- Email: [email protected]
Description of Endozoicomonas ascidiicola sp. nov., isolated from Scandinavian ascidians
Accepted Manuscript Title: Description of Endozoicomonas ascidiicola sp. nov., isolated from Scandinavian ascidians Author: Lars Schreiber Kasper Urup Kjeldsen Matthias Obst Peter Funch Andreas Schramm PII: DOI: Reference:
S0723-2020(16)30040-6 http://dx.doi.org/doi:10.1016/j.syapm.2016.05.008 SYAPM 25777
To appear in: Received date: Revised date: Accepted date:
17-3-2016 20-5-2016 25-5-2016
Please cite this article as: L. Schreiber, K.U. Kjeldsen, M. Obst, P. Funch, A. Schramm, Description of Endozoicomonas ascidiicola sp. nov., isolated from Scandinavian ascidians, Systematic and Applied Microbiology (2016), http://dx.doi.org/10.1016/j.syapm.2016.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Confidential information:
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Revised manuscript for Systematic and Applied Microbiology, 19 May 2016
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Description of Endozoicomonas ascidiicola sp. nov., isolated from
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Scandinavian ascidians
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1Lars
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Schramm
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1Center
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University, Denmark
1Kasper
Urup Kjeldsen,
2Matthias
Obst,
3Peter
Funch, 1Andreas
cr
Schreiber,
ip t
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2Systematics
and Biodiversity, Department of Biological and Environmental Sciences,
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Göteborg University, Sweden
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3Section
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Denmark
an
10
us
for Geomicrobiology & Section for Microbiology, Department of Bioscience, Aarhus
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for Genetics, Ecology, and Evolution, Department of Bioscience, Aarhus University,
14 *Corresponding author:
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Lars Schreiber, Center for Geomicrobiology, Department of Bioscience, Aarhus
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University, Ny Munkegade 114, 8000 Aarhus C, Denmark;
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Phone: +45 871 56549
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Email: [email protected]
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Running title: Endozoicomonas ascidiicola sp. nov.
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Key words: marine, symbiosis, Ascidia, Endozoicomonas, tunicates, sea squirts
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The work was conducted at Aarhus University, 8000 Aarhus C, Denmark
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Abstract
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Two gram-negative, facultative anaerobic, chemoorganoheterotrophic, motile and rod-
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shaped bacteria, strains AVMART05T and KASP37, were isolated from ascidians
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(Tunicata, Ascidiaceae) of the genus Ascidiella collected at Gullmarsfjord, Sweden. The
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strains are the first cultured representatives of an ascidian-specific lineage within the
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genus Endozoicomonas (Gammaproteobacteria, Oceanospirillales, Hahellaceae). Both
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strains feature three distinct 16S rRNA gene paralogs, with identities of 98.9-99.1%
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(AVMART05T) and 97.7-98.8% (KASP37) between paralogs. The strains are closely
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related to Endozoicomonas atrinae and Endozoicomonas elysicola, with which they share
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97.3-98.0% 16S rRNA gene sequence identity. Digital DNA-DNA hybridization, average
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nucleotide identity, and tetra-nucleotide correlation analysis indicate that both strains
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belong to a single species distinct from their closest relatives. Both strains feature
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similar DNA G+C contents of 46.70 mol% (AVMART05T) and 44.64 mol% (KASP37). The
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fatty acid patterns of AVMART05T and KASP37 are most similar to those of
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Endozoicomonas euniceicola and Endozoicomonas gorgoniicola. Based on the polyphasic
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approach, we propose the species Endozoicomonas ascidiicola sp. nov. to accommodate
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the newly isolated strains. Endozoicomonas ascidiicola sp. nov. is represented by the
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type strain AVMART05T (=DSM 100913T= LMG 29095T) and strain KASP37 (=DSM
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100914= LMG 29096).
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Main text
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Bacteria affiliated with the gammaproteobacterial Endozoicomonas (Oceanospirillales;
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Hahellaceae) clade are almost exclusively associated with marine invertebrates; to date
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more than 99% of available Endozoicomonas 16S rRNA sequences originate from this
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host group (SILVA database release 123, Ref NR 99; [17]). At present, six
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Endozoicomonas species have been validly described: Endozoicomonas elysicola [11],
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isolated from a sea slug; E. numazuensis [14], isolated from a marine sponge; E.
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montiporae [35], E. euniceicola [16], and E. gorgoniicola [16], all isolated from corals;
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and E. atrinae [8], isolated from a mussel. In addition to the above hosts,
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Endozoicomonas have also been detected in sea squirts (Tunicata, Ascidiaceae) by
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cultivation-independent methods [22]. Ascidian-associated Endozoicomonas mainly
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affiliate with a specific subclade within the Endozoicomonas [22]. Here we describe the
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first two cultured representatives of this ascidian-specific subclade.
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Strains AVMART05T and KASP37 were isolated from ascidians of the species Ascidiella
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sp. and Ascidiella scabra, respectively, collected at Gullmarsfjord (Sweden; 58.2658 N,
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11.4175 E) as reported earlier [22]. In brief, pharynx tissues of ascidian specimens were
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dissected and washed with filter-sterilized (pore size: 0.2 µm) seawater. Tissue samples
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were added to 50 µL sterile seawater and homogenized in 1.5 mL tubes using sterile
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polypropylene pestles. The homogenate was diluted 1:14 with filter-sterilized seawater
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and 80 µL aliquots of this suspension were spread on full (AVMART05T) or half-strength
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(KASP37) Difco marine agar (MA; BD, Franklin Lakes, USA) plates. Plates were incubated
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at 22°C for 1 week in the dark. Colonies of AVMART05T and KASP37 were purified by
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repeated streaking.
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Gram reaction was determined as negative based on gram staining [24] and the KOH
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test [4]. Strains were identified by 16S rRNA gene sequencing: Single colonies were
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suspended in 100 µL PCR-grade H2O. Of this suspension, 1 µL was used as template for
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PCR with primers GM3 and GM4 [13]. HotStar Taq Master Mix (Qiagen; Venlo, The
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Netherlands) and the following conditions were used for PCR amplification: initial
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denaturing at 95°C for 5 min; 36 cycles at 95°C for 1 min, 42°C for 1 min, and 72°C for 3
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min; final elongation at 72°C for 10 min. PCR products were cloned using the pGEM®-T
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vector System (Promega; Fitchberg, USA), and eight clones of AVMART05T and three
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clones of KASP37 were subsequently sequenced from both directions by Macrogen Inc
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(Seoul, South Korea). Sequences were assembled using Sequencher version 5.0.1 (Gene
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Codes; Ann Arbor, USA). Sequences were trimmed using the online SINA aligner of
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SILVA [17], which removed all bases at either sequence end that could not be aligned.
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One representative, nearly full-length 16S rRNA gene sequence for each of the three
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paralogs per strain was deposited at Genbank with the accession numbers KT364255-
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KT364257 (AVMART05T) and KT364258-KT364260 (KASP37).
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Both strains feature at least three distinct 16S rRNA gene paralogs (1,502-1,521 bp
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long) with identities of 98.9-99.1% (AVMART05T) and 97.7-98.8% (KASP37) between
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paralogs. The differences between paralogs are most prominent in the 206-213
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(hypervariable region V2) and 456-476 (V3) regions and are also reflected in different
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gene lengths (Figure S1). Two divergent 16S rRNA gene paralogs (98.0% sequence
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identity between paralogs) are also present in E. montiporae (Figure S1). Based on the
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predominant paralog present in public databases (827/1,216 16S rRNA sequences of
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the Endozoicomonas clade represent this paralog; SILVA database release 123, NR 99
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[17]), the shared 16S rRNA gene sequence identity between AVMART05T and KASP37 is
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98.9%. Phylogenetic analysis was performed using the software packages MEGA 6.06
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[26], MrBayes 3.2.5 [20], and FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/)
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after a multiple sequence alignment by MAFFT-qinsi 7.221 [10]. The phylogeny was
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reconstructed by Bayesian inference (MrBayes) based on 3 million generations (trees
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sampled every 1,000 generations) after a burn-in of 25%. The closest relatives of
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AVMART05T and KASP37 are E. atrinae (97.5-98.0% sequence identity) and E. elysicola
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(97.3-97.9%)(Figure 1). Lower sequence similarities (<96%) were found with all other
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validly described species of the genus Endozoicomonas. Single colonies of AVMART05T, KASP37, and E. atrinae DSM-100061 were grown in
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Difco marine broth (MB; BD) at 25°C for 72-96 h. The DNA of the cultures was extracted
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using the DNA Extraction Midi kit (QIAGEN) with the Genomic Tip 100 system
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(QIAGEN). The genomes of AVMART05T, KASP37, and E. atrinae were sequenced using a
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MiSeq sequencer with 250bp reads in paired-end (average insert sizes: 554bp,
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AVMART05T; 562bp, KASP37; 333bp, E. atrinae) and mate-pair (8,567bp, AVMART05T;
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10,262bp, KASP37; no mate-pair library for E. atrinae) mode. Reads were clipped and
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trimmed using Trimmomatic 0.32 [3]. Trimmed reads of E. atrinae were assembled
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using SPADes 3.5 [2]. Trimmed paired-end reads of AVMART05T and KASP37 were
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filtered (discarding all reads <150bp) and down-sampled to 350Mbp using BBMap 34.94
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(http://sourceforge.net/projects/bbmap/). Down-sampled reads were split into
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overlapping 150bp reads using a custom Perl script and BBTools 34.94 to simulate
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Allpaths-LG-compatible reads [18]. Genome assemblies of AVMART05T and KASP37
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were performed using Allpaths-LG release 44870 [18]. The assembled genomes were
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submitted to Genbank WGS with the accession numbers: LUTV00000000 (AVMART05T),
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LUTW00000000 (KASP37), and LUKQ00000000 (E. atrinae). Genome completeness was
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estimated using CheckM 1.0.3 [15] with conserved genes of the order Oceanospirillales
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as reference: AVMART05T, 99.3% complete; KASP37, 98.9%; E. atrinae, 99.6%. The
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genomes of AVMART05T and KASP37 were compared to the genomes of E. atrinae (this
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study) and E. elysicola (GenBank assembly accession number: GCA_000710775.1) via
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average nucleotide identity analysis (ANI; [5]), tetra-nucleotide pattern analysis as
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implemented in JSpecies [19, 27] and digital DNA-DNA hybridization (DDH) as provided
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by the Genome-to-Genome Distance Calculator (GGDC) 2.1 hosted by the DSMZ
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(Braunschweig, Germany )[12]. All three analyses indicate that AVMART05T and
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KASP37 (i) are not clonal, (ii) belong to the same species, and (iii) form a separate
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species distinct from their closest relatives E. atrinae and E. elysicola (Table 1). G+C
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contents were determined using GGDC 2.1 [12], and were 46.70mol% (AVMART05T)
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and 46.64mol% (KASP37). These G+C contents are similar, but clearly distinct from
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those inferred from genomic information of other Endozoicomonas species (44.88-
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48.46mol%; Table 2).
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Colonies of strain AVMART05T are on average smaller (0.5-1 mm) than those of KASP37
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(2-3 mm) when grown on MA at 25°C for 72 h. Colony morphologies also clearly differ
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between both strains, with AVMART05T forming beige, circular and convex colonies
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with a smooth surface and entire margins; and KASP37 forming transparent-white,
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circular and flat colonies with a smooth surface and undulate margins. Cell
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morphologies and sizes were observed by phase contrast microscopy under an
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Observer.Z1 microscope (Carl Zeiss, Jena, Germany) after reaching the stationary
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growth phase; after growth at 25°C for 120h in MB. Lengths and widths of 100 cells
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were measured using the ZEN 2012 software (blue edition; Carl Zeiss) and averaged.
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Cells were fixed using paraformaldehyde, and subsequently stored in PBS/ethanol (1:1,
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v/v) prior to microscopy. Cells were identified as rod-shaped (Figure S2); cell sizes are
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found in the species description. Motility was tested by phase contrast microscopy
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throughout a period of 120 h during growth at 25°C in MB.
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Detailed results of chemotaxonomic analyses are given in the species description. The
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following analytical procedures were performed by the DSMZ Identification Service
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(DSMZ) as described earlier: respiratory quinones [29, 30], polar lipids [32], fatty acids
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[9], and peptidoglycan [23]. The quinone system supports the affiliation of AVMART05T
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and KASP37 to the Gammaproteobacteria, where the majority of species including all
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previously described Endozoicomonas have Q-9 as the major quinone [7, 8, 11, 14, 16,
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35]. In contrast to previously described Endozoicomonas species, no quinone Q-8 was
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detected in AVMART05T and KASP37 [8, 11, 14, 16, 35](Table 2). The polar lipid profiles
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of AVMART05T and KASP37 are consistent between the strains (Figure S3); they differ,
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however, clearly from profiles reported for E. atrinae and E. elysicola (Table 2). The
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major fatty acids of AVMART05T and KASP37 were C18 : 1ω7c (24.3%, AVMART05T;
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23.0%, KASP37), summed feature 3 (SF3) = C16:1 ω7c / C16:1 ω6c (49.6%; 52.9%), and
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C16 : 0 (15.7%; 16.0%). The obtained fatty acid patterns were compared to those reported
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for other Endozoicomonas species. Comparative analyses were limited to fatty acids with
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percentages of >1% in at least half of the samples (Table S1) and were performed using
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principal component analysis (PCA) and an un-constrained, unweighted pair group
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method with arithmetic mean (UPGMA); both implemented in the PAST software
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version 3.04 [6]. Robustness of the UPGMA analysis was tested using bootstrapping with
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1,000 re-samplings. Based on these analyses, the fatty acid patterns of AVMART05T and
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KASP37 are most similar to the patterns of E. euniceicola and E. gorgoniicola, and are
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clearly distinct from those of their closest phylogenetic relatives E. elysicola and E.
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atrinae (Figure 2); i.e. AVMART05T and KASP37 produce lower amounts of C18 : 1ω7c and
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C16 : 0, and higher amounts of SF3 than E. elysicola and E. atrinae (Table S1). The detected
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peptidoglycan type supports the affiliation of AVMART05T and KASP37 with the Gram-
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negative bacteria, which appear to predominantly feature the A1γ variation [21].
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Both strains grow well on full and half-strength MA and MB, as well as on porcine mucin
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agar [22]. Growth spectra were tested in MB with NaCl concentrations of 0-9% (0%,
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0.5% and 1–9% [w/v] in 1% increments), at temperatures from -6 to 40°C (3-4°C
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increments), and pH values adjusted to 4.2, 4.8, 5.3, 6.2, 6.9, 7.7, 8.3, 8.6, and 8.9.
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Cultures were grown at 25°C for 10 and 14 days for NaCl and pH value testing,
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respectively, and for 17 days for temperature testing. Results are given in the species
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description. Anaerobic growth was confirmed after incubation on MA at 25°C for 35
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days using the Oxoid AnaeroGen system (Fischer Scientific). Assimilation tests were
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performed with artificial seawater (salinity: 2%, pH 7; Instant Ocean; Blacksburg, USA)
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supplemented with 1 ml L-1 vitamin solution [34], 1 ml L-1 vitamin B12 solution [34], 2
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ml L-1 trace metal SL-10 solution [33], 0.2 g L-1 NH3Cl, and 10 mM of either D-glucose or
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DL-malate. Assimilation test setups were incubated at 21°C for 120 h in the dark. The
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following additional tests were performed by the DSMZ Identification Service (DSMZ)
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according to standard protocols: the ability to reduce nitrate (API20NE kit; bioMerieux,
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Marcy-l'Étoile, France), potential growth substrates (API20NE), spectrum of substrates
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that can be metabolized (BIOLOG GEN III assay; BIOLOG, Hayward, USA), and enzymatic
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activities (API20NE and API ZYM, bioMerieux). Incubation for BIOLOG assays were
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performed at 25°C for 22 h for AVMART05T and KASP37, and for 72 h for the reference
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species E. atrinae and E. elysicola. The ability to metabolize DNA as well as DNase,
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antibacterial, and hemolytic activities were tested previously [22]. Results are given in
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the species description and Tables S2-S4.
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Sensitivity to antibiotics was tested by the DSMZ Identification Service according to
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DIN58940 (Deutsches Institut für Normung e. V., Berlin, Germany) using a disc diffusion
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assay on MA. Discs were impregnated with antibiotics (see concentrations below). Zones
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of inhibition indicating antibiotic susceptibility were measured after 4 days of
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incubation at 25°C. Susceptibility was interpreted according to the Enterobacteriaceae
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breakpoint tables published by EUCAST [29][28] as either susceptible (S), moderately
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susceptible (MS), or resistant (R). In cases where no breakpoints were given a clearance
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zone of ≥50 mm was interpreted as S, <50 mm as MS, and no clearance zone as R. Strains
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AVMART05T and KASP37 were resistant to amakacin (30 μg) and bacitracin (10
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international units [IU]); moderately susceptible to clindamycin (10 μg), colistin (10 μg),
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doxycyclin (30 μg), fosfomycin (50 μg), kanamycin (30 μg), lincomycin (15 μg),
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neomycin (30 μg), penicillin G (6 μg [=10 IU]), pipemid acid (20 μg), and polymyxin B
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(300 IE); and susceptible to ampicillin (10 μg), aztreonam (30 μg), cefazolin (30 μg),
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cefotaxim (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), erythromycin (15 μg),
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imipenem (10 μg), linezolid (10 μg), moxifloxacin (5 μg), nitrofurantoin (100 μg),
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norfloxacin (10 μg), ofloxacin (5 μg), piperacillin/tazobactam (40 μg), tetracyclin (30
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μg), and ticarcillin (75 μg). Susceptibility to cefalotin (30 μg; AVMART05T: MS/KASP37:
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S), gentamycin (10 μg; MS/R), mezlocillin (30 μg; MS/S), nystatin (100 IU; S/R), oxacillin
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(5 μg; R/MS), quinupristin/dalfopristin (15 μg; MS/S), teicoplanin (30 μg; R/MS), and
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vancomycin (30 μg; R/MS) differed between strains AVMART05T and KASP37. An
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overview of the susceptibility patterns of both strains is given in Table S5.
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Based on the phenotypic and genotypic differences to their closest described relatives,
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strains AVMART05T and KASP37 unambiguously represent a novel species of the genus
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Endozoicomonas, for which we propose the name Endozoicomonas ascidiicola sp. nov.
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Description of Endozoicomonas ascidiicola sp. nov.
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Endozoicomonas ascidiicola sp. nov. ( as.ci.di.i'co.la. N.L. n. Ascidium, a genus of
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Ascidiidae; L. suff. -cola, an inhabitant, dweller; N.L. fem. n. ascidiicola, an Ascidian-
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dweller).
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Cells are Gram-negative and KOH test positive. Cells are motile rods, and are 0.3-0.7 μm
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in diameter and 1.2-11.3 μm long. Colony morphology on marine agar is variable, and
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can be beige, circular and convex with a smooth surface and entire margins
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(AVMART05T) or transparent-white, circular and flat with a smooth surface and
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undulate margins (KASP37). Colonies are 0.5-1 mm (AVMART05T) or 2-3 mm (KASP37)
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in diameter on marine agar after 72 h of incubation at 25°C. E. ascidiicola is mesophilic,
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neutrophilic, and slightly halophilic, with growth occurring at 5-27°C (AVMART05T) or
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7-31°C (KASP37), at pH 6.2-8.3 (AVMART05T) or 6.3-8.9 (KASP37) and in the presence
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of 0.5-5% NaCl. Optimal growth occurs at 23-25°C, pH 6-7, and in the presence of 1-2%
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NaCl. The peptidoglycan type is A1γ. Cells are facultative anaerobic and reduce nitrate
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(API20NE). Grows well on full and half-strength marine agar and marine broth. Grows
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well on porcine mucin agar. Demonstrated activities of the following enzymes:
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aminopeptidase, β-galactosidase (API20NE), DNase and oxidase. Weakly positive
230
activity for catalase. Additionally with API ZYM the following enzyme activities were
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detected: positive for alkaline phosphatase, leucine-arylamidase, trypsin, and N-acetyl-β
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–glucosaminidase; weakly positive for esterase, esterase lipase, valine-arylamidase, and
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naphtol-AS-BI-phosphohydrolase. Negative for lipase, cystine-arylamidase,
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chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-
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glucosidase, β-glucosidase, α-mannosidase, α-fucosidase (all API ZYM); as well as
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arginindihydrolase, urease, esculin hydrolysis, and gelatine hydrolysis (all API20NE). No
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acid is produced from glucose. The following carbon substrates are metabolized
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(BIOLOG GEN III): D-galactose, glycerol, inosine, L-alanine, L-aspartic acid, L-glutamic
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acid, L-malic acid, L-serine, N-acetyl neuraminic acid, N-acetyl-D-glucosamine, propionic
240
acid, and α-D-glucose; inconclusive results for metabolizing of acetoacetic acid, D-
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saccharic acid, L-arginine and α-keto-glutaric acid. The following substrates are not
242
metabolized: 3-methyl glucose, bromo-succinic acid, citric acid, D-arabitol, D-aspartic
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acid, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-fucose, D-galacturonic acid, D-
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gluconic acid, D-glucose-6-phosphate, D-glucuronic acid, D-lactic acid methyl ester, D-
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malic acid, D-maltose, D-mannitol, D-mannose, D-melibiose, D-raffinose, D-salicin, D-
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serine, D-sorbitol, D-trehalose, D-turanose, formic acid, gelatin, gentiobiose,
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glucuronamide, L-fucose, L-galactonic acid lactone, L-histidine, L-lactic acid, L-
248
rhamnose, methyl pyruvate, mucic acid, myo-inositol, N-acetyl-D-galactosamine, p-
249
hydroxy-phenylacetic acid, quinic acid, stachyose, sucrose, α-D-lactose, α-hydroxy-
250
butyric acid, β-hydroxy-D,L-butyric acid, β-methyl-D-glucoside. Metabolizing of acetic
251
acid, dextrin, and Tween 40 is variable. Growth was observed via the assimilation of D-
252
glucose, but not via the assimilation of DL-malate. Negative for the assimilation of
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adipate, arabinose, caprate, citrate, gluconate, maltose, mannitol, mannose, N-
254
acetylglucosamin, and phenylacetate (all API20NE). E. ascidiicola is non-hemolytic,
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exhibits no antibacterial activities against strains Escherichia coli K-12 JM109, Bacillus
256
cereus ATCC 10987, or Staphylococcus epidermidis DSM 20044, and does not grow on
257
nucleotides or DNA. The major cellular fatty acids are C18 : 1ω7c, C16 : 1ω7c, and C16 : 0. The
258
only detected respiratory quinone is Q-9. The polar lipids comprise
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phosphatidylglycerol (PG), phosphatidylethanolamin (PE), and one phosphoaminolipid
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(PN). Contains at least three distinct 16S rRNA gene paralogs with 0.9-2.3% sequence
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divergence between paralogs. Most prominent sites of sequence divergence are located
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in the V2 and V3 region of the 16S rRNA genes. The DNA G+C content is 46.64-46.70
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mol%. E. ascidiicola is represented by the type strain AVMART05T (=DSM 100913T=
264
LMG 29095T) and strain KASP37 (=DSM 100914= LMG 29096). Strain AVMART05T was
265
isolated from the pharynx tissue of an ascidian of the genus Ascidiella collected at the
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Gullmarsfjord (58.2658 N, 11.4175 E), Sweden, in November 2010. Strain KASP37, was
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isolated from the pharynx tissue of an ascidian of the species Ascidiella scabra collected
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at the Gullmarsfjord (58.2658 N, 11.4175 E), Sweden, in October 2011.
269
Acknowledgements
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We thank Anne Stentebjerg, Susanne Nielsen, Trine Bech Søgaard, Tove Wiegers, and
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Britta Poulsen for technical help during work in the laboratory. This project was funded
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by the Danish Council for Independent Research / Natural Sciences (DFF FNU; 09-
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064111), the Aarhus University Research Foundation (AU Ideas Program 2013; R9-
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A995-13-S833), the European Community (ASSEMBLE grant agreement no. 227799),
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and the Max Planck Society. We are grateful to B. Schink for advice on nomenclature.
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276 277 278 279
References
280 281 282 283
[2] A. Bankevich, S. Nurk, D. Antipov, A.A. Gurevich, M. Dvorkin, A.S. Kulikov, V.M. Lesin, S.I. Nikolenko, S. Pham, A.D. Prjibelski, A.V. Pyshkin, A.V. Sirotkin, N. Vyahhi, G. Tesler, M.A. Alekseyev, P.A. Pevzner, SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing, J. Comput. Biol., 19 (2012) 455-477.
284 285
[3] A.M. Bolger, M. Lohse, B. Usadel, Trimmomatic: a flexible trimmer for Illumina sequence data, Bioinformatics, 30 (2014) 2114-2120.
286 287
[4] G.M. Carlone, M.J. Valadez, M.J. Pickett, Methods for distinguishing gram-positive from gram-negative bacteria, J. Clin. Microbiol., 16 (1982) 1157-1159.
288 289 290
[5] J. Goris, K.T. Konstantinidis, J.A. Klappenbach, T. Coenye, P. Vandamme, J.M. Tiedje, DNA–DNA hybridization values and their relationship to whole-genome sequence similarities, Int. J. Syst. Evol. Microbiol., 57 (2007) 81-91.
291 292
[6] Ø. Hammer, D.A.T. Harper, P.D. Ryan, PAST: Paleontological statistics software package for education and data analysis., Palaeontologia Electronica, 4 (2001) 1-9.
293 294
[7] A. Hiraishi, Isoprenoid quinones as biomarkers of microbial populations in the environment, J. Biosci. Bioeng., 88 (1999) 449-460.
295 296 297
[8] D.-W. Hyun, N.-R. Shin, M.-S. Kim, S.J. Oh, P.S. Kim, T.W. Whon, J.-W. Bae, Endozoicomonas atrinae sp. nov., isolated from the intestine of a comb pen shell Atrina pectinata, Int. J. Syst. Evol. Microbiol., 64 (2014) 2312-2318.
298 299
[9] P. Kämpfer, R.M. Kroppenstedt, Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa, Can. J. Microbiol., 42 (1996) 989-1005.
300 301
[10] K. Katoh, K.-i. Kuma, H. Toh, T. Miyata, MAFFT version 5: improvement in accuracy of multiple sequence alignment, Nucleic Acids Res., 33 (2005) 511-518.
302 303 304
[11] M. Kurahashi, A. Yokota, Endozoicomonas elysicola gen. nov., sp. nov., a γproteobacterium isolated from the sea slug Elysia ornata, Syst. Appl. Microbiol., 30 (2007) 202-206.
305 306 307
[12] J.P. Meier-Kolthoff, A.F. Auch, H.-P. Klenk, M. Göker, Genome sequence-based species delimitation with confidence intervals and improved distance functions, BMC Bioinformatics, 14 (2013) 1-14.
308 309 310 311
[13] G. Muyzer, A. Teske, C. Wirsen, H. Jannasch, Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments, Arch. Microbiol., 164 (1995) 165-172.
312 313 314 315
[14] M. Nishijima, K. Adachi, A. Katsuta, Y. Shizuri, K. Yamasato, Endozoicomonas numazuensis sp. nov., a gammaproteobacterium isolated from marine sponges, and emended description of the genus Endozoicomonas Kurahashi and Yokota 2007, Int. J. Syst. Evol. Microbiol., 63 (2013) 709-714.
Ac ce
pt
ed
M
an
us
cr
ip t
[1] A.F. Auch, M. von Jan, H.-P. Klenk, M. Göker, Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison, Stand. Genomic Sci., 2 (2010) 117-134.
13
Page 13 of 22
[15] D.H. Parks, M. Imelfort, C.T. Skennerton, P. Hugenholtz, G.W. Tyson, CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes, Genome Res., (2015).
319 320 321 322
[16] R.E. Pike, B. Haltli, R.G. Kerr, Description of Endozoicomonas euniceicola sp. nov. and Endozoicomonas gorgoniicola sp. nov., bacteria isolated from the octocorals Eunicea fusca and Plexaura sp., and an emended description of the genus Endozoicomonas, Int. J. Syst. Evol. Microbiol., 63 (2013) 4294-4302.
323 324 325
[17] C. Quast, E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, F.O. Glöckner, The SILVA ribosomal RNA gene database project: improved data processing and web-based tools, Nucleic Acids Res., 41 (2013) D590-D596.
326 327 328 329
[18] F.J. Ribeiro, D. Przybylski, S. Yin, T. Sharpe, S. Gnerre, A. Abouelleil, A.M. Berlin, A. Montmayeur, T.P. Shea, B.J. Walker, S.K. Young, C. Russ, C. Nusbaum, I. MacCallum, D.B. Jaffe, Finished bacterial genomes from shotgun sequence data, Genome Res., 22 (2012) 2270-2277.
330 331
[19] M. Richter, R. Rosselló-Móra, Shifting the genomic gold standard for the prokaryotic species definition, Proc. Natl. Acad. Sci. USA, 106 (2009) 19126-19131.
332 333
[20] F. Ronquist, J.P. Huelsenbeck, MrBayes 3: Bayesian phylogenetic inference under mixed models, Bioinformatics, 19 (2003) 1572-1574.
334 335
[21] K.H. Schleifer, O. Kandler, Peptidoglycan types of bacterial cell walls and their taxonomic implications, Bacteriol. Rev., 36 (1972) 407-477.
336 337 338
[22] L. Schreiber, K.U. Kjeldsen, P. Funch, J. Jensen, M. Obst, S. Lopez-Legentil, A. Schramm, Endozoicomonas are specific, facultative symbionts of sea squirts, Front. Microbiol., Under review (2016).
339 340
[23] P. Schumann, Peptidoglycan Structure, Methods in Microbiology, 38 (2011) 101129.
341 342
[24] H.W. Seeley Jr, P.J. VanDemark, J.J. Lee, Microbes in action. A laboratory manual of microbiology (4th Edition), W. H. Freeman, New York, 1991.
343 344
[25] E. Stackebrandt, J. Ebers, Taxonomic parameters revisited: tarnished gold standards, Microbiol. Today, 33 (2006) 152.
345 346
[26] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0, Mol. Biol. Evol., 30 (2013) 2725-2729.
347 348 349
[27] H. Teeling, A. Meyerdierks, M. Bauer, R. Amann, F.O. Glöckner, Application of tetranucleotide frequencies for the assignment of genomic fragments, Environ. Microbiol., 6 (2004) 938-947.
350 351
[28] The European Committee on antimicrobial susceptibility testing, Clinical breakpoints - bacteria (v 5.0), in, 2015.
352 353
[29] B.J. Tindall, A comparative study of the lipid composition of Halobacterium saccharovorum from various sources, Syst. Appl. Microbiol., 13 (1990) 128-130.
354 355
[30] B.J. Tindall, Lipid composition of Halobacterium lacusprofundi, FEMS Microbiol. Lett., 66 (1990) 199-202.
Ac ce
pt
ed
M
an
us
cr
ip t
316 317 318
14
Page 14 of 22
[31] B.J. Tindall, R. Rosselló-Móra, H.-J. Busse, W. Ludwig, P. Kämpfer, Notes on the characterization of prokaryote strains for taxonomic purposes, Int. J. Syst. Evol. Microbiol., 60 (2010) 249-266.
359 360 361
[32] B.J. Tindall, J. Sikorski, R.A. Smibert, N.R. Krieg, Phenotypic characterization and the principles of comparative systematics, in: Methods for General and Molecular Microbiology, Third Edition, American Society of Microbiology, 2007.
362 363 364
[33] F. Widdel, F. Bak, Gram-negative mesophilic sulfate-reducing bacteria, in: A. Balows, H. Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (Eds.) The Prokaryotes, Springer New York, 1992, pp. 3352-3378.
365 366
[34] F. Widdel, G.-W. Kohring, F. Mayer, Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids, Arch. Microbiol., 134 (1983) 286-294.
367 368 369
[35] C.-S. Yang, M.-H. Chen, A.B. Arun, C.A. Chen, J.-T. Wang, W.-M. Chen, Endozoicomonas montiporae sp. nov., isolated from the encrusting pore coral Montipora aequituberculata, Int. J. Syst. Evol. Microbiol., 60 (2010) 1158-1162.
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356 357 358
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Figure captions
371
Figure 1. Phylogeny of the Hahellaceae based on the 16S rRNA gene and Bayesian
372
inference. Node support is expressed as posterior probabilities. Sequences of E.
373
ascidiicola are shown in bold. Accession numbers are shown in parentheses. For E.
374
montiporae (*) the sequence identifier of the genomic contig and the sequence
375
coordinates of the 16S rRNA gene are shown in parentheses. Sequences of the genus
376
Marinobacter were used as outgroup (not shown). Scale bar: 3% estimated sequence
377
divergence.
us
cr
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370
an
378
Figure 2. Multivariate analysis of fatty acid patterns. (A) UPGMA analysis of fatty
380
acid patterns of Endozoicomonas species. Numbers at nodes indicate bootstrap support.
381
The E. ascidiicola strains described in this study are shown in boldface. Fatty acids
382
patterns obtained in the present study are marked with an asterisk (*). (B) PCA of fatty
383
acid patterns of Endozoicomonas species. Fatty acid patterns are indicated by dots. Dots
384
from the same species are connected by lines and share the same color. Loadings of the
385
principal components are shown in brackets as part of the axis labels. Fatty acids
386
patterns obtained in the present study are marked with a black circle.
ed
pt
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387
M
379
16
Page 16 of 22
Tables Table 1 Summary of genomic taxonomy
AVMART05T
KASP37
ANIa
99.2%
-
Digital DDHb
91.8
-
Tetra correlationa
0.999
-
16S rRNA identityc
98.9%
-
ANIa
79.2%
Digital DDHb
22%d
an
403 404 405
Tetra correlationa
0.953
0.956
16S rRNA identityc
97.9%
97.5%
Reference & analysis
M
22%d
78.6%
pt
ANIa
406
78.9%
22.5%d
22.3%d
Tetra correlationa
0.958
0.958
16S rRNA identityc
98.0%
97.3%
Ac ce
Digital DDHb
cr
79.3%
ed
E. elysicola
us
E. atrinae
ip t
KASP37
407
Footnotes:
408
a
409
tetranucleotide regression value >0.999 [19]
410
b
411
formula applicable for incomplete genomes [1]. DNA-DNA hybridization threshold for
412
species delineation is 70% [31].
The current recommendation for genomic circumscription of species is: ANI >94–96%,
The here reported value was calculated with the digital DDH formula 2; the only
18
Page 17 of 22
413
c
The arbitrary, but recommended threshold for circumscription of a species is ≥97%
414
[31]. However systematic analysis of species demarcation based on DNA-DNA
415
hybridization values and 16S rRNA gene identity analyses suggests a higher threshold of
416
≥98.7-99% [25]
417 418 419
d Probability
Ac ce
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us
cr
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that actual DDH is >70% is 0
19
Page 18 of 22
ip t cr
E. ascidiicola
E. atrinae
E. elysicola
E. numazuensis
E. montiporae
AVMART05T
KASP37
DSM 100061
DSM 22380
DSM-25634
LMG 24815
Isolation source
Ascidiella sp.
Ascidiella scabra
Atrina pectinata
Elysia ornate
Marine sponge
(Ascidian)
(Ascidian)
(Pen shell)
(Sea slug)
Colony color
Beige
Transparentwhite
Beigea
Beigeb
Pale creamy white and opaquec
Colony morphology
Convex, circular, smooth surface with entire margins +
Flat, circular, smooth surface, with undulate margins
Convex, circular, with entire marginsa
Convex, circular, smooth, shinyb
+
-a
46.64
Motility
E. gorgoniicola NCCB 100438
E. euniceicola
Montipora aequituberculata
Plexaura sp.
Eunicea fusca
(Coral)
(Coral)
(Coral)
Beiged
Whitee
Creamy whitee
Low-convex, circular to irregular, with undulate marginsc
Convex, circular, with entire marginsd
Convex, circulare
Convex, circulare
+b
ND
+d
+e
+e
47.91
46.75
47.02
48.46
ND
ND
PG, PE, PN, AL, DPG, PL (2) (a) Q-9, Q-8
ND
ND
ND
ND
Q-9, Q-8
Q-9, Q-8
Q-9, Q-8
+d
-a,e
-a,e
d
M
an
E. ascidiicola
ep te
Strain
us
Table 2 Comparison of differential morphological, physiological, and molecular properties between E. ascidiicola and other Endozoicomonas species. Data was obtained in this study unless indicated otherwise. G+C content was calculated based on sequenced genomes. Abbreviations: FAN = facultative anaerobe, A = Aerobe, var = variable, + = positive, - = negative, ND = no data available, PG = Phosphatidylglycerol, PE = Phosphatidylethanolamin, PN = Unidentified phosphoaminolipid, AL = Unidentified aminolipid, DPG = Diphosphatidylglycerol, L = Unidentified lipid, PL = Unidentified phospholipid.
DSM-26535
G+C content (mol%)
46.70
Polar lipids
PG, PE, PN
PG, PE, PN
Respiratory quinones Oxidase
Q-9
Q-9
PG, PE, PN, AL, DPG, L, PL (3)(a) Q-9, Q-8
+
+
+
+
Q-9, Q-8, MK9, MK-8 +c
Gelatin hydrolysis
-
-
-
-
vara,c
-a,d
+a,e
-a,e
Esculin hydrolysis
-
-
+
+
-a,c
+a,d
-a,e
-a,e
Ac c
420 421 422 423
20
Page 19 of 22
ip t 15-37/30a
4-37/25-30b
pH range/optimum
6.2-8.3/6-7
6.3-8.9/6-8
6-9/7a
ND
NaCl% range/optimum
0.5-5/1
0.5-5/1-2
1-4/2a
>0/>0b
Relation to oxygen
FAN
FAN
Aa
cr
7-31/25
15-37/25c
15-35/25d
15-30/22-30e
15-30/22-30e
5.5-9/7.5-8c
6-10/8d
7-9/8e
7-8/8e
1-5/2c
1-3/2-3d
1-4/2-3e
1-4/2-3e
FANc
Ad
FANa
FANa
an
us
5-27/23
Ab
M
424 425
°C range/optimum
Footnotes: (a) data from [8]; (b) data from [11]; (c) data from [14]; (d) data from [35]; (e) data from [16]
d
426
Ac c
ep te
427
21
Page 20 of 22
ip t cr us an M d te
Hahella antarctica (EF495227)
1
Ac ce p
Hahella chejuensis (CP000155)
0.999
Hahella ganghwensis (AY676463)
0.997
0.977
Zooshikella ganghwensis (AY676463) Kistimonas asteriae (EU599216)
1
Kistimonas scarphacae (JF811908)
1
Candidatus Endonucleobacter bathymodioli (FM162188)
Endozoicomonas numazuensis (AB695088)
1
Endozoicomonas gorgoniicola (JX488685)
1
Endozoicomonas euniceicola (JX488684)
0.997 1
Endozoicomonas montiporae (JOKG01000007.7100.8645)* 0.965
Endozoicomonas elysicola (AB196667) 1
Endozoicomonas atrinae (KC878324) 1
Endozoicomonas ascidiicola AVMART05 T (KT364257) 1
Page 21 of 22
Endozoicomonas ascidiicola KASP37 (KT364259) 0.03
Component 2 (10%) 27
B 1.6
1.2
0.8
0.4
-2.0 -2.0
d
30
te
24 44
30
9 48
-1.6 46
12
15
18 46
21
E. numazuensis
-1.2
-0.8
-0.4
cr
64
69
48
us
3
an
M
6
Ac ce p
Distance
95 95
46 38
0.0
-0.8
E. ascidiicola KASP37
0.0
0.4
Component 1 (81%)
ip t
un ice ico E. la eu ni ce ico E. la go rg on iic E. ol go a rg on iic E. ol as a cid iic ol E. a as AV cid M iic AR ol E. T0 a ely K 5T sic AS * ol P3 a E. 7 * ely sic ol a E. ely sic ol a* E. ely sic ol a E. ely sic ol a E. m on tip or E. ae m on tip or E. ae m on tip or E. ae m on tip or E. ae nu m az ue E. ns nu is m st az .H u C5 en E. 0 sis nu m st az .H ue C4 E. ns 6 at is rin st ae .H C5 E. 0 at rin ae *
E. e
A
47
0.8
1.2
77
37 53
27
54
100
E. atrinae
E. elysicola
E. euniceicola
-0.4
E. montiporae
E. ascidiicola AVMART05T
E. gorgoniicola
-1.2
-1.6
1.6
Page 22 of 22
S0723-2020(16)30040-6 http://dx.doi.org/doi:10.1016/j.syapm.2016.05.008 SYAPM 25777
To appear in: Received date: Revised date: Accepted date:
17-3-2016 20-5-2016 25-5-2016
Please cite this article as: L. Schreiber, K.U. Kjeldsen, M. Obst, P. Funch, A. Schramm, Description of Endozoicomonas ascidiicola sp. nov., isolated from Scandinavian ascidians, Systematic and Applied Microbiology (2016), http://dx.doi.org/10.1016/j.syapm.2016.05.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Confidential information:
2
Revised manuscript for Systematic and Applied Microbiology, 19 May 2016
3
Description of Endozoicomonas ascidiicola sp. nov., isolated from
5
Scandinavian ascidians
6
1Lars
7
Schramm
8
1Center
9
University, Denmark
1Kasper
Urup Kjeldsen,
2Matthias
Obst,
3Peter
Funch, 1Andreas
cr
Schreiber,
ip t
4
2Systematics
and Biodiversity, Department of Biological and Environmental Sciences,
11
Göteborg University, Sweden
12
3Section
13
Denmark
an
10
us
for Geomicrobiology & Section for Microbiology, Department of Bioscience, Aarhus
M
for Genetics, Ecology, and Evolution, Department of Bioscience, Aarhus University,
14 *Corresponding author:
16
Lars Schreiber, Center for Geomicrobiology, Department of Bioscience, Aarhus
17
University, Ny Munkegade 114, 8000 Aarhus C, Denmark;
18
Phone: +45 871 56549
19
Email: [email protected]
pt
Ac ce
20
ed
15
21
Running title: Endozoicomonas ascidiicola sp. nov.
22
Key words: marine, symbiosis, Ascidia, Endozoicomonas, tunicates, sea squirts
23 24
The work was conducted at Aarhus University, 8000 Aarhus C, Denmark
25
1
Page 1 of 22
Abstract
27
Two gram-negative, facultative anaerobic, chemoorganoheterotrophic, motile and rod-
28
shaped bacteria, strains AVMART05T and KASP37, were isolated from ascidians
29
(Tunicata, Ascidiaceae) of the genus Ascidiella collected at Gullmarsfjord, Sweden. The
30
strains are the first cultured representatives of an ascidian-specific lineage within the
31
genus Endozoicomonas (Gammaproteobacteria, Oceanospirillales, Hahellaceae). Both
32
strains feature three distinct 16S rRNA gene paralogs, with identities of 98.9-99.1%
33
(AVMART05T) and 97.7-98.8% (KASP37) between paralogs. The strains are closely
34
related to Endozoicomonas atrinae and Endozoicomonas elysicola, with which they share
35
97.3-98.0% 16S rRNA gene sequence identity. Digital DNA-DNA hybridization, average
36
nucleotide identity, and tetra-nucleotide correlation analysis indicate that both strains
37
belong to a single species distinct from their closest relatives. Both strains feature
38
similar DNA G+C contents of 46.70 mol% (AVMART05T) and 44.64 mol% (KASP37). The
39
fatty acid patterns of AVMART05T and KASP37 are most similar to those of
40
Endozoicomonas euniceicola and Endozoicomonas gorgoniicola. Based on the polyphasic
41
approach, we propose the species Endozoicomonas ascidiicola sp. nov. to accommodate
42
the newly isolated strains. Endozoicomonas ascidiicola sp. nov. is represented by the
43
type strain AVMART05T (=DSM 100913T= LMG 29095T) and strain KASP37 (=DSM
44
100914= LMG 29096).
45
Main text
46
Bacteria affiliated with the gammaproteobacterial Endozoicomonas (Oceanospirillales;
47
Hahellaceae) clade are almost exclusively associated with marine invertebrates; to date
48
more than 99% of available Endozoicomonas 16S rRNA sequences originate from this
Ac ce
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2
Page 2 of 22
host group (SILVA database release 123, Ref NR 99; [17]). At present, six
50
Endozoicomonas species have been validly described: Endozoicomonas elysicola [11],
51
isolated from a sea slug; E. numazuensis [14], isolated from a marine sponge; E.
52
montiporae [35], E. euniceicola [16], and E. gorgoniicola [16], all isolated from corals;
53
and E. atrinae [8], isolated from a mussel. In addition to the above hosts,
54
Endozoicomonas have also been detected in sea squirts (Tunicata, Ascidiaceae) by
55
cultivation-independent methods [22]. Ascidian-associated Endozoicomonas mainly
56
affiliate with a specific subclade within the Endozoicomonas [22]. Here we describe the
57
first two cultured representatives of this ascidian-specific subclade.
58
Strains AVMART05T and KASP37 were isolated from ascidians of the species Ascidiella
59
sp. and Ascidiella scabra, respectively, collected at Gullmarsfjord (Sweden; 58.2658 N,
60
11.4175 E) as reported earlier [22]. In brief, pharynx tissues of ascidian specimens were
61
dissected and washed with filter-sterilized (pore size: 0.2 µm) seawater. Tissue samples
62
were added to 50 µL sterile seawater and homogenized in 1.5 mL tubes using sterile
63
polypropylene pestles. The homogenate was diluted 1:14 with filter-sterilized seawater
64
and 80 µL aliquots of this suspension were spread on full (AVMART05T) or half-strength
65
(KASP37) Difco marine agar (MA; BD, Franklin Lakes, USA) plates. Plates were incubated
66
at 22°C for 1 week in the dark. Colonies of AVMART05T and KASP37 were purified by
67
repeated streaking.
68
Gram reaction was determined as negative based on gram staining [24] and the KOH
69
test [4]. Strains were identified by 16S rRNA gene sequencing: Single colonies were
70
suspended in 100 µL PCR-grade H2O. Of this suspension, 1 µL was used as template for
71
PCR with primers GM3 and GM4 [13]. HotStar Taq Master Mix (Qiagen; Venlo, The
72
Netherlands) and the following conditions were used for PCR amplification: initial
Ac ce
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3
Page 3 of 22
denaturing at 95°C for 5 min; 36 cycles at 95°C for 1 min, 42°C for 1 min, and 72°C for 3
74
min; final elongation at 72°C for 10 min. PCR products were cloned using the pGEM®-T
75
vector System (Promega; Fitchberg, USA), and eight clones of AVMART05T and three
76
clones of KASP37 were subsequently sequenced from both directions by Macrogen Inc
77
(Seoul, South Korea). Sequences were assembled using Sequencher version 5.0.1 (Gene
78
Codes; Ann Arbor, USA). Sequences were trimmed using the online SINA aligner of
79
SILVA [17], which removed all bases at either sequence end that could not be aligned.
80
One representative, nearly full-length 16S rRNA gene sequence for each of the three
81
paralogs per strain was deposited at Genbank with the accession numbers KT364255-
82
KT364257 (AVMART05T) and KT364258-KT364260 (KASP37).
83
Both strains feature at least three distinct 16S rRNA gene paralogs (1,502-1,521 bp
84
long) with identities of 98.9-99.1% (AVMART05T) and 97.7-98.8% (KASP37) between
85
paralogs. The differences between paralogs are most prominent in the 206-213
86
(hypervariable region V2) and 456-476 (V3) regions and are also reflected in different
87
gene lengths (Figure S1). Two divergent 16S rRNA gene paralogs (98.0% sequence
88
identity between paralogs) are also present in E. montiporae (Figure S1). Based on the
89
predominant paralog present in public databases (827/1,216 16S rRNA sequences of
90
the Endozoicomonas clade represent this paralog; SILVA database release 123, NR 99
91
[17]), the shared 16S rRNA gene sequence identity between AVMART05T and KASP37 is
92
98.9%. Phylogenetic analysis was performed using the software packages MEGA 6.06
93
[26], MrBayes 3.2.5 [20], and FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/)
94
after a multiple sequence alignment by MAFFT-qinsi 7.221 [10]. The phylogeny was
95
reconstructed by Bayesian inference (MrBayes) based on 3 million generations (trees
96
sampled every 1,000 generations) after a burn-in of 25%. The closest relatives of
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AVMART05T and KASP37 are E. atrinae (97.5-98.0% sequence identity) and E. elysicola
98
(97.3-97.9%)(Figure 1). Lower sequence similarities (<96%) were found with all other
99
validly described species of the genus Endozoicomonas. Single colonies of AVMART05T, KASP37, and E. atrinae DSM-100061 were grown in
101
Difco marine broth (MB; BD) at 25°C for 72-96 h. The DNA of the cultures was extracted
102
using the DNA Extraction Midi kit (QIAGEN) with the Genomic Tip 100 system
103
(QIAGEN). The genomes of AVMART05T, KASP37, and E. atrinae were sequenced using a
104
MiSeq sequencer with 250bp reads in paired-end (average insert sizes: 554bp,
105
AVMART05T; 562bp, KASP37; 333bp, E. atrinae) and mate-pair (8,567bp, AVMART05T;
106
10,262bp, KASP37; no mate-pair library for E. atrinae) mode. Reads were clipped and
107
trimmed using Trimmomatic 0.32 [3]. Trimmed reads of E. atrinae were assembled
108
using SPADes 3.5 [2]. Trimmed paired-end reads of AVMART05T and KASP37 were
109
filtered (discarding all reads <150bp) and down-sampled to 350Mbp using BBMap 34.94
110
(http://sourceforge.net/projects/bbmap/). Down-sampled reads were split into
111
overlapping 150bp reads using a custom Perl script and BBTools 34.94 to simulate
112
Allpaths-LG-compatible reads [18]. Genome assemblies of AVMART05T and KASP37
113
were performed using Allpaths-LG release 44870 [18]. The assembled genomes were
114
submitted to Genbank WGS with the accession numbers: LUTV00000000 (AVMART05T),
115
LUTW00000000 (KASP37), and LUKQ00000000 (E. atrinae). Genome completeness was
116
estimated using CheckM 1.0.3 [15] with conserved genes of the order Oceanospirillales
117
as reference: AVMART05T, 99.3% complete; KASP37, 98.9%; E. atrinae, 99.6%. The
118
genomes of AVMART05T and KASP37 were compared to the genomes of E. atrinae (this
119
study) and E. elysicola (GenBank assembly accession number: GCA_000710775.1) via
120
average nucleotide identity analysis (ANI; [5]), tetra-nucleotide pattern analysis as
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implemented in JSpecies [19, 27] and digital DNA-DNA hybridization (DDH) as provided
122
by the Genome-to-Genome Distance Calculator (GGDC) 2.1 hosted by the DSMZ
123
(Braunschweig, Germany )[12]. All three analyses indicate that AVMART05T and
124
KASP37 (i) are not clonal, (ii) belong to the same species, and (iii) form a separate
125
species distinct from their closest relatives E. atrinae and E. elysicola (Table 1). G+C
126
contents were determined using GGDC 2.1 [12], and were 46.70mol% (AVMART05T)
127
and 46.64mol% (KASP37). These G+C contents are similar, but clearly distinct from
128
those inferred from genomic information of other Endozoicomonas species (44.88-
129
48.46mol%; Table 2).
130
Colonies of strain AVMART05T are on average smaller (0.5-1 mm) than those of KASP37
131
(2-3 mm) when grown on MA at 25°C for 72 h. Colony morphologies also clearly differ
132
between both strains, with AVMART05T forming beige, circular and convex colonies
133
with a smooth surface and entire margins; and KASP37 forming transparent-white,
134
circular and flat colonies with a smooth surface and undulate margins. Cell
135
morphologies and sizes were observed by phase contrast microscopy under an
136
Observer.Z1 microscope (Carl Zeiss, Jena, Germany) after reaching the stationary
137
growth phase; after growth at 25°C for 120h in MB. Lengths and widths of 100 cells
138
were measured using the ZEN 2012 software (blue edition; Carl Zeiss) and averaged.
139
Cells were fixed using paraformaldehyde, and subsequently stored in PBS/ethanol (1:1,
140
v/v) prior to microscopy. Cells were identified as rod-shaped (Figure S2); cell sizes are
141
found in the species description. Motility was tested by phase contrast microscopy
142
throughout a period of 120 h during growth at 25°C in MB.
143
Detailed results of chemotaxonomic analyses are given in the species description. The
144
following analytical procedures were performed by the DSMZ Identification Service
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(DSMZ) as described earlier: respiratory quinones [29, 30], polar lipids [32], fatty acids
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[9], and peptidoglycan [23]. The quinone system supports the affiliation of AVMART05T
147
and KASP37 to the Gammaproteobacteria, where the majority of species including all
148
previously described Endozoicomonas have Q-9 as the major quinone [7, 8, 11, 14, 16,
149
35]. In contrast to previously described Endozoicomonas species, no quinone Q-8 was
150
detected in AVMART05T and KASP37 [8, 11, 14, 16, 35](Table 2). The polar lipid profiles
151
of AVMART05T and KASP37 are consistent between the strains (Figure S3); they differ,
152
however, clearly from profiles reported for E. atrinae and E. elysicola (Table 2). The
153
major fatty acids of AVMART05T and KASP37 were C18 : 1ω7c (24.3%, AVMART05T;
154
23.0%, KASP37), summed feature 3 (SF3) = C16:1 ω7c / C16:1 ω6c (49.6%; 52.9%), and
155
C16 : 0 (15.7%; 16.0%). The obtained fatty acid patterns were compared to those reported
156
for other Endozoicomonas species. Comparative analyses were limited to fatty acids with
157
percentages of >1% in at least half of the samples (Table S1) and were performed using
158
principal component analysis (PCA) and an un-constrained, unweighted pair group
159
method with arithmetic mean (UPGMA); both implemented in the PAST software
160
version 3.04 [6]. Robustness of the UPGMA analysis was tested using bootstrapping with
161
1,000 re-samplings. Based on these analyses, the fatty acid patterns of AVMART05T and
162
KASP37 are most similar to the patterns of E. euniceicola and E. gorgoniicola, and are
163
clearly distinct from those of their closest phylogenetic relatives E. elysicola and E.
164
atrinae (Figure 2); i.e. AVMART05T and KASP37 produce lower amounts of C18 : 1ω7c and
165
C16 : 0, and higher amounts of SF3 than E. elysicola and E. atrinae (Table S1). The detected
166
peptidoglycan type supports the affiliation of AVMART05T and KASP37 with the Gram-
167
negative bacteria, which appear to predominantly feature the A1γ variation [21].
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Both strains grow well on full and half-strength MA and MB, as well as on porcine mucin
169
agar [22]. Growth spectra were tested in MB with NaCl concentrations of 0-9% (0%,
170
0.5% and 1–9% [w/v] in 1% increments), at temperatures from -6 to 40°C (3-4°C
171
increments), and pH values adjusted to 4.2, 4.8, 5.3, 6.2, 6.9, 7.7, 8.3, 8.6, and 8.9.
172
Cultures were grown at 25°C for 10 and 14 days for NaCl and pH value testing,
173
respectively, and for 17 days for temperature testing. Results are given in the species
174
description. Anaerobic growth was confirmed after incubation on MA at 25°C for 35
175
days using the Oxoid AnaeroGen system (Fischer Scientific). Assimilation tests were
176
performed with artificial seawater (salinity: 2%, pH 7; Instant Ocean; Blacksburg, USA)
177
supplemented with 1 ml L-1 vitamin solution [34], 1 ml L-1 vitamin B12 solution [34], 2
178
ml L-1 trace metal SL-10 solution [33], 0.2 g L-1 NH3Cl, and 10 mM of either D-glucose or
179
DL-malate. Assimilation test setups were incubated at 21°C for 120 h in the dark. The
180
following additional tests were performed by the DSMZ Identification Service (DSMZ)
181
according to standard protocols: the ability to reduce nitrate (API20NE kit; bioMerieux,
182
Marcy-l'Étoile, France), potential growth substrates (API20NE), spectrum of substrates
183
that can be metabolized (BIOLOG GEN III assay; BIOLOG, Hayward, USA), and enzymatic
184
activities (API20NE and API ZYM, bioMerieux). Incubation for BIOLOG assays were
185
performed at 25°C for 22 h for AVMART05T and KASP37, and for 72 h for the reference
186
species E. atrinae and E. elysicola. The ability to metabolize DNA as well as DNase,
187
antibacterial, and hemolytic activities were tested previously [22]. Results are given in
188
the species description and Tables S2-S4.
189
Sensitivity to antibiotics was tested by the DSMZ Identification Service according to
190
DIN58940 (Deutsches Institut für Normung e. V., Berlin, Germany) using a disc diffusion
191
assay on MA. Discs were impregnated with antibiotics (see concentrations below). Zones
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of inhibition indicating antibiotic susceptibility were measured after 4 days of
193
incubation at 25°C. Susceptibility was interpreted according to the Enterobacteriaceae
194
breakpoint tables published by EUCAST [29][28] as either susceptible (S), moderately
195
susceptible (MS), or resistant (R). In cases where no breakpoints were given a clearance
196
zone of ≥50 mm was interpreted as S, <50 mm as MS, and no clearance zone as R. Strains
197
AVMART05T and KASP37 were resistant to amakacin (30 μg) and bacitracin (10
198
international units [IU]); moderately susceptible to clindamycin (10 μg), colistin (10 μg),
199
doxycyclin (30 μg), fosfomycin (50 μg), kanamycin (30 μg), lincomycin (15 μg),
200
neomycin (30 μg), penicillin G (6 μg [=10 IU]), pipemid acid (20 μg), and polymyxin B
201
(300 IE); and susceptible to ampicillin (10 μg), aztreonam (30 μg), cefazolin (30 μg),
202
cefotaxim (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), erythromycin (15 μg),
203
imipenem (10 μg), linezolid (10 μg), moxifloxacin (5 μg), nitrofurantoin (100 μg),
204
norfloxacin (10 μg), ofloxacin (5 μg), piperacillin/tazobactam (40 μg), tetracyclin (30
205
μg), and ticarcillin (75 μg). Susceptibility to cefalotin (30 μg; AVMART05T: MS/KASP37:
206
S), gentamycin (10 μg; MS/R), mezlocillin (30 μg; MS/S), nystatin (100 IU; S/R), oxacillin
207
(5 μg; R/MS), quinupristin/dalfopristin (15 μg; MS/S), teicoplanin (30 μg; R/MS), and
208
vancomycin (30 μg; R/MS) differed between strains AVMART05T and KASP37. An
209
overview of the susceptibility patterns of both strains is given in Table S5.
210
Based on the phenotypic and genotypic differences to their closest described relatives,
211
strains AVMART05T and KASP37 unambiguously represent a novel species of the genus
212
Endozoicomonas, for which we propose the name Endozoicomonas ascidiicola sp. nov.
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Description of Endozoicomonas ascidiicola sp. nov.
214
Endozoicomonas ascidiicola sp. nov. ( as.ci.di.i'co.la. N.L. n. Ascidium, a genus of
215
Ascidiidae; L. suff. -cola, an inhabitant, dweller; N.L. fem. n. ascidiicola, an Ascidian-
216
dweller).
217
Cells are Gram-negative and KOH test positive. Cells are motile rods, and are 0.3-0.7 μm
218
in diameter and 1.2-11.3 μm long. Colony morphology on marine agar is variable, and
219
can be beige, circular and convex with a smooth surface and entire margins
220
(AVMART05T) or transparent-white, circular and flat with a smooth surface and
221
undulate margins (KASP37). Colonies are 0.5-1 mm (AVMART05T) or 2-3 mm (KASP37)
222
in diameter on marine agar after 72 h of incubation at 25°C. E. ascidiicola is mesophilic,
223
neutrophilic, and slightly halophilic, with growth occurring at 5-27°C (AVMART05T) or
224
7-31°C (KASP37), at pH 6.2-8.3 (AVMART05T) or 6.3-8.9 (KASP37) and in the presence
225
of 0.5-5% NaCl. Optimal growth occurs at 23-25°C, pH 6-7, and in the presence of 1-2%
226
NaCl. The peptidoglycan type is A1γ. Cells are facultative anaerobic and reduce nitrate
227
(API20NE). Grows well on full and half-strength marine agar and marine broth. Grows
228
well on porcine mucin agar. Demonstrated activities of the following enzymes:
229
aminopeptidase, β-galactosidase (API20NE), DNase and oxidase. Weakly positive
230
activity for catalase. Additionally with API ZYM the following enzyme activities were
231
detected: positive for alkaline phosphatase, leucine-arylamidase, trypsin, and N-acetyl-β
232
–glucosaminidase; weakly positive for esterase, esterase lipase, valine-arylamidase, and
233
naphtol-AS-BI-phosphohydrolase. Negative for lipase, cystine-arylamidase,
234
chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, β-glucuronidase, α-
235
glucosidase, β-glucosidase, α-mannosidase, α-fucosidase (all API ZYM); as well as
236
arginindihydrolase, urease, esculin hydrolysis, and gelatine hydrolysis (all API20NE). No
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acid is produced from glucose. The following carbon substrates are metabolized
238
(BIOLOG GEN III): D-galactose, glycerol, inosine, L-alanine, L-aspartic acid, L-glutamic
239
acid, L-malic acid, L-serine, N-acetyl neuraminic acid, N-acetyl-D-glucosamine, propionic
240
acid, and α-D-glucose; inconclusive results for metabolizing of acetoacetic acid, D-
241
saccharic acid, L-arginine and α-keto-glutaric acid. The following substrates are not
242
metabolized: 3-methyl glucose, bromo-succinic acid, citric acid, D-arabitol, D-aspartic
243
acid, D-cellobiose, D-fructose, D-fructose-6-phosphate, D-fucose, D-galacturonic acid, D-
244
gluconic acid, D-glucose-6-phosphate, D-glucuronic acid, D-lactic acid methyl ester, D-
245
malic acid, D-maltose, D-mannitol, D-mannose, D-melibiose, D-raffinose, D-salicin, D-
246
serine, D-sorbitol, D-trehalose, D-turanose, formic acid, gelatin, gentiobiose,
247
glucuronamide, L-fucose, L-galactonic acid lactone, L-histidine, L-lactic acid, L-
248
rhamnose, methyl pyruvate, mucic acid, myo-inositol, N-acetyl-D-galactosamine, p-
249
hydroxy-phenylacetic acid, quinic acid, stachyose, sucrose, α-D-lactose, α-hydroxy-
250
butyric acid, β-hydroxy-D,L-butyric acid, β-methyl-D-glucoside. Metabolizing of acetic
251
acid, dextrin, and Tween 40 is variable. Growth was observed via the assimilation of D-
252
glucose, but not via the assimilation of DL-malate. Negative for the assimilation of
253
adipate, arabinose, caprate, citrate, gluconate, maltose, mannitol, mannose, N-
254
acetylglucosamin, and phenylacetate (all API20NE). E. ascidiicola is non-hemolytic,
255
exhibits no antibacterial activities against strains Escherichia coli K-12 JM109, Bacillus
256
cereus ATCC 10987, or Staphylococcus epidermidis DSM 20044, and does not grow on
257
nucleotides or DNA. The major cellular fatty acids are C18 : 1ω7c, C16 : 1ω7c, and C16 : 0. The
258
only detected respiratory quinone is Q-9. The polar lipids comprise
259
phosphatidylglycerol (PG), phosphatidylethanolamin (PE), and one phosphoaminolipid
260
(PN). Contains at least three distinct 16S rRNA gene paralogs with 0.9-2.3% sequence
261
divergence between paralogs. Most prominent sites of sequence divergence are located
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in the V2 and V3 region of the 16S rRNA genes. The DNA G+C content is 46.64-46.70
263
mol%. E. ascidiicola is represented by the type strain AVMART05T (=DSM 100913T=
264
LMG 29095T) and strain KASP37 (=DSM 100914= LMG 29096). Strain AVMART05T was
265
isolated from the pharynx tissue of an ascidian of the genus Ascidiella collected at the
266
Gullmarsfjord (58.2658 N, 11.4175 E), Sweden, in November 2010. Strain KASP37, was
267
isolated from the pharynx tissue of an ascidian of the species Ascidiella scabra collected
268
at the Gullmarsfjord (58.2658 N, 11.4175 E), Sweden, in October 2011.
269
Acknowledgements
270
We thank Anne Stentebjerg, Susanne Nielsen, Trine Bech Søgaard, Tove Wiegers, and
271
Britta Poulsen for technical help during work in the laboratory. This project was funded
272
by the Danish Council for Independent Research / Natural Sciences (DFF FNU; 09-
273
064111), the Aarhus University Research Foundation (AU Ideas Program 2013; R9-
274
A995-13-S833), the European Community (ASSEMBLE grant agreement no. 227799),
275
and the Max Planck Society. We are grateful to B. Schink for advice on nomenclature.
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276 277 278 279
References
280 281 282 283
[2] A. Bankevich, S. Nurk, D. Antipov, A.A. Gurevich, M. Dvorkin, A.S. Kulikov, V.M. Lesin, S.I. Nikolenko, S. Pham, A.D. Prjibelski, A.V. Pyshkin, A.V. Sirotkin, N. Vyahhi, G. Tesler, M.A. Alekseyev, P.A. Pevzner, SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing, J. Comput. Biol., 19 (2012) 455-477.
284 285
[3] A.M. Bolger, M. Lohse, B. Usadel, Trimmomatic: a flexible trimmer for Illumina sequence data, Bioinformatics, 30 (2014) 2114-2120.
286 287
[4] G.M. Carlone, M.J. Valadez, M.J. Pickett, Methods for distinguishing gram-positive from gram-negative bacteria, J. Clin. Microbiol., 16 (1982) 1157-1159.
288 289 290
[5] J. Goris, K.T. Konstantinidis, J.A. Klappenbach, T. Coenye, P. Vandamme, J.M. Tiedje, DNA–DNA hybridization values and their relationship to whole-genome sequence similarities, Int. J. Syst. Evol. Microbiol., 57 (2007) 81-91.
291 292
[6] Ø. Hammer, D.A.T. Harper, P.D. Ryan, PAST: Paleontological statistics software package for education and data analysis., Palaeontologia Electronica, 4 (2001) 1-9.
293 294
[7] A. Hiraishi, Isoprenoid quinones as biomarkers of microbial populations in the environment, J. Biosci. Bioeng., 88 (1999) 449-460.
295 296 297
[8] D.-W. Hyun, N.-R. Shin, M.-S. Kim, S.J. Oh, P.S. Kim, T.W. Whon, J.-W. Bae, Endozoicomonas atrinae sp. nov., isolated from the intestine of a comb pen shell Atrina pectinata, Int. J. Syst. Evol. Microbiol., 64 (2014) 2312-2318.
298 299
[9] P. Kämpfer, R.M. Kroppenstedt, Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa, Can. J. Microbiol., 42 (1996) 989-1005.
300 301
[10] K. Katoh, K.-i. Kuma, H. Toh, T. Miyata, MAFFT version 5: improvement in accuracy of multiple sequence alignment, Nucleic Acids Res., 33 (2005) 511-518.
302 303 304
[11] M. Kurahashi, A. Yokota, Endozoicomonas elysicola gen. nov., sp. nov., a γproteobacterium isolated from the sea slug Elysia ornata, Syst. Appl. Microbiol., 30 (2007) 202-206.
305 306 307
[12] J.P. Meier-Kolthoff, A.F. Auch, H.-P. Klenk, M. Göker, Genome sequence-based species delimitation with confidence intervals and improved distance functions, BMC Bioinformatics, 14 (2013) 1-14.
308 309 310 311
[13] G. Muyzer, A. Teske, C. Wirsen, H. Jannasch, Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments, Arch. Microbiol., 164 (1995) 165-172.
312 313 314 315
[14] M. Nishijima, K. Adachi, A. Katsuta, Y. Shizuri, K. Yamasato, Endozoicomonas numazuensis sp. nov., a gammaproteobacterium isolated from marine sponges, and emended description of the genus Endozoicomonas Kurahashi and Yokota 2007, Int. J. Syst. Evol. Microbiol., 63 (2013) 709-714.
Ac ce
pt
ed
M
an
us
cr
ip t
[1] A.F. Auch, M. von Jan, H.-P. Klenk, M. Göker, Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison, Stand. Genomic Sci., 2 (2010) 117-134.
13
Page 13 of 22
[15] D.H. Parks, M. Imelfort, C.T. Skennerton, P. Hugenholtz, G.W. Tyson, CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes, Genome Res., (2015).
319 320 321 322
[16] R.E. Pike, B. Haltli, R.G. Kerr, Description of Endozoicomonas euniceicola sp. nov. and Endozoicomonas gorgoniicola sp. nov., bacteria isolated from the octocorals Eunicea fusca and Plexaura sp., and an emended description of the genus Endozoicomonas, Int. J. Syst. Evol. Microbiol., 63 (2013) 4294-4302.
323 324 325
[17] C. Quast, E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, F.O. Glöckner, The SILVA ribosomal RNA gene database project: improved data processing and web-based tools, Nucleic Acids Res., 41 (2013) D590-D596.
326 327 328 329
[18] F.J. Ribeiro, D. Przybylski, S. Yin, T. Sharpe, S. Gnerre, A. Abouelleil, A.M. Berlin, A. Montmayeur, T.P. Shea, B.J. Walker, S.K. Young, C. Russ, C. Nusbaum, I. MacCallum, D.B. Jaffe, Finished bacterial genomes from shotgun sequence data, Genome Res., 22 (2012) 2270-2277.
330 331
[19] M. Richter, R. Rosselló-Móra, Shifting the genomic gold standard for the prokaryotic species definition, Proc. Natl. Acad. Sci. USA, 106 (2009) 19126-19131.
332 333
[20] F. Ronquist, J.P. Huelsenbeck, MrBayes 3: Bayesian phylogenetic inference under mixed models, Bioinformatics, 19 (2003) 1572-1574.
334 335
[21] K.H. Schleifer, O. Kandler, Peptidoglycan types of bacterial cell walls and their taxonomic implications, Bacteriol. Rev., 36 (1972) 407-477.
336 337 338
[22] L. Schreiber, K.U. Kjeldsen, P. Funch, J. Jensen, M. Obst, S. Lopez-Legentil, A. Schramm, Endozoicomonas are specific, facultative symbionts of sea squirts, Front. Microbiol., Under review (2016).
339 340
[23] P. Schumann, Peptidoglycan Structure, Methods in Microbiology, 38 (2011) 101129.
341 342
[24] H.W. Seeley Jr, P.J. VanDemark, J.J. Lee, Microbes in action. A laboratory manual of microbiology (4th Edition), W. H. Freeman, New York, 1991.
343 344
[25] E. Stackebrandt, J. Ebers, Taxonomic parameters revisited: tarnished gold standards, Microbiol. Today, 33 (2006) 152.
345 346
[26] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0, Mol. Biol. Evol., 30 (2013) 2725-2729.
347 348 349
[27] H. Teeling, A. Meyerdierks, M. Bauer, R. Amann, F.O. Glöckner, Application of tetranucleotide frequencies for the assignment of genomic fragments, Environ. Microbiol., 6 (2004) 938-947.
350 351
[28] The European Committee on antimicrobial susceptibility testing, Clinical breakpoints - bacteria (v 5.0), in, 2015.
352 353
[29] B.J. Tindall, A comparative study of the lipid composition of Halobacterium saccharovorum from various sources, Syst. Appl. Microbiol., 13 (1990) 128-130.
354 355
[30] B.J. Tindall, Lipid composition of Halobacterium lacusprofundi, FEMS Microbiol. Lett., 66 (1990) 199-202.
Ac ce
pt
ed
M
an
us
cr
ip t
316 317 318
14
Page 14 of 22
[31] B.J. Tindall, R. Rosselló-Móra, H.-J. Busse, W. Ludwig, P. Kämpfer, Notes on the characterization of prokaryote strains for taxonomic purposes, Int. J. Syst. Evol. Microbiol., 60 (2010) 249-266.
359 360 361
[32] B.J. Tindall, J. Sikorski, R.A. Smibert, N.R. Krieg, Phenotypic characterization and the principles of comparative systematics, in: Methods for General and Molecular Microbiology, Third Edition, American Society of Microbiology, 2007.
362 363 364
[33] F. Widdel, F. Bak, Gram-negative mesophilic sulfate-reducing bacteria, in: A. Balows, H. Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (Eds.) The Prokaryotes, Springer New York, 1992, pp. 3352-3378.
365 366
[34] F. Widdel, G.-W. Kohring, F. Mayer, Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids, Arch. Microbiol., 134 (1983) 286-294.
367 368 369
[35] C.-S. Yang, M.-H. Chen, A.B. Arun, C.A. Chen, J.-T. Wang, W.-M. Chen, Endozoicomonas montiporae sp. nov., isolated from the encrusting pore coral Montipora aequituberculata, Int. J. Syst. Evol. Microbiol., 60 (2010) 1158-1162.
Ac ce
pt
ed
M
an
us
cr
ip t
356 357 358
15
Page 15 of 22
Figure captions
371
Figure 1. Phylogeny of the Hahellaceae based on the 16S rRNA gene and Bayesian
372
inference. Node support is expressed as posterior probabilities. Sequences of E.
373
ascidiicola are shown in bold. Accession numbers are shown in parentheses. For E.
374
montiporae (*) the sequence identifier of the genomic contig and the sequence
375
coordinates of the 16S rRNA gene are shown in parentheses. Sequences of the genus
376
Marinobacter were used as outgroup (not shown). Scale bar: 3% estimated sequence
377
divergence.
us
cr
ip t
370
an
378
Figure 2. Multivariate analysis of fatty acid patterns. (A) UPGMA analysis of fatty
380
acid patterns of Endozoicomonas species. Numbers at nodes indicate bootstrap support.
381
The E. ascidiicola strains described in this study are shown in boldface. Fatty acids
382
patterns obtained in the present study are marked with an asterisk (*). (B) PCA of fatty
383
acid patterns of Endozoicomonas species. Fatty acid patterns are indicated by dots. Dots
384
from the same species are connected by lines and share the same color. Loadings of the
385
principal components are shown in brackets as part of the axis labels. Fatty acids
386
patterns obtained in the present study are marked with a black circle.
ed
pt
Ac ce
387
M
379
16
Page 16 of 22
Tables Table 1 Summary of genomic taxonomy
AVMART05T
KASP37
ANIa
99.2%
-
Digital DDHb
91.8
-
Tetra correlationa
0.999
-
16S rRNA identityc
98.9%
-
ANIa
79.2%
Digital DDHb
22%d
an
403 404 405
Tetra correlationa
0.953
0.956
16S rRNA identityc
97.9%
97.5%
Reference & analysis
M
22%d
78.6%
pt
ANIa
406
78.9%
22.5%d
22.3%d
Tetra correlationa
0.958
0.958
16S rRNA identityc
98.0%
97.3%
Ac ce
Digital DDHb
cr
79.3%
ed
E. elysicola
us
E. atrinae
ip t
KASP37
407
Footnotes:
408
a
409
tetranucleotide regression value >0.999 [19]
410
b
411
formula applicable for incomplete genomes [1]. DNA-DNA hybridization threshold for
412
species delineation is 70% [31].
The current recommendation for genomic circumscription of species is: ANI >94–96%,
The here reported value was calculated with the digital DDH formula 2; the only
18
Page 17 of 22
413
c
The arbitrary, but recommended threshold for circumscription of a species is ≥97%
414
[31]. However systematic analysis of species demarcation based on DNA-DNA
415
hybridization values and 16S rRNA gene identity analyses suggests a higher threshold of
416
≥98.7-99% [25]
417 418 419
d Probability
Ac ce
pt
ed
M
an
us
cr
ip t
that actual DDH is >70% is 0
19
Page 18 of 22
ip t cr
E. ascidiicola
E. atrinae
E. elysicola
E. numazuensis
E. montiporae
AVMART05T
KASP37
DSM 100061
DSM 22380
DSM-25634
LMG 24815
Isolation source
Ascidiella sp.
Ascidiella scabra
Atrina pectinata
Elysia ornate
Marine sponge
(Ascidian)
(Ascidian)
(Pen shell)
(Sea slug)
Colony color
Beige
Transparentwhite
Beigea
Beigeb
Pale creamy white and opaquec
Colony morphology
Convex, circular, smooth surface with entire margins +
Flat, circular, smooth surface, with undulate margins
Convex, circular, with entire marginsa
Convex, circular, smooth, shinyb
+
-a
46.64
Motility
E. gorgoniicola NCCB 100438
E. euniceicola
Montipora aequituberculata
Plexaura sp.
Eunicea fusca
(Coral)
(Coral)
(Coral)
Beiged
Whitee
Creamy whitee
Low-convex, circular to irregular, with undulate marginsc
Convex, circular, with entire marginsd
Convex, circulare
Convex, circulare
+b
ND
+d
+e
+e
47.91
46.75
47.02
48.46
ND
ND
PG, PE, PN, AL, DPG, PL (2) (a) Q-9, Q-8
ND
ND
ND
ND
Q-9, Q-8
Q-9, Q-8
Q-9, Q-8
+d
-a,e
-a,e
d
M
an
E. ascidiicola
ep te
Strain
us
Table 2 Comparison of differential morphological, physiological, and molecular properties between E. ascidiicola and other Endozoicomonas species. Data was obtained in this study unless indicated otherwise. G+C content was calculated based on sequenced genomes. Abbreviations: FAN = facultative anaerobe, A = Aerobe, var = variable, + = positive, - = negative, ND = no data available, PG = Phosphatidylglycerol, PE = Phosphatidylethanolamin, PN = Unidentified phosphoaminolipid, AL = Unidentified aminolipid, DPG = Diphosphatidylglycerol, L = Unidentified lipid, PL = Unidentified phospholipid.
DSM-26535
G+C content (mol%)
46.70
Polar lipids
PG, PE, PN
PG, PE, PN
Respiratory quinones Oxidase
Q-9
Q-9
PG, PE, PN, AL, DPG, L, PL (3)(a) Q-9, Q-8
+
+
+
+
Q-9, Q-8, MK9, MK-8 +c
Gelatin hydrolysis
-
-
-
-
vara,c
-a,d
+a,e
-a,e
Esculin hydrolysis
-
-
+
+
-a,c
+a,d
-a,e
-a,e
Ac c
420 421 422 423
20
Page 19 of 22
ip t 15-37/30a
4-37/25-30b
pH range/optimum
6.2-8.3/6-7
6.3-8.9/6-8
6-9/7a
ND
NaCl% range/optimum
0.5-5/1
0.5-5/1-2
1-4/2a
>0/>0b
Relation to oxygen
FAN
FAN
Aa
cr
7-31/25
15-37/25c
15-35/25d
15-30/22-30e
15-30/22-30e
5.5-9/7.5-8c
6-10/8d
7-9/8e
7-8/8e
1-5/2c
1-3/2-3d
1-4/2-3e
1-4/2-3e
FANc
Ad
FANa
FANa
an
us
5-27/23
Ab
M
424 425
°C range/optimum
Footnotes: (a) data from [8]; (b) data from [11]; (c) data from [14]; (d) data from [35]; (e) data from [16]
d
426
Ac c
ep te
427
21
Page 20 of 22
ip t cr us an M d te
Hahella antarctica (EF495227)
1
Ac ce p
Hahella chejuensis (CP000155)
0.999
Hahella ganghwensis (AY676463)
0.997
0.977
Zooshikella ganghwensis (AY676463) Kistimonas asteriae (EU599216)
1
Kistimonas scarphacae (JF811908)
1
Candidatus Endonucleobacter bathymodioli (FM162188)
Endozoicomonas numazuensis (AB695088)
1
Endozoicomonas gorgoniicola (JX488685)
1
Endozoicomonas euniceicola (JX488684)
0.997 1
Endozoicomonas montiporae (JOKG01000007.7100.8645)* 0.965
Endozoicomonas elysicola (AB196667) 1
Endozoicomonas atrinae (KC878324) 1
Endozoicomonas ascidiicola AVMART05 T (KT364257) 1
Page 21 of 22
Endozoicomonas ascidiicola KASP37 (KT364259) 0.03
Component 2 (10%) 27
B 1.6
1.2
0.8
0.4
-2.0 -2.0
d
30
te
24 44
30
9 48
-1.6 46
12
15
18 46
21
E. numazuensis
-1.2
-0.8
-0.4
cr
64
69
48
us
3
an
M
6
Ac ce p
Distance
95 95
46 38
0.0
-0.8
E. ascidiicola KASP37
0.0
0.4
Component 1 (81%)
ip t
un ice ico E. la eu ni ce ico E. la go rg on iic E. ol go a rg on iic E. ol as a cid iic ol E. a as AV cid M iic AR ol E. T0 a ely K 5T sic AS * ol P3 a E. 7 * ely sic ol a E. ely sic ol a* E. ely sic ol a E. ely sic ol a E. m on tip or E. ae m on tip or E. ae m on tip or E. ae m on tip or E. ae nu m az ue E. ns nu is m st az .H u C5 en E. 0 sis nu m st az .H ue C4 E. ns 6 at is rin st ae .H C5 E. 0 at rin ae *
E. e
A
47
0.8
1.2
77
37 53
27
54
100
E. atrinae
E. elysicola
E. euniceicola
-0.4
E. montiporae
E. ascidiicola AVMART05T
E. gorgoniicola
-1.2
-1.6
1.6
Page 22 of 22