- Short genome report
- Open Access
High quality draft genome sequence of Brachymonas chironomi AIMA4T (DSM 19884T) isolated from a Chironomus sp. egg mass
Standards in Genomic Sciencesvolume 10, Article number: 29 (2015)
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.
Strain AIMA4T (= LGM 24400T = DSM 19884T), 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.  while characterizing rhodoquinone-containing bacteria that had been isolated from soybean crude waste sludge in Japan. Strain AIMA4T, was isolated from an insect egg mass (Chironomus sp.) that was sampled from a waste stabilization pond in northern Israel . 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) . 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. [3-10]. V. cholerae degrades chironomid egg masses by the secreted haemagglutinin protease (HAP) [11,12]. Strain AIMA4T 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 AIMA4T didn’t show the ability to degrade the egg masses like it was found for V. cholerae.
Classification and features
A taxonomic study using a polyphasic approach placed B. chironomi strain AIMA4T 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].
B. chironomi strain AIMA4T is a Gram-negative, non-motile coccobacillus or rod (Figure 2). After 48 h incubation on LB agar at 30°C, colonies are beige colored (opaque) that turn to light brown after few days of incubation. Strain AIMA4T is aerobic, chemoorganotrophic and does not produce acid from carbohydrates (including glucose) . Growth is observed at 18–37°C (optimum 30°C), with 0–2.5% (w/v) NaCl (optimum 0.5% NaCl) and at pH 5.0–9.0 (optimum pH 6.0–8.0) (Table 1). The following enzymatic activities were observed in strain AIMA4T: catalase and oxidase, alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, trypsin and naphthol-AS-BI-phosphohydrolase. Strain AIMA4T produces acetoin and reduces nitrate to nitrite .
The dominant cellular fatty acids are C16:1 ω7c, C16:0 and C18:1 ω7c. The main isoprenoid quinone is Q-8. Phosphatidylglycerol, phosphatidylethanolamine and phosphatidylserine occur as polar lipids .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [17-19]. Sequencing of B. chironomi strain AIMA4T is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes project  which aims in increasing the sequencing coverage of key reference microbial genomes . The genome project is deposited in the Genomes OnLine Database  and the permanent draft genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology . A summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation
B. chironomi strain AIMA4T, DSM 19884T, was grown in DSMZ medium 1 (Nutrient Agar), at 28°C . 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 .
Genome sequencing and assembly
The draft genome of B. chironomi strain AIMA4T 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 . All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts . Following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet , (2) 1–3 Kbp simulated paired end reads were created from Velvet Contigs using wgsim , (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG . Parameters for assembly steps were: (1) Velvet (velveth: 63 –shortPaired and velvetg: −very clean yes –export-Filtered yes –min contig lgth 500 –scaffolding no –cov cutoff 10) (2) wgsim (−e 0 –1 100 –2 100 –r 0 –R 0 –X 0) (3) Allpaths–LG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25 LONG JUMP COV = 50, RunAllpathsLG: THREADS = 8 RUN = std shredpairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True). The final draft assembly contained 36 contigs in 36 scaffolds. The total size of the genome is 2.5 Mbp and the final assembly is based on 249.2 Mbp of Illumina data, which provides an average 99.6 × coverage of the genome.
Genes were identified using Prodigal  as part of the DOE-JGI genome annotation pipeline [33,34], following by a round of manual curation using the JGI GenePRIMP pipeline . 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 .
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 AIMA4T 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 . Chironomid tolerance towards pollution is well documented . 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 of this species to protect its host in polluted environments. Genes encoding arsenate detoxification are present in B. chironomi strain AIMA4T. 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 AIMA4T 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 AIMA4T 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 AIMA4T by Halpern et al. . 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 AIMA4T and demonstrate quorum sensing skills.
In the current study, we characterized the genome of B. chironomi strain AIMA4T that was isolated from a chironomid egg mass . 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) . Likewise, the genome of strain AIMA4T 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.
One thousand microbial genomes
Hiraishi A, Shin YK, Sugiyama J. Brachymonas denitrificans gen. nov., sp. nov., an aerobic chemoorganotrophic bacterium which contains rhodoquinones, and evolutionary relationships of rhodoquinone producers to bacterial species with various quinone classes. J Gen Appl Microbiol. 1995;41:99–117.
Halpern M, Shaked T, Schumann P. Brachymonas chironomi sp. nov., isolated from a chironomid egg mass, and emended description of the genus Brachymonas. Int J Syst Evol Microbiol. 2009;59:3025–9.
Halpern M, Landsberg O, Raats D, Rosenberg E. Culturable and VBNC Vibrio cholerae: interactions with chironomid egg masses and their bacterial population. Microb Ecol. 2007;53:285–93.
Broza M, Halpern M. Chironomid egg masses and Vibrio cholerae. Nature. 2001;412:40.
Halpern M, Broza YB, Mittler S, Arakawa E, Broza M. Chironomid egg masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater habitats. Microb Ecol. 2004;47:341–9.
Halpern M, Raats D, Lavion R, Mittler S. Dependent population dynamics between chironomids (non-biting midges) and Vibrio cholerae. FEMS Microbiol Ecol. 2006;55:98–104.
Senderovich Y, Gershtein Y, Halewa E, Halpern M. Vibrio cholerae and Aeromonas; do they share a mutual host? ISME J. 2008;2:276–83.
Figueras MJ, Beaz-Hidalgo R, Senderovich Y, Laviad S, Halpern M. Re-identification of Aeromonas isolates from chironomid egg masses as the potential pathogenic bacteria Aeromonas aquariorum. Environ Microbiol Rep. 2011;3:239–44.
Beaz-Hidalgo R, Shakèd T, Laviad S, Halpern M, Figueras M. Chironomid egg masses harbour the clinical species Aeromonas taiwanensis and Aeromonas sanarellii. FEMS Microbiol Lett. 2012;337:48–54.
Halpern M. Chironomids and Vibrio cholerae. In: Rosenberg E, Gophna U, editors. Beneficial Microorganisms in Multicultural Life Forms. Berlin Heidelberg: Springer; 2011. p. 43–56.
Halpern M, Gancz H, Broza M, Kashi Y. Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses. Appl Environ Microbiol. 2003;69:4200–4.
Halpern M. Novel insights into hemagglutinin protease (HAP) gene regulation in Vibrio cholerae. Mol Ecol. 2010;19:4108–12.
Raats D, Halpern M. Oceanobacillus chironomi sp. nov., a halotolerant and facultative alkaliphilic species isolated from a chironomid egg mass. Int J Syst Evol Microbiol. 2007;57:255–9.
Halpern M, Senderovich Y, Snir S. Rheinheimera chironomi sp. nov., isolated from a chironomid (Diptera; Chironomidae) egg mass. Int J Syst Evol Microbiol. 2007;57:1872–5.
Willems A, Pot B, Falsen E, Vandamme P, Gillis M, Kersters K, et al. Polyphasic taxonomic study of the emended genus Comamonas: relationship to Aquuspirillum aquaticurn, E. Falsen group 10, and other clinical isolates. Int J Syst Bacteriol. 1991;41:427–244.
Weiss M, Kesberg A, LaButti K, Pitluck S, Bruce D, Hauser L, et al. Permanent draft genome sequence of Comamonas testosteroni KF-1. Stand Genomic Sci. 2013;8:2.
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, et al. A phylogeny-driven Genomic Encyclopaedia of Bacteria and Archaea. Nature. 2009;462:1056–60.
Klenk HP, Göker M. En route to a genome - based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010;33:175–82.
Göker M, Klenk HP. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects. Stand Genomic Sci. 2013;8:360–74.
Kyrpides NC, Woyke T, Eisen JA, Garrity G, Lilburn TG, Beck BJ, et al. Genomic encyclopedia of type strains, phase I: the one thousand microbial genomes (KMG-I) project. Stand Genomic Sci. 2013;9:628–6234.
Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Göker M, Parker CT, et al. Genomic Encyclopedia of Bacteria and Archaea: sequencing a myriad of type strains. PLoS Biol. 2014;8:e1001920.
Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, et al. The genomes OnLine database (GOLD) v. 4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012;40:D571–9.
Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, et al. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS One. 2012;7:e48837.
List of growth media used at DSMZ [http://www.dsmz.de/catalogues/catalogue-microorganisms/culture-technology/list-of-media-for-microorganisms.html]
Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, et al. The DNA bank network: the start from a German initiative. Biopreserv Biobank. 2011;9:51–5.
Bennett S. Solexa Ltd. Pharmacogenomics. 2004;5:433–8.
JGI web page [http://www.jgi.doe.gov]
Mingkun L, Copeland A, Han J. DUK, unpublished, 2011
Zerbino D, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18:821–9.
Gnerre S, MacCallum I. High–quality draft assemblies of mammalian genomes from massively parallel sequence data. Proc Natl Acad Sci U S A. 2011;108:1513–8.
Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiati on site identification. BMC Bioinformatics. 2010;11:119.
Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM, Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009;1:63–7.
Chen IM, Markowitz VM, Chu K, Anderson I, Mavromatis K, Kyrpides NC, et al. Improving microbial genome annotations in an integrated database context. PLoS One. 2013;8:e54859.
Pati A, Ivanova NN, Mikhailova N, Ovchinnikova G, Hooper SD, Lykidis A, et al. GenePRIMP: a gene prediction improvement pipeline for prokaryotic genomes. Nat Methods. 2010;7:455–7.
Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009;25:2271–8.
Senderovich Y, Halpern M. The protective role of endogenous bacterial communities in chironomid egg masses and larvae. ISME J. 2013;7:2147–58.
Armitage P, Cranston PS, Pinder LCV. The Chironomidae: The Biology and Ecology of Non-biting Midges. London: Chapman and Hall; 1995.
Senderovich Y, Halpern M. Bacterial community composition associated with chironomid egg masses. J Insect Sci. 2012;12:148.
Field D, Garrity GM, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008;26:541–7.
Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, et al. The Genomic Standards Consortium. PLoS Biol. 2011;9:e1001088.
Garrity GM. Namesforlife browser tool takes expertise out of the database and puts it right in the browser. Microbiol Today. 2010;37:9.
Woese CR, Kandler O, Weelis ML. Towards a natural system of organisms. Proposal for the domains Archaea and Bacteria. Proc Natl Acad Sci U S A. 1990;87:4576–9.
Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl nov. In: Brenner DJ, Krieg NR, Stanley JT, Garrity GM, editors. Bergey’s Manual of Systematic Bacteriology. volume 2 (The Proteobacteria part B The Gammaproteobacteria). 2nd ed. New York: Springer; 2005. p. 1.
Garrity GM, Bell JA, Lilburn T. Class II. Betaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Stanley JT, Garrity GM, editors. Bergey’s Manual of Systematic Bacteriology. Volume 2 (the Proteobacteria part C the alpha-, beta-, delta-, and Epsilonproteobacteria). 2nd ed. New York: Springer; 2005. p. 575.
Garrity GM, Bell JA, Lilburn T. Order I. Burkholderiales ord. nov. In: Brenner DJ, Krieg NR, Stanley JT, Garrity GM, Brenner DJ, Krieg NR, Stanley JT, Garrity GM, editors. Bergey’s Manual of Systematic Bacteriology. Volume 2 (The Proteobacteria part C The Alpha-, Beta-, Delta-, and Epsilonproteobacteria). 2nd ed. New York: Springer; 2005. p. 575.
Willems A, De Ley J, Gillis M, Kersters K. Comamonadaceae, a new family encompassing the acidovorans rRNA complex, including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969). Int J Syst Bacteriol. 1991;41:445–50.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Gene Ontology Consortium. Nat Genet. 2000;25:25–9.
We would like to gratefully acknowledge the help of Nicole Reimann for growing B. chironomi cultures, and Evelyne-Marie Brambilla for DNA extraction and quality control (both at DSMZ). This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344 Genome analysis was supported by the National Basic Research Program of China (No. 2010CB833801). A.L. was supported in part by Russian Ministry of Science Mega-grant no.11.G34.31.0068 (PI Dr Stephen J O’Brien). M. H. was supported in part by a grant from the US Civilian Research and Development Foundation (CRDF grant no. ILB1-7045-HA).
The authors declare that they have no competing interests.
MH (Halpern) isolated and characterized strain B. chironomi AIMA4T; SL, MH (Halpern), HPK and NCK drafted the manuscript. AL, JH, MH (Haynes), TBKR, MH (Huntemann), AP, NNI, KM, VM and TW sequenced, assembled and annotated the genome. EL provided the biomass for DNA extraction and collected data about the organism. MR performed electron microscopy. All authors read and approved the final manuscript.