High quality draft genome sequence of Brachymonas chironomi AIMA4T (DSM 19884T) isolated from a Chironomus sp. egg mass

Brachymonas chironomi strain AIMA4T (Halpern et al., 2009) is a Gram-negative, non-motile, aerobic, chemoorganotroph bacterium. B. chironomi is a member of the Comamonadaceae, a family within the class Betaproteobacteria. This species was isolated from a chironomid (Diptera; Chironomidae) egg mass, sampled from a waste stabilization pond in northern Israel. Phylogenetic analysis based on the 16S rRNA gene sequences placed strain AIMA4T in the genus Brachymonas. Here we describe the features of this organism, together with the complete genome sequence and annotation. The DNA GC content is 63.5%. The chromosome length is 2,509,395 bp. It encodes 2,382 proteins and 68 RNA genes. Brachymonas chironomi genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.


Introduction
Strain AIMA4 T (= LGM 24400 T = DSM 19884 T ), is the type strain of Brachymonas chironomi, one out of two species in the genus Brachymonas. The genus Brachymonas was formed by Hiraishi et al. [1] while characterizing rhodoquinone-containing bacteria that had been isolated from soybean crude waste sludge in Japan. Strain AIMA4 T , was isolated from an insect egg mass (Chironomus sp.) that was sampled from a waste stabilization pond in northern Israel [2]. Chironomids (Arthropoda; Insecta; Diptera; Chironomidae; Chironomus sp.) inhabit virtually every type and condition of aquatic habitats. They undergo a complete metamorphosis of four life stages (egg, larva, pupa and adult that emerges into the air) [3]. Eggs are laid in an egg mass at the water's edge. Each egg mass contains hundreds of eggs. Chironomid egg masses were found to harbor Vibrio cholerae and Aeromonas spp. [3][4][5][6][7][8][9][10]. V. cholerae degrades chironomid egg masses by the secreted haemagglutinin protease (HAP) [11,12]. Strain AIMA4 T was isolated in the course of a study that investigated endogenous bacterial communities that inhabit chironomid egg masses [2,13,14]. The species epithet chironomi was derived from the non-biting midge insect Chironomus (Diptera; Chironomidae), from where this species was isolated. Strain AIMA4 T didn't show the ability to degrade the egg masses like it was found for V. cholerae.
Here we describe a summary classification and a set of the features of Brachymonas chironomi strain AIMA4 T (DSM 19884 T ), together with the genome sequence description and annotation.

Classification and features
A taxonomic study using a polyphasic approach placed B. chironomi strain AIMA4 T in the genus Brachymonas within the family Comamonadaceae ( Figure 1). The family Comamonadaceae comprises a larger number of genera (as shown in Figure 1) and a larger variety of species and phenotypes [15,16].

Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [17][18][19]. Sequencing of B. chironomi strain AIMA4 T is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project [20] which aims in increasing the sequencing coverage of key reference microbial genomes [21]. The genome project is deposited in the Genomes On-Line Database [22] and the permanent draft genome sequence is deposited in GenBank. Sequencing, finishing and Figure 1 Phylogenetic tree highlighting the position of Brachymonas chironomi relative to the type strains of the other species within the family Comamonadaceae. The sequence alignments were performed by using the CLUSTAL W program and the tree was generated using the maximum likelihood method in MEGA 5 software. Bootstrap values (from 1,000 replicates) greater than 50% are shown at the branch points. The bar indicates a 1% sequence divergence. annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [23]. A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation B. chironomi strain AIMA4 T , DSM 19884 T , was grown in DSMZ medium 1 (Nutrient Agar), at 28°C [24]. DNA was isolated from 0.5-1 g of cell paste using JetFlex Genomic DNA Purification Kit (GENOMED) following the standard protocol as recommended by the manufacturer, however with additional 50 μl protease K (20 mg/ml) during digest for 60 min. at 58°C. Protein precipitation was done with additional 200 μl Protein Precipitation Buffer, followed by over night incubation on ice. DNA is available through the DNA Bank Network [25].

Genome sequencing and assembly
The draft genome of B. chironomi strain AIMA4 T was generated using the Illumina technology [23,26]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 14,014,260 reads totaling 2,102.1 Mb. All general aspects of library construction and sequencing performed at the JGI can be found at the institute website [27]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts [28]. Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [29], (2) 1-3 Kbp simulated paired end reads were created from Velvet Contigs using wgsim [30], (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG [31]. Parameters for assembly steps were: (1) Velvet (velveth: 63 -shortPaired and velvetg: −very clean yes -export-

Genome annotation
Genes were identified using Prodigal [32] as part of the DOE-JGI genome annotation pipeline [33,34], following by a round of manual curation using the JGI GenePRIMP pipeline [35]. The predicted CDSs were translated and searched against the Integrated Microbial Genomes (IMG) non-redundant database, UniProt, TIGERFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes-Expert Review (IMG-ER) platform [36].

Genome properties
The assembly of the draft genome sequence consists of 36 scaffolds amounting to 2,509,395 bp, and the G + C content is 63.5% (Table 3). Of the 2,450 genes predicted, 2,382 were protein-coding genes, and 68 RNAs. The majority of the protein-coding genes (85.5%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Insights from the genome sequence
Strain AIMA4 T was isolated from chironomid egg masses. Using pyrosequencing method, we have recently shown that the prevalence of Brachymonas in the endogenous bacterial communities of chironomid egg masses and larva was 0.04% and 0.006%, respectively [37]. Chironomid tolerance towards pollution is well documented [38]. Senderovich and Halpern [37,39], demonstrated by using Koch's postulates that endogenous bacteria in chironomids have a role in protecting the insect from toxicants. Although B. chironomi was isolated from chironomid egg masses, its features regarding its protective potential have never been examined. Nevertheless, its genome reveals the potential   a Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). Evidence codes are from the Gene Ontology project [48]. b The only carbon source that was positive for this strain, out of all carbon sources that were tested (strain AIMA4 T does not use carbohydrates, not even glucose) [2].
of this species to protect its host in polluted environments. Genes encoding arsenate detoxification are present in B. chironomi strain AIMA4 T . These genes include an arsenical resistance gene cluster with candidates for transcriptional regulator, ArsR; arsenical resistance operon trans-acting repressor, ArsD; arsenite efflux ATP-binding protein, ArsA and a hypothetical arsenic resistance protein (ACR3 family). A gene for arsenate reductase (ArsC family) is present in a different operon. More genes which may indicate the potential of this bacterium to tolerate or detoxify metals are: copper resistance protein D, CopD; copper chaperone, copper-resistance protein, CopA; copper (or silver) translocating P-type ATPase; uncharacterized lipoprotein NlpE involved in copper resistance; magnesium Mg(2+) and cobalt Co(2+) transport protein, CorA. Moreover, two genes encoding ABC-type transport system involved in resistance to organic solvents, auxiliary and periplasmic components are also present.
The genome of B. chironomi strain AIMA4 T reveals the potential of the species to produce a polysaccharide capsule. It includes two gene clusters with candidates for capsule polysaccharide export protein, periplasmic protein involved in polysaccharide export, ABC-type polysaccharide/polyol phosphate transport system, ATPase component, ABC-type polysaccharide/polyol phosphate export systems, permease component and predicted glycosyltransferase involved in capsule biosynthesis. Another feature that is found in the genome of B. chironomi AIMA4 T is its potential to produce a pilus (or pili). The following predicted genes indicate this ability; type IV pilus assembly protein PilB; type IV pilus secretin PilQ; Tfp pilus assembly proteins PilP, PilO and PilV; type IV prepilin peptidase; prepilin-type N-terminal cleavage/ methylation domain and pilus retraction ATPase PilT (indicating the ability of twitching motility).
Tolerance of 2.5% NaCl was described for strain AIMA4 T by Halpern et al. [2]. The presence of ABC-type proline/glycine betaine transport system in the genome may explain the way this species can tolerate high salt concentrations. In respect to the ampicillin (beta-lactam) antibiotic resistance, the genome encodes one beta-lactamase class B and a negative regulator of beta-lactamase expression. Three genes encoding two component transcriptional regulators (LuxR family), can be found in the genome of strain AIMA4 T and demonstrate quorum sensing skills.

Conclusions
In the current study, we characterized the genome of B. chironomi strain AIMA4 T that was isolated from a chironomid egg mass [2]. B. chironomi belongs to the family Comamonadaceae (order Bukholderiales; class Betaproteobacteria) (Figure 1). Members of this family are known for their ability to cope with harsh environmental condition such as high concentration of toxic metals and other pollutants like aromatic compounds or polymers [e.g. poly (3-hydroxybutyrate-co-3-hydroxyvalerate) [16]. Likewise, the genome of strain AIMA4 T reveals the potential of this species to cope with toxic metals. These demonstrate that B. chironomi may have a role in protecting its aquatic host (chironomids) in polluted environments.

Competing interest
The authors declare that they have no competing interests.