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Contents lists available at ScienceDirect
Systematic and Applied Microbiology
journal homepage: www.elsevier.de/syapm
New Taxa: Proteobacteria
Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi
sp. nov. and Rosenbergiella epipactidis sp. nov., three novel bacterial
species isolated from floral nectar夽
Marijke Lenaerts a,1 , Sergio Álvarez-Pérez b,∗∗,1 , Clara de Vega c , Ado Van Assche a ,
Steven D. Johnson d , Kris A. Willems a , Carlos M. Herrera c , Hans Jacquemyn e ,
Bart Lievens a,∗
a
Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Cluster for Bioengineering Technology (CBet), Department of
Microbial and Molecular Systems (M2 S), KU Leuven, Campus De Nayer, B-2860 Sint-Katelijne-Waver, Belgium
b
Department of Animal Health, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, E-28040 Madrid, Spain
c
Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas (CSIC), E-41092 Sevilla, Spain
d
School of Life Sciences, University of KwaZulu-Natal, Scottsville, Pietermaritzburg 3209, South Africa
e
Division of Plant Ecology and Systematics, Biology Department, KU Leuven, B-3001 Heverlee, Belgium
a r t i c l e
i n f o
Article history:
Received 6 December 2013
Received in revised form 17 March 2014
Accepted 21 March 2014
Keywords:
Enterobacteriaceae
Nectar
Rosenbergiella
a b s t r a c t
The taxonomic status of nine strains of the family Enterobacteriaceae isolated from floral nectar of wild
Belgian, French, South African and Spanish insect-pollinated plants was investigated following a polyphasic approach. Confirmation that these strains belonged to the genus Rosenbergiella was obtained from
comparative analysis of partial sequences of the 16S rRNA gene and other core housekeeping genes (atpD
[ATP synthase ␤-chain], gyrB [DNA gyrase subunit B] and rpoB [RNA polymerase ␤-subunit]), DNA–DNA
reassociation data, determination of the DNA G+C content and phenotypic profiling. Two strains belonged
to the recently described species Rosenbergiella nectarea, while the other seven strains represented
three novel species within the genus Rosenbergiella. The names Rosenbergiella australoborealis sp. nov.
(with strain CdVSA 20.1T [LMG 27954T = CECT 8500T ] as the type strain), Rosenbergiella collisarenosi sp.
nov. (with strain 8.8AT [LMG 27955T = CECT 8501T ] as the type strain) and Rosenbergiella epipactidis sp.
nov. (with strain 2.1AT [LMG 27956T = CECT 8502T ] as the type strain) are proposed. Additionally, the
description of the genus Rosenbergiella is updated on the basis of new phenotypic and molecular data.
© 2014 Elsevier GmbH. All rights reserved.
Introduction
Floral nectar is regarded as the key component in the mutualism
between animal-pollinated plants and their pollinators, which use
this sugar-rich solution as a reward for their pollination services
Abbreviations: BI, Bayesian inference; ML, maximum-likelihood; MLSA, multilocus sequence analysis; MP, maximum-parsimony; NJ, neighbour-joining; PP,
posterior probabilities; SH, Shimodaira–Hasegawa.
夽 Note: The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequences determined in this study are KF876184–KF876192. Those for the partial
atpD, gyrB and rpoB gene sequences are KF876193–KF876201, KF876202–KF876210
and KF876211–KF876219, respectively.
∗ Corresponding author. Tel.: +32 15 305590; fax: +32 15 305599.
∗∗ Corresponding author. Tel.: +34 91 394 3717; fax: +34 91 394 3908.
E-mail addresses: [email protected] (S. Álvarez-Pérez),
[email protected] (B. Lievens).
1
These authors contributed equally to this work.
[14,37]. Floral nectar is assumed to be initially sterile [15], but floral
visitors can actively vector different microorganisms, most often
yeasts and bacteria [1,8,41]. Some of these yeasts and bacteria,
are particularly well adapted to flourish in this rather unique
harsh environment of high sugar concentrations and antimicrobial
compounds [4,41].
Although it has already been known for decades that microbes
are common inhabitants of floral nectars [48], the ecological importance of nectar-associated microorganisms for the plant–pollinator
mutualisms is only now beginning to be understood. Floral
microbes can have a negative impact on plant–pollinator mutualisms by decreasing floral attractiveness through a reduction of
nectar nutritional value [24,38] and interfering with pollen germination and damaging pollen tubes [27]. However, it has also
been suggested that floral microorganisms enhance pollination by
producing volatiles or fermentation by-products that attract pollinators [57,59] and by raising flower temperature [40]. In this
regard, Herrera et al. [42] recently demonstrated that nectar yeasts
http://dx.doi.org/10.1016/j.syapm.2014.03.002
0723-2020/© 2014 Elsevier GmbH. All rights reserved.
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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2
Table 1
Isolates used in this study.
Isolate
Host plant species (family)
Geographic origin
Latitude/longitude
Date of isolation
Reference
1.12A
Epipactis palustris
(Orchidaceae)
Epipactis palustris
(Orchidaceae)
Epipactis palustris
(Orchidaceae)
Epipactis palustris
(Orchidaceae)
Narcissus papyraceus
(Amaryllidaceae)
Iris xiphium (Iridaceae)
Dune Dewulf, Gijvelde,
France
Dune du Perroquet,
Bray-Dunes, France
Dune du Perroquet,
Bray-Dunes, France
Ter Yde, Oostduinkerke,
Belgium
Hinojos, Huelva, Spain
51◦ 3 48 N,
2◦ 28 9 E
51◦ 4 48 N,
2◦ 32 28 E
51◦ 4 48 N,
2◦ 32 28 E
51◦ 7 4 N, 2◦ 40 6 E
July 2012
This study
July 2012
This study
July 2012
This study
July 2012
This study
January 2011
Protea roupelliae
(Proteaceae)
Protea roupelliae
(Proteaceae)
Protea subvestita
(Proteaceae)
Mount Gilboa, South Africa
January 2011
Álvarez-Pérez and
Herrera (2013)
Álvarez-Pérez and
Herrera (2013)
This study
January 2011
This study
January 2011
This study
2.1AT
2.6A
8.8AT
SAP 86.2
SAP 817.2
CdVSA 20.1T
CdVSA 21.1
CdVSA 50.1
Hinojos, Huelva, Spain
Mount Gilboa, South Africa
Sani Pass, Southern
Drakensberg, South Africa
increased pollinator visitation, and may have important consequences for the fecundity of plants. In particular, Vannette et al. [68]
further showed that nectar bacteria, rather than yeasts, reduced
pollination success, seed set and nectar consumption by pollinators,
thereby weakening the plant–pollinator mutualism.
Current studies have highlighted that nectar-associated microbial communities are generally species-poor (but see [29]), but may
nevertheless represent a reservoir of unexplored microbial biodiversity [15,16,22,39,46,55,58]. However, research efforts to find
new species associated with floral nectar have been significantly
more intensive for yeasts (e.g. [23,30,62]) than for bacteria and to
date only two studies have described novel bacterial species in nectar [4,35]. The newly described bacterial species all belonged to the
Gammaproteobacteria, and include two new species of the family
Moraxellaceae, Acinetobacer nectaris and A. boissieri [4], and one of
the family Enterobacteriaceae, Rosenbergiella nectarea [35].
The Enterobacteriaceae are a large family that live in a wide variety of habitats, including plants, food and environmental sources
and clinical samples [11,12,43,50]. With regard to flowers, Junker
et al. [49] demonstrated that bacteria of this family dominate the
epiphytic bacterial communities in petals. Additionally, several
members of this family have also been found inhabiting the floral nectar of some cultivated plant species from Northern Israel
[1,29] and diverse wild plant species from Belgium, Spain and
South Africa [2,3,46,47]. Strikingly, some Enterobacteriaceae isolates were found that could not be identified conclusively to the
genus or species level based on 16S ribosomal RNA (rRNA) gene
sequence analysis. In this study, we investigated the taxonomic
status of nine of these Enterobacteriaceae isolates obtained from
floral nectar from phylogenetically diverse wild plant species across
different study sites on two continents, Europe (Belgium, France
and Spain) and Africa (South Africa). Additionally, we provide an
updated description of the genus Rosenbergiella on the basis of new
physiological and molecular data.
◦
37 18 14 N,
06◦ 26 15 E
37◦ 18 14 N,
06◦ 26 15 E
29◦ 16 58 S,
30◦ 17 32 E
29◦ 16 58 S,
30◦ 17 32 E
29◦ 36 40 S,
29◦ 21 40 E
May 2011
reference strains of the most closely related species to our strains
were included in the study for comparative phenotypic analysis,
including R. nectarea (LMG 26121T , further referred to as 8N4T ),
Phaseolibacter flectens (LMG 2186) and Tatumella citrea (LMG
22049T ). Cultures were preserved at −80 ◦ C in trypticase soy broth
(Oxoid, Basingstoke, UK), containing 32.5% glycerol. Genomic DNA
was extracted from five-day old cultures grown on trypticase soy
agar (TSA; Oxoid) using the phenol–chloroform extraction method
described previously [53].
RAPD fingerprinting
In order to assess whether our isolates represented different strains, each of the nine isolates was subjected to Random
Amplified Polymorphic DNA (RAPD) analysis. Amplification was
performed using a Bio-Rad T100 thermal cycler in a total volume
of 20 ␮l containing 0.5 ␮M of primer OPB-10 (5 -CTGCTGGGAC-3 )
(Operon, Huntsville, USA), 0.15 mM of each dNTP, 1.0 unit Titanium
Taq DNA polymerase, 1× Titanium Taq PCR buffer (Clontech Laboratories, Palo Alto, CA, USA), and 5 ng genomic DNA (as measured
by a Nanodrop spectrophotometer). Samples were subjected to the
following PCR conditions: denaturation at 94 ◦ C for 2 min, followed
by 35 cycles of 1 min at 94 ◦ C, 1 min at 30 ◦ C and 2 min at 72 ◦ C, with
a final extension at 72 ◦ C for 10 min. Obtained PCR products were
separated by loading 6.5 ␮l of the reaction volume on 1.5% agarose
gels followed by gel electrophoresis in 1× Tris/acetate EDTA (TAE)
buffer at 100 V for three hours. Gels were stained with ethidium
bromide and visualized with UV light. A 200–10,000 bp DNA ladder (Smartladder, Eurogentec, Seraing, Belgium) was used as size
marker for comparison. The BioChemi System (UVP, Upland, CA,
USA) was used to acquire image data. The analysis was performed
twice with identical fingerprints, and indicated that our isolates
represented different strains (Fig. S1).
PCR amplification and sequencing of selected loci
Materials and methods
Bacterial strains and DNA extraction
Nine strains isolated from floral nectar according to the procedure described by Álvarez-Pérez et al. [2] were used in this
study (Table 1). Strains were isolated in 2011 and 2012 from
five insect-pollinated plant species, including Epipactis palustris
(Orchidaceae; Belgium and France), Iris xiphium (Iridaceae; Spain),
Narcissus papyraceus (Amaryllidaceae; Spain), Protea roupelliae
and P. subvestita (Proteaceae; South Africa) (Table 1). In addition,
For each of the nine nectar strains, the almost complete 16S
rRNA gene was PCR amplified using the primers 27F and 1492R
[52]. PCR amplification was performed in a reaction volume of
20 ␮l, containing 312.5 ␮M of each dNTP, 1.0 ␮M of each primer,
1.25 units TaKaRa Ex Taq Polymerase, 1x Ex Taq Buffer (Clontech
Laboratories, Palo Alto, CA, USA) and 5 ng genomic DNA. Before
amplification, DNA samples were denatured at 94 ◦ C for 2 min, followed by 35 cycles of 45 s at 94 ◦ C, 45 s at 59 ◦ C and 45 s at 72 ◦ C, with
a final extension at 72 ◦ C for 10 min. Following agarose gel electrophoresis, target amplicons were cut from the gel and purified
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA).
Purified PCR products were cloned into Escherichia coli plasmids
using the pCR2.1 vector and the Topo-TA cloning kit (InvitrogenTM
Life TechnologiesTM , Carlsbad, CA, USA) and sequenced with the
primers M13F and M13R. To improve the accuracy of the sequencing, inserts of at least three individual clones were sequenced.
Subsequently, obtained sequences were individually trimmed for
quality, using a minimum Phred score of 20. For each target fragment, accurate consensus sequences were obtained for each isolate
by aligning the acquired sequences with MEGA v.5 [67] and manual sequence editing of ambiguous base calls based on the obtained
electropherograms, resulting in a sequence length of 1501 bp.
Partial fragments of the housekeeping genes atpD (ATP
synthase ␤-chain), gyrB (DNA gyrase subunit B) and rpoB
(RNA polymerase ␤-subunit) were amplified using the primer
pairs atpD 01-F/atpD 02-R [10], gyrB 01-F/gyrB 02-R [21] and
rpoB ML01-F (5 -AGTTTATGGACCAGAACAACC-3 )/rpoB ML02-R
(5 -TTGCATGTTTGCACCCATCA-3 ), respectively. In all cases, PCR
amplification was performed in a reaction volume of 20 ␮l, consisting of 0.15 mM of each dNTP, 0.5 ␮M of each primer, 1 unit
Titanium Taq DNA polymerase, 1x Titanium Taq PCR buffer (Clontech Laboratories), and 5 ng genomic DNA. PCR conditions were as
described above, with exception of the annealing time and temperature, which were 1 min at 55 ◦ C for atpD and gyrB, and 45 s at 63 ◦ C
for rpoB. Cloning, sequencing (using M13F) and subsequent assembly of consensus sequences were performed as described above
and resulted in sequences of 856, 914 and 507 bp for atpD, gyrB and
rpoB, respectively.
Taxonomy of nectar isolates and phylogenetic reconstruction
In order to determine the taxonomic affiliation of our nectar strains, the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/,
last accessed 15 November 2013; [51]) was used to search for
neighbours among validly named bacterial species on the basis
of 16S rRNA gene sequences. Closest relatives of the studied nectar strains were also identified by a BLAST search of 16S rRNA
sequences in GenBank [5]. Nucleotide sequences of the nectar isolates and type strains of closely related species for the four studied
loci were included in multiple alignments generated by MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/, [26]). The resulting
alignments were trimmed with BioEdit v.7.0.9.0 [33] to ensure that
all sequences had the same start and end point.
Phylogenetic trees for each individual gene and a concatenation
of the three protein-encoding genes (atpD, gyrB and rpoB, in this
order) were constructed using four different methods in order to
provide solid evidence of the classification: neighbour-joining (NJ),
maximum parsimony (MP), Bayesian inference (BI) and maximum
likelihood (ML). NJ and MP trees were obtained using MEGA v.5.
In NJ analyses, the evolutionary distances were computed by the
Tamura-Nei method [66], and the rate of variation among sites was
modelled by a gamma distribution, with shape parameters set at
the values estimated by PhyML in ML analyses (see below). MP trees
were obtained using the close-neighbour-interchange (CNI) algorithm with search level 3, in which the initial trees were obtained
with the random addition of sequences (ten replicates). In both the
NJ and MP methods, 1000 bootstrap replications were used to infer
consensus trees.
BI analyses were performed with MrBayes v.3.1.2 [61]. The
simplest models of sequence evolution among those available in
MrBayes that best fitted the sequence data were determined using
the jModeltest v.0.1.1 package [56], and resulted in selection of
the following: HKY+G+I for the 16S rRNA gene, HKY+G for gyrB,
SYM+G+I for atpD, and GTR+G+I for rpoB. A partitioned model
was used to obtain the atpD + gyrB + rpoB concatenated tree. Four
Metropolis-coupled Markov chains (five for the 16S rRNA dataset)
3
were run twice, until average standard deviation of split frequencies fell below 0.01 (1.4 × 106 to 1.5 × 107 generations). The chains
were sampled each 100 generations and chain temperature was set
to 0.2. Fifty percent majority rule consensus trees were calculated
using the sumt command and discarding the first 25% of the trees
to yield the final Bayesian estimates of phylogeny. Posterior probabilities (PP) from the 50% majority rule consensus trees were used
as estimates of robustness.
ML analyses were carried out using the online version of
PhyML v.3.0 (http://www.atgc-montpellier.fr/phyml/, [32]), under
the HKY+G+I (16S rRNA), HKY+G (gyrB) or the GTR+G+I (atpD, rpoB
and concatenated dataset) models of molecular evolution, with four
substitution rate categories, starting trees generated by BIONJ, and
SPR tree search algorithms. Model parameters were estimated from
the dataset and support for the inferred topologies was tested using
1000 bootstrap replications.
The confidence of alternative tree topologies based on single gene and concatenated datasets was evaluated by the
Shimodaira–Hasegawa (SH) test [64]. This analysis was performed with TREE-PUZZLE v.5.2 [63], and tree topologies with
p-values < 0.05 were considered to be incongruent with the dataset
under analysis.
DNA–DNA hybridisation and DNA G+C content analysis
In order to perform DNA–DNA hybridisations between the
strains 1.12A, 2.1AT , 8.8AT , CdVSA20.1T , and R. nectarea 8N4T as well
as to determine the DNA G+C content of the novel type strains, highquality DNA was prepared by the method of Wilson [70], with some
minor adjustments [19]. DNA–DNA hybridisations were performed
at 41 ◦ C using the microplate method [28] with some modifications [19,31]. Reciprocal reactions were performed for every pair.
For determination of the DNA G+C content the DNA was enzymatically degraded into nucleosides and the DNA base composition was
determined by HPLC [54]. Three independent analyses were conducted for each DNA sample, and mean DNA G+C content values
were calculated.
Physiological and biochemical characterisation
For physiological and biochemical characterisation, strains were
grown aerobically on TSA at 25 ◦ C for 24 h unless otherwise indicated. Tests for motility, hydrogen sulphide production and indole
production were performed in SIM (Sulphide Indole Motility)
medium (Becton, Dickinson and Company, Le Pont de Claix, France)
at 25 ◦ C. Growth at 4, 22, 25, 30, 37 and 42 ◦ C, haemolysis of sheep
erythrocytes on Columbia blood agar (bioMérieux, Marcy l’Etoile,
France) and sucrose tolerance (0, 10, 20, 30, 40, 50, 60% sucrose
(w/v, Sigma–Aldrich) in Luria–Bertani (LB) broth (Difco)) was determined as described previously [4]. Catalase activity was determined
by evaluating bubble production with a 3% (v/v) hydrogen peroxide solution [17]. Oxidase activity was tested with oxidase test
strips (Oxoid) and DNase plates (Oxoid) were used for investigating
DNase activity. Acetoin (3-hydroxy-2-butanone) production was
determined with the Voges–Proskauer reaction [7]. Production of
acid from glucose, fructose and sucrose in Hugh–Leifson medium
was performed as described previously [44]. Fermentation of lactose was examined using MacConkey agar (TecLaim, Madrid, Spain)
with confirmation in Phenol Red Lactose Broth [7]. Further, the ability to hydrolyse gelatin was determined by the gelatin agar plate
assay described by Atlas [7] and growth on Brain Heart Infusion
agar (Panreac, Castellar del Vallès, Barcelona) was evaluated as
well. Finally, Phenotype MicroArray (PM) tests (Biolog, Hayward,
CA, USA) were performed [9] using PM Plate 1, containing 95 different single carbon sources in addition to a negative control. Plates
were incubated during four days at 25 ◦ C in the OmniLog automated
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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4
incubator-reader (Biolog) and were read every 15 min. Interpretation of the results was performed using Omnilog PM software
according to the manufacturer’s instructions.
Table 2
DNA–DNA relatedness between strains 1.12A, 2.1AT , 8.8AT , CdVSA 20.1T and the
type strain of R. nectarea (8N4T ).
Isolate
Results and discussion
BLAST analysis of the 16S rRNA gene sequences against GenBank
revealed that all nectar strains studied belonged to the Enterobacteriaceae. Additionally, using the EzTaxon server, R. nectarea 8N4T
was found to be the most closely related species to our nectar
strains, followed by Phaseolibacter flectens ATCC12775T (Table S1).
The 16S rRNA gene sequence identity level between all our strains
varied between 99.27% and 100%. Whereas bacterial species delineation is generally based on a 16S rRNA gene similarity cut-off level
of 97% [65], different Enterobacteriaceae species may share over
99% 16S rRNA gene sequence similarity [12], while 98% is generally considered as a reasonable cut-off value to delineate different
genera in this family [25,45]. On the other hand, Halpern et al.
[35] defined a specific signature region in the 16S rRNA gene for
the genus Rosenbergiella. While this signature perfectly matched
sequences for the strains 1.12A, 2.1AT , 2.6A and 8.8AT , four mismatches were found when compared with sequences of the other
five nectar strains (SAP 86.2, SAP 817.2, CdVSA 20.1T , CdVSA 21.1
and CdVSA 50.1). It has to be noted, however, that the proposed
signature region was found based on a single Rosenbergiella species
only. To further assess the phylogenetic position of our strains, partial atpD, gyrB and rpoB gene sequences were analysed. For these
individual markers, it has been shown that species from different Enterobacteriaceae genera generally share less than 94% gene
similarity [25,60]. Nevertheless, specific limits for species or genus
differentiation of the Enterobacteriaceae based on these genes are
not defined so far [13]. Highest levels of sequence similarity were
again found with R. nectarea 8N4T , ranging from 94.86 to 97.30%,
from 82.12 to 98.47% and from 84.84 to 96.21% for atpD (740 bp),
gyrB (850 bp) and rpoB (343 bp), respectively (Table S2).
Phylogenetic analysis of the 16S rRNA gene sequences showed
that the Belgian (8.8AT ) and French (1.12A, 2.1AT and 2.6A) nectar strains clustered with R. nectarea 8N4T , but that grouping was
only well-supported in the NJ tree (bootstrap support for the clade
was ≤75% in the MP and ML trees, and the posterior probability
was 50% in the BI tree; Fig. 1). The Spanish (SAP 86.2 and SAP
817.2) and South African (CdVSA 20.1T , CdVSA 21.1 and CdVSA 50.1)
nectar strains formed another poorly supported clade in the 16S
rRNA trees obtained by all phylogenetic methods (<75% bootstrap
support or posterior probability). These strains formed separate
clusters, but were closely together to R. nectarea 8N4T and the
other nectar strains. The combined analysis of atpD, gyrB and rpoB
sequences provided a better resolution and allowed classifying
the studied strains in four clades (Fig. 2). The first clade, which
was highly supported by all the phylogenetic methods, included
strains 1.12A and 2.6A, as well as R. nectarea 8N4T . Strain 2.1AT
was close to but clustered apart from the former strains in all concatenated trees, whereas the Spanish and South African strains
showed limited sequence variability (as inferred from the length
of tree branches) and formed a third robust cluster. Strain 8.8AT
was basal to the rest of nectar strains in the concatenated NJ and
MP trees with ≥99% bootstrap support, but clustered with the Spanish and South African strains in the trees obtained by the ML and
BI methods (75.5% bootstrap support and 88% posterior probability, respectively; data not shown). These four clusters were also
shown in the independent analyses of the three protein-encoding
genes, although their support values and phylogenetic relations
were variable (Fig. S2a–S2c). The results of the SH test performed on
individual gene sequences and on the concatenated dataset showed
that most NJ trees were incongruent with each other, but were
1.12A
2.1AT
8.8AT
CdVSA 20.1T
8N4T
% DNA–DNA relatedness
1.12A
2.1AT
8.8AT
CdVSA 20.1T
8N4T
100
53 ± 9
33 ± 7
28 ± 2
77 ± 7
100
30 ± 1
23 ± 15
53 ± 6
100
28 ± 6
32 ± 1
100
30 ± 7
100
not significantly different from the NJ tree based on the alignment
of concatenated sequences (Table S3). Furthermore, most datasets
supported the trees obtained by the ML and BI methods for the
concatenation of individual loci, and the gyrB and rpoB datasets also
supported the concatenated MP tree (Table S3). Therefore, although
the studied genes may not exhibit the same evolutionary history,
in general terms the trees based on the alignment of concatenated
sequences did not contradict the information brought by each individual locus. DNA–DNA hybridisation confirmed the MLSA analysis.
Strains 2.1AT , 8.8AT and CdVSA 20.1T showed between 23 ± 15% and
53 ± 9% DNA–DNA relatedness among each other and with 8N4T
(Table 2). Strain 1.12A showed 77 ± 7% DNA–DNA relatedness with
the type strain of R. nectarea (Table 2), which is above the generally
accepted limit for species delineation (i.e. 70%; [69]).
All strains tested were facultative anaerobic, catalase-positive,
oxidase-negative and DNase-negative, and could grow at 22, 25
and 30 ◦ C, but not at 4 ◦ C (Table S4). Most strains also grew at 37 ◦ C,
with the exception of strains 8.8AT and 2.1AT (Tables 3 and S4). All
nectar strains were able to grow at sucrose concentrations up to
50% (w/v), while no growth was observed at 60% sucrose. In contrast to Halpern et al. [35], who observed growth for R. nectarea
8N4T up to 60% sucrose concentration, in our assay 8N4T was only
found to grow up to 50% sucrose. All strains were non-haemolytic
on Columbia blood agar and were motile in SIM medium. None
of them produced indole or hydrogen sulphide, nor were able to
hydrolyse gelatin. All strains produced acid from sucrose, glucose
and fructose. When streaked on MacConkey agar, all strains were
positive for lactose fermentation, which was also confirmed using
Phenol Red Lactose Broth. Further, all strains were able to produce
acetoin (positive Voges–Proskauer reaction) (Table S4). Clear differences were observed among our nectar strains and between these
and the reference strains of R. nectarea, P. flectens and T. citrea when
phenotyped by the Biolog system (Tables 3 and S4).
In contrast to the other nectar strains, strains 1.12A, 2.6A, R.
nectarea 8N4T and T. citrea LMG 22049T were the only strains able to
oxidise 2 -deoxyadenosine. Oxidation of phenylethylamine could
only be performed by the strains 1.12A and 2.6A. T. citrea LMG
22049T , and strains 1.12A and 2.6A were the only strains that could
oxidise citric acid. Together with R. nectarea 8N4T and T. citrea LMG
22049T , strain 8.8AT was the only nectar isolate that could oxidise
d-glucose-6-phosphate. Further, together with the Spanish and
South African strains (CdVSA 20.1T , CdVSA 21.1, CdVSA 50.1, SAP
86.2, and SAP 817.2), these four isolates could oxidise ␤-methyld-glucoside. The Spanish and South African strains differed from
the other nectar strains in the inability to oxidise glycyl-l-aspartic
acid (Gly-Asp), glycyl-l-glutamic acid (Gly-Glu), d,l-␣-glycerol
phosphate, uridine and d-ribose. Some key biochemical features
(namely d-glucose-6-phosphate, ␤-methyl-d-glucoside, glycyl-laspartic acid, glycyl-l-glutamic acid, uridine and d-ribose) were
also assessed using more conventional tests using the basal mineral medium of Cruze et al. [20], supplemented with 0.1% (w/v)
of the tested carbon source (incubated for 10 days at 25 ◦ C), and
confirmed the Biolog fingerprinting results.
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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5
Table 3
Distinctivea phenotypic characteristics for the three novel Rosenbergiella species in comparison with their most related species.
Characteristic
Growth (72 h) on TSA at:
4 ◦C
37 ◦ C
42 ◦ C
Growth on MacConkey agar
(48 h)
Oxidation ofb :
Ala-Gly
d-Alanine
Gly-Asp
Gly-Glu
Gly-Pro
l-Alanine
l-Aspartic acid
l-Glutamine
l-Proline
l-Serine
2 -Deoxyadenosine
Adenosine
␣-d-Lactose
␤-Methyl-d-glucoside
d,l-␣-Glycerol phosphate
d-Cellobiose
d-Fructose-6-phosphate
d-Glucose-1-phosphate
d-Glucose-6-phosphate
d-Mannitol
d-Mannose
d-Psicose
d-Ribose
d-Trehalose
d-Xylose
Lactulose
l-Arabinose
l-Lyxose
l-Rhamnose
Maltose
Maltotriose
m-Inositol
N-Acetyl-d-glucosamine
Thymidine
Uridine
Bromosuccinic acid
Citric acid
d,l-Malic acid
d-Galactonic acid-␥-lactone
d-Galacturonic acid
d-Gluconic acid
d-Glucuronic acid
d-Malic acid
d-Saccharic acid
Fumaric acid
l-Galactonic acid-␥-lactone
l-Lactic acid
l-Malic acid
Mono-methylsuccinate
Mucic acid
Pyruvic acid
Succinic acid
Methylpyruvate
R. epipactidis
2.1AT
R. collisarenosi
8.8AT
R. australoborealis
CdVSA 20.1T
R. nectarea
8N4T
P. flectens LMG
2186
T. citrea LMG
22049T
−
−
−
+
−
−
−
+
−
+
−
+
−
+
−
+
+
+
w
w
−
+
−
+
−
−
+
+
+
−
+
−
+
−
−
+
−
−
+
−
−
−
−
−
+
−
+
−
−
−
+
−
−
−
−
−
+
−
+
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
−
+
+
+
−
−
+
−
+
+
−
−
−
+
−
+
−
+
−
−
−
+
−
−
−
−
−
+
−
+
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
+
+
+
−
−
+
−
+
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
−
−
+
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
−
+
+
+
−
+
+
−
+
+
−
−
−
+
−
+
−
+
−
−
−
+
−
−
−
−
−
+
−
+
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
−
−
−
+
+
−
+
−
−
−
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
+
+
+
+
+, positive reaction; −, negative reaction; w, weak growth.
a
A comprehensive overview of the results obtained for the different phenotypic tests performed in this study is given in Table S4.
b
Oxidation of carbon sources was determined by Phenotype MicroArray (PM) technology (Biolog) using PM Plate 1. Plates were incubated in the OmniLog® incubatorreader for 4 days at 25 ◦ C and were read every 15 minutes. OmniLog® PM software was used to calculate the area under the curve. Reactions were considered positive when the
net area under the curve exceeded the value for the blank three times. All isolates were positive for oxidation of the following substrates not shown in the table: l-asparagine,
l-glutamic acid, ␣-d-glucose, d-fructose, d-galactose, glycerol, inosine and sucrose. All isolates were negative for oxidation of the following substrates not shown in the
table: 1,2-propanediol, 2-aminoethanol, glucuronamide, phenylethylamine, tyramine, d-aspartic acid, d-serine, d-threonine, l-threonine, adonitol, ␣-methyl-d-galactoside,
d-melibiose, d-sorbitol, dulcitol, l-fucose, N-acetyl-d-mannosamine, acetic acid, acetoacetic acid, ␣-hydroxybutyric acid, ␣-hydroxyglutaric acid-␥-lactone, ␣-ketobutyric
acid, ␣-ketoglutaric acid, d-glucosaminic acid, formic acid, glycolic acid, glyoxylic acid, m-hydroxyphenyl acetic acid, m-tartaric acid, p-hydroxyphenyl acetic acid, propionic
acid, tricarballylic acid, Tween 20, Tween 40 and Tween 80.
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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SYAPM-25614; No. of Pages 10
6
ARTICLE IN PRESS
M. Lenaerts et al. / Systematic and Applied Microbiology xxx (2014) xxx–xxx
Fig. 1. Neighbour-joining (NJ) consensus tree, based on 16S rRNA gene sequences, showing the relationships of the nectar strains of Rosenbergiella australoborealis sp. nov.,
Rosenbergiella collisarenosi sp. nov., Rosenbergiella epipactidis sp. nov. and Rosenbergiella nectarea characterised in this study with respect to R. nectarea 8N4T and representatives
of closely related genera within the family Enterobacteriaceae. Evolutionary distances were computed using the Tamura-Nei method and are in the units of number of base
substitutions per site. There were a total of 1331 positions in the final dataset. Node support values (bootstrap percentages, based on 1000 simulations) ≥80% are shown
next to the branches. Maximum parsimony (MP) and maximum likelihood (ML) bootstrap node support values ≥ 90% and Bayesian inference (BI) posterior probabilities ≥
90% are marked by asterisks, filled squares and long crosses, respectively. GenBank accession numbers are given in parentheses. The tree was rooted with the 16S rRNA gene
sequence of Pseudomonas aeruginosa LMG 1242T .
Taken together, the results of these genotypic and phenotypic
analyses support the classification of 1.12A and 2.6A within the
species R. nectarea. Additionally, our results support the distinction
of three additional Rosenbergiella species isolated from floral nectar.
For these species, we propose the names Rosenbergiella australoborealis sp. nov., Rosenbergiella epipactidis sp. nov. and Rosenbergiella
collisarenosi sp. nov., represented by the type strains CdVSA 20.1T ,
2.1AT and 8.8AT , respectively. The DNA G+C content of the novel
type strains CdVSA 20.1T , 2.1AT and 8.8AT was 45.3, 47.2 and
47.6 mol%, respectively, which is similar to the DNA G+C content
of R. nectarea 8N4T (46.8 mol%) and slightly higher than that of the
related species P. flectens (44.3 mol%) [34,35]. The DNA G+C content
of strains 1.12A and 2.6A was 47 mol%, which is very close to the
G+C content of the type strain of R. nectarea, supporting again that
both strains belong to the same species.
The results of the present study also provide new insights
into the taxonomy and phylogenetic affiliation of nectar-dwelling
prokaryotes, which complement the recent description of bacteria
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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7
Fig. 2. Neighbour-joining (NJ) consensus tree, based on phylogenetic analysis of concatenated atpD + gyrB + rpoB sequences, displaying the relationships of the nectar strains
of Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov., Rosenbergiella epipactidis sp. nov. and Rosenbergiella nectarea characterised in this study with
respect to R. nectarea 8N4T and other representatives belonging to the family Enterobacteriaceae. Evolutionary distances were computed using the Tamura-Nei method and
are in the units of number of base substitutions per site. There were a total of 1404 positions in the final dataset. Node support values (bootstrap percentages, based on 1000
simulations) ≥80% are shown next to the branches. Maximum parsimony (MP) and maximum likelihood (ML) bootstrap node support values ≥90% and Bayesian inference
(BI) posterior probabilities ≥90% are marked by asterisks, filled squares and long crosses, respectively. Pseudomonas aeruginosa LMG 1242T was used as an outgroup to root
the tree. GenBank accession numbers are provided in Figure S2.
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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8
in the Gammaproteobacteria of similar ecology [4,35]. Rosenbergiella is a recently described new genus of Enterobacteriaceae
that was previously isolated from floral nectar of two cultivated
plant species in Israel, Amygdalus communis (Almond) and Citrus paradisi (Grapefruit). In this study, we report the description
of novel Rosenbergiella species isolated from wild phylogenetically diverse plant species with a diverse array of pollinators
including bees, beetles, birds, butterflies, flies, and wasps [6,18,36].
As microbes are vectored from flower to flower by floral visitors [1,8,41] and the observed vectors are common in nature, it
may be assumed that the bacteria they are transporting are also
widespread among different plant hosts. The diverse vector and
host sources together with its wide geographical distribution in
both hemispheres suggests that Rosenbergiella is a well-established
genus, which has adapted to the conditions of nectar, being able to
withstand high sugar concentrations (up to 50%, w/v) and to oxidise the major sugars of nectar (i.e. sucrose, glucose and fructose).
The ecological role of Rosenbergiella species in the multi-kingdom
interactions taking place within and around floral nectar remains
unclear to date, but clearly deserves further studies.
Update of Rosenbergiella Halpern et al. 2013
The genus Rosenbergiella (Ro.sen.ber.gi.ell’a. N.L. fem. dim. n.,
named after Prof. E. Rosenberg, an Israeli microbiologist) has been
described previously by Halpern et al. [35] based on three strains
and a single species (R. nectarea), with 8N4T as the type species
[35]. Our study allows updating the description of the genus. Cells
are Gram-negative, yellow/orange/(pale) beige pigmented, rods
that are facultatively anaerobic and motile. Strains grow well at
22–30 ◦ C, some also at 37 ◦ C, but not at 4 ◦ C (but see Halpern et al.
[35]) and 42 ◦ C. Catalase-positive, oxidase-, DNase- and gelatinasenegative. Strains grow on Brain Heart Infusion agar, MacConkey
agar and Columbia blood agar but are not haemolytic on the latter
medium. Sucrose is tolerated up to a concentration of 50% (w/v).
Indole and H2 S are not produced. Positive for Voges-Proskauer reaction. Produce acid from sucrose, glucose and fructose and oxidise
glycyl-l-proline (Gly-Pro), l-asparagine, l-aspartic acid, l-glutamic
acid, l-proline, adenosine, ␣-d-glucose, d-fructose, d-galactose,
glycerol, inosine, l-arabinose, N-acetyl-d-glucosamine, sucrose and
d-gluconic acid. Lactose is fermented. At the genetic level, all Rosenbergiella strains (12 investigated, including nine from this study
and the three strains used by Halpern et al. [35]) have over 99%
(≥99.4% on a total of ∼1350 bp) sequence similarity for the 16S
rRNA gene. Additionally, all strains share >80% sequence similarity
for the rpoB (343 bp investigated), gyrB (850 bp) and atpD (740 bp)
genes, respectively. G+C content for the different species varies
between 45.3 and 47.6 mol%. Major cellular fatty acids, as determined for the type species R. nectarea 8N4T , are C16:0, C17:0, CYCLO
C18:1 ␻7c and C16:1 ␻7c/iso-C15:0 2OH [35].
Description of R. australoborealis sp. nov.
R. australoborealis [aus.tra.lo.bo.re.a’lis L. gen., referring to the
fact that strains of this species were discovered in floral nectar
from different plants from the southern (australis) and the northern
(borealis) hemispheres].
Cells are Gram-negative rods that are facultatively anaerobic and motile. After 24 h aerobic incubation at 25 ◦ C on TSA
medium, colonies are pale beige pigmented, convex and smooth
with entire margins. Catalase-positive, oxidase-, DNase- and
gelatinase- negative. Colonies grow on Brain Heart Infusion agar,
MacConkey agar and Columbia blood agar but all strains are not
haemolytic on the latter medium. R. australoborealis strains produce acid from sucrose, glucose and fructose, and ferment lactose.
Indole and H2 S are not produced. Positive for Voges–Proskauer
reaction. Colonies grow well at 22–37 ◦ C, but not at 4 ◦ C and 42 ◦ C.
Sucrose is tolerated at concentrations ranging from 10% to 50%
(w/v). Oxidation of glycyl-l-proline, l-asparagine, l-aspartic acid,
l-glutamic acid, l-proline, adenosine, ␣-d-glucose, ␤-methyl-dglucoside, d-fructose, d-galactose, glycerol, inosine, l-arabinose,
N-acetyl-d-glucosamine, sucrose and d-gluconic acid as Biolog PM
1 substrates, while negative for the other tested sources. Oxidation
of l-glutamine and d-mannose is strain-dependent.
The description of the species is based on the characteristics of
five strains which were isolated from floral nectar of different plant
species from Spain and South Africa. The type strain is CdVSA 20.1T
(=LMG 27954T = CECT 8500T ), isolated from floral nectar of Protea
roupelliae (Proteaceae) in South Africa (Mount Gilboa). The DNA G+C
content of the type strain is 45.3 mol%.
Description of R. collisarenosi sp. nov.
R. collisarenosi [co.llis.a.re.no’si L. gen. collisarenosi, named after
the environment of the isolation source, found in nectar of an Epipactis palustris plant living in a dune habitat].
Cells are Gram-negative rods that are facultative anaerobic and motile. After 24 h aerobic incubation at 25 ◦ C on TSA
medium, colonies are pale beige pigmented, convex and smooth
with entire margins. Catalase-positive, oxidase-, DNase- and
gelatinase-negative. Colonies grow on Brain Heart Infusion agar,
MacConkey agar and Columbia blood agar without haemolysis
activity. Acid is produced from sucrose, glucose, and fructose.
Lactose is fermented, indole and H2 S are not produced. Positive for Voges–Proskauer reaction. Strain 8.8AT grows well at
22–30 ◦ C, but not at 4 ◦ C, 37 ◦ C and 42 ◦ C. Sucrose is tolerated at concentrations ranging from 10% to 50% (w/v). The
studied strain oxidises glycyl-l-aspartic acid, glycyl-l-glutamic
acid, glycyl-l-proline, l-asparagine, l-aspartic acid, l-glutamic
acid, l-glutamine, l-proline, adenosine, ␣-d-glucose, ␤-methyld-glucoside, d,l-␣-glycerol phosphate, d-fructose, d-galactose,
d-glucose-6-phosphate, d-mannose, d-ribose, glycerol, inosine,
l-arabinose, N-acetyl-d-glucosamine, sucrose, uridine and dgluconic acid as determined with the Biolog PM 1 assay.
The type strain is 8.8AT (=LMG 27955T = CECT 8501T ), isolated
from floral nectar of Epipactis palustris (Orchidaceae) collected in
Belgium (Ter Yde, Oostduinkerke). The DNA G+C content of the type
strain is 47.6 mol%.
Description of R. epipactidis sp. nov.
R. epipactidis [epi.pac.ti’dis. L. gen. f. s. Epipactis -idis, referring
to the genus name of the host (Epipactis); the first strain of this
bacterial species found in nectar of this host plant].
Cells are Gram-negative rods that are facultative anaerobic,
motile and catalase positive. After 24 h aerobic incubation at 25 ◦ C
on TSA, colonies are beige-pigmented, convex and smooth with
entire margins. Growth is observed on Brain Heart Infusion agar,
MacConkey agar and Columbia blood agar without haemolysis
activity. Strain 2.1AT grows well at 22–30 ◦ C, but not at 4 ◦ C, 37 ◦ C
and 42 ◦ C. Acid is produced from sucrose, glucose and fructose.
Lactose is fermented, indole and H2 S are not produced. Positive
for Voges–Proskauer reaction. Sucrose is tolerated at concentrations ranging from 10% to 50% (w/v). Liquefaction of gelatine,
oxidase and DNase activity is absent. The tested strain oxidises
the following Biolog PM 1 substrates: glycyl-l-aspartic acid, glycyll-glutamic acid, glycyl-l-proline, l-asparagine, l-aspartic acid,
l-glutamic acid, l-proline, adenosine, ␣-d-glucose, d,l-␣-glycerol
phosphate, d-fructose, d-galactose, d-mannose, d-ribose, glycerol,
inosine, l-arabinose, N-acetyl-d-glucosamine, sucrose, uridine and
d-gluconic acid.
Please cite this article in press as: M. Lenaerts, et al., Rosenbergiella australoborealis sp. nov., Rosenbergiella collisarenosi sp. nov.
and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
http://dx.doi.org/10.1016/j.syapm.2014.03.002
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SYAPM-25614; No. of Pages 10
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The type strain is 2.1AT (=LMG 27956T = CECT 8502T ) and was
isolated from floral nectar of Epipactis palustris (Orchidaceae) growing in Dune du Perroquet, France. The G+C content of the type strain
is 47.2 mol%.
Acknowledgements
This work was supported by the Flemish Fund for Scientific
Research (FWO) (project G.0652.13 N), the European Research
Council (ERC starting grant 260601-MYCASOR) and the Spanish
Ministry of Economy and Competitiveness, through the Severo
Ochoa Programme for Centres of Excellence in R&D&I (SEV-20120262). We are grateful to Ken Meerbergen for assistance with the
phenotypic assays.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.syapm.2014.
03.002.
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and Rosenbergiella epipactidis sp. nov., three novel bacterial species isolated from floral nectar, Syst. Appl. Microbiol. (2014),
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G Model
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