Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts

Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts OPENACCESS The bacterial communities colonizing amphibian skin have been intensively studied due Citation: Bletz MC, Bunk B, Spro ¨er C, Biwer P, to their interactions with pathogenic chytrid fungi that are causing drastic amphibian pop- Reiter S, Rabemananjara FCE, et al. (2019) Amphibian skin-associated Pigmentiphaga: ulation declines. Bacteria of the family Alcaligenaceae, and more specifically of the Genome sequence and occurrence across genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. geography and hosts. PLoS ONE 14(10): Here we analyze their occurrence in a previously assembled global skin microbiome e0223747. https://doi.org/10.1371/journal. dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total num- pone.0223747 ber of reads in this global dataset. They were mostly found in unrelated arboreal frogs Editor: Bi-Song Yue, Sichuan University, CHINA from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances Received: May 17, 2019 on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a Accepted: September 29, 2019 clade within Pigmentiphaga not assignable to any type strains of the five described spe- Published: October 11, 2019 cies of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome Copyright:© 2019 Bletz et al. This is an open access article distributed under the terms of the sequence of this bacterium which, based on 16S sequences of>97% similarity, has pre- Creative Commons Attribution License, which viously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its permits unrestricted use, distribution, and genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted reproduction in any medium, provided the original author and source are credited. coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with Data Availability Statement: The complete environmental information processing and cellular processes. Furthermore, it has a bio- genome sequence of Pigmentiphaga aceris Mada1488 has been deposited at NCBI GenBank synthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosyn- under the accession no CP043046. (BioProject thetic genes can be found, but clusters forβ-lactones present in other comparative number PRJNA561098). Pigmentiphaga genomes are lacking. Funding: The sampling and culturing of amphibian skin bacteria in Madagascar was supported by a grant from the Mohamed bin Zayed Conservation Fund to MB and RH, a grant from the Amphibian Survival Alliance to MB, RH, and MV, a grant from Chester Zoo to MB and RH, a scholarship of the PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 1 / 14 Genome of amphibian skin-associated Pigmentiphaga German Academic Exchange Service (DAAD) to Introduction MB, and a grant from the Deutsche The cutaneous microbiome of amphibians has become a well-studied system, triggered by the Forschungsgemeinschaft (DFG) to MV (VE247/9- rise of the pathogenic fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans 1). The funders had no role in study design, data collection and analysis, decision to publish, or (Bsal). These fungi colonize the amphibian skin and are causing drastic population declines preparation of the manuscript. and extinctions in this class of animals [1,2]. The bacterial communities associated to amphibi- ans interact with these fungi and some of these bacteria have the potential to inhibit the growth Competing interests: The authors have declared that no competing interests exist. of Bd and Bsal, thus providing protection to their hosts [3]. Recent research based on next-generation amplicon sequencing of the 16S rRNA gene show that bacterial communities on the skin of amphibians are predominantly composed of common bacteria recruited from environmental reservoirs [4], and their dominant members can be readily cultured [5]. Unsurprisingly these communities are strongly controlled by environmental factors, e.g., bioclimate [6] and microhabitat [7–9]. However, clear differences have also been found between co-occurring hosts [7,10,11], suggesting that the skin mucosal differences among amphib- ian species act as filters determining which bacterial species are recruited into the community. Considering the strong environmental influences on the amphibian cutaneous micro- biome, it is of particular interest to analyze in more depth those bacteria that regularly colonize this habitat but are restricted to certain host taxa or host ecomorphs. An in-depth understand- ing of the genomic background, variation, phylogenetic relationships, and distribution of these bacteria may offer clues to understand which traits predispose them to successfully colonize this particular habitat. This study was triggered by the observation that operational taxonomic units (OTUs) of the family Alcaligenaceae were strongly associated to arboreal ecomorphs in a study on Madagas- can amphibians [12], and were also found to be a common member of the cutaneous micro- biome of several Central American tree frogs, such as Agalychnis callidryas [13,14]. The family was represented by a pure culture isolate identified as Pigmentiphaga by 16S sequences in our bacterial culture collection from Madagascar frog skin [15], and also the sequences of Alcali- genaceae OTUs identified by amplicon sequences from Madagascar frogs. Pigmentiphaga is a genus of the family Alcaligenaceae, assigned to the order Burkholderiales order within the Betaproteobacteria, and currently containing five species: the type species, P. kullae, plus P. aceris, P. daeguensis, P. litoralis, and P. solis [16–20]. According to these descrip- tions, the genus contains gram-negative, facultatively anaerobic, motile or nonmotile, catalase- and oxidase-positive, rod-shaped bacteria, found in diverse environments: P. daeguensis from dye wastewater, P. litoralis from tidal sediment, P. soli from soil, P, aceris from tree sap [17– 20], and an unidentified species from tree-associated nematodes [21]. Furthermore, Pigmenti- phaga have also been isolated from human clinical material [22], and genome sequences are available from these isolates [23]. Pigmentiphaga have been studied in the context of their abil- ity to degrade azo dyes and aniline [16,24], and their role also has been discussed in the context of biphenyl-degradation [25]. Here, we analyze the occurrence of OTUs assigned to Pigmentiphaga on the skin of amphibians across taxa, geography and ecomorphs, assemble the full genome sequence of one Pigmentiphaga isolate obtained from a Madagascan frog, and analyze the phylogenetic rela- tionships and differentiation of this isolate. Methods Analysis of amplicon data To explore the distribution of the focal bacterial groups on amphibian hosts we used a recently published global dataset of amphibian skin microbiomes [6], and extracted all OTUs assigned PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 2 / 14 Genome of amphibian skin-associated Pigmentiphaga to the family Alcaligenaceae and genus Pigmentiphaga. This global dataset is a compilation of targeted amplicon sequencing data of the 16S rRNA gene (V4 region) generated on Illumina sequencing platforms. It contains skin microbiome samples from 2,349 post-metamorphic amphibians, comprising 27 amphibian families (205 species) collected from 12 countries (5 con- tinents) [6]. In this study, quality-filtered Illumina reads were classified into sub-operational taxonomic units (sOTUs) using the deblur pipeline [26]. These data were subsequently rarified and taxonomy was assigned using the Ribosomal Database Classifier [27] in QIIME [28] (see [6] for detailed methods). We examined the distribution of the genus Pigmentiphaga across locations (i.e. countries), host species, and host ecomorphological classes. To explore the distri- bution of the focal bacteria across host phylogeny, a phylogenetic tree of arboreal and scansorial host genera was built using timetree.org [29]. All plots were produced with ggplot2 [30]. Isolate sampling The bacterium for which we here report the full genome sequence was isolated on 1% tryptone agar from a skin swab of a single individual of the amphibian species, Mantella crocea at Toro- torofotsy "Prolemur Camp" near Andasibe, Madagascar (-18.7709 S, 48.43222 E). This individ- ual frog was captured by gloved hands and kept in a sterile Whirl-Pak bag for no longer than 1 hour before sampling. Frog skin microbiota were sampled by first rinsing the skin to remove transient microbes and then swabbing the ventral skin with a sterile rayon tipped swab 10 times. Swabs were stored in Tryptic Soy Yeast Extract media with 20% Glycerol and frozen until processing. The frog was immediately release at the site of capture after sampling. DNA extraction and complete genome sequencing DNA was isolated using Qiagen Genomic-tip 100/G (Qiagen, Hilden Germany) according to manufacturer instructions. SMRTbell™ template library was prepared according to the instruc- tions from PacificBiosciences, Menlo Park, CA, USA, following the Procedure & Checklist– Greater Than 10 kb Template Preparation. Briefly, for preparation of 15kb libraries 8μg geno- mic DNA was sheared using g-tubes™ from Covaris, Woburn, MA, USA according to the instructions of the manufacturer. DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosci- ences, Menlo Park, CA, USA. Reactions were carried out according to the manufacturer instructions. BluePippin™ Size-Selection to greater than 4 kb was performed according to the manufacturer’s instructions (Sage Science, Beverly, MA, USA). Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote, PacificBiosciences, Menlo Park, CA, USA. 1 SMRT cell was sequenced on the PacBio RSII (PacificBiosciences, Menlo Park, CA, USA) taking one 240-min- utes movie. SMRT sequencing revealed a total number of 85,590 reads with a mean read length of 12,138 bp and a N50 value of 16,449 bp. From the same batches of DNA, short insert librar- ies were created using the Illumina Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA) resulting in 7,916,070 paired-end reads of 2x76 bp. Genome assembly and annotation Genome assembly was performed applying the RS_HGAP_Assembly.3 protocol included in SMRT Portal version 2.3.0 using default parameters. The assembly revealed a single circular chromosome with a coverage of 133x. The chromosome was circularized, artificial redundan- cies at the ends of the contigs were removed and adjusted to dnaA as the first gene. Error-cor- rection was performed by a mapping of Illumina short reads onto finished genome using PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 3 / 14 Genome of amphibian skin-associated Pigmentiphaga Burrows-Wheeler Alignment bwa 0.6.2 in paired-end (sample) mode using default setting [31] with subsequent variant and consensus calling using VarScan 2.3.6 (Parameters: mpileup2cns —min-coverage 10—min-reads2 6—min-avg-qual 20—min-var-freq 0.8—min-freq-for-hom 0.75—p-value 0.01—strand-filter 1—variants 1—output-vcf 1) [32]. A consensus concordance of QV60 could be reached. Automated genome annotation was carried out using Prokka [33] and NCBI PGAP [34]. Genome comparisons We used Mauve software [35] to compare gene arrangement of the newly sequenced genome with those of other available Pigmentiphaga genomes. We compared the general gene functional characterization (KEGG functional categories) of the newly sequenced genomes with all other available Pigmentiphaga genomes using BlastKOALA [36]. On average, 50% of genomes’ pro- tein coding sequences were annotated. Identification of natural product gene clusters was per- formed with the antibiotics and Secondary Metabolite Analysis SHell (antiSMASH version beta5; https://antismash.secondarymetabolites.org) [37] AntiSMASH is an online platform that allows for a genome-wide identification and analysis of secondary metabolite BGCs in bacterial genomes, by integrating and cross-linking with a large number of in silico secondary metabolite analysis tools like CLUSEAN [38], BAGEL2 [39], ClustScan [40], and NORINE [41]. Phylogenetic analysis We performed BLAST searches against the NCBI database, using the full 16S rRNA gene sequence from the sequenced genome to understand the distribution of this bacterium outside of amphibian hosts. We also used the MOLE-BLAST tool of NCBI to retrieve from GenBank the sequences of bacterial taxa most closely related to the Alcaligenaceae sOTUs in our ampli- con data set. MOLE-BLAST is an experimental tool to find closest database neighbors of sub- mitted query sequences, by computing a multiple sequence alignment (MSA) between the query sequences along with their top BLAST database hits. The obtained sequences, along with sequences of our isolates and amplicon-derived sOTU sequences, were aligned with the MAFFT 7.0 algorithm [42] and phylogenetically analyzed in MEGA v. 7 [43]. We successively filtered the data set to remove highly deviant sequences retrieved from the database (as well as sOTU sequences associated to them), as identified by obvious alignment artefacts and excessively long branches in exploratory phylogenetic trees. The final Maximum Likelihood (ML) tree was computed under the GTR+G model of sequence evolution as determined under the Bayesian Information criterion in MEGA 7. Volatile compound analysis A bacterial culture of the target bacterial strain was incubated on 1% tryptone agar for seven days at room temperature. Headspace extracts were obtained using a vacuum pump to draw clean air (purification by active charcoal filter) through a 250 mL glass vessel containing the culture plate. The air was then passed through a thermal desorption tube filled with an absor- bent (Tenax TA Tube; GERSTEL, Mu ¨ lheim an der Ruhr, Germany) for 5 h (three replicates). Thermal desorption tubes were desorbed using a thermal desorption unit (TDU), cooled injection system (CIS) and a MultiPurposeSampler (MPS) autosampler (GERSTEL, Mu ¨ lheim an der Ruhr, Germany) connected to an Agilent 7890B gas chromatograph. The gas chromato- graph was equipped with a HP-5 MS fused silica capillary column (30 m, 0.25 i. d., 0.25 μm film, Hewlett-Packard, Wilmington, USA) connected to an Agilent 5977A mass-selective detector. Conditions: transfer line 300˚C, electron energy 70 eV. Thermal desorption: 30˚C, increasing at 60˚C/min to 280˚C (10 min isothermal). Cooled injection: -150˚C, increasing at PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 4 / 14 Genome of amphibian skin-associated Pigmentiphaga 12˚C/min to 300˚C (3 min isothermal). Gas chromatographic method: 50˚C (5 min isother- mal), increasing at 5˚C/min to 320˚C, and operated in splitless mode. Helium was used as car- rier gas at 1.2 ml/min. GC retention indices (RI) were determined from a homologous series of n-alkanes (C -C ). Compounds were identified by comparison of mass spectra and retention 8 30 indices with those of authentic samples. Results and discussion Representation of Alcaligenaceae in amphibian cutaneous microbiomes In the global dataset of amplicon sequences from 205 amphibian species [6] Alcaligenaceae sOTUs made up 284,771 out of 5,872,500 total rarified reads in the final data set (4.8%). Alcali- genaceae sOTUs (at a minimum threshold of 5 reads) were found in a total of 119 amphibian species from eight countries, and Pigmentiphaga sOTUs in 95 amphibian species. In a culture database of amphibian skin bacteria [22, Bletz & Woodhams unpublished data] 28 of 5938 iso- lates were from the Alcaligenaceae, and our isolate was the sole member from the genus Pig- mentiphaga. Therefore, apparently, Alcaligenaceae and more specifically Pigmentiphaga appear to be underrepresented in culture databases of amphibian skin microbiota and thus might be less readily culturable than other bacteria from this habitat [5]. The family Alcaligenaceae currently contains 27 genera (UniProt 2019); in the amphibian microbiome data set, 338 out of a total of 124,348 sOTUs were assigned to this family, and of these, 35 to the genus Pigmentiphaga (reads = 266,723). The remaining Alcaligenaceae reads were assigned to the genera Achromobacter (n = 3,355), Alcaligenes (n = 666), Sutterella (n = 20) Oligella (n = 16), Denitrobacter (n = 3), Candidimonas (n = 12) or were left unassigned to a specific genus (n = 13,976). Confirming previous findings [12–14], in our global skin microbiome dataset [6] Pigmenti- phaga was predominantly found on arboreal species as well as scansorial hosts within the amphibian clades Mantellidae (mean: 7.7% + 1.5%SD) and Hyperoliidae (mean: 7.9% + 4.7% SD, which are distributed in Madagascar, and (Hyperoliidae only) in mainland Africa (Fig 1). The genus also appears on amphibians from the genus Pseudacris (Fig 1). Overall, Pigmenti- phaga was more common on amphibians from Madagascar; however, this could be associated with extensive sampling of arboreal hosts within this country. Phylogenetic diversity of Pigmentiphaga in amphibian cutaneous microbiomes We aligned short amplicon-based consensus sequences of Alcaligenaceae amplicons with the longer 16S sequences of all Madagascan amphibian-derived isolates from a previous work [15] belonging to this family. We then used a series of BLAST and MOLE-BLAST searches, allowing for hits with and without environmental sequences, and restricting searches to type strains or not, to retrieve a representative set of 273 homologous Alcaligenaceae sequences of the 16S rRNA gene for analysis of phylogeny and environmental distribution of our focal bacterial taxa. The exploratory analysis of these sequences along with our Alcaligenaceae sOTU and isolate sequences placed 92 sOTUs, 8 isolates and 82 related sequences retrieved from GenBank in a clade with the Pigmentiphaga type strains. A ML tree calculated on this restricted dataset (Fig 2) reveals a large diversity of Pigmentiphaga, many of which are not assignable to any of the described species. A large number of 61 additional sOTUs from the amphibian skin, as well as one isolate from the skin of a fire salamander (DE946; accession number MH512662) and two from Madagascar frogs (Mada281, Mada1835; accession numbers MF526411, MF523827), are placed in a large subclade of putative Pigmentiphaga sequences that does not contain any type PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 5 / 14 Genome of amphibian skin-associated Pigmentiphaga Fig 1. Distribution of Pigmentiphaga spp. across amphibian hosts. relative abundance within amphibian skin microbiomes across host eco-morphology classes (A), countries (B), and phylogeny of arboreal and scansorial amphibian hosts (C). https://doi.org/10.1371/journal.pone.0223747.g001 strain sequences. This subclade also contains sOTU7503, the most widespread Alcaligenaceae sOTU in our global amplicon-derived data set (45,697 reads). Various sequences retrieved from GenBank and included in this subclade are named P. daguensis but are unlikely to belong to this species, given that the type strain is placed in another, phylogenetically distant clade. Whether this diverse subclade is to be assigned to Pigmentiphaga definitively, or to another, possibly undescribed genus in the Alcaligenaceae, will require additional study. Genome characteristics of Pigmentiphaga aceris isolated from amphibian skin One of our isolates (Mada1488) was placed close to P. aceris and had >99% sequence similarity with the type strain of this species (Table 1). The sequence obtained by direct Sanger PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 6 / 14 Genome of amphibian skin-associated Pigmentiphaga Fig 2. Maximum likelihood phylogenetic tree of selected Pigmentiphaga based on DNA sequences of up to 1478 bp of the bacterial 16S rRNA gene. Sequences of amphibian skin bacteria from an Illumina-based amplicon survey[6] are colored purple; isolates from amphibian skin are colored blue. Red color highlights the sequences of the P. aceris strain used for genome sequencing. Sequences from type or reference strains are boldfaced. Alcaligenes faecalis, the type species of the type genus of Alcaligenaceae, was used as the outgroup. results of a bootstrap analysis (100 replicates) are marked by gray (bootstrap proportion >50%) and black circles (>70%). Note that due to the inclusion of many short sequences from Illumina amplicon analysis, most nodes did not receive strong bootstrap support. https://doi.org/10.1371/journal.pone.0223747.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 7 / 14 Genome of amphibian skin-associated Pigmentiphaga Table 1. NCBI database matches (97–100% sequence identity) to the 16S rRNA gene of Pigmentiphaga aceris (strain Mada1488). Accession # Description Query cover Percent match Isolation Source HM276147.1 Uncultured bacterium clone ncd518h02c1 89% 99.4% Human skin NR_157990.1 Pigmentiphaga aceris strain SAP-32 94% 99.3% tree sap HM270483.1 Uncultured bacterium clone ncd266g01c1 89% 99.2% human skin FJ849553.1 Uncultured bacterium clone SedUMA34 95% 99.2% arctic stream sediment; ultramafic lithology JX067670.1 Alcaligenaceae bacterium SAP706.3 98% 99.1% floral nectar FN421876.1 Uncultured bacterium, clone 2_F01 92% 99.1% phyllosphere of clover HM316492.1 Uncultured bacterium clone ncd307b11c1 89% 99.0% human skin JX067692.1 Alcaligenaceae bacterium SAP773.2 98% 99.0% floral nectar HM316454.1 Uncultured bacterium clone ncd306e07c1 89% 99.0% human skin FJ849534.1 Uncultured bacterium clone SedUMA17 1 95% 98.4% arctic stream sediment; ultramafic lithology MH667611.1 Pigmentiphaga sp. strain IMT-318 94% 97.1% soil (USA) https://doi.org/10.1371/journal.pone.0223747.t001 sequencing of DNA extracted from this isolate agreed fully with the sequence from both 16S copies found in the assembled genome. One amplicon-derived sOTU also the sequence of this isolate. In our global amphibian data set, 139 of these reads came from the sOTU matching the Mada1488 isolate. This sOTU was found on frogs of the genera Anaxyrus, Boophis, Colos- tethus, Craugastor, Gephyromantis, Eleutherodactylus, Lithobates, Mantidactylus, Mantella, and Plethodontohyla. Thus, the bacterium represented by our culture (Mada1488) was not Fig 3. The circular genome of 6,165,255 bp of Pigmentiphaga aceris (strain Mada1488). In blue (circle 1) genes lying on the forward strand are shown and in red (circle 2) those on the reverse strand. In circle 3 tRNA genes are shown in brown, often clustered together with green rRNA genes. Circle 4 shows the GC content (G+C)/(A+T+G+C), whereas in circle 5 a GC skew (G−C)/(G+C) is shown. This map has been created using DNAplotter [44]. https://doi.org/10.1371/journal.pone.0223747.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 8 / 14 Genome of amphibian skin-associated Pigmentiphaga very common in our Illumina dataset. As it was the only cultured Pigmentiphaga from amphibian skin assignable to a known species we nevertheless chose this isolate for genome sequencing, to obtain first data of the genomic background of these bacteria. In culture, this bacterium forms white, glossy colonies with circular form; the elevation is raised and the margins are entire. In a growth inhibition assay (see [15] for methods) the metabolites produced by this bacterium reduced the growth of the amphibian skin pathogen Batrachochytrium dendrobatidis by 60%. BLAST searches in the NCBI nucleotide database revealed that bacterial strains with >97% 16S sequence identity to Mada1488 have been found on human skin, in floral nectar, tree sap, artic stream sediment, and soil (Table 1). The highest identity was found with an uncultured isolate from human skin (99.4% identity), directly fol- lowed by the P. aceris type strain (SAP-32) with 99.3% identity. The complete genome of Pigmentiphaga aceris (Mada1488) consists of a single circular chromosome with 6,165,255 bp and a GC content of 62.1%. Prokka predicted 5,300 coding sequences, 57 tRNA genes, and three rRNA operons (Fig 3). A multiple genome alignment suggests that the new genome shows only limited similarities to the five congeneric genomes available (S1 Fig); however, the five available genomes all belong to closely related strains (all >98% 16S similarity to the type strains of P. kullae and P. daeguensis, which themselves show 99.6% similarity, questioning the distinctness of these two taxa at the spe- cies level). The newly sequenced genome of P. aceris (Mada1488) differs in its gene functions com- pared to other known Pigmentiphaga genomes; more specifically, genes associated with envi- ronmental information processing and cellular processes were more abundant in Mada1488 (S2 Fig). Natural product biosynthetic gene clusters (BGCs) are moderately represented in the newly sequenced genome, and the cluster content differs from that in the other available Pig- mentiphaga genomes (S1 Table). Amongst others, P. aceris (Mada1488) contains a nonriboso- mal peptide syntethase (NRPS) cluster most likely involved in the production of a peptide siderophore similar to enterobactin [45], and a BGC coding for bacteriocin biosynthetic genes, both missing in the other Pigmentiphaga. Bacteriocines are widely occurring, ribosomally pro- duced antimicrobial peptides, presumably with an anti-competitor function [46] and an often narrow activity range against Gram-positive as well as Gram-negative bacteria [47,48]. The most noticeable feature in the comparative Pigmentiphaga genomes but absent in P. aceris (Mada1488) are clusters forβ-lactones, a class of protein-inhibiting natural products with a wide range of activities [49]. Furthermore, P. aceris (Mada1488) lacks genes involved in ectoin biosynthesis. Because ectoins are usually produced to protect the bacteria from environmental extremes like hyperosmotic conditions [50], the lack of ectoins might support this strain being adapted to rather stable environmental conditions. Volatile compounds produced by Pigmentiphaga aceris The ability to produce volatile organic compounds (VOCs) is widespread among bacteria [51] and our data demonstrate this ability also in Pigmentiphaga aceris (Mada1488). GC/MS analy- sis of headspace extracts from this bacterium revealed the release of 21 compounds (S1 Table), among them sulfur-containing volatiles such as methanethiole, dimethyl disulfide (1), dimethyl trisulfide (2), S-methyl ethanethioate (3), S-methyl propanethioate (4), S-methyl 2-methylpropanethioate (5), S-methyl 3-methylbutanethioate (6) and S-methyl phenyletha- nethioate (7), as well asγ-decalactone (8) (S3 Fig). Some these compounds, for example, dimethyl trisulfide and S-methyl 3-methylbutanethioate, have been associated with inhibition of a variety of plant pathogens [52,53], suggesting they may play a role in suppression of amphibian fungal pathogens, such as Bd and Bsal. PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 9 / 14 Genome of amphibian skin-associated Pigmentiphaga Conclusions To our knowledge, Pigmentiphaga aceris (Mada1488) is the first amphibian skin-derived bac- terial isolate with a full genome sequenced. Overall, the genome does not present any out- standing characteristics, in line with the hypothesis that the amphibian cutaneous microbiome mainly consists of generalist species recruited from environmental reservoirs. It is remarkable that bacterial strains most similar to Mada1488 have multiple times been found in plant-asso- ciated microbiomes (tree sap, nectar, phyllosphere). The finding of a Pigmentiphaga similar to the strain described herein on human skin may be explained by a bias of information in genetic databases towards human-derived microbes, but also confirms that these bacteria are not strictly associated to plants only. Yet, it is tempting to relate the apparent common occur- rence of Pigmentiphaga on plants to its high abundance in treefrogs which may have acquired it from plant-associated reservoirs. To test this hypothesis, in-depth analysis of additional bac- teria differentially abundant on arboreal vs. terrestrial amphibians may be rewarding. A wider sampling of genomes represented in amphibian cutaneous microbiomes will be a crucial step to better understand functional properties of these bacterial communities and their potential role in defense against pathogens. Data availability The complete genome sequence of Pigmentiphaga aceris Mada1488 has been deposited at NCBI GenBank under the accession no. CP043046. The version described in this paper is the first version. (BioProject no. PRJNA561098). The 16S sequence of this isolate is archived under accession no. MF525803.1. Accession numbers of sequences used in the phylogenetic analysis are given in the respective tree. Accession numbers for the raw data of amplicon analy- ses are summarized in a previous study [6]. Supporting information S1 Fig. Comparison of genomic composition of the Pigmentiphaga aceris strain isolated from amphibian skin (A) to the five other Pigmentiphaga genomes available: (A) Pigmenti- phaga sp. H8, (B) P. sp. NML030171, (C) P. sp. NML080357, (D) P. kullae K24, (E) P. sp. IMT- 318. The figure shows a multiple genome alignment calculated with Mauve (Darling et al. 2004), using A as reference. Colinear blocks are indicated by identical colors and indicate homologous DNA regions shared by two or more genomes without sequence rearrangements, and are indicated below the black horizontal line if representing reverse complements of the respective sequence of the reference. Note similarities between genomes A-C, larger differ- ences of D and E, and massive differences in the arrangement of the newly sequenced P. aceris genome (F). (JPG) S2 Fig. Comparative summary of gene function across the newly sequenced Pigmentiphaga aceris (red box) and other available genomes from this genus. Pie charts were created directly from BlastKOALA. Colors for a given functional categories are consistent across each chart; categories are ordered by abundance within a given pie chart. (PDF) S3 Fig. Selected volatile compounds released by Pigmentiphaga aceris (Mada1488): metha- nethiole, dimethyl disulfide (1), dimethyl trisulfide (2), S-methyl ethanethioate (3), S-methyl propanethioate (4), S-methyl 2-methylpropanethioate (5), S-methyl 3-methylbutanethioate (6) and S-methyl phenylethanethioate (7), as well asγ-decalactone (8). (PDF) PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 10 / 14 Genome of amphibian skin-associated Pigmentiphaga S1 Table. Natural product biosynthetic gene clusters (BGCs) in available Pigmentiphaga genomes as predicted by AntiSMASH. (DOCX) S2 Table. Volatile compounds released by Pigmentiphaga aceris (Mada1488). Numbered compounds 1–8 are those shown in S2 Fig. (DOCX) Acknowledgments We are grateful to the Malagasy authorities for issuing research and export permits for this research. We are indebted to numerous local guides and field assistants that help during field work. We thank Simone Severitt and Carola Berg for technical assistance with laboratory work related to genome sequencing. We thank the Malagasy authorities for permits to collect, export and analyze the amphibian-skin derived bacteria, including full genome sequencing (research authorizations 105N-EA04/MG17from 25 April 2017; collecting permits 182/13/MEF/SG/ DGF/DCB.SA/SCB from 1 August 2013, 248/16/MEEF/SG/DGF/DSAP/SCB.Re from 14 October 2016, and 282/16/MEEF/SG/DGF/DSAP/SCB from 28 November 2016). The work was carried out in the framework of a collaboration accord of the Technische Universita ¨t Braunschweig with the Cellule d’Urgence Chytride de Madagascar and the Universite ´ de Mad- agascar (Mention Biodiversite ´ Animale), with a Material Transfer Agreement (002/ZBA/17/ ZR) from 3 January 2017. Author Contributions Conceptualization: Molly C. Bletz, Boyke Bunk, Cathrin Spro ¨er, Falitiana C. E. Rabemanan- jara, Stefan Schulz, Jo ¨rg Overmann, Miguel Vences. Data curation: Molly C. Bletz, Boyke Bunk, Cathrin Spro ¨er, Silke Reiter, Miguel Vences. Formal analysis: Molly C. Bletz, Boyke Bunk, Peter Biwer, Silke Reiter, Stefan Schulz, Miguel Vences. Funding acquisition: Falitiana C. E. Rabemananjara, Miguel Vences. Methodology: Cathrin Spro ¨ er, Peter Biwer, Silke Reiter, Stefan Schulz. Project administration: Jo ¨rg Overmann. Visualization: Molly C. Bletz. Writing – original draft: Molly C. Bletz, Miguel Vences. Writing – review & editing: Molly C. Bletz, Boyke Bunk, Peter Biwer, Silke Reiter, Falitiana C. E. Rabemananjara, Stefan Schulz, Jo ¨ rg Overmann, Miguel Vences. References 1. Fisher MC, Garner TW, Walker SF. Global emergence of Batrachochytrium dendrobatidis and amphib- ian chytridiomycosis in space, time, and host. Annu Rev Microbiol. 2009; 63: 291–310. https://doi.org/ 10.1146/annurev.micro.091208.073435 PMID: 19575560 2. Stegen G, Pasmans F, Schmidt BR, Rouffaer LO, Van Praet S, Schaub M, et al. 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Amphibian skin-associated Pigmentiphaga: Genome sequence and occurrence across geography and hosts

PLoS ONE, Volume 14 (10) – Oct 11, 2019

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Copyright: © 2019 Bletz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The complete genome sequence of Pigmentiphaga aceris Mada1488 has been deposited at NCBI GenBank under the accession no CP043046. (BioProject number PRJNA561098). Funding: The sampling and culturing of amphibian skin bacteria in Madagascar was supported by a grant from the Mohamed bin Zayed Conservation Fund to MB and RH, a grant from the Amphibian Survival Alliance to MB, RH, and MV, a grant from Chester Zoo to MB and RH, a scholarship of the German Academic Exchange Service (DAAD) to MB, and a grant from the Deutsche Forschungsgemeinschaft (DFG) to MV (VE247/9-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
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Abstract

OPENACCESS The bacterial communities colonizing amphibian skin have been intensively studied due Citation: Bletz MC, Bunk B, Spro ¨er C, Biwer P, to their interactions with pathogenic chytrid fungi that are causing drastic amphibian pop- Reiter S, Rabemananjara FCE, et al. (2019) Amphibian skin-associated Pigmentiphaga: ulation declines. Bacteria of the family Alcaligenaceae, and more specifically of the Genome sequence and occurrence across genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. geography and hosts. PLoS ONE 14(10): Here we analyze their occurrence in a previously assembled global skin microbiome e0223747. https://doi.org/10.1371/journal. dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total num- pone.0223747 ber of reads in this global dataset. They were mostly found in unrelated arboreal frogs Editor: Bi-Song Yue, Sichuan University, CHINA from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances Received: May 17, 2019 on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a Accepted: September 29, 2019 clade within Pigmentiphaga not assignable to any type strains of the five described spe- Published: October 11, 2019 cies of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome Copyright:© 2019 Bletz et al. This is an open access article distributed under the terms of the sequence of this bacterium which, based on 16S sequences of>97% similarity, has pre- Creative Commons Attribution License, which viously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its permits unrestricted use, distribution, and genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted reproduction in any medium, provided the original author and source are credited. coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with Data Availability Statement: The complete environmental information processing and cellular processes. Furthermore, it has a bio- genome sequence of Pigmentiphaga aceris Mada1488 has been deposited at NCBI GenBank synthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosyn- under the accession no CP043046. (BioProject thetic genes can be found, but clusters forβ-lactones present in other comparative number PRJNA561098). Pigmentiphaga genomes are lacking. Funding: The sampling and culturing of amphibian skin bacteria in Madagascar was supported by a grant from the Mohamed bin Zayed Conservation Fund to MB and RH, a grant from the Amphibian Survival Alliance to MB, RH, and MV, a grant from Chester Zoo to MB and RH, a scholarship of the PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 1 / 14 Genome of amphibian skin-associated Pigmentiphaga German Academic Exchange Service (DAAD) to Introduction MB, and a grant from the Deutsche The cutaneous microbiome of amphibians has become a well-studied system, triggered by the Forschungsgemeinschaft (DFG) to MV (VE247/9- rise of the pathogenic fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans 1). The funders had no role in study design, data collection and analysis, decision to publish, or (Bsal). These fungi colonize the amphibian skin and are causing drastic population declines preparation of the manuscript. and extinctions in this class of animals [1,2]. The bacterial communities associated to amphibi- ans interact with these fungi and some of these bacteria have the potential to inhibit the growth Competing interests: The authors have declared that no competing interests exist. of Bd and Bsal, thus providing protection to their hosts [3]. Recent research based on next-generation amplicon sequencing of the 16S rRNA gene show that bacterial communities on the skin of amphibians are predominantly composed of common bacteria recruited from environmental reservoirs [4], and their dominant members can be readily cultured [5]. Unsurprisingly these communities are strongly controlled by environmental factors, e.g., bioclimate [6] and microhabitat [7–9]. However, clear differences have also been found between co-occurring hosts [7,10,11], suggesting that the skin mucosal differences among amphib- ian species act as filters determining which bacterial species are recruited into the community. Considering the strong environmental influences on the amphibian cutaneous micro- biome, it is of particular interest to analyze in more depth those bacteria that regularly colonize this habitat but are restricted to certain host taxa or host ecomorphs. An in-depth understand- ing of the genomic background, variation, phylogenetic relationships, and distribution of these bacteria may offer clues to understand which traits predispose them to successfully colonize this particular habitat. This study was triggered by the observation that operational taxonomic units (OTUs) of the family Alcaligenaceae were strongly associated to arboreal ecomorphs in a study on Madagas- can amphibians [12], and were also found to be a common member of the cutaneous micro- biome of several Central American tree frogs, such as Agalychnis callidryas [13,14]. The family was represented by a pure culture isolate identified as Pigmentiphaga by 16S sequences in our bacterial culture collection from Madagascar frog skin [15], and also the sequences of Alcali- genaceae OTUs identified by amplicon sequences from Madagascar frogs. Pigmentiphaga is a genus of the family Alcaligenaceae, assigned to the order Burkholderiales order within the Betaproteobacteria, and currently containing five species: the type species, P. kullae, plus P. aceris, P. daeguensis, P. litoralis, and P. solis [16–20]. According to these descrip- tions, the genus contains gram-negative, facultatively anaerobic, motile or nonmotile, catalase- and oxidase-positive, rod-shaped bacteria, found in diverse environments: P. daeguensis from dye wastewater, P. litoralis from tidal sediment, P. soli from soil, P, aceris from tree sap [17– 20], and an unidentified species from tree-associated nematodes [21]. Furthermore, Pigmenti- phaga have also been isolated from human clinical material [22], and genome sequences are available from these isolates [23]. Pigmentiphaga have been studied in the context of their abil- ity to degrade azo dyes and aniline [16,24], and their role also has been discussed in the context of biphenyl-degradation [25]. Here, we analyze the occurrence of OTUs assigned to Pigmentiphaga on the skin of amphibians across taxa, geography and ecomorphs, assemble the full genome sequence of one Pigmentiphaga isolate obtained from a Madagascan frog, and analyze the phylogenetic rela- tionships and differentiation of this isolate. Methods Analysis of amplicon data To explore the distribution of the focal bacterial groups on amphibian hosts we used a recently published global dataset of amphibian skin microbiomes [6], and extracted all OTUs assigned PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 2 / 14 Genome of amphibian skin-associated Pigmentiphaga to the family Alcaligenaceae and genus Pigmentiphaga. This global dataset is a compilation of targeted amplicon sequencing data of the 16S rRNA gene (V4 region) generated on Illumina sequencing platforms. It contains skin microbiome samples from 2,349 post-metamorphic amphibians, comprising 27 amphibian families (205 species) collected from 12 countries (5 con- tinents) [6]. In this study, quality-filtered Illumina reads were classified into sub-operational taxonomic units (sOTUs) using the deblur pipeline [26]. These data were subsequently rarified and taxonomy was assigned using the Ribosomal Database Classifier [27] in QIIME [28] (see [6] for detailed methods). We examined the distribution of the genus Pigmentiphaga across locations (i.e. countries), host species, and host ecomorphological classes. To explore the distri- bution of the focal bacteria across host phylogeny, a phylogenetic tree of arboreal and scansorial host genera was built using timetree.org [29]. All plots were produced with ggplot2 [30]. Isolate sampling The bacterium for which we here report the full genome sequence was isolated on 1% tryptone agar from a skin swab of a single individual of the amphibian species, Mantella crocea at Toro- torofotsy "Prolemur Camp" near Andasibe, Madagascar (-18.7709 S, 48.43222 E). This individ- ual frog was captured by gloved hands and kept in a sterile Whirl-Pak bag for no longer than 1 hour before sampling. Frog skin microbiota were sampled by first rinsing the skin to remove transient microbes and then swabbing the ventral skin with a sterile rayon tipped swab 10 times. Swabs were stored in Tryptic Soy Yeast Extract media with 20% Glycerol and frozen until processing. The frog was immediately release at the site of capture after sampling. DNA extraction and complete genome sequencing DNA was isolated using Qiagen Genomic-tip 100/G (Qiagen, Hilden Germany) according to manufacturer instructions. SMRTbell™ template library was prepared according to the instruc- tions from PacificBiosciences, Menlo Park, CA, USA, following the Procedure & Checklist– Greater Than 10 kb Template Preparation. Briefly, for preparation of 15kb libraries 8μg geno- mic DNA was sheared using g-tubes™ from Covaris, Woburn, MA, USA according to the instructions of the manufacturer. DNA was end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosci- ences, Menlo Park, CA, USA. Reactions were carried out according to the manufacturer instructions. BluePippin™ Size-Selection to greater than 4 kb was performed according to the manufacturer’s instructions (Sage Science, Beverly, MA, USA). Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell™ template were assessed with the Calculator in RS Remote, PacificBiosciences, Menlo Park, CA, USA. 1 SMRT cell was sequenced on the PacBio RSII (PacificBiosciences, Menlo Park, CA, USA) taking one 240-min- utes movie. SMRT sequencing revealed a total number of 85,590 reads with a mean read length of 12,138 bp and a N50 value of 16,449 bp. From the same batches of DNA, short insert librar- ies were created using the Illumina Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) and sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA) resulting in 7,916,070 paired-end reads of 2x76 bp. Genome assembly and annotation Genome assembly was performed applying the RS_HGAP_Assembly.3 protocol included in SMRT Portal version 2.3.0 using default parameters. The assembly revealed a single circular chromosome with a coverage of 133x. The chromosome was circularized, artificial redundan- cies at the ends of the contigs were removed and adjusted to dnaA as the first gene. Error-cor- rection was performed by a mapping of Illumina short reads onto finished genome using PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 3 / 14 Genome of amphibian skin-associated Pigmentiphaga Burrows-Wheeler Alignment bwa 0.6.2 in paired-end (sample) mode using default setting [31] with subsequent variant and consensus calling using VarScan 2.3.6 (Parameters: mpileup2cns —min-coverage 10—min-reads2 6—min-avg-qual 20—min-var-freq 0.8—min-freq-for-hom 0.75—p-value 0.01—strand-filter 1—variants 1—output-vcf 1) [32]. A consensus concordance of QV60 could be reached. Automated genome annotation was carried out using Prokka [33] and NCBI PGAP [34]. Genome comparisons We used Mauve software [35] to compare gene arrangement of the newly sequenced genome with those of other available Pigmentiphaga genomes. We compared the general gene functional characterization (KEGG functional categories) of the newly sequenced genomes with all other available Pigmentiphaga genomes using BlastKOALA [36]. On average, 50% of genomes’ pro- tein coding sequences were annotated. Identification of natural product gene clusters was per- formed with the antibiotics and Secondary Metabolite Analysis SHell (antiSMASH version beta5; https://antismash.secondarymetabolites.org) [37] AntiSMASH is an online platform that allows for a genome-wide identification and analysis of secondary metabolite BGCs in bacterial genomes, by integrating and cross-linking with a large number of in silico secondary metabolite analysis tools like CLUSEAN [38], BAGEL2 [39], ClustScan [40], and NORINE [41]. Phylogenetic analysis We performed BLAST searches against the NCBI database, using the full 16S rRNA gene sequence from the sequenced genome to understand the distribution of this bacterium outside of amphibian hosts. We also used the MOLE-BLAST tool of NCBI to retrieve from GenBank the sequences of bacterial taxa most closely related to the Alcaligenaceae sOTUs in our ampli- con data set. MOLE-BLAST is an experimental tool to find closest database neighbors of sub- mitted query sequences, by computing a multiple sequence alignment (MSA) between the query sequences along with their top BLAST database hits. The obtained sequences, along with sequences of our isolates and amplicon-derived sOTU sequences, were aligned with the MAFFT 7.0 algorithm [42] and phylogenetically analyzed in MEGA v. 7 [43]. We successively filtered the data set to remove highly deviant sequences retrieved from the database (as well as sOTU sequences associated to them), as identified by obvious alignment artefacts and excessively long branches in exploratory phylogenetic trees. The final Maximum Likelihood (ML) tree was computed under the GTR+G model of sequence evolution as determined under the Bayesian Information criterion in MEGA 7. Volatile compound analysis A bacterial culture of the target bacterial strain was incubated on 1% tryptone agar for seven days at room temperature. Headspace extracts were obtained using a vacuum pump to draw clean air (purification by active charcoal filter) through a 250 mL glass vessel containing the culture plate. The air was then passed through a thermal desorption tube filled with an absor- bent (Tenax TA Tube; GERSTEL, Mu ¨ lheim an der Ruhr, Germany) for 5 h (three replicates). Thermal desorption tubes were desorbed using a thermal desorption unit (TDU), cooled injection system (CIS) and a MultiPurposeSampler (MPS) autosampler (GERSTEL, Mu ¨ lheim an der Ruhr, Germany) connected to an Agilent 7890B gas chromatograph. The gas chromato- graph was equipped with a HP-5 MS fused silica capillary column (30 m, 0.25 i. d., 0.25 μm film, Hewlett-Packard, Wilmington, USA) connected to an Agilent 5977A mass-selective detector. Conditions: transfer line 300˚C, electron energy 70 eV. Thermal desorption: 30˚C, increasing at 60˚C/min to 280˚C (10 min isothermal). Cooled injection: -150˚C, increasing at PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 4 / 14 Genome of amphibian skin-associated Pigmentiphaga 12˚C/min to 300˚C (3 min isothermal). Gas chromatographic method: 50˚C (5 min isother- mal), increasing at 5˚C/min to 320˚C, and operated in splitless mode. Helium was used as car- rier gas at 1.2 ml/min. GC retention indices (RI) were determined from a homologous series of n-alkanes (C -C ). Compounds were identified by comparison of mass spectra and retention 8 30 indices with those of authentic samples. Results and discussion Representation of Alcaligenaceae in amphibian cutaneous microbiomes In the global dataset of amplicon sequences from 205 amphibian species [6] Alcaligenaceae sOTUs made up 284,771 out of 5,872,500 total rarified reads in the final data set (4.8%). Alcali- genaceae sOTUs (at a minimum threshold of 5 reads) were found in a total of 119 amphibian species from eight countries, and Pigmentiphaga sOTUs in 95 amphibian species. In a culture database of amphibian skin bacteria [22, Bletz & Woodhams unpublished data] 28 of 5938 iso- lates were from the Alcaligenaceae, and our isolate was the sole member from the genus Pig- mentiphaga. Therefore, apparently, Alcaligenaceae and more specifically Pigmentiphaga appear to be underrepresented in culture databases of amphibian skin microbiota and thus might be less readily culturable than other bacteria from this habitat [5]. The family Alcaligenaceae currently contains 27 genera (UniProt 2019); in the amphibian microbiome data set, 338 out of a total of 124,348 sOTUs were assigned to this family, and of these, 35 to the genus Pigmentiphaga (reads = 266,723). The remaining Alcaligenaceae reads were assigned to the genera Achromobacter (n = 3,355), Alcaligenes (n = 666), Sutterella (n = 20) Oligella (n = 16), Denitrobacter (n = 3), Candidimonas (n = 12) or were left unassigned to a specific genus (n = 13,976). Confirming previous findings [12–14], in our global skin microbiome dataset [6] Pigmenti- phaga was predominantly found on arboreal species as well as scansorial hosts within the amphibian clades Mantellidae (mean: 7.7% + 1.5%SD) and Hyperoliidae (mean: 7.9% + 4.7% SD, which are distributed in Madagascar, and (Hyperoliidae only) in mainland Africa (Fig 1). The genus also appears on amphibians from the genus Pseudacris (Fig 1). Overall, Pigmenti- phaga was more common on amphibians from Madagascar; however, this could be associated with extensive sampling of arboreal hosts within this country. Phylogenetic diversity of Pigmentiphaga in amphibian cutaneous microbiomes We aligned short amplicon-based consensus sequences of Alcaligenaceae amplicons with the longer 16S sequences of all Madagascan amphibian-derived isolates from a previous work [15] belonging to this family. We then used a series of BLAST and MOLE-BLAST searches, allowing for hits with and without environmental sequences, and restricting searches to type strains or not, to retrieve a representative set of 273 homologous Alcaligenaceae sequences of the 16S rRNA gene for analysis of phylogeny and environmental distribution of our focal bacterial taxa. The exploratory analysis of these sequences along with our Alcaligenaceae sOTU and isolate sequences placed 92 sOTUs, 8 isolates and 82 related sequences retrieved from GenBank in a clade with the Pigmentiphaga type strains. A ML tree calculated on this restricted dataset (Fig 2) reveals a large diversity of Pigmentiphaga, many of which are not assignable to any of the described species. A large number of 61 additional sOTUs from the amphibian skin, as well as one isolate from the skin of a fire salamander (DE946; accession number MH512662) and two from Madagascar frogs (Mada281, Mada1835; accession numbers MF526411, MF523827), are placed in a large subclade of putative Pigmentiphaga sequences that does not contain any type PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 5 / 14 Genome of amphibian skin-associated Pigmentiphaga Fig 1. Distribution of Pigmentiphaga spp. across amphibian hosts. relative abundance within amphibian skin microbiomes across host eco-morphology classes (A), countries (B), and phylogeny of arboreal and scansorial amphibian hosts (C). https://doi.org/10.1371/journal.pone.0223747.g001 strain sequences. This subclade also contains sOTU7503, the most widespread Alcaligenaceae sOTU in our global amplicon-derived data set (45,697 reads). Various sequences retrieved from GenBank and included in this subclade are named P. daguensis but are unlikely to belong to this species, given that the type strain is placed in another, phylogenetically distant clade. Whether this diverse subclade is to be assigned to Pigmentiphaga definitively, or to another, possibly undescribed genus in the Alcaligenaceae, will require additional study. Genome characteristics of Pigmentiphaga aceris isolated from amphibian skin One of our isolates (Mada1488) was placed close to P. aceris and had >99% sequence similarity with the type strain of this species (Table 1). The sequence obtained by direct Sanger PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 6 / 14 Genome of amphibian skin-associated Pigmentiphaga Fig 2. Maximum likelihood phylogenetic tree of selected Pigmentiphaga based on DNA sequences of up to 1478 bp of the bacterial 16S rRNA gene. Sequences of amphibian skin bacteria from an Illumina-based amplicon survey[6] are colored purple; isolates from amphibian skin are colored blue. Red color highlights the sequences of the P. aceris strain used for genome sequencing. Sequences from type or reference strains are boldfaced. Alcaligenes faecalis, the type species of the type genus of Alcaligenaceae, was used as the outgroup. results of a bootstrap analysis (100 replicates) are marked by gray (bootstrap proportion >50%) and black circles (>70%). Note that due to the inclusion of many short sequences from Illumina amplicon analysis, most nodes did not receive strong bootstrap support. https://doi.org/10.1371/journal.pone.0223747.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 7 / 14 Genome of amphibian skin-associated Pigmentiphaga Table 1. NCBI database matches (97–100% sequence identity) to the 16S rRNA gene of Pigmentiphaga aceris (strain Mada1488). Accession # Description Query cover Percent match Isolation Source HM276147.1 Uncultured bacterium clone ncd518h02c1 89% 99.4% Human skin NR_157990.1 Pigmentiphaga aceris strain SAP-32 94% 99.3% tree sap HM270483.1 Uncultured bacterium clone ncd266g01c1 89% 99.2% human skin FJ849553.1 Uncultured bacterium clone SedUMA34 95% 99.2% arctic stream sediment; ultramafic lithology JX067670.1 Alcaligenaceae bacterium SAP706.3 98% 99.1% floral nectar FN421876.1 Uncultured bacterium, clone 2_F01 92% 99.1% phyllosphere of clover HM316492.1 Uncultured bacterium clone ncd307b11c1 89% 99.0% human skin JX067692.1 Alcaligenaceae bacterium SAP773.2 98% 99.0% floral nectar HM316454.1 Uncultured bacterium clone ncd306e07c1 89% 99.0% human skin FJ849534.1 Uncultured bacterium clone SedUMA17 1 95% 98.4% arctic stream sediment; ultramafic lithology MH667611.1 Pigmentiphaga sp. strain IMT-318 94% 97.1% soil (USA) https://doi.org/10.1371/journal.pone.0223747.t001 sequencing of DNA extracted from this isolate agreed fully with the sequence from both 16S copies found in the assembled genome. One amplicon-derived sOTU also the sequence of this isolate. In our global amphibian data set, 139 of these reads came from the sOTU matching the Mada1488 isolate. This sOTU was found on frogs of the genera Anaxyrus, Boophis, Colos- tethus, Craugastor, Gephyromantis, Eleutherodactylus, Lithobates, Mantidactylus, Mantella, and Plethodontohyla. Thus, the bacterium represented by our culture (Mada1488) was not Fig 3. The circular genome of 6,165,255 bp of Pigmentiphaga aceris (strain Mada1488). In blue (circle 1) genes lying on the forward strand are shown and in red (circle 2) those on the reverse strand. In circle 3 tRNA genes are shown in brown, often clustered together with green rRNA genes. Circle 4 shows the GC content (G+C)/(A+T+G+C), whereas in circle 5 a GC skew (G−C)/(G+C) is shown. This map has been created using DNAplotter [44]. https://doi.org/10.1371/journal.pone.0223747.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 8 / 14 Genome of amphibian skin-associated Pigmentiphaga very common in our Illumina dataset. As it was the only cultured Pigmentiphaga from amphibian skin assignable to a known species we nevertheless chose this isolate for genome sequencing, to obtain first data of the genomic background of these bacteria. In culture, this bacterium forms white, glossy colonies with circular form; the elevation is raised and the margins are entire. In a growth inhibition assay (see [15] for methods) the metabolites produced by this bacterium reduced the growth of the amphibian skin pathogen Batrachochytrium dendrobatidis by 60%. BLAST searches in the NCBI nucleotide database revealed that bacterial strains with >97% 16S sequence identity to Mada1488 have been found on human skin, in floral nectar, tree sap, artic stream sediment, and soil (Table 1). The highest identity was found with an uncultured isolate from human skin (99.4% identity), directly fol- lowed by the P. aceris type strain (SAP-32) with 99.3% identity. The complete genome of Pigmentiphaga aceris (Mada1488) consists of a single circular chromosome with 6,165,255 bp and a GC content of 62.1%. Prokka predicted 5,300 coding sequences, 57 tRNA genes, and three rRNA operons (Fig 3). A multiple genome alignment suggests that the new genome shows only limited similarities to the five congeneric genomes available (S1 Fig); however, the five available genomes all belong to closely related strains (all >98% 16S similarity to the type strains of P. kullae and P. daeguensis, which themselves show 99.6% similarity, questioning the distinctness of these two taxa at the spe- cies level). The newly sequenced genome of P. aceris (Mada1488) differs in its gene functions com- pared to other known Pigmentiphaga genomes; more specifically, genes associated with envi- ronmental information processing and cellular processes were more abundant in Mada1488 (S2 Fig). Natural product biosynthetic gene clusters (BGCs) are moderately represented in the newly sequenced genome, and the cluster content differs from that in the other available Pig- mentiphaga genomes (S1 Table). Amongst others, P. aceris (Mada1488) contains a nonriboso- mal peptide syntethase (NRPS) cluster most likely involved in the production of a peptide siderophore similar to enterobactin [45], and a BGC coding for bacteriocin biosynthetic genes, both missing in the other Pigmentiphaga. Bacteriocines are widely occurring, ribosomally pro- duced antimicrobial peptides, presumably with an anti-competitor function [46] and an often narrow activity range against Gram-positive as well as Gram-negative bacteria [47,48]. The most noticeable feature in the comparative Pigmentiphaga genomes but absent in P. aceris (Mada1488) are clusters forβ-lactones, a class of protein-inhibiting natural products with a wide range of activities [49]. Furthermore, P. aceris (Mada1488) lacks genes involved in ectoin biosynthesis. Because ectoins are usually produced to protect the bacteria from environmental extremes like hyperosmotic conditions [50], the lack of ectoins might support this strain being adapted to rather stable environmental conditions. Volatile compounds produced by Pigmentiphaga aceris The ability to produce volatile organic compounds (VOCs) is widespread among bacteria [51] and our data demonstrate this ability also in Pigmentiphaga aceris (Mada1488). GC/MS analy- sis of headspace extracts from this bacterium revealed the release of 21 compounds (S1 Table), among them sulfur-containing volatiles such as methanethiole, dimethyl disulfide (1), dimethyl trisulfide (2), S-methyl ethanethioate (3), S-methyl propanethioate (4), S-methyl 2-methylpropanethioate (5), S-methyl 3-methylbutanethioate (6) and S-methyl phenyletha- nethioate (7), as well asγ-decalactone (8) (S3 Fig). Some these compounds, for example, dimethyl trisulfide and S-methyl 3-methylbutanethioate, have been associated with inhibition of a variety of plant pathogens [52,53], suggesting they may play a role in suppression of amphibian fungal pathogens, such as Bd and Bsal. PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 9 / 14 Genome of amphibian skin-associated Pigmentiphaga Conclusions To our knowledge, Pigmentiphaga aceris (Mada1488) is the first amphibian skin-derived bac- terial isolate with a full genome sequenced. Overall, the genome does not present any out- standing characteristics, in line with the hypothesis that the amphibian cutaneous microbiome mainly consists of generalist species recruited from environmental reservoirs. It is remarkable that bacterial strains most similar to Mada1488 have multiple times been found in plant-asso- ciated microbiomes (tree sap, nectar, phyllosphere). The finding of a Pigmentiphaga similar to the strain described herein on human skin may be explained by a bias of information in genetic databases towards human-derived microbes, but also confirms that these bacteria are not strictly associated to plants only. Yet, it is tempting to relate the apparent common occur- rence of Pigmentiphaga on plants to its high abundance in treefrogs which may have acquired it from plant-associated reservoirs. To test this hypothesis, in-depth analysis of additional bac- teria differentially abundant on arboreal vs. terrestrial amphibians may be rewarding. A wider sampling of genomes represented in amphibian cutaneous microbiomes will be a crucial step to better understand functional properties of these bacterial communities and their potential role in defense against pathogens. Data availability The complete genome sequence of Pigmentiphaga aceris Mada1488 has been deposited at NCBI GenBank under the accession no. CP043046. The version described in this paper is the first version. (BioProject no. PRJNA561098). The 16S sequence of this isolate is archived under accession no. MF525803.1. Accession numbers of sequences used in the phylogenetic analysis are given in the respective tree. Accession numbers for the raw data of amplicon analy- ses are summarized in a previous study [6]. Supporting information S1 Fig. Comparison of genomic composition of the Pigmentiphaga aceris strain isolated from amphibian skin (A) to the five other Pigmentiphaga genomes available: (A) Pigmenti- phaga sp. H8, (B) P. sp. NML030171, (C) P. sp. NML080357, (D) P. kullae K24, (E) P. sp. IMT- 318. The figure shows a multiple genome alignment calculated with Mauve (Darling et al. 2004), using A as reference. Colinear blocks are indicated by identical colors and indicate homologous DNA regions shared by two or more genomes without sequence rearrangements, and are indicated below the black horizontal line if representing reverse complements of the respective sequence of the reference. Note similarities between genomes A-C, larger differ- ences of D and E, and massive differences in the arrangement of the newly sequenced P. aceris genome (F). (JPG) S2 Fig. Comparative summary of gene function across the newly sequenced Pigmentiphaga aceris (red box) and other available genomes from this genus. Pie charts were created directly from BlastKOALA. Colors for a given functional categories are consistent across each chart; categories are ordered by abundance within a given pie chart. (PDF) S3 Fig. Selected volatile compounds released by Pigmentiphaga aceris (Mada1488): metha- nethiole, dimethyl disulfide (1), dimethyl trisulfide (2), S-methyl ethanethioate (3), S-methyl propanethioate (4), S-methyl 2-methylpropanethioate (5), S-methyl 3-methylbutanethioate (6) and S-methyl phenylethanethioate (7), as well asγ-decalactone (8). (PDF) PLOS ONE | https://doi.org/10.1371/journal.pone.0223747 October 11, 2019 10 / 14 Genome of amphibian skin-associated Pigmentiphaga S1 Table. Natural product biosynthetic gene clusters (BGCs) in available Pigmentiphaga genomes as predicted by AntiSMASH. (DOCX) S2 Table. Volatile compounds released by Pigmentiphaga aceris (Mada1488). Numbered compounds 1–8 are those shown in S2 Fig. (DOCX) Acknowledgments We are grateful to the Malagasy authorities for issuing research and export permits for this research. We are indebted to numerous local guides and field assistants that help during field work. We thank Simone Severitt and Carola Berg for technical assistance with laboratory work related to genome sequencing. We thank the Malagasy authorities for permits to collect, export and analyze the amphibian-skin derived bacteria, including full genome sequencing (research authorizations 105N-EA04/MG17from 25 April 2017; collecting permits 182/13/MEF/SG/ DGF/DCB.SA/SCB from 1 August 2013, 248/16/MEEF/SG/DGF/DSAP/SCB.Re from 14 October 2016, and 282/16/MEEF/SG/DGF/DSAP/SCB from 28 November 2016). The work was carried out in the framework of a collaboration accord of the Technische Universita ¨t Braunschweig with the Cellule d’Urgence Chytride de Madagascar and the Universite ´ de Mad- agascar (Mention Biodiversite ´ Animale), with a Material Transfer Agreement (002/ZBA/17/ ZR) from 3 January 2017. Author Contributions Conceptualization: Molly C. Bletz, Boyke Bunk, Cathrin Spro ¨er, Falitiana C. E. Rabemanan- jara, Stefan Schulz, Jo ¨rg Overmann, Miguel Vences. Data curation: Molly C. Bletz, Boyke Bunk, Cathrin Spro ¨er, Silke Reiter, Miguel Vences. Formal analysis: Molly C. Bletz, Boyke Bunk, Peter Biwer, Silke Reiter, Stefan Schulz, Miguel Vences. Funding acquisition: Falitiana C. E. Rabemananjara, Miguel Vences. Methodology: Cathrin Spro ¨ er, Peter Biwer, Silke Reiter, Stefan Schulz. Project administration: Jo ¨rg Overmann. Visualization: Molly C. Bletz. Writing – original draft: Molly C. Bletz, Miguel Vences. Writing – review & editing: Molly C. Bletz, Boyke Bunk, Peter Biwer, Silke Reiter, Falitiana C. E. Rabemananjara, Stefan Schulz, Jo ¨ rg Overmann, Miguel Vences. References 1. Fisher MC, Garner TW, Walker SF. Global emergence of Batrachochytrium dendrobatidis and amphib- ian chytridiomycosis in space, time, and host. Annu Rev Microbiol. 2009; 63: 291–310. https://doi.org/ 10.1146/annurev.micro.091208.073435 PMID: 19575560 2. Stegen G, Pasmans F, Schmidt BR, Rouffaer LO, Van Praet S, Schaub M, et al. 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