1997; Chu et al., 1999; Dijkshoorn et al., 2005 A. junii Activated sludge Germany Pcr fingerprinting Wiedmann-al-Ahmad et al., 1994 Sewage, water Denmark, Croitia 16S-rRNA Geiger et al., 2009; Maravić et al., 2015 Animal Lebanon rpoB Rafei et al., 2015 Soil China ARDRA 16S-rRNA rpoB Wang and Sun, 2015 Human skin Germany Hong Kong India UK, Netherland Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD, AFLP Seifert et al., 1997; Chu et al., 1999; Patil and Chopade, 2001; Dijkshoorn et al., 2005 A. koukii Soil, beet field, sediment Korea, Germany, Netherland, Malaysia, Thailand Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Choi et al., 2013 A. kyonggiensis Sewage Korea Phenotypic, G+C, fatty acids, 16S-rRNA, DNA-DNA hybridization Lee and Lee, 2010 A. lwoffii Activated sludge Germany PCR fingerprinting Wiedmann-al-Ahmad et al., 1994 Sewage, water, sea food Denmark 16S-rRNA Geiger et al., 2009 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Animal Lebanon, Croitia rpoB, 16S-RNA Rafei et al., 2015; Sun et al., 2015 Vegetables UK ARDRA Berlau et al., 1999a Human skin Germany UK Hong Kong India Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD Seifert et al., 1997; Berlau et al., 1999b; Chu et al., 1999; Patil and Chopade, 2001 (Continued) Frontiers in Microbiology | www.frontiersin.org 4 February 2016 | Volume 7 | Article 49 TABLE 1 | Continued Acinetobacter species Origin of isolation Country of isolation Identification method References A. marinus Water Korea G+C, 16S-RNA, DNA-DNA hybridization Yoon et al., 2007 A. nectaris Floral nectar Spain Phenotypic, G+C, fatty acids, 16S-rRNA, rpoB, DNA-DNA hybridization Álvarez-Pérez et al., 2013 A. nosocomialis Sewage Denmark 16S-rRNA Geiger et al., 2009 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Vegetables UK ARDRA Berlau et al., 1999a Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 A. oleivorans Rice paddy Korea % G+C, fatty acid analysis, 16S-RNA, DNA-DNA hybridization Kang et al., 2011 A. pakistanensis Wastewater Pakistan Phenotypic, fatty acids, 16S-rRNA, gyrB, rpoB, atpD, DNA-DNA hybridization Abbas et al., 2014 A. parvus Soil Korea 16S-rRNA rpoB Choi et al., 2012 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 A. pittii Sewage Denmark 16S-rRNA Geiger et al., 2009 Soil Hong Kong, Lebanon ARDRA rpoB Houang et al., 2001; Rafei et al., 2015 Vegetables Hong Kong Lebanon UK ARDRA rpoB Berlau et al., 1999a; Houang et al., 2001; Rafei et al., 2015 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Water Lebanon rpoB Rafei et al., 2015 Cheese, Meat Lebanon rpoB Rafei et al., 2015 Animal Lebanon rpoB Rafei et al., 2015 Human skin Germany Hong Kong India Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD Seifert et al., 1997; Chu et al., 1999; Patil and Chopade, 2001 A. populi Populus bark China Phenotypic,16S-RNA, gyrB, rpoB, DNA-DNA hybridization Li et al., 2015b A. puyangensis Populus bark China Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Li et al., 2013 A. qingfengensis Populus bark China Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Li et al., 2014a A. radioresistens Soil, cotton, water Australia, Croitia Dortet et al., 2006; Maravić et al., 2015 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Animal Lebanon rpoB Rafei et al., 2015; Sunantaraporn et al., 2015 Human skin Germany UK Hong Kong Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD Seifert et al., 1997; Berlau et al., 1999b; Chu et al., 1999 (Continued) Frontiers in Microbiology | www.frontiersin.org 5 February 2016 | Volume 7 | Article 49 TABLE 1 | Continued Acinetobacter species Origin of isolation Country of isolation Identification method References A. refrigeratoris Life environment surface China 16S-rRNA, rpoB DNA-DNA hybridization Feng et al., 2014a A. rudis Wastewter, raw milk Portugal, Israel Phenotypic, G+C, fatty acids, 16S-rRNA, gyrB, rpoB, DNA-DNA hybridization Vaz-Moreira et al., 2011 A. seifertii/genomspecies close 13 TU Life environment surface Korea 16S-RNA, rpoB Choi et al., 2012 Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 A. seohaensis Water Korea G+C, 16S-RNA, DNA-DNA hybridization Yoon et al., 2007 A. shindleri Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Animal Lebanon rpoB Rafei et al., 2015; Sunantaraporn et al., 2015 A. soli Soil Korea Phenotypic, fatty acids, G+C content, 16S-rRNA gyrB, DNA-DNA hybridization Kim et al., 2008 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 Vegetables Lebanon rpoB Rafei et al., 2015 A. tandoii Activated sludge plant Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 Soil Korea 16S-rRNA rpoB Choi et al., 2012 Life environment surface Korea 16S-rRNA rpoB Choi et al., 2012 A. tjernbergiae Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. towneri Activated sludge Australia 16S-rRNA DNA-DNA hybridization Carr et al., 2003 A. variabilis /(genomspecies 15TU) Sewage, water, sea food Denmark 16S-rRNA Geiger et al., 2009 Life environment surface Korea rpoB Choi et al., 2012 Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 Animal France Phenotypic, gyrA, gyrB, rpoB Poirel et al., 2012 Animal – Phenotypic, rpoB, gyrB, Maldi-Tof, whole genome analysis Nishimura et al., 1988 A. venetianus Water Oil vegetables Israel, Italy, Denmark, Hong Kong, japan Phenotypic, DNA-DNA hybridization, AFLP, rpoB, ARDRA, tDNA PCR Vaneechoutte et al., 2009 Acinetobacter spp. Water China Malaysia, Thailand Vietnam 16S-rRNA Fuhs and Chen, 1975; Huys et al., 2007; Krizova et al., 2015b; Xiong et al., 2015 Soil France-Kuwait 16S-rRNA Bordenave et al., 2007; Obuekwe et al., 2009 Meat Hong Kong ARDRA Houang et al., 2001 (Continued) Frontiers in Microbiology | www.frontiersin.org 6 February 2016 | Volume 7 | Article 49 TABLE 1 | Continued Acinetobacter species Origin of isolation Country of isolation Identification method References Fish, shrimps Hong Kong ARDRA Houang et al., 2001; Huys et al., 2007 Sediment China Malaysia, Thailand Vietnam 16S-rRNA Huys et al., 2007; Xiong et al., 2015 Plants nectar Israel, Spain Pyrosequencing, 16S-rRNA Fridman et al., 2012; Álvarez-Pérez and Herrera, 2013 Milk United states Kenya Korea Phenotypic Jayarao and Wang, 1999; Ndegwa et al., 2001; Nam et al., 2009; Gurung et al., 2013 Animal Angola 16S-rRNA Guardabassi et al., 1999 Human skin Germany Hong Kong India UK, Netherland Phenotypic, ARDRA, SDS-PAGE, ribotyping, DNA-DNA hybridization, RAPD, AFLP Seifert et al., 1997; Chu et al., 1999; Patil and Chopade, 2001; Dijkshoorn et al., 2005 genomspecies 14 BJ Sewage Denmark 16S-rRNA Geiger et al., 2009 Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 A. genospecies 15 BJ Human skin UK Hong Kong Phenotypic, ADRA, RAPD Berlau et al., 1999b; Chu et al., 1999 genomspecies 16 Sewage Denmark 16S-rRNA Geiger et al., 2009 Vegetables Hong Kong ARDRA Houang et al., 2001 Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 A. genospecies 17 Human skin Hong Kong ARDRA, RAPD Chu et al., 1999 A. genospecies 13 BJ, 14 TU Human skin Hong Kong ARDRA, RAPD Chu et al., 1999...

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Journal ArticleDOI
TL;DR: Analysis of the whole-body bacterial flora of An.
Abstract: The intolerable burden of malaria has for too long plagued humanity and the prospect of eradicating malaria is an optimistic, but reachable, target in the 21st century. However, extensive knowledge is needed about the spatial structure of mosquito populations in order to develop effective interventions against malaria transmission. We hypothesized that the microbiota associated with a mosquito reflects acquisition of bacteria in different environments. By analyzing the whole-body bacterial flora of An. gambiae mosquitoes from Burkina Faso by 16 S amplicon sequencing, we found that the different environments gave each mosquito a specific bacterial profile. In addition, the bacterial profiles provided precise and predicting information on the spatial dynamics of the mosquito population as a whole and showed that the mosquitoes formed clear local populations within a meta-population network. We believe that using microbiotas as proxies for population structures will greatly aid improving the performance of vector interventions around the world.

82 citations


References
More filters

Journal ArticleDOI
TL;DR: The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models, inferring ancestral states and sequences, and estimating evolutionary rates site-by-site.
Abstract: Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.

37,583 citations


"Acinetobacter nectaris sp. nov. and..." refers methods in this paper

  • ...Following these procedures, 1320 nt positions (98 % of the original alignment) remained for subsequent phylogenetic analysis using the neighbour-joining (NJ) method as implemented in the MEGA 5 software package (Tamura et al., 2011)....

    [...]



Journal ArticleDOI
TL;DR: A computerized method is presented that reduces to a certain extent the necessity of manually editing multiple alignments, makes the automation of phylogenetic analysis of large data sets feasible, and facilitates the reproduction of the final alignment by other researchers.
Abstract: The use of some multiple-sequence alignments in phylogenetic analysis, particularly those that are not very well conserved, requires the elimination of poorly aligned positions and divergent regions, since they may not be homologous or may have been saturated by multiple substitutions. A computerized method that eliminates such positions and at the same time tries to minimize the loss of informative sites is presented here. The method is based on the selection of blocks of positions that fulfill a simple set of requirements with respect to the number of contiguous conserved positions, lack of gaps, and high conservation of flanking positions, making the final alignment more suitable for phylogenetic analysis. To illustrate the efficiency of this method, alignments of 10 mitochondrial proteins from several completely sequenced mitochondrial genomes belonging to diverse eukaryotes were used as examples. The percentages of removed positions were higher in the most divergent alignments. After removing divergent segments, the amino acid composition of the different sequences was more uniform, and pairwise distances became much smaller. Phylogenetic trees show that topologies can be different after removing conserved blocks, particularly when there are several poorly resolved nodes. Strong support was found for the grouping of animals and fungi but not for the position of more basal eukaryotes. The use of a computerized method such as the one presented here reduces to a certain extent the necessity of manually editing multiple alignments, makes the automation of phylogenetic analysis of large data sets feasible, and facilitates the reproduction of the final alignment by other researchers.

7,620 citations


"Acinetobacter nectaris sp. nov. and..." refers methods in this paper

  • ...0 (Hall, 1999) to ensure that all sequences had the same start and end point, and analysed with Gblocks (Castresana, 2000) to eliminate ambiguously aligned regions, using ‘allow gap positions5with half’, ‘minimum length of a block559 and default settings for all other options....

    [...]

  • ...…alignment was trimmed with BioEdit v. 7.0.9.0 (Hall, 1999) to ensure that all sequences had the same start and end point, and analysed with Gblocks (Castresana, 2000) to eliminate ambiguously aligned regions, using ‘allow gap positions5with half’, ‘minimum length of a block559 and default settings…...

    [...]

  • ...As with the 16S rRNA gene sequence, concatenated sequences of rpoB zones 1 and 2 of the nectar strains and type strains of other species of the genus Acinetobacter were included in a multiple alignment and analysed with Gblocks, which resulted in the selection of 843 nucleotide positions (99 % of the original alignment)....

    [...]

  • ...The resulting alignment was trimmed with BioEdit v. 7.0.9.0 (Hall, 1999) to ensure that all sequences had the same start and end point, and analysed with Gblocks (Castresana, 2000) to eliminate ambiguously aligned regions, using ‘allow gap positions5with half’, ‘minimum length of a block559 and default settings for all other options....

    [...]


Journal ArticleDOI
TL;DR: The Clustal series of programs, widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees, are extended.
Abstract: The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute) (http://www.ebi.

5,140 citations


"Acinetobacter nectaris sp. nov. and..." refers methods in this paper

  • ...The 16S rRNA gene sequences of the novel nectar strains and reference strains of members of the genus Acinetobacter and the family Moraxellaceae were included in a multiple alignment generated by CLUSTAL W (Chenna et al., 2003)....

    [...]


Journal ArticleDOI
TL;DR: High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA) and may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods.
Abstract: High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by P1 nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G+C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C content. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.

4,667 citations


"Acinetobacter nectaris sp. nov. and..." refers methods in this paper

  • ...The DNA was enzymically degraded into nucleosides and the nucleotidic base composition was determined by HPLC, according to the method of Mesbah et al. (1989)....

    [...]