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Complete genome sequence of Brachyspira murdochii type strain (56-150T)
Standards in Genomic Sciences volume 2, pages 260–269 (2010)
Brachyspira murdochii Stanton et al. 1992 is a non-pathogenic, host-associated spirochete of the family Brachyspiraceae. Initially isolated from the intestinal content of a healthy swine, the ‘group B spirochaetes’ were first described as Serpulina murdochii. Members of the family Brachyspiraceae are of great phylogenetic interest because of the extremely isolated location of this family within the phylum ‘Spirochaetes’. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a type strain of a member of the family Brachyspiraceae and only the second genome sequence from a member of the genus Brachyspira. The 3,241,804 bp long genome with its 2,893 protein-coding and 40 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain 56–150T (= DSM 12563 = ATCC 51284 = CIP 105832) is the type strain of the species Brachyspira murdochii. This strain was first described as Serpulina murdochii [1,2], and later transferred to the genus Brachyspira . The genus Brachyspira currently consists of seven species, with Brachyspira aalborgi as the type species [4,5]. The genus Brachyspira is the only genus in the not yet formally described family ‘Brachyspiraceae’ [6,7]. The generic name derives from ‘brachys’, Greek for short, and ‘spira’, Latin for a coil, a helix, to mean ‘a short helix’ . The species name for B. murdochii derives from the city of Murdoch, in recognition of work conducted at Murdoch University in Western Australia, where the type strain was identified . Some species of the genus Brachyspira cause swine dysentery and porcine intestinal spirochetosis. Swine dysentery is a severe, mucohemorrhagic disease that sometimes leads to death of the animals . B. murdochii is generally not considered to be a pathogen, although occasionally it has been seen in association with colitis in pigs [3,8], and was also associated with clinical problems on certain farms [9–11].
In 1992, a user-friendly and robust novel PCR-based restriction fragment length polymorphism analysis of the Brachyspira nox-gene was developed, which allows one to identify, with high specificity, members of B. murdochii using only two restriction endonucleases . More recently, a multi-locus sequence typing scheme was developed that facilitates the identification of Brachyspira species and reveals the intraspecies diversity of B. murdochii  (see also http://pubmlst.org/brachyspira/).
Only one genome of a member of the family ‘Brachyspiraceae’ been sequenced to date: B. hyodysenteriae strain WA1 ,. It is an intestinal pathogen of pigs. Based on 16S rRNA sequence this strain is 0.8% different from strain 56–150T. Here we present a summary classification and a set of features for B. murdochii 56–150T, together with the description of the complete genomic sequencing and annotation.
Classification and features
Brachyspira species colonize the lower intestinal tract (cecum and colons) of animals and humans . The type of B. murdochii, 56–150T, was isolated from a healthy swine in Canada [1,15]. Other isolates have been obtained from wild rats in Ohio, USA, from laboratory rats in Murdoch, Western Australia , and from the joint fluid of a lame pig . Further isolates have been obtained from the feces or gastrointestinal tract of pigs in Canada, Tasmania, Queensland, and Western Australia [2,15]. The type strains of the other species of the genus Brachyspira share 95.9-99.4% 16S rRNA sequence identity with strain 56–150T. GenBank contains 16S rRNA sequences for about 250 Brachyspira isolates, all of which share at least 96% sequence identity with strain 56–150T . The closest related type strain of a species outside of the Brachyspira, but within the order Spirochaetales, is Turneriella parva , which exhibits only 75% 16S rRNA sequence similarity . 16S rRNA sequences from environmental samples and metagenomic surveys do not exceed 78–79% sequence similarity to strain 56–150T, with the sole exception of one clone from a metagenome analysis of human diarrhea , indicating that members of the species, genus and even family are poorly represented in the habitats outside of various animal intestines screened thus far (status March 2010).
Figure 1 shows the phylogenetic neighborhood of B. murdochii 56–150T in a 16S rRNA based tree. The sequence of the single 16S rRNA gene in the genome sequence is identical with the previously published 16S rRNA gene sequence generated from DSM 12563 (AY312492).
The cells of B. murdochii 56-l50T were 5–8 by 0.35–0.4 µm in size (Table 1 and Figure 2), and each cell possessed 22 to 26 flagella (11 to 13 inserted at each end) . In brain/heart infusion broth containing 10% calf serum (BHIS) under an N2-O2 (99::l) atmosphere, strain 56–150T had optimum growth temperatures of 39 to 42°C (shortest population doubling times and highest final population densities) . In BHIS broth at 39°C, the doubling times of strain 56–150T were 2 to 4 h, and the final population densities were 0.5 x l09 to 2.0 x l09 cells/ml. Strain 56–150T did not grow at 32 or 47°C .
Substrates that support growth of strain 56–150T in HS broth (basal heart infusion broth containing 10% fetal calf serum) include glucose, fructose, sucrose, N-acetylglucosamine, pyruvate, L-fucose, cellobiose, trehalose, maltose, mannose, and lactose, but not galactose, D-fucose, glucosamine, ribose, raffinose, rhamnose, or xylose . In HS broth supplemented with 0.4% glucose under an N2-O2 (99:l) atmosphere, the metabolic end products of strain 56–150T are acetate, butyrate, ethanol, CO2, and H2. Strain 56–150T produces more H2 than CO2 , which is indicative of NADH-ferredoxin oxidoreductase reaction . The ethanol is likely to be formed from acetyl-CoA by the enzymes acetaldehyde dehydrogenase and alcohol dehydrogenase . Strain 56–150T is weakly hemolytic, negative for indole production, does not hydrolyze hippurate, is negative for α-galactosidase and α-glucosidase activity, but positive for β-glucosidase activity . Strain 56–150T is anaerobic but aerotolerant .
Minimal inhibitory concentrations have been determined for strain 56–150T for tiamulin hydrogen fumarate, tylosin tartrate, erythromycin, clindamycin hydrochloride, virginiamycin, and carbadox . Several strains of B. murdochii have been described to be naturally resistant against the rifampicin [7,32]. Also, a ring test for quality assessment for diagnostics and antimicrobial susceptibility testing of the genus Brachyspira has been reported .
At present there are no reports on the chemotaxonomy of B. murdochii. However, some data are available for B. innocens (formerly classified as Treponema innocens ), the species that is currently most closely related to B. murdochii . B. innocens cellular phospholipids and glycolipids were found to contain acyl (fatty acids with ester linkage) with alkenyl (unsaturated alcohol with ether linkage) side chains [6,38]. The glycolipid of B. innocens contains monoglycosyldiglyceride (MGDG) and, in most strains, acylMGDG is also found, with galactose as the predominant sugar moiety .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position , and is part of the Genomic Encyclopedia of Bacteria and Archaea project . The genome project is deposited in the Genome OnLine Database  and the complete genome sequence is deposited in GenBank Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
B. murdochii, strain 56–150T, DSM 12563, was grown anaerobically in DSMZ medium 840 (Serpulina murdochii medium)  at 37°C. DNA was isolated from 0.5–1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with lysis modification st/L according to Wu et al. .
Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed can be found at the JGI website (http://www.jgi.doe.gov/). In total, 861,386 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 3,554 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible misassemblies were corrected with Dupfinisher or transposon bombing of bridging clones . A total of 300 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 68.6× coverage of the genome. The final assembly contains 79,829 Sanger reads and 861,386 pyrosequencing reads.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform .
The genome is 3,241,804 bp long and comprises one main circular chromosome with an overall GC content of 27.8% (Table 3 and Figure 3). Of the 2,893 genes predicted, 2,853 were protein-coding genes, and 40 RNAs. A total of 44 pseudogenes were identified. The majority of the protein-coding genes (66.2%) were assigned a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
Stanton TB, Fournie-Amazouz E, Postic D, Trott DJ, Grimont PAD, Baranton G, Hampson DJ, Saint Girons I. Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov. Int J Syst Bacteriol 1997; 47:1007–1012. PubMed doi:10.1099/00207713-47-4-1007
Lee JI, Hampson DJ. Genetic characterisation of intestinal spirochaetes and their association with disease. J Med Microbiol 1994; 40:365–371. PubMed doi:10.1099/00222615-40-5-365
Hampson DJ, La T. Reclassification of Serpulina intermedia and Serpulina murdochii in the genus Brachyspira as Brachyspira intermedia comb. nov. and Brachyspira murdochii comb. nov. Int J Syst Evol Microbiol 2006; 56:1009–1012. PubMed doi:10.1099/ijs.0.64004-0
Euzéby JP. List of bacterial names with standing in nomenclature: A folder available on the Internet. Int J Syst Bacteriol 1997; 47:590–592. PubMed doi:10.1099/00207713-47-2-590
Hovind-Hougen K, Birch-Andersen A, Henrik-Nielsen R, Orholm M, Pedersen JO, Teglbjaerg PS, Thaysen EH. Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov. J Clin Microbiol 1982; 16:1127–1136. PubMed
Stanton TB. 2006. The genus Brachyspira. In M Dworkin, S Falkow, E Rosenberg, KH Schleifer E Stackebrandt (eds), The Prokaryotes, 3. ed, vol. 7. Springer, New York, p. 330–356.
Paster BJ, Dewhirst FE. Phylogenetic foundation of spirochetes. J Mol Microbiol Biotechnol 2000; 2:341–344. PubMed
Weissenböck H, Maderner A, Herzog AM, Lussy H, Nowotny N. Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii. Vet Microbiol 2005; 111:67–75. PubMed doi:10.1016/j.vetmic.2005.09.002
Stephens CP, Hampson DJ. Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia. 1999; 28:447–454.
Stephens CP, Oxberry SL, Phillips ND, La T, Hampson DJ. The use of multilocus enzyme electrophoresis to characterise intestinal spirochaetes (Brachyspira spp.) colonising hens in commercial flocks. Vet Microbiol 2005; 107:149–157. PubMed doi:10.1016/j.vetmic.2005.01.011
Feberwee A, Hampson DJ, Phillips ND, La T, van der Heijden HMJF, Wellenberg GJ, Dwars RM, Landman WJM. Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both. J Clin Microbiol 2008; 46:593–600. PubMed doi:10.1128/ICM.01829-07
Rohde J, Rothkamp A, Gerlach GF. Differentiation of porcine Brachyspira species by a novel nox PCR-based restriction fragment length polymorphism analysis. J Clin Microbiol 2002; 40:2598–2600. PubMed doi:10.1128/JCM.40.7.2598-2600.2002
Råsbäck T, Johansson KE, Jansson DS, Fellstrom C, Alikhani MY, La T, Dunn DS, Hampson DJ. Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira. Microbiology 2007; 153:4074–4087. PubMed doi:10.1099/mic.0.2007/008540-0
Bellgard MI, Eanchanthuek P, La T, Ryan K, Moolhuijzen P, Albertyn Z, Shaban B, Motro Y, Dunn DS, Schibeci D, et al. Genome sequence of the pathogenic intestinal spirochaete Brachyspira hyodysenteriae reveals adapations to its lifestyle in the porcine large intestions. PLoS ONE 2009; 4:e4641. PubMed doi:10.1371/journal.pone.0004641
Lee JI, Hampson DJ, Lymbery AJ, Harders SJ. The porcine intestinal spirochaetes: identification of new genetic groups. Vet Microbiol 1993; 34:273–285. PubMed doi:10.1016/0378-1135(93)90017-2
Trott DJ, Atyeo RF, Lee JI, Swayne DA, Stoutenbgurg JW, Hampson DJ. Genetic relatedness amongst intestinal spirochaetes isolated from rate and birds. Lett Appl Microbiol 1996; 23:431–436. PubMed doi:10.1111/j.1472-765X.1996.tb01352.x
Hampson DJ, Robertson ID, Oxberry SL. Isolation of Serpulina murdochii from the joint fluid of a lame pig. Aust Vet J 1999; 77:48. PubMed doi:10.1111/j.1751-0813.1999.tb12430.x
Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 2007; 57:2259–2261. PubMed doi:10.1099/ijs.0.64915-0
Levett PN, Morey RE, Galloway R, Steigerwalt AG, Ellis WA. Reclassification of Leptospira parva Hovind-Hougen et al. 1982 as Turneriella parva gen. nov., comb. nov. Int J Syst Evol Microbiol 2005; 55:1497–1499. PubMed doi:10.1099/ijs.0.63088-0
Finkbeiner SR, Allred AF, Tarr PI, Klenin EJ, Kirkwood CD, Wang D. Metagenomic analysis of human iarrhea: viral detection and discovery. PLoS Pathog 2008; 4:e1000011. PubMed doi:10.1371/journal.ppat.1000011
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552. PubMed
Lee C, Grasso C, Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics 2002; 18:452–464. PubMed doi:10.1093/bioinformatics/18.3.452
Stamatakis A, Hoover P, Rougemont J. A Rapid Bootstrap algorithm for the RAxML web servers. Syst Biol 2008; 57:758–771. PubMed doi:10.1080/10635150802429642
Pattengale ND, Alipour M, Bininda-Emonds ORP, Stamatakis A. How many bootstrap replicates are necessary? Lect Notes Comput Sci 2009; 5541:184–200. doi:10.1007/978-3-642-02008-713
Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM, Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res 2010; 38:D346–D354. PubMed doi:10.1093/nar/gkp848
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ, Angiuoli SV, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed doi:10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed doi:10.1073/pnas.87.12.4576
Garrity GM, Lilburn TG, Cole JR, Harrison SH, Euzéby J, Tindall BJ. Taxonomic outline of the Bacteria and Archaea, Release 7.7 March 6, 2007. Part 11 — The Bacteria: Phyla “Planctomycetes”, “Chlamydiae”, “Spirochaetes”, “Fibrobacteres”, “Acidobacteria”, “Bacteroidetes”, “Fusobacteria”, “Verrucomicrobia”, “Dictyoglomi”, “Gemmatimonadetes”, and “Lentisphaerae”. http://www.taxonomicoutline.org/index.php/toba/article/viewFile/188/220 2007.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. doi:10.1099/00207713-30-1-225
Buchanan RE. Studies in the nomenclature and classification of Bacteria. II. The primary subdivisions of the Schizomycetes. J Bacteriol 1917; 2:155–164. PubMed
Paster BJ, Dewhirst FE. Phylogenetic foundation of spirochetes. J Mol Microbiol Biotechnol 2000; 2:341–344. PubMed
Imachi H, Sakai S, Hirayama H, Nakagawa S, Nunoura T, Takai K, Horikoshi K. Exilispira thermophila gen. nov., sp. nov., an anaerobic, thermophilic spirochaete isolated from a deep-sea hydrothermal vent chimney. Int J Syst Evol Microbiol 2008; 58:2258–2265. PubMed doi:10.1099/ijs.0.65727-0
Stanton TB, Postic D, Jensen NS. Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens. Int J Syst Bacteriol 1998; 48:669–676. PubMed doi:10.1099/00207713-48-3-669
Classification of Bacteria and Archaea in risk groups. www.baua.de TRBA 466.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene Ontology: tool for the unification of biology. Nat Genet 2000; 25:25–29. PubMed doi:10.1038/75556
Karlsson M, Fellstrom C, Gunnarsson A, Landen A, Franklin A. Antimicrobial susceptibility testing of porcine brachyspira (Serpulina) species isolates. J Clin Microbiol 2003; 41:2596–2604. PubMed doi:10.1128/JCM.41.6.2596-2604.2003
Råsbäck T, Fellström C, Bergsjø B, Cizek A, Collin K, Gunnarsson A, Jensen SM, Mars A, Thomson J, Vyt P, et al. Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test. Vet Microbiol 2005; 109:229–243. PubMed doi:10.1016/j.vetmic.2005.05.009
Matthews HM, Kinyon JM. Cellular lipid comparisons between strains of Treponema hyodysenteriae and Treponema innocens. Int J Syst Bacteriol 1984; 34:160–165. doi:10.1099/00207713-34-2-160
Klenk HP, Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol (In press).
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, et al. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature 2009; 462:1056–1060. PubMed doi:10.1038/nature08656
List of growth media used at DSMZ: http://www.dsmz.de/microorganisms/media_list.php
Sims D, Brettin T, Detter J, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F, Lucas S, et al. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci 2009; 1:12–20. doi:10.4056/sigs.761
Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics 2010; 11:119. PubMed doi:10.1186/1471-2105-11-119
Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A, Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods (Epub). doi:10.1038/nmeth.1457
Markowitz VM, Ivanova NN, Chen IMA, Chu K, Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 2009; 25:2271–2278. PubMed doi:10.1093/bioinformatics/btp393
We would like to gratefully acknowledge the help of Sabine Welnitz for growing B. murdochii cells and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). This work was performed under the auspices of the US Department of Energy 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, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, UT-Battelle, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and SI 1352/1-2.
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Pati, A., Sikorski, J., Gronow, S. et al. Complete genome sequence of Brachyspira murdochii type strain (56-150T). Stand in Genomic Sci 2, 260–269 (2010). https://doi.org/10.4056/sigs.831993