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Stand Genomic Sci. Jun 30, 2010; 2(3): 260–269.
Published online Jun 15, 2010. doi:  10.4056/sigs.831993
PMCID: PMC3035287

Complete genome sequence of Brachyspira murdochii type strain (56-150T)

Abstract

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.

Keywords: host-associated, non-pathogenic, motile, anaerobic, Gram-negative, Brachyspiraceae, Spirochaetes, GEBA

Introduction

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 [3]. 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’ [5]. 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 [1]. 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 [1]. 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 [12]. 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 [13] (see also http://pubmlst.org/brachyspira/).

Only one genome of a member of the family ‘Brachyspiraceae’ been sequenced to date: B. hyodysenteriae strain WA1 [14],. 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 [6]. 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 [16], and from the joint fluid of a lame pig [17]. 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 [18]. The closest related type strain of a species outside of the Brachyspira, but within the order Spirochaetales, is Turneriella parva [19], which exhibits only 75% 16S rRNA sequence similarity [18]. 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 [20], 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).

Figure 1
Phylogenetic tree highlighting the position of B. murdochii 56-150T relative to the other type strains within the genus and to the type strains of the other genera within the class Spirochaetes (excluding members of the Spirochaetaceae). The tree was ...

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) [1]. 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) [1]. 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 [1].

Table 1
Classification and general features of B. murdochii 56-150T according to the MIGS recommendations [26]
Figure 2
Scanning electron micrograph of B. murdochii 56-150T

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 [1]. 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 [1], which is indicative of NADH-ferredoxin oxidoreductase reaction [6]. The ethanol is likely to be formed from acetyl-CoA by the enzymes acetaldehyde dehydrogenase and alcohol dehydrogenase [6]. 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 [1]. Strain 56-150T is anaerobic but aerotolerant [1].

Minimal inhibitory concentrations have been determined for strain 56-150T for tiamulin hydrogen fumarate, tylosin tartrate, erythromycin, clindamycin hydrochloride, virginiamycin, and carbadox [36]. 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 [37].

Chemotaxonomy

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 [6]), the species that is currently most closely related to B. murdochii [13]. 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 [38].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [39], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [40]. The genome project is deposited in the Genome OnLine Database [25] 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.

Table 2
Genome sequencing project information

Growth conditions and DNA isolation

B. murdochii, strain 56-150T, DSM 12563, was grown anaerobically in DSMZ medium 840 (Serpulina murdochii medium) [41] 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. [40].

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 [42]. 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.

Genome annotation

Genes were identified using Prodigal [43] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [44]. 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 [45].

Genome properties

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.

Table 3
Genome Statistics
Figure 3
Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.
Table 4
Number of genes associated with the general COG functional categories

Acknowledgements

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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