Complete genome sequence of Streptobacillus moniliformis type strain (9901T)

Streptobacillus moniliformis Levaditi et al. 1925 is the type and sole species of the genus Streptobacillus, and is of phylogenetic interest because of its isolated location in the sparsely populated and neither taxonomically nor genomically much accessed family 'Leptotrichiaceae' within the phylum Fusobacteria. The 'Leptotrichiaceae' have not been well characterized, genomically or taxonomically. S. moniliformis,is a Gram-negative, non-motile, pleomorphic bacterium and is the etiologic agent of rat bite fever and Haverhill fever. Strain 9901T, the type strain of the species, was isolated from a patient with rat bite fever. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is only the second completed genome sequence of the order Fusobacteriales and no more than the third sequence from the phylum Fusobacteria. The 1,662,578 bp long chromosome and the 10,702 bp plasmid with a total of 1511 protein-coding and 55 RNA genes are part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 9901 T (= DSM 12112 = ATCC 14647 = NCTC 10651) is the type strain of Streptobacillus moniliformis, which also represents the type species of the genus first described in 1925 by Levaditi et al. [1,2] The taxonomic history of S. moniliformis; affiliated several genera such as 'Haverhillia [1]' and was only placed recently in the family "Leptotrichiaceae" (unpublished). It has also been suggested that S. moniliformis be placed within the Mycoplasmatales due to its similarity to some members based on the low G+C content of 24-26%, the fastidious requirements for growth and the production of L-form organisms [3]. S. moniliformis is commonly found in the nasopharynx of feral rats as well as in laboratory or pet rats. Between 50 and 100% of wild rats carry the commensal and secrete it with their urine [4]. The organism has been associated with rat bite fever and Haverhill fever in humans, following a bite or contamination of food by rat urine, respectively. Be-fore it could be demonstrated that both diseases are caused by the same organism, the etiologic agent for Haverhill fever was called 'Haverhillia multiformis' [5]. Both are systemic illnesses characterized by fever, rigors and migratory polyarthralgias and nearly 75% of patients develop a rash. Untreated, rat bite fever has a mortality rate of approximately 10%, with most deaths occurring due to endocarditis [6]. S. moniliformis is only the second species from the phylum Fusobacteria for which a complete genome sequence is described. Here we present a summary classification and a set of features for S. moniliformis strain 9901 T ( Table 1), together with the description of the complete genomic sequencing and annotation.

Classification and features
Isolate H2730, from a clinical case of fatal rat bite fever in the US [13] perfectly matches the 16S rRNA gene sequence of the genome of strain 9901 T described in this report; other recently described strains (TSD4, IKB1, IKC1, and IKC5) isolated from feral rats in Japan differ in just 1-4 nucleotides [14]. No phylotypes from environmental screening or genomic surveys could be linked with more than 90% 16S rRNA sequence similarity to S. moniliformis (status May 2009), indicating that the strain is rarely found in the environment outside of its natural hosts. Figure 1 shows the phylogenetic neighborhood of S. moniliformis strain 9901 T in a 16S rRNA based tree. The sequences of the five 16S rRNA gene copies in the genome of S. moniliformis 9901 T do not differ from each other, and differ by six nucleotides from the previously published 16S rRNA sequence generated from ATCC 14647 (Z35305). The difference between the genome data and the reported 16S rRNA gene sequence is most likely due to sequencing errors in the previously reported sequence data.  [15,16] of the 16S rRNA sequence under the maximum likelihood criterion [17] and rooted with the type strain of the family 'Fusobacteriaceae' The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [18] are shown in blue, published genomes in bold, e.g. the GEBA type strain Leptotrichia buccalis [19].
S. moniliformis is a Gram-negative, non-motile, fastidious, slow-growing and facultatively anaerobic organism that grows as elongated rods (0.3-0.7 µm by 1-5 µm in length) which tend to form chains or filaments with occasional bulbar swellings leading to a necklace-like appearance ("moniliformis" means necklace-shaped) ( Figure 2). The organism exists in two variants: the bacillary form and a cell wall-deficient L-form, which is considered nonpathogenic [20]. The primary habitat of S. moniliformis is small rodents, including rats (dominant reservoir) and more rarely gerbils, squirrels and mice. Rat-eating carnivores such as dogs, cats, ferrets and pigs can also become hosts and thus transfer the pathogen to humans. However, the organism is not directly transmitted from person to person and thus presents a typical zoonotic agent. A large number of case reports of S. moniliformis infections have been published (references in [4]). For cultivation, complex media containing blood, serum or ascitis fluid are necessary and increased CO2 concentration enhances growth. The organism is extremely sensitive to sodium polyanethol sulfonate ("Liquoid"), an anticoagulant used in commercial blood culture bottles, which can lead to problems during primary isolation [21]. S. moniliformis is catalase and oxidase negative and is biochemically rather inert. The metabolism is fermentative. Acid but no gas is produced from glucose, fructose, maltose and starch; H2S is produced. Arginine dihydrolase is synthesized [22,23]. S. moniliformis is susceptible to all β-lactam antibiotics, no β-lactamase activity could be demonstrated thus far [10].

Chemotaxonomy
No data are available about the murein composition of strain 9901 T . The fatty acid pattern of S. moniliformis can be used for its rapid identifica-tion and comprises a mixture of saturated and unsaturated straight-chain acids: C16:0, C18:0, C18:1 and C18:2. The type of menaquinones and polar lipids used by S. moniliformis has not been described yet.  Altitude not reported Evidence codes -IDA: Inferred from Direct Assay (first time in publication); 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). These evidence codes are from the Gene Ontology project [12]. If the evidence code is IDA, then the property was observed for a living isolate by one of the authors or an expert mentioned in the acknowledgements.

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 Genomes OnLine Database [18] and the complete genome sequence 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.  [25].

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 at the JGI can be found at the JGI website (http://www.jgi.doe.gov). 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 1,459 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 to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [26]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. 1,081 Sanger finishing reads were produced to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. The final assembly consists of 22,979 Sanger and 326,576 pyrosequence reads. Together all se-quence types provided 36.4× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [27] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org) [28]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant 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 (http://img.jgi.doe.ogv/er) [29].

Genome properties
The genome is 1,673,280 bp long and comprises one circular chromosome and one plasmid with a 26.3% GC content ( Table 3 and Figure 3). Of the 1,566 genes predicted, 1,511 were protein coding genes, and 55 RNAs. A total of 69 pseudogenes were also identified. The majority of the proteincoding genes (67.3%) genes were assigned with a putative function, while the remaining ones were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.