Introduction

The genus Chitinophaga belongs to the family Chtiniphagaceae (phylum Bacteroidetes ) alongside with the genera Arachidicoccus , Asinibacterium , Balneola , Cnuella , Crenotalea , Ferruginibacter , Filimonas , Flaviaesturariibacter , Flavihumibacter , Flavisolibacter , Flavitalea , Gracilimonas , Heliimonas , Hydrotalea , Lacibacter , Niabella , Niastella , Parasediminibacterium , Parasegetibacter , Sediminibacterium , Segetibacter , Taibaiella , Terrimonas , Thermoflavifilum and Vibriomonas. The genus Chitinophaga is widely distributed in the environment and strains of this genus have been isolated from pine trees, soil, rhizosphere soil, roots, vermicompost and weathered rock [1]. Twenty-four species belonging to the genus Chitinophaga have been described [2], and only the type species of the genus C. pinensis has the complete genome sequenced [3].

Pinus pinaster trees from Central Portugal present a diverse endophytic microbial community. Strain A37T2T was isolated as part of the endophytic microbiome of pine trees affected by Pine Wilt Disease (PWD) which is a world devastating disease, consequence of Bursaphelenchus xylophilus colonization in pine trees [4]. Here, we show the second genome of the genus Chitinophaga , a draft genome of Chitinophaga costaii A37T2T, previously isolated as endophyte of Pinus pinaster affected by PWD [1].

Organism information

Classification and features

The type strain A37T2T (=CIP 110584 T =LMG 27458 T), was isolated from tree trunk of a Pinus pinaster tree affected by PWD and it described as Chitinophaga costaii (family Chitinophagaceae , phylum Bacteroidetes ) [1]. It was Gram-stain-negative, facultative anaerobic, non-motile, formed rod-shaped cells, 0-5-1 μm in diameter and 1-8 μm in length after 48 h on R2A agar media (Fig. 1). Showed capacity to grow on R2A agar medium at 15-45 °C (optimum, 26-30 °C), at pH 5.5-8.0 (optimum, pH 7) and supplemented with up to 1% (w/v) NaCl (optimum without NaCl). The major fatty acids (>25%) showed by the strain A37T2T are saturated iso-C15: 0 and unsaturated C16: 1 ω5c . The major polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids and one unidentified lipid. No glycolipid was detected. The menaquinone 7 (MK-7) was shown as the major respiratory lipoquinone. The determined DNA G + C content of the C. costaii A37T2T was 46.6 mol%. Key features of this microorganism are summarized in Table 1. A phylogenetic tree based on the 16S rRNA gene sequence of this strain and its closest relative members are given in Fig. 2. The sequences were aligned by SINA (v1.2.9) using the SILVA SEED as reference alignment [5]. Sequences were included in 16S rRNA-based Living Tree Project (LTP) release 115 database [6] by parsimony implemented in the ARB software package version 5.5 [7]. Evolutionary distances were calculated [8] and phylogenetic dendrograms were constructed using the neighbor-joining [9] and Randomized Axelerated Maximum Likelihood (RAxML) method with GTRGAMMA model [10] included in the ARB software [7]. Trees topologies were evaluated by performing bootstrap analysis [11] of 1000 data sets by using ARB software package.

Fig. 1
figure 1

Scanning electron micrograph of C. costaii A37T2T after 48 h of growth on R2A agar plates at 30 °C

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Table 1 Classification and general features of Chitinophaga costaii A37T2T according to the MIGS recommendations [26]
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Fig. 2
figure 2

Phylogenetic tree based on a comparison of the 16S rRNA gene sequence of strain A37T2T and the other type strains within the family Chitinophagaceae. The tree was created using the maximum likelihood method (RAxML). The numbers on the tree indicate the percentages of bootstrap sampling, derived from 1000 replications; values below 50% are not shown. Symbol (•) indicates node branches conserved when the tree was reconstructed using the neighbor-joining method. The isolate characterized in this study is indicated in bold. Scale bar, 1 inferred nucleotide substitution per 100 nucleotides

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Genome sequencing information

Genome project history

This Whole Genome Shotgun project has been deposited at ENA under the accession numbers FMAR01000001-FMAR01000056 and in the Integrated Microbial Genomes database (IMG) with Biosample ID SAMN05216457 [12]. The genome sequencing of this organism is part of the Genomic Encyclopedia of Bacteria and Archaea [13], 1000 Microbial Genomes project, phase III (KMG-III) [14], at the U.S. Department of Energy, Joint Genome Institute (JGI). The project information and its association with the MIGS is summarized in Table 2.

Table 2 Project information
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Growth conditions and genomic DNA preparation

The strain A37T2T was grown on R2A agar media at 30 °C during 48 h and its genomic DNA was extracted using the E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions.

Genome sequencing and assembly

The draft genome of C. costaii A37T2T was generated at the DOE Joint Genome Institute (JGI) using the Illumina technology [15]. An Illumina 300 bp insert standard shotgun library was constructed and sequenced using the Illumina HiSeq–2500 1 TB platform, generating 9,965,394 reads totaling 1494.8 Mbp. All general aspects of library construction and sequencing performed at the JGI can be found at [16]. All raw Illumina sequence data was filtered using BBDuk [17], which removes known Illumina artifacts and PhiX. Reads with more than one “N” or with quality scores (before trimming) averaging less than 8 or reads shorter than 51 bp (after trimming) were discarded. Remaining reads were mapped to masked versions of human, cat and dog references using BBMAP [17] and discarded if identity exceeded 95%. Sequence masking was performed with BBMask [17]. Following steps were then performed for assembly: (1) artifact filtered Illumina reads were assembled using SPAdes (version 3.6.2) [18]; (2) assembled contigs were discarded if length was <1 kbp. Parameters for the SPAdes assembly were ––cov–cutoff auto ––phred–offset 33 –t 8 –m 40 ––careful –k 25,55,95 ––12.

Genome annotation

Protein-coding genes were identified using Prodigal [19], as part of the DOE-JGI genome annotation pipeline [20]. Additional gene prediction analysis and manual functional annotation were performed within the Integrated Microbial Genomes Expert Review system (IMG-ER), which provides tools for analyzing and reviewing the structural and functional annotations of genomes in a comparative context [12, 21]. Genome annotation procedures are detailed in Markowitz et al. [12] and references therein. Briefly, the predicted CDSs were translated and used to search the NCBI nonredundant database, UNIProt, TIGRFam, Pfam, KEGG, COG and InterPro databases. Transfer RNA genes were identified using the tRNAScan-SE tool and other non-coding RNAs were found using INFERNAL. Ribosomal RNA genes were predicted using hmmsearch against the custom models generated for each type of rRNA.

Genome properties

The draft genome sequence of C. costaii strain A37T2T comprised 5,074,440 bp, based on 1494.8 Mbp of Illumina data with a mapped coverage of 297.2-fold of the genome. The final draft assembly contained 56 contigs in 56 scaffolds with more than 1052 bp. The G + C content was 47.6%. The genome encoded 4204 putative coding sequences (CDSs) (Table 3). Fifty four % of the CDSs, corresponding to 2284 proteins, could be assigned to Cluster of Orthologous Groups (COG) families [22] (Table 4). The draft genome sequence contained four ribosomal RNAs and 50 tRNAs loci (Table 3).

Table 3 General genome features of Chitinophaga costaii A37T2T
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Table 4 Number of genes associated with general COG functional categories
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The Average Nucleotide Identity between C. costaii A37T2T and C. pinensis DSM 2588 T was 70.9 based on 1593 of total Bidirectional Best Hits, using MiSI [23]. Figure 3 shows the circular graph of the genome of C. costaii A37T2T query to the only available complete genome of the genus Chitinophaga , C. pinensis DSM 2588 T [2].

Fig. 3
figure 3

The genome of Chitinophaga costaii A37T2T. From outside to the center: genes of genome of C. pinensis DSM 2588T and its similarity with the genome of C. costaii (50-100%), GC content of C. costaii A37T2T, GC skew of C. costaii A37T2T, genome of C. costaii A37T2T

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The comparison between the draft genome of C. costaii A37T2T and the complete genome of C. pinensis DSM 2588 T showed 1145 unique genes only present in the genome of C. costaii A37T2T and 3493 unique genes only present in the genome of C. pinensis DSM 2588 T. Focused on the unique genes present on the genome of strain A37T2T it was possible to assigned 109 COG, summarized in Table 5.

Table 5 Unique Cluster Orthologous Groups present in the genome of C. costaii A37T2T
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Insights from the genome sequence

The draft genome sequence of C. costaii A37T2T carries multiple genes involved in cellulolytic activity, including one gene encoding the enzyme cellulase (SCC15587) and six genes encoding for β-glucosidase (SCB82491, SCB92249, SCB95191, SCC15475, SCC57293, SCC61957), which might be involved in cellulose degradation in the environment and in biotechnological processes [24]. As expected for this genus, four genes encoding chitinases (SCC19468, SCC19522, SCC23114, SCC34676) were found. Six genes encoded lysophospholipase L1, including representatives of both of size groups, i.e. less than 300aa (SCB77875, SCC28514, SCC37316, SCC54197) and less than 500aa (SCB98645, SCC50813). Moreover, the genome of strain A37T2T encoded 1-aminocyclopropane-1-carboxylate deaminase (SCB80758), a hydrolase that might be involved in lowering ethylene levels in the plant [25]. In summary, the genome sequence suggested multiple potentials for the strain to interact with the plant metabolism.

Conclusions

This work contributed to the knowledge of the genome sequence of the type species of C. costaii A37T2T (=CIP 110584 T, =LMG 27458 T), an endophyte of P. pinaster affected by PWD. The genome encoded multiple genes involved in cellulolytic activity and the sequence provided insights into the role of bacteria in PWD. As there are only a few bacterial genomes related to PWD, this work provides a contribution to this field.