High quality draft genome sequence of Leucobacter chironomi strain MM2LBT (DSM 19883T) isolated from a Chironomus sp. egg mass

Leucobacter chironomi strain MM2LBT (Halpern et al., Int J Syst Evol Microbiol 59:665-70 2009) is a Gram-positive, rod shaped, non-motile, aerobic, chemoorganotroph bacterium. L. chironomi belongs to the family Microbacteriaceae, a family within the class Actinobacteria. Strain MM2LBT was isolated from a chironomid (Diptera; Chironomidae) egg mass that was sampled from a waste stabilization pond in northern Israel. In a phylogenetic tree based on 16S rRNA gene sequences, strain MM2LBT formed a distinct branch within the radiation encompassing the genus Leucobacter. Here we describe the features of this organism, together with the complete genome sequence and annotation. The DNA GC content is 69.90%. The chromosome length is 2,964,712 bp. It encodes 2,690 proteins and 61 RNA genes. L. chironomi genome is part of the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) project.

L. chironomi MM2LB T , was isolated from an insect egg mass (Chironomus sp.) that was sampled from a waste stabilization pond in northern Israel [1]. Chironomids (Insecta; Diptera; Chironomidae; Chironomus sp.), also known as the non biting midges, are aquatic insects. They undergo a complete metamorphosis of four life stages; egg, larva, pupa and adult that emerges into the air. The eggs are deposited by the adult female at the water's edge in egg masses which contain hundreds of eggs [11]. Chironomid egg masses were found as natural reservoirs of Vibrio cholerae and Aeromonas species [11][12][13][14][15][16][17]. Strain MM2LB T was isolated in the course of a study that explored the endogenous bacterial communities in chironomid egg masses [1]. Using 454-pyrosequencing technique, Senderovich & Halpern [18], showed that the prevalence of Leucobacter in chironomid egg masses and larval endogenous bacterial communities is 0.1% and 0.2%, respectively.
Here we describe a summary classification and a set of the features of L. chironomi, together with the genome sequence description and annotation.

Organism Information
Classification and features A taxonomic study using a polyphasic approach placed L. chironomi strain MM2LB T in the genus Leucobacter within the family Microbacteriaceae (order; Actinomyecetales, class; Actinobacteria, phylum; Actinobacteria) ( Figure 1). The family Microbacteriaceae comprises more than 40 genera and a large variety of species and phenotypes.

Genome sequencing information
Genome project history L. chironomi MM2LB T , was selected for sequencing due to its phylogenetic position [19][20][21], and is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG) study [22] which aims not only to increase the sequencing coverage of key reference microbial genomes [23] but also to generate a large genomic basis for the discovery of genes encoding novel enzymes [24]. The sequencing project is accessible in the Genomes OnLine Database [25] and the genome sequence is deposited in GenBank. Sequencing, finishing and annotation were accomplished by the DOE Joint Genome Institute (JGI) [26] using state of the art  Figure 1 Phylogenetic tree highlighting the position of Leucobacter chironomi relative to the type strains of the other species within the genus Leucobacter. 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 [41]. Bootstrap values (from 1,000 replicates) greater than 50% are shown at the branch points. The bar indicates a 1% sequence divergence. genome sequencing technology [27]. The project information is summarized in Table 2.
Growth conditions and genomic DNA preparation L. chironomi MM2LB T , DSM 19883, was grown in Trypticase Soy Yeast Extract medium (DSMZ medium 92) at 28°C [28]. DNA was isolated from 0.5-1.0 g of cell paste using Masterpure DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with additional 7.5 units of each of the following enzymes achromopeptidase, lysostaphin, mutanolysin and 2100 units of lysozyme, incubated for one hour at 37°C, followed by addition of 1 μl proteinase K and incubation for 20 min at 70°C for cell lysis. DNA is available through the DNA Bank Network [29].

Genome sequencing and assembly
The draft genome of L. chironomi DSM 19883 T was generated at the DOE Joint genome Institute (JGI) using the Illumina technology [30]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 13,901,154 reads totaling 2,085.2 Mb. All general aspects of library construction and sequencing performed at the JGI can be found at the Institute web site [25]. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts (Mingkun L, et al., unpublished, 2011). Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet [31], (2) 1-3 kb simulated paired end reads 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 [53]. b The only carbon source that was positive for this strain, out of all carbon sources that were tested (strain MM2LB T does not use carbohydrates, not even glucose) [1].

Genome annotation
Genes were detected using the Prodigal software [34] at the DOE-JGI Genome Annotation pipeline [35,36]. The CDSs predicted were translated and searched against the Integrated Microbial Genomes (IMG) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction and functional annotation analysis was carried out in the Integrated Microbial Genomes (IMG-ER) platform [37].

Genome properties
The assembly of the draft genome sequence consists of 27 scaffolds amounting to 2,964,712 bp, and the G + C content is 69.9% (Table 3). Of the 2,751 genes predicted, 2,690 were protein-coding genes, and 61 RNAs. The majority of the protein-coding genes (79.5%) 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
Senderovich and Halpern [18,38], demonstrated that endogenous bacteria in chironomids have a role in protecting their insect host from toxic metals. L. chironomi strain MM2LB T , which was isolated from a chironomid egg mass was found to tolerate up to 18 mM Cr(VI) [1]. Other Leucobacter species like L. alluvii, L. aridicollis, L. chromiireducens, L. chromiiresistens, L. komagatae, L. luti and L. salisicius, have also been found to be resistant to hexavalent chromium [1,2,5,39,40]. A chromate membrane transport protein A (ChrA) was detected in the genome of the chromate-resistant bacterium, L. salsicius M1-8 T [40]. However, this gene or other genes with chromium reduction predicted functions were not identified in L. chironomi MM2LB T genome. Nevertheless, three genes for ABC-type metal ion transport system (permease, ATPase and periplasmic components), were detected in the genome of strain MM2LB T . These genes may have a role in L. chironomi chromium tolerance.
More genes that may indicate the potential of strain MM2LB T to tolerate or detoxify metals, were also detected. Among them are genes for arsenical resistance: arsenical-resistance protein (arsB); arsenite efflux pump ACR3 and related permeases. Other genes suggest the potential of L. chironomi to survive in the presence of other toxic metals: copper chaperone; copper-(or silver)-translocating P-type ATPase; heavy metal-(Cd/Co/Hg/Pb/Zn)translocating P-type ATPase and transcriptional regulator (ArsR family) which is involved in stress-response to heavy metal ions.
Three genes encoding drug resistance transporters are found in strain MM2LB T genome: drug resistance transporter Bcr/CflA subfamily; multidrug resistance  efflux transporter and drug resistance transporter EmrB/QacA subfamily. Four copies of Beta-lactamase class C and other penicillin binding proteins were also found in three different domains of strain's MM2LB T genome.
One gene encoding the two component transcriptional regulator LuxR family is present in the genome of strain MM2LB T and demonstrates quorum sensing skills.
Tolerance of up to 7.0% NaCl was described for strain MM2LB T [1]. Three genes for ABC-type proline/glycine betaine transport system (ATP binding subunit, permease and periplasmatic components), that seem to be located in the same operon, are present in strain MM2LB T genome. The accumulation of glycine betaine and other solutes offer osmoprotection, thus, this transport system is probably involved in osmoregulation.
Three genes in L. chironomi had best hits with genes from Eukaryotes, indicating a possible horizontal transfer of genes from Eukaryotes to L. chironomi. These genes were: Exodeoxyribonuclease VII small subunit and a protein from PAC2 family, both form Anopheles gambiae origin and a hypothetical protein from Drosophila willistoni origin. Anopheles and Drosophila as well as Chironomids belong to the Diptera order. L. chironomi was isolated from chironomids. Since chironomid species have not yet been sequenced, the horizontal gene transfer from the Diperan origin to L. chironomi may point toward the ancient relationships between this bacterium and its chironomid host.
The genome sequences of three more Leucobacter isolates have recently been published; L. chromiiresistens, isolated from a soil sample [40]; Leucobacter sp. UCD-THU isolated from a residential toilet [54]; and Leucobacter salsicius isolated from Korean salt-fermented seafood [39]. Chromate resistance was reported for some of these species (L. chironomi, L. chromiiresistens and L. salsicius) [1,39,40]. The genome analysis of L. salsicius detected chromate transport protein A (ChrA) that confers heavy metal tolerance via chromate ion efflux from the cytoplasm [39]. In contrast, this gene is not present in the genome of L. chironomi and L. chromiiresistens. However, in both strains, other genes for metals tolerance or ion efflux, are present. Interestingly, we have detected a chromate transporter (Chr) gene in the genome of Leucobacter sp. UCD-THU, although no evidence for chromate resistance was reported in vivo for this strain [54]. Another interesting feature is the differences in the horizontal gene transfer found in all four Leucobacter species genomes. While no horizontal gene transfer from Eukaryotes was detected for Leucobacter sp. UCD-THU, we detected horizontal gene transfer from fungi belonging to the phyla Basidiomycota and Ascomycota in L. salsicius and L. chromiiresistens genomes, respectively. For L. chromiiresistens, which was isolated from seafood, genes transfer from the phylum Chordata was also found. Horizontal gene transfer from insects was detected for L. chironomi in the current study, confirming the fact that chironomid insects are L. chironomi hosts.

Conclusions
In the current study, we characterized the genome of L. chironomi strain MM2LB T that was isolated from a chironomid egg mass [1]. Recently, we have demonstrated that endogenous bacteria in chironomids have a role in protecting their insect host from toxic metals [18,38]. Genes indicating the potential role of strain L. chironomi to tolerate or detoxify metals, where detected in its genome, demonstrating that indeed, L. chironomi which inhabits chironomids has a part in protecting its host from toxicants. Genes for ABC-type proline/glycine betaine transport system that were found in the genome may explain the salt tolerance properties of L. chironomi. Evidence of horizontal transfer of genes from Diperan origin to L. chironomi, implies toward an ancient relationships between L. chironomi and its chironomid host.
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