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

Sergio A´ lvarez-Pe´rez,13 Bart Lievens,2,33 Hans Jacquemyn4 and Carlos M. Herrera1

Correspondence 1Estacio´n Biolo´gica de Don˜ana, Consejo Superior de Investigaciones Cient´ı ficas (CSIC), Avda. S. A´ lvarez-Pe´rez Ame´rico Vespucio, E-41092 Sevilla, Spain [email protected] 2Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), Lessius B. Lievens University College, De Nayer Campus, Consortium for Industrial Microbiology and Biotechnology [email protected] (CIMB), Department of Microbial and Molecular Systems (M2S), KU Leuven Association, B-2860 Sint-Katelijne-Waver, Belgium 3Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium 4Division of Plant Ecology and Systematics, Biology Department, KU Leuven, B-3001 Heverlee, Belgium

The taxonomic status of 14 strains of members of Acinetobacterthe genus 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 Acinetobacterthe genus was obtained from comparative analysis of the partial sequences of the 16S rRNA gene and the gene encoding theb-subunit of RNA polymeraserpoB (), DNA–DNA reassociation data, determination of the DNA +GC content and physiological tests. The Acinetobacternames nectarissp. nov. and boissierisp. nov. are proposed. The type strainA. nectarisof sp. nov. is SAP 763.2T (5LMG 26958T5CECT 8127T) and that A.of boissierisp. nov. is SAP 284.1T (5LMG 26959T5CECT 8128T).

Members of the genus Acinetobacter are generally regarded environmental sources, such as forest soils (Kim et al., as common, free-living saprophytes that show extensive 2008), seawater (Vaneechoutte et al., 2009) and wetlands metabolic versatility and potential to adapt to different (Anandham et ,al 2010).. Nevertheless, except for those human-associated and natural environments (Towner, species with clinical importance, the distribution and 2006; Doughari et ,al 201. 1; Sand et al., 2011). Apart from ecological role(s) of the ‘acinetobacters’ in most environ- the well-known human and animal-pathogenic species of ments are largely unknown (Carr et al., 2003; Towner, the genus Acinetobacte,r several novel species within this 2006). In particular, the possible associations of members genus have been described during recent years to of this bacterial group with plant hosts remain to be accommodate isolates from agricultural soils (Kang et ,al. addressed. 2011), activated sludge (Carr et ,al 20. 03), raw wastewater Floral nectar is the key component in the mutualism (Vaz-Moreira et al., 2011), a hexachlorocyclohexane between angiosperms and their animal pollinators, which dumpsite (Malhotra et ,al 2012). and diverse natural take this sugary solution as a reward for their pollination services (Brandenburg et al., 2009; Heil, 2011). While 3These authors contributed equally to this work. foraging on flowers, pollinators can contaminate floral Abbreviations: BI, Bayesian inference; ML, maximum-likelihood; NJ, nectar with different prokaryotic and eukaryotic micro- neighbour-joining; OTU, operational taxonomical unit; PM, Phenotype organisms, some of which are particularly well-adapted to MicroArray. thrive in this ephemeral habitat characterized by high The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene osmotic pressure and the presence of plant secondary sequences determined in this study are JQ771129–JQ771142 and metabolites with defensive functions (Herrera et ,al 2010;. those for the partial rpoB gene sequences are JQ771143–JQ771156. Pozo et ,al .2012). Nectar micro-organisms can alter A supplementary figure and four supplementary tables are available with pollinators’ foraging behaviour in different ways, for the online version of this paper. example by reducing the nutritional value of floral nectar and/or changing other physico-chemical conditions within test strips (MB 0266 A; Oxoid,). All strains were aerobic, the floral microenvironment, and thus potentially interfere -positive and oxidase-negative. Whereas no growth with plant sexual reproduction, as has been recently was observed on TSA in an anaerobic jar, all nectar strains suggested for nectar yeasts (Canto et ,al 2008;. Herrera were able to grow at decreased oxygen levels, as assessed by et al., 2008; de Vega et , 200al. 9; Herrera & Pozo, 2010; visual inspection of bacterial cultures grown on TSA in a Peay et al., 2012). candle jar for 5 days at 25 uC. Growth at 4, 25, 30, 37 and 41 uC, haemolysis of sheep blood, gelatin hydrolysis and Recently, species of the genus Acinetobacter have been production of acid from glucose and sucrose in Hugh– identified as representing the main bacterial genus Leifson medium were examined as described previously inhabiting the floral nectar of some cultivated plant species (Hugh & Leifson, 1953; Skerman, 1959; Bouvet & Grimont, from Northern Israel (Fridman et ,al 2012). and phylo- 1986; Carr et al., 2003). All nectar strains grew at 25 and genetically diverse wild Mediterranean plants from 30 uC, but not at 37 or 41 uC. Strains SAP219.2, SAP239.2, Southern Spain (A´ lvarez-Pe´rez & Herrera, 2013). In this SAP240.2, SAP241.2, SAP242.2, SAP284.1T and SAP320.1 latter study, nectar isolates of members of the genus were able to grow at 4 uC but strains SAP220.2, SAP249.1, Acinetobacter grouped into a single operational taxonom- SAP 305.1, SAP763.2T, SAP 956.2, SAP970.1 and SAP971.1 ical unit (OTU) defined on the basis of a 3 % dissimilarity could not. All strains were non-haemolytic on Columbia cut-off in the 16S rRNA gene sequence, but into two agar supplemented with sheep blood. Strains SAP 305.1 different OTUs when this threshold was lowered to 1 % and SAP 970.1 were found to grow poorly on this medium. (A´ lvarez-Pe´rez & Herrera, 2013). Such differentiation was None of the tested isolates were able to hydrolyse gelatin. also supported by some differences in colony morphology All nectar strains produced acid from sucrose and glucose. and growth rate in plate cultures, but no additional tests were conducted to further characterize those isolates. In Carbon source oxidation was determined by Phenotype this study we explore the taxonomic status of these two MicroArray (PM) technology (Biolog) using PM plate 1. nectar groups of acinetobacters associated with wild Using this technology, kinetic profiles are generated by Mediterranean plants. continuously monitoring the metabolic activity during incubation (Bochner et al., 2001). Plates were incubated in The 14 strains investigated in this study are listed in Table the OmniLog automated incubator-reader (Biolog) for S1, available in IJSEM Online. These strains were isolated 5 days at 25 uC and were read every 15 min. Interpretation on different dates from nectar samples of several plant of results was performed using OmniLog PM software species collected at different places within the surroundings according to the manufacturer’s instructions. Clear differ- of Don˜ana’s Natural Park (Huelva province, southwest ences were observed between the two groups of nectar Spain), using the procedure described by A´ lvarez-Pe´rez strains and between these nectar strains and the type strains et al. (2012). Additionally, for comparative taxonomic of A. bayly, iA. calcoaceticu, sA. gerneri and A. radioresisten. s analysis, Acinetobacter calcoaceticus DSM 30006T, In contrast to the type strains of the related species of the Acinetobacter baylyi DSM 14961T, Acinetobacter gerneri genus Acinetobacte,r all nectar strains were able to oxidize DSM 14967T and Acinetobacter radioresistens DSM 6976T sucrose and D-fructose as the only carbon source. D- were included in some phenotypic and genotypic assays. mannose, on the other hand, was only oxidized by the The inclusion of Acinetobacter calcoaceticus in those tests T group which included strain SAP763.2 . In addition, D- was justified by its status as the type species for the genus glucose was only oxidized by some strains of that group. In Acinetobacter, while the other three taxa were identified as addition to sucrose and D-fructose, L-malic acid was the the most closely related species to our nectar strains on the only carbon source that could be oxidized by the majority basis of 16S rRNA gene sequence data (see below). of the strains of the group containing strain SAP284.1T. For Colonies of all the nectar strains grown on trypticase soy the group including strain SAP763.2T, all strains were agar (TSA; Panreac) were circular, convex to umbilicate, found to oxidize L-aspartic acid, bromosuccinic acid, smooth and slightly opaque with entire margins. After fumaric acid, L-glutamic acid, L-malic acid, DL-malic acid, 5 days of incubation at 25 uC, colonies of these strains were succinic acid, L-asparagine and L-proline; and some of the variable in diameter, ranging from 0.5 to 2.5 mm, although seven strains in this group were able to oxidize D-xylose, D- smaller colonies were also observed for some strains. On gluconic acid, a-ketoglutaric acid, mono-methylsuccinate microscope images, cells appeared as non-motile coccoba- or L-alanine (Table S2). In order to compare the results cilli and commonly occurred in pairs, but also alone or in obtained by PM fingerprinting with more conventional short chains. No spores were observed. The main assimilation tests commonly used for classification of phenotypic properties of the type strains of all studied members of the genus Acinetobacte,r some key biochemical taxa are summarized in Table 1, and those of all studied features were also assessed using the phenotypic system nectar isolates are shown in Table S2. All tests were carried described by Bouvet & Grimont (1986) and adapted by out at 25 uC unless otherwise indicated. Catalase activity Nemec et al. (2009). More specifically, tests were was determined by evaluating bubble production with a performed for sucrose, D-glucose, succinic acid and 3 % (v/v) hydrogen peroxide solution (Cappuccino & phenylacetate. Briefly, assimilation tests were performed Sherman, 2002). Oxidase activity was tested using oxidase using the basal mineral medium of Cruze et al. (1979) Table 1.Differential phenotypic characteristics of the type Table 1.cont. strains of all studied species of the Acinetobactergenus Characteristic 1 2 3 4 5 6 Strains: 1, A. nectaris sp. nov. SAP 763.2T;2,A. boissieri sp. nov. SAP 284.1T;3,A. calcoaceticus DSM 30006T;4,A. baylyi DSM 14961T;5,A. Mucic acid 22 2+ 22 gerneri DSM 14967T;6,A. radioresistens DSM 6976T. A. nectaris sp. Propionic acid 22 2++2 ++ nov. and A. boissieri sp. nov. can be separated from the type strains of Pyruvic acid 22 2 2 + A. calcoaceticu,s A. bayly, iA. gerneri and A. radioresistens by some D-Saccharic acid 22 2 22 + +++ basic phenotypic characteristics, such as their ability to oxidize Succinic acid 2 2 + fructose and sucrose, their tolerance to sucrose concentrations above Tricarballylic acid 22 2 22 Methylpyruvate 22 +++2 30 % (w/v) and their inability to grow at ¢37 uC and assimilate Mono-methylsuccinate 22 2++2 acetate. +, Positive reaction; 2, negative reaction; W, weak growth; a-Hydroxyglutaric acid-g-Lactone 22 2 2+ 2 ND, no data available. 2-Aminoethanol 222222 Dulcitol 22 + 222 Characteristic 1 2 3 4 5 6 D-Alanine 22 +++2 Growth on TSA at: L-Alanine 22 2++2 4 uC 2 + WW2 + L-Asparagine + 22 + 22 25 uC ++ + + + + L-Glutamine + 22 ++2 30 uC ++ + + + + Gly–Pro 22 2 2+ 2 37 uC 22 ++++ Phenylethylamine 22 2 2+ 2 41 uC 22 2W ++ L-Proline + 22 ++2 Anaerobic growth 222222 L-Threonine 22 + 222 Growth at decreased oxygen levels ++ ND ND ND ND Tween 20 22 +++2 Haemolysis on Columbia blood agar 222222 Tween 40 22 +++2 Gelatin hydrolysis 222222 Tween 80 22 ++++ Catalase activity ++ + + + + Oxidase activity 222222 *Oxidation of carbon sources was determined by Phenotype Growth on LB broth plus sucrose at: MicroArray (PM) technology (Biolog) using PM plate 1. Further 10 % (w/v) ++ + + + + details on the procedure and the results obtained for all the novel 20 % (w/v) ++ + + + + nectar isolates characterized in this work are provided in Table S2. 30 % (w/v) ++ + + +2 40 % (w/v) ++ 2222 50 % (w/v) + W 2222 Acid production from glucose ++ + + +2 supplemented with 0.1 % (w/v) of the tested carbon source. ++ + + + Acid production from sucrose 2 The basal medium consisted of the following (l21): 10.0 g Oxidation of: * KH2PO4,5.0gNa2HPO4, 2.0 g (NH4)2SO4, 0.2 g D-Glucose 22 2+ 22 MgSO4 .7H2O, 0.001 g CaCl2 .2H2O and 0.001 g D-Fructose ++ 2222 FeSO4 .7H2O (pH 7.0). 5 ml of this medium was dispensed D-Mannose + 22222 into tubes, inoculated with washed bacterial cells and Sucrose ++ 2222 incubated at 25 uC under agitation. Growth on the different D-Xylose 222222 Acetic acid 22 ++++ carbon sources was evaluated after 2, 4, 6 and 10 days by D-Aspartic acid 22 2+ 22 means of visual comparison between inoculated tubes L-Aspartic acid + 22 + 22 containing carbon sources and control tubes containing Bromosuccinic acid + 22 ++2 only inoculated basal medium. In general, results obtained Citric acid 22 2++2 by these assays confirmed the results obtained by the PM Fumaric acid + 22 ++2 technology assays (data not shown). From the two groups of D-Galacturonic acid 22 + 222 nectar isolates, all isolates were again able to assimilate D-Gluconic acid + 22 + 22 sucrose, whereas D-glucose and succinic acid were only L-Glutamic acid + 22 ++2 assimilated by (some of) the isolates of the group which a-Hydroxybutyric acid 22 2++2 included SAP763.2T. In contrast to other known species of m-Hydroxyphenyl acetic acid 22 2 2+ 2 the genus Acinetobacte,r none of the nectar isolates were able + p-Hydroxyphenyl acetic acid 22 2 2 2 to assimilate acetate. a-Ketobutyric acid 22 2++2 a-Ketoglutaric acid 22 2++2 Sucrose tolerance was determined by culturing the studied L-Lactic acid 22 2++2 strains in transparent plastic vials containing Luria–Bertani D-Malic acid 222222 (LB) broth (Difco) supplemented with 0 (positive control), L-Malic acid ++ 2 ++2 10, 20, 30, 40 or 50 % sucrose (w/v, Sigma–Aldrich). All DL-Malic acid + 22 ++2 these liquid media were filter-sterilized and kept at 4 uC until use. The range of sugar concentrations tested closely matched the range of naturally occurring variation in floral The 16S rRNA gene sequences of the novel nectar strains nectars of the wild Mediterranean plant communities from and reference strains of members of the genus Acinetobacter which our nectar strains were recovered (S. A´ lvarez-Pe´rez and the family were included in a multiple & C. M. Herrera, unpublished results). Single colonies alignment generated by CLUSTAL W (Chenna et al., 2003). picked from 5-day cultures on TSA medium were used to The resulting alignment was trimmed with BioEdit v. inoculate the tubes and these were incubated at 25 uC for 7.0.9.0 (Hall, 1999) to ensure that all sequences had the up to 10 days. The turbidity of the cultures with respect to same start and end point, and analysed with Gblocks negative controls (i.e. tubes containing no inoculated (Castresana, 2000) to eliminate ambiguously aligned media) was recorded as a positive result. At the end of the regions, using ‘allow gap positions5with half’, ‘minimum experiment, an aliquot of each test tube was plated on length of a block559 and default settings for all other TSA medium to check for possible contaminations. options. Following these procedures, 1320 nt positions Furthermore, acid production by bacterial strains when (98 % of the original alignment) remained for subsequent growing at different sucrose concentrations was tested by phylogenetic analysis using the neighbour-joining (NJ) adding 40 ml methyl red (Panreac) to each tube. All nectar method as implemented in the MEGA 5 software package strains were able to grow at sucrose concentrations ranging (Tamura et ,al 2011).. Pairwise evolutionary distances were from 10 to 50 % (w/v), although the growth of strains of computed by the Jukes–Cantor method, and reliability of the group containing SAP284.1T at 40 % and 50 % sucrose nodes in the NJ phylogram was assessed by running 1000 was very weak. Acidification of the culture media was bootstrap replicates. In the NJ phylogram based on 16S observed in all tubes containing sucrose, but not in the rRNA gene sequences (Fig. 1) the novel nectar strains positive control (no sucrose). In contrast, A. baylyi DSM clustered with other members of the genus Acinetobacte,r 14961T, A. calcoaceticus DSM 30006T and A. gerneri DSM but stood apart from the recognised species of this genus 14967T only grew at sucrose concentrations up to 30 %, by forming a consistently differentiated group with 99 % and A. radioresistens DSM 6976T only tolerated 10 % and bootstrap support. Furthermore, all strains of the group 20 % sucrose. Furthermore, these four reference strains did which included strain SAP 763.2T clustered together with a not acidify either sucrose-containing culture broths or the 100 % bootstrap support, as did all those strains corres- positive control. ponding to the group which included strain SAP 284.1T (Fig. 1). Methods for genotypic characterization of the studied strains included comparative sequence analysis of the 16S Comparative sequence analysis of two variable regions – rRNA and the b-subunit of RNA polymerase (rpoB)- zone 1 (approximately 397 bp) and zone 2 (approximately encoding genes, assessment of overall genomic relatedness 544 bp) – of the rpoB gene was used to confirm both the by DNA–DNA hybridizations and determination of the within-species relatedness of the two groups of novel DNA G+C content. strains, and their separation from each other and from previously described species of the genus Acinetobacte.r An almost complete fragment of the 16S rRNA gene Primer sequences and PCR conditions were as previously was amplified and subsequently sequenced as described described (Khamis et ,al 200. 4; La Scola et al., 2006), with by A´ lvarez-Pe´rez et al. (2012). Preliminary sequence some minor modifications: 250 mM of each dNTP (Sigma– comparisons with the 16S rRNA gene sequences stored in Aldrich), 0.4 mM of each of the corresponding forward and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and the reverse primers (Sigma–Aldrich) and 561022 U Taq Ribosomal Database Project (RDP, http://rdp.cme.msu. polymerase ml21 (Bioline) were used in reaction mixtures edu/) databases showed that the nectar strains belonged to and 52 uC was the temperature for primer annealing in the family Moraxellaceae in the PCR cycles. As with the 16S rRNA gene sequence, subdivision, and the best hits for all sequences were the concatenated sequences of rpoB zones 1 and 2 of the putative isolates of members of the genus Acinetobacter nectar strains and type strains of other species of the genus recovered by Fridman et al. (2012) from floral nectar of Acinetobacter were included in a multiple alignment and cultivated plants (99 % and 97–98 % overall similarity to analysed with Gblocks, which resulted in the selection of strains SAP763.2T and SAP284.1T, respectively). The 843 nucleotide positions (99 % of the original alignment). SimTable tool available at the EzTaxon server v. 2.1 A phylogenetic tree was inferred by the NJ method using (http://www.eztaxon.org/, last accessed 10 December 2011; the Jukes–Cantor method, and again, the two groups of Chun et ,al 2007). was used to search for neighbours nectar strains formed two different clusters with bootstrap among species of the genus Acinetobacter with validly values supporting their distinctness from each other and published names on the basis of 16S rRNA gene sequences, from the other acinetobacters (Fig. S1). identifying A. baylyi B2T, A. gerneri 9A01T and A. radioresistens DSM 6976T as the species most closely The genomic DNA–DNA relatedness between the strains related to the novel nectar strains, but with a sequence SAP 763.2T and SAP 284.1T and between these and the type similarity value ¡96.3 % in all cases (Table S3). The strains of A. calcoaceticu, s A. baylyi and A. gerneri was sequence similarity value between strains SAP 763.2T and evaluated by DNA–DNA hybridizations. High-molecular- SAP 284.1T, as determined through the EzTaxon server, mass total genomic DNA was extracted by the method of was 97.7 %. Wilson (1987). DNA–DNA hybridizations were carried out Acinetobacter beijerinckii LUH 4759T (AJ626712)

Acinetobacter haemolyticus ATCC 17906T (Z93437)

Acinetobacter bouvetii CCM 7196T (HQ180181)

Acinetobacter johnsonii ATCC 17909T (Z93440)

Acinetobacter gyllenbergii RUH 442T (AJ293694)

Acinetobacter tjemberguae CCM 7200T (HQ180190)

Acinetobacter schindleri LUH 5832T (AJ278311)

Acinetobacter. Parvus LUH 4616T (NR 025425)

Acinetobacter tandoii CCM 7199T (HQ180189)

Acinetobacter Iwoffii DSM 2403T (NR 026209)

Acinetobacter brisouii 5YN5-8T (DQ832256)

Acinetobacter nosocomialis RUH 2376T (HQ180192)

99 Acinetobacter calcoaceticus DSM 30006T (AJ633632)

Acinetobacter pittii LMG 1035T (HQ180184)

Acinetobacter ursingii LUH 3792T (AJ275038)

Acinetobacter bereziniae ATCC 17924T (Z93443)

100 T Acinetobacter guillouiae DSM 590 (X81659)

Acinetobacter baumannii ATCC 19606T (Z93435)

Acinetobacter junii ATCC 17908T (Z93435)

Acinetobacter gemeri CCM 7197T (HQ180188)

Acinetobacter venetianus ATCC 31012T (AJ295007)

Acinetobacter towneri CCM 7201T (HQ180191)

Acinetobacter baylyi CCM 7195T (AM410709) 98 Acinetobacter soli B1T (EU290155)

Acinetobacter indicus A648T (HM047743) Acinetobacter radioresistens M 17694T (Z93445) Acinetobacter rudis G30T (EF204258)

SAP 305.1 (JQ771131)

100 SAP 956.2 (JQ771133)

0.005 SAP 970.1 (JQ771134) SAP 971.1 (JQ771135) Acinetobacter nectaris

100 SAP 220.2 (JQ771129)

SAP 763.2T (JQ771132)

SAP 249.1 (JQ771130)

SAP 240.2 (JQ771138) 99 SAP 239.2 (JQ771137)

SAP 284.1T (JQ771141)

SAP 242.2 (JQ771140) Acinetobacter boissieri 100 100 SAP 3 20.1 (JQ771142)

SAP 219.2 (JQ771136)

SAP 241.2 (JQ771139)

Alkanindiges illinoisensis MVAB Hex1T (AF513979)

Perlucidibaca piscinae lMCC1704T (DQ664237)

Moraxella lawnala ATCC 17967T (AF005160)

Psychrobacter immobilis ATCC 43116T (U39399)

Fig. 1.Neighbour-joining tree, based on 16S rRNA gene sequences, showing the relationships of nectarA. nectaris strains of sp. nov. andA. boissierisp. nov. with respect to other members of theAcinetobacter genus and representatives of closely related genera within the familyMoraxellaceae. 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 containing gaps and missing data were eliminated. Node support values (bootstrap percentages, based on 1000 simulations) ¢90 % are shown next to the branches. GenBank accession numbers are given in parentheses. The tree was rooted with the 16S rRNA gene sequencePsychrobacter of immobilisATCC 43116T. according to a modification of the microplate method relevant driving force in the ecology and evolution of described by Ezaki et al. (1989) (Goris et, 19al9.8; nectar-inhabiting microbiota. Cleenwerck et ,al 200. 2). The hybridization temperature was 38 uC and the assay was repeated at least seven times Description of Acinetobacter nectaris sp. nov. for each pair of strains to calculate the mean percentages Acinetobacter nectaris (nec.ta9ris. L. n. nectar -aris the drink of DNA–DNA relatedness. Reciprocal reactions were of the gods, and also the sugary solution secreted by some performed and the variation between them was within flowers to attract pollinators; L. gen. n. nectaris of nectar, acceptable limits (Goris et al., 1998). Strain SAP 763.1T isolated from floral nectar of plants). and strain SAP 284.1T showed very low DNA–DNA relatedness values to each other and with the type The description is based on the characteristics of seven strains of the other species of the genus Acinetobacter strains which were isolated from the floral nectar of plants tested (¡30 % of relative binding in all cases; Table S4), collected in the surroundings of Don˜ana’s Natural Park thus supporting the conclusion that the novel nectar (Huelva province, SW Spain). Cells are Gram-negative, strains group into two novel genomic lineages within their aerobic, oxidase-negative, catalase-positive, non-motile genus. coccobacilli, generally occurring in pairs. All strains tested can grow at decreased oxygen concentrations, but not Finally, the DNA G+C content was determined for strain under anaerobic conditions. Growth occurs at moderate SAP 763.2T and strain SAP 284.1T using the same DNA temperatures (e.g. 25 and 30 uC), but not at 4, 37 and extract as in the DNA–DNA hybridizations. The DNA was 41 uC. Colonies on TSA medium are round and smooth, enzymically degraded into nucleosides and the nucleotidic convex to umbilicate, slightly opaque, with entire margins base composition was determined by HPLC, according to and (,)0.5–2.5 mm in diameter after 5 days of incubation the method of Mesbah et al. (1989). Three independent at 25 uC. Some isolates grow poorly on Columbia agar analyses were conducted for each DNA sample, and mean supplemented with sheep blood, and all strains are non- DNA G+C content values were calculated. The DNA haemolytic on this medium. Gelatin is not hydrolysed. All G+C content of strain SAP 763.2T and strain SAP 284.1T strains produce acid from sucrose and glucose and oxidize was 36.6 and 37.8 mol%, respectively. These values are D-fructose, D-mannose and sucrose as the sole carbon slightly lower than those commonly found for strains of source. Some isolates can grow on D-glucose, though at other species of the genus Acinetobacter (Bouvet & slow growth rates. In addition, all strains oxidize L-aspartic Grimont, 1986; Nishimura et ,al 198. 8; Nemec et ,al. acid, bromosuccinic acid, fumaric acid, L-glutamic acid, L- 2003; Vaneechoutte et al., 2009; Kang et al., 2011). malic acid, DL-malic acid, succinic acid, L-asparagine and Taken together, the results of the present study support L-proline. The type strain oxidizes D-gluconic acid but not a clear genotypic and phenotypic differentiation of the D-xylose, a-ketoglutaric acid, mono-methylsuccinate or L- two groups of acinetobacters isolated from floral nectar alanine. Other isolates showed different reactions for these of wild Mediterranean plants, as well as their indepen- latter substrates. None of the strains assimilated acetate. dence from other currently recognized species of the genus Sucrose is tolerated at concentrations ranging from 10 % to Acinetobacter. The studied strains should therefore be 50 % (w/v). classified into two novel species, for which we propose the T T The type strain is SAP 763.2 (5LMG 26958 5CECT names Acinetobacter nectaris sp. nov. and Acinetobacter T 8127 ), isolated from floral nectar of the plant species boissieri sp. nov. Furthermore, the habitat from which these Muscari comosum (L.) Mill. (Hyacinthaceae) collected in species were recovered is also a novelty for the genus, as no the surroundings of Don˜ana’s Natural Park (Huelva other nectar-dwelling species of the genus Acinetobacter province, SW Spain). The DNA G+C content of the type have been described so far. Future research should be strain is 36.6 mol%. aimed at clarifying if A. nectaris sp. nov. and A. boissieri sp. nov. have any relevant role within plant–pollinator systems, as suggested in the case of nectar yeasts. In this Description of Acinetobacter boissieri sp. nov. respect, the carbon source oxidation profiles observed in Acinetobacter boissieri[bois.si9e.ri. N.L. masc. gen. n. boissieri this work for A. nectaris sp. nov. and A. boissieri sp. nov. of Boissier, named after the Swiss botanist Pierre-Edmond seem to be complementary to those of nectarivorous yeasts Boissier (1810–1885), in recognition of his great contribution that quickly deplete glucose in nectar and enrich this floral to the description of the flora of Southern Spain]. reward in fructose (Canto et al., 2008; Herrera et, 2008).al. If confirmed, such a scenario would suggest that interac- The description is based on the characteristics of seven tions between fungal and bacterial kingdoms might be a strains which were isolated from the floral nectar of plants s collected in the surroundings of Don˜ana’s Natural Park Canto, A., Herrera, C. M., Medrano, M., Pe´ rez, R. & Garcı´a, I. M. (Huelva province, SW Spain). Cells are Gram-negative, (2008). Pollinator foraging modifies nectar sugar composition in aerobic, oxidase-negative, catalase-positive, non-motile Helleborus foetidus (Ranunculacea):Ane experimental test. Am J Bot 95, 315–320. coccobacilli, generally occurring in pairs. All strains can Cappuccino, J. G. & Sherman, N. (2002). grow at decreased oxygen concentrations, but not under Microbiology: a Laboratory Manual, 6th edn. Menlo Park, CA: Pearson Education/Benjamin anaerobic conditions. Growth occurs between 4 uC and Cummings. u u higher temperatures such as 30 C, but not at 37 Cor Carr, E. L., Ka¨ mpfer, P., Patel, B. K., Gu¨ rtler, V. & Seviour, R. J. (2003). 41 uC. Colonies on TSA medium are round, convex to Seven novel species of Acinetobacter isolated from activated sludge. Int umbilicate, smooth, slightly opaque, with entire margins J Syst Evol Microbiol 53, 953–963. and (,)0.5–2.5 mm in diameter after 5 days of incubation Castresana, J. (2000). Selection of conserved blocks from multiple at 25 uC. Haemolysis on sheep blood agar is negative. alignments for their use in phylogenetic analysis. Mol Biol Evol 17, Gelatin is not hydrolysed. All strains produce acid from 540–552. sucrose and glucose and oxidize D-fructose and sucrose as Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, the sole carbon source. In addition, the type strain and the D. G. & Thompson, J. D. (2003). Multiple sequence alignment with majority of the other tested strains oxidize L-malic acid. the CLUSTAL series of programs. Nucleic Acids Res 31, 3497–3500. None of the strains assimilated acetate. Sucrose is tolerated Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y. W. at concentrations ranging from 10 % to 50 % (w/v) but, for (2007). EzTaxon: a web-based tool for the identification of most strains, growth at 40 % or higher values is slow. prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57, 2259–2261. T T The type strain is SAP 284.1 (5LMG 26959 5CECT Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. T 8128 ), isolated from floral nectar of the plant species (2002). Re-examination of the genus Acetobacte,r with descriptions of Fritillaria lusitanica Wikstr. (Liliaceae) collected in the Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J surroundings of Don˜ana’s Natural Park (Huelva province, Syst Evol Microbiol 52, 1551–1558. SW Spain). The DNA G+C content of the type strain is Cruze, J. A., Singer, J. T. & Finnerty, W. R. (1979). Conditions for 37.8 mol%. quantitative transformation in Acinetobacter calcoaceticus. Curr Microbiol 3, 129–132. de Vega, C., Herrera, C. M. & Johnson, S. D. (2009). Yeasts in floral Acknowledgements nectar of some South African plants: quantification and associations with pollinator type and sugar concentration. S Afr J Bot 75, 798–806. This work was supported by funds from Consejerı´a de Innovacio´n, Doughari, H. J., Ndakidemi, P. A., Human, I. S. & Benade, S. (2011). Ciencia y Empresa, Junta de Andalucı´a (Proyecto de Excelencia P09- The ecology, biology and pathogenesis of Acinetobacter spp.: an RNM-4517 to C. M. H.) and the European Research Council (ERC overview. Microbes Environ 26, 101–112. starting grant 260601 - MYCASOR to H. J.). We are grateful to Ezaki, T., Hashimoto, T. & Yabuuchi, E. (1989). Fluorometric Annelies Juste´, Marijke Lenaerts, Christel Verreth and Ado Van deoxyribonucleic acid-deoxyribonucleic acid hybridisation in micro- Assche for their help with the phenotypic assays. dilution wells as an alternative to membrane filter hybridisation in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229. References Fridman, S., Izhaki, I., Gerchman, Y. & Halpern, M. (2012). Bacterial A´ lvarez-Pe´ rez, S. & Herrera, C. M. (2013). Composition, richness and communities in floral nectar. Environ Microbiol Rep 4, 97–104. nonrandom assembly of culturable bacterial–microfungal communit- Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). ies in floral nectar of Mediterranean plants. FEMS Microbiol Ecol 83, Evaluation of a microplate DNA–DNA hybridization method 685–699. compared with the initial renaturation method. Can J Microbiol 44, A´ lvarez-Pe´ rez, S., Herrera, C. M. & de Vega, C. (2012). Zooming-in 1148–1153. on floral nectar: a first exploration of nectar-associated in Hall, T. A. (1999). BioEdit: a user-friendly biological sequence wild plant communities. FEMS Microbiol Ecol 80, 591–602. alignment editor and analysis program for Windows 95/98/NT. Anandham, R., Weon, H. Y., Kim, S. J., Kim, Y. S., Kim, B. Y. & Kwon, Nucleic Acids Symp Ser, 95–98.41 S. W. (2010). Acinetobacter brisouii sp. nov., isolated from a wetland in Heil, M. (2011). Nectar: generation, regulation and ecological Korea. J Microbiol , 36–39.48 functions. Trends Plant Sci, 191–200.16 Bochner, B. R., Gadzinski, P. & Panomitros, E. (2001). Phenotype Herrera, C. M. & Pozo, M. I. (2010). Nectar yeasts warm the flowers of microarrays for high-throughput phenotypic testing and assay of gene a winter-blooming plant. Proc Biol Sci , 1827–1834.277 function. Genome Res , 1246–1255.11 Herrera, C. M., Garcı´a, I. M. & Pe´ rez, R. (2008). Invisible floral Bouvet, P. J. M. & Grimont, P. A. D. (1986). of the genus larcenies: microbial communities degrade floral nectar of bumble - Acinetobacter with the recognition of Acinetobacter baumannii sp. pollinated plants. Ecology 89, 2369–2376. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. Herrera, C. M., Canto, A., Pozo, M. I. & Bazaga, P. (2010). nov., and Acinetobacter junii sp. nov. and emended descriptions of Inhospitable sweetness: nectar filtering of pollinator-borne inocula Acinetobacter calcoaceticus and Acinetobacter lwofi. Inti J Syst Bacteriol leads to impoverished, phylogenetically clustered yeast communities. 36, 228–240. Proc Biol Sci 277, 747–754. Brandenburg, A., Dell’Olivo, A., Bshary, R. & Kuhlemeier, C. (2009). Hugh, R. & Leifson, E. (1953). The taxonomic significance of The sweetest thing: advances in nectar research. Curr Opin Plant Biol fermentative versus oxidative metabolism of carbohydrates by various 12, 486–490. Gram-negative bacteria. J Bacteriol , 24–26.66

1 Kang, Y. S., Jung, J., Jeon, C. O. & Park, W. (2011). Acinetobacter Pozo, M. I., Lachance, M.-A. & Herrera, C. M. (2012). Nectar yeasts of oleivorans sp. nov. is capable of adhering to and growing on diesel-oil. two southern Spanish plants: the roles of immigration and J Microbiol ,4 29–34.9 physiological traits in community assembly. FEMS Microbiol Ecol Khamis, A., Raoult, D. & La Scola, B. (2004). rpoB gene sequencing for 80, 281–293. identification of Corynebacterium species. JClinMicrob42iol, 3925–3931. Sand, M., de Berardinis, V., Mingote, A., Santos, H., Go¨ ttig, S., Kim, D., Baik, K. S., Kim, M. S., Park, S. C., Kim, S. S., Rhee, M. S., Mu¨ ller, V. & Averhoff, B. (2011). Salt adaptation in Acinetobacter Kwak, Y. S. & Seong, C. N. (2008). Acinetobacter soli sp. nov., isolated baylyi: identification and characterization of a secondary glycine from forest soil. J Microbiol , 396–401.46 betaine transporter. Arch Microbiol 193, 723–730. La Scola, B., Gundi, V. A., Khamis, A. & Raoult, D. (2006). Sequencing Skerman, V. B. D. (1959). A Guide to the Identification of the Genera of of the rpoB gene and flanking spacers for molecular identification of Bacteria. Baltimore, MA: The Williams & Wilkins Co. Acinetobacter species. J Clin Microbiol 44, 827–832. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, Malhotra, J., Anand, S., Jindal, S., Rajagopal, R. & Lal, R. (2012). S. (2011). MEGA5: molecular evolutionary genetics analysis using Acinetobacter indicus sp. nov., isolated from a hexachlorocyclohexane maximum likelihood, evolutionary distance, and maximum par- dump site. Int J Syst Evol Microbiol 62, 2883–2890. simony methods. Mol Biol Evol 28, 2731–2739. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise Towner, K. (2006). The genus Acinetobacte.r InThe Prokaryotes measurement of the G+C content of deoxyribonucleic acid by high (, Gamma Subclas), s3rd edn, vol. 6, pp. 746–758. performance liquid chromathography. Int J Syst Bacteriol 39, 159–167. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & Nemec, A., Dijkshoorn, L., Cleenwerck, I., De Baere, T., Janssens, D., E. Stackebrandt. New York: Springer-Verlag. Van Der Reijden, T. J., Jezek, P. & Vaneechoutte, M. (2003). Vaneechoutte, M., Nemec, A., Musı´lek, M., van der Reijden, T. J., van Acinetobacter parvus sp. nov., a small-colony-forming species isolated den Barselaar, M., Tjernberg, I., Calame, W., Fani, R., De Baere, T. & from human clinical specimens. Int J Syst Evol Microb, 1i5ol63– 513567. Dijkshoorn, L. (2009). Description of Acinetobacter venetianus ex Di Nemec, A., Musı´lek, M., Maixnerova´ , M., De Baere, T., van der Cello et al. 1997 sp. nov. Int J Syst Evol Microbiol 59, 1376–1381. Reijden, T. J. K., Vaneechoutte, M. & Dijkshoorn, L. (2009). Vaz-Moreira, I., Novo, A., Hantsis-Zacharov, E., Lopes, A. R., Gomila, Acinetobacter beijerinckii sp. nov. and Acinetobacter gyllenbergii sp. M., Nunes, O. C., Manaia, C. M. & Halpern, M. (2011). Acinetobacter nov., haemolytic organisms isolated from humans. Int J Syst Evol rudis sp. nov., isolated from raw milk and raw wastewater. Int J Syst Microbiol 5,9 118–124. Evol Microbiol ,6 2837–2843.1 Nishimura, Y., Ino, T. & Iizuka, H. (1988). Acinetobacter radioresistens Wilson, K. (1987). Preparation of genomic DNA from bacteria. In sp. nov. isolated from cotton and soil. Int J Syst Bacteriol 38, 209–211. Current Protocols in Molecular Biolog, pp.y 241–245. Edited by F. Peay, K. G., Belisle, M. & Fukami, T. (2012). Phylogenetic relatedness M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. predicts priority effects in nectar yeast communities. Proc Biol Sci 279, A. Smith & K. Struhl. New York: Green Publishing and Wiley- 749–758. Interscience.