Draft genome of the Arabidopsis thaliana phyllosphere bacterium, Williamsia sp. ARP1
© Horn et al. 2016
Received: 16 April 2015
Accepted: 21 December 2015
Published: 16 January 2016
The Gram-positive actinomycete Williamsia sp. ARP1 was originally isolated from the Arabidopsis thaliana phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus Williamsia and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat.
KeywordsDraft genome Phyllosphere Williamsia sp. ARP1 Adaption Whole genome sequencing Next generation sequencing Assembly Annotation Arabidopsis thaliana
The genus Williamsia was originally proposed by Kämpfer et al. in 1999  to accommodate an unusual mycolic-acid containing actinomycete. Members of the genus Williamsia are Gram-positive, non-spore forming, and form round, orange colonies. Their cell shape is coccoid- or rod-like . The genus Williamsia forms a distinct group within actinomycetes of the suborder Corynebacterineae , which also comprises the genera Corynebacterium , Dietzia , Gordonia , Mycobacterium , Nocardia , Rhodococcus , Skermania , Tsukamurella and Turicella . Based on the mycolic-acid profile with carbon chain lengths ranging from 50 to 56, the genus Williamsia is likely to be placed between the genera Gordonia and Rhodococcus . At the time of writing, only one other draft genome of Williamsia sp. D3 was publicly available  and nine species of this taxon were recognized with valid scientific names: Williamsia deligens , Williamsia faeni , Williamsia limnetica , Williamsia marianensis , Williamsia maris , Williamsia muralis , Williamsia phyllosphaerae , Williamsia serinedens  and Williamsia sterculiae . Further this genus has been linked with the degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine in soils as a sole nitrogen source , the degradation of carbonyl sulfide in soils  and polychlorinated biphenyls in tree habitats . Williamsia was isolated from various sources, including indoor building material , human blood  and following pulmonary infections , oil-contaminated and Antarctic soils [4, 11], extreme environments as glacier ice , deep sea sediments of the Mariana Trench , hay meadows , and the rare soil biosphere . Besides, Williamsia was also reported as an endophyte of grey box eucalyptus tree roots  and as an epiphytic bacterium residing in the phyllosphere of white clover .
The phyllosphere, known as the aerial surface of plant leaves, is a short-lived environment  to diverse microorganisms of various taxonomic groups comprising bacteria, filamentous fungi, yeasts, viruses and protists. The phyllosphere presents a challenging environment for microbial colonizers with respect to climatic conditions, UV radiation, desiccation, water availability, reactive oxygen species, and in terms of antimicrobial compounds produced by the plant or possibly also microbes [21–25]. Additionally, the wax composition of the cuticle, surface characteristics such as stomata and veins affect nutrient availability and leaching, as they are likely to retain more water [23, 26].
Here, we present a summary, classification and general physiological features of the strain Williamsia sp. ARP1 together with the genomic sequencing, assembly, annotation, and its putative adaptions to the phyllosphere.
Classification and features
Classification and general features of Williamsia sp. ARP1 
Species Williamsia sp.
(Type) strain: ARP1
Coccoid to rod-like
pH range; Optimum
198 m above sea level
Genome sequencing information
Genome project history
One Illumina paired-end library (400 bp insert size)
SPAdes 3.0, SSPACE 3.0
Gene calling method
GenBank Date of Release
July 1, 2015
Source Material Identifier
Growth conditions and genomic DNA preparation
Several plants were collected from a Landsberg erecta (Ler) population of Arabidopsis thaliana from the Botanical Garden (University of Würzburg, June 2012). Leaf washings  were used for inoculation of minimal media with C16 alkane (Sigma-Aldrich) as the sole carbon source in order to enrich for bacteria with the ability to degrade long-chain hydrocarbons. Aliquots were streaked (in duplicate) on agar plates prepared with minimal media and supplemented with C22 alkane (Sigma-Aldrich). This procedure provided a total of 17 isolates, of which most belonged to the genus Rhodococcus and two to genus Williamsia .
Williamsia sp. ARP1 was grown in 10 ml Luria-Bertani broth medium (10 g peptone, 5 g yeast extract, 5 g NaCl in 1000 ml demineralized water) for 24 h at 30 °C and rotary shaking at 180 rpm. For genomic DNA isolation, 2 ml of overnight culture were centrifuged at 8000 rpm for 5 min at room temperature. The pellet was rinsed in 1 ml TNE (1 ml 1 M Tris pH 8, 0.2 ml 5 M NaCl, 2 ml 0.5 M EDTA pH8, and 100 ml demineralized water) and resuspended in 270 μl TNEx (TNE, 1 % v/v TritonX-100) and 25 μl lysozyme (10 mg/ml). After a 30 min incubation at 37 °C, 50 μl of proteinase K (20 mg/ml) were added. After an incubation of 2 h and 55 °C, 15 μl of 5 M NaCl and 500 μl of 100 % EtOH were added. The mixture was then centrifuged at 13,000 rpm for 15 min at room temperature, rinsed with 70 % EtOH, air dried and resuspended in 150 μl TE buffer. The quality and quantity of the extracted DNA was evaluated by 0.8 % (w/v) agarose gel electrophoresis, by measuring absorption ratios 260/280 and 260/230 with a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific) and an additional Qubit dsDNA HS assay (Life Technologies).
Genome sequencing and assembly
High molecular weight DNA was cleaned with the DNA Clean & Concentrator kit (Zymo Research). The genomic DNA library for the Illumina platform was generated using Nextera XT (Illumina Inc.) according to the manufacturer’s instructions. After tagmentation, size-selection was performed using NucleoMag NGS Clean-up and Size Select (Macherey-Nagel) to obtain a library with median insert-size around 400 bp. After PCR enrichment, the library was validated with a high-sensitivity DNA chip and Bioanalyzer 2100 (both Agilent Technologies, Inc.) and additionally quantified using the Qubit dsDNA HS assay (Life Technologies). Sequencing was performed on a MiSeq device using v2 2 × 250 bp chemistry, and the genome was multiplexed together with ten other bacterial genomes from other sources. Multiplexing was done via dual indexing, with the official Nextera indices N706 and S503 for Williamsia sp. ARP1.
In total, 1,304,294 (mean length 237.86 bp) raw paired-end sequences were subjected to the Trimmomatic software  for adapter and quality trimming (mean Phred quality score ≥30), filtering of sequences containing ambiguous bases and a minimum length of 200 bp. Subsequently, human and viral decontamination was excluded using DeconSeq . The 1,287,247 (mean length 236.95 bp) remaining paired-end sequences were assembled with five different tools: a5-miseq , IDBA-UD , MaSuRCA , SPAdes  and Velvet . In order to obtain the most reliable contigs, all assemblies were evaluated with QUAST , REAPR , ALE  and Feature Response Curves . According to those evaluations, we have selected SPAdes assembler with enabled pre-correction and k-mer sizes ranging from 15 to 125 (step size of 10) as the best assembly. Obtained contigs were extended with remaining reads where possible. This led to 50 large contigs (≥1000 bp, N50: 140,970 bp, longest contig: 428,355 bp) and an overall genome size of 4,745,080 bp (GC content: 68.63 %). As a final step, the contigs were ordered according to the nearest related complete genome by functional content using Mauve in 12 iterations . As Williamsia sp. D3 was only available as a draft genome, Gordonia bronchialis was used for this step.
Open reading frames were identified using Prodigal  followed by manual correction. The predicted coding sequences were translated into amino acid sequences and searched against COG position-specific scoring matrices obtained from the Conserved Domains Database  using RPS-BLAST . Comparisons with TIGRFAM, Pfam, and PANTHER databases were performed with the InterProScan pipeline . Only matches with an e-value ≤1∗10−2, ≥25 % identity and a minimum of 70 % alignment length to the target sequence were maintained. During this run, matches were also mapped to Gene Ontology terms. Additional gene prediction and functional annotation was performed with the Integrated Microbial-Genomes Expert Review  and the Rapid Annotation using Subsystem Technology webserver [52, 53]. Features as tRNA, rRNA, ncRNA, transmembrane helices, signal peptides, CRISPR elements and secondary metabolite gene clusters were predicted using tRNAscan-SE , RNAmmer , INFERNAL  and Prokka’s prokaryotic RNA covariance models , TMHMM , SignalP  PILER-CR  and antiSMASH . Searching for essential genes  was performed using HMMER3 . Ortholog detection between Williamsia sp. ARP1 and three other genomes were carried out with InParanoid  whereas the mean percentage of nucleotide identity among the found orthologous genes was calculated using BLASTn. Average nucleotide identities between Williamsia sp. ARP1 and reference genomes were calculated with JSpecies .
% of total
Genome size (bp)
DNA coding (bp)
DNA G+C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with TIGRFAM domains
Genes with signal peptides
Genes with transmembrane helices
Insights from the genome sequence
Number of genes associated with general COG functional categories
Translation, ribosomal structure, and biogenesis
RNA processing and modification
Replication, recombination, and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Used actinomycete reference genomes in this study
Genome Size [Mbp]
UV radiation may impose stress on bacteria inhabiting plant leaves. In this context, a cluster of genes synthesizing mycosporins was found. These secondary metabolites are known to protect cells by absorbing UV light without generating reactive oxygen species (ROS) [67, 68]. Additionally, genes involved in the repair of UV-damaged DNA were found, which comprise DNA photolyases, the UvrABC endonuclease enzyme complex, and the DNA helicase II UvrD of the UvrABC system. The red color of Williamsia sp. ARP1 might protect it against photo-oxidative stress as pigmentation is known to be a common feature of phyllosphere colonizers . All genes of the carotenoid biosynthetic pathway were found, consisting of a geranylgeranyl diphosphate synthase, a phytoene synthase, a phytoene desaturase, a carotene desaturase and a lycopene-β-cyclase. The products of this pathway are lycopene and β-carotene, both producing orange to red pigments.
Further adaptions to an epiphytic lifestyle are encoded on genes responding to reactive oxygen species (ROS; e.g. hydrogen peroxide, superoxide, hydroperoxil radical), which are products of the plant defense [70, 71]. Here, two genes encoding for glutathione peroxidases, two superoxide dismutases with copper/zinc or manganese as active site, two glutaredoxins, three thioredoxins, and one catalase were found.
Regarding temperature shifts, the heatshock chaperones DnaK, DnaJ and GrpE and the cold shock protein CspC were identified.
ABC transporters for the uptake of carbohydrates such as ribose, glycerol or maltose, amino acids such as methionine, known plant photosynthates such as fructose, and enzymes for fructose utilization were identified. Also, genes mediating the uptake of choline and subsequent biosynthesis (choline dehydrogenase, betaine-aldehyde dehydrogenase) of the osmoprotectant betaine were found.
Trehalose is a compatible solute and known to prevent cells from desiccation and water loss . Eight genes encoding for the biosynthesis pathway (Malto-oligosyltrehalose synthase, 1,4-alpha-glucan (glycogen) branching enzyme, GH-13-type trehalose-6-phosphate phosphatase, putative glucanase glgE, malto-oligosyltrehalose trehalohydrolase, glycogen debranching enzyme alpha, alpha-trehalose-phosphate synthase, glucoamylase) were identified.
The isolate ARP1 was isolated from the Arabidopsis thaliana phyllosphere. Phylogenetic analysis based on the 16S rRNA gene confirmed its affiliation to the genus Williamsia . However genomic properties also showed close similarities to Gordonia , as derived from GC content, COGs, and average nucleotide identities. Thus, an unequivocal delinearization based on the functional genomics level was not possible, which may be due to the underrepresentation of genomes from this genus. The genomic features of strain ARP1 would be consistent with a lifestyle within the phyllosphere, including putative adaptions to UV radiation, heat and cold shock, desiccation and oxidative stress. With this study, we provide novel genomic insights into the rarely sequenced genus Williamsia and discuss its putative adaptations to the phyllosphere habitat.
We thank Dr. Eva E. Reisberg for original isolation of the strain, Christine Gernert and Srikkanth Balasubramanian for technical assistance, Wiebke Sickel for library preparation, the Department of Human Genetics for access to the MiSeq device, and Daniela Bunsen for help with the electron microscope (all University of Würzburg). This work was financially supported by the DFG Graduiertenkolleg GK1342 (TP A8).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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