Complete genome sequence of Arcanobacterium haemolyticum type strain (11018T)

Arcanobacterium haemolyticum (ex MacLean et al. 1946) Collins et al. 1983 is the type species of the genus Arcanobacterium, which belongs to the family Actinomycetaceae. The strain is of interest because it is an obligate parasite of the pharynx of humans and farm animal; occasionally, it causes pharyngeal or skin lesions. It is a Gram-positive, nonmotile and non-sporulating bacterium. The strain described in this study was isolated from infections amongst American soldiers of certain islands of the North and West Pacific. This is the first completed sequence of a member of the genus Arcanobacterium and the ninth type strain genome from the family Actinomycetaceae. The 1,986,154 bp long genome with its 1,821 protein-coding and 64 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 11018 T (= DSM 20595 = CCM 5947 = ATCC 9345 = NBRC 15585) is the type strain of the species A. haemolyticum, which is the type species of its genus Arcanobacterium [1]. Arcanobacterium is one of six genera in the family Actinomycetaceae [2][3][4]. The genus currently consists of nine validly described species. The strain was first described in 1946 by MacLean as 'Corynebacterium haemolyticum' [5]. Based on chemical features and the presence of unique phenotypic characteristics, the strain was subsequently transferred to the new genus Arcanobacterium as A. haemolyticum [1] and emended by Lehnen et al. in 2006 [6]. The generic name drives from the Latin word 'arcanus', meaning 'secretive' and the Latin word 'bacterium', a small rod, meaning 'secretive bacterium' [1]. The species epithet is derived from the Latin word 'haema' meaning 'blood' and the Neo-Latin word 'lyticus' meaning 'able to loose or able to dissolve' referring to blood-dissolving or hemolytic when the cells grow on blood agar [1]. There are many medical case reports that A. haemolyticum is occasionally isolated in patients with brain abscess [7][8][9], cellulitis [10,11], endocarditis [12], meningitis [13], peritonitis [14], post-traumatic ankle joint infection [15], septic arthritis [16], septicemia [17], sinusitis [11], soft tissue infections [18], venous ulcer infection [19], vertebral osteomyelitis [20] and wound infection [21,22]. Only rarely are cases reported in animals, where pathogenicity of A. haemolyticum has not been well documented [23][24][25]. Here we present a summary classification and a set of features for A. haemolyticum strain 11018 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
Strain 11018 T is an obligate parasite of the pharynx of human and farm animals; occasionally it causes pharyngeal or skin lesions [26]. The strain was isolated from infections in American soldiers [5]. The 16S rRNA gene sequence of strain 11018 T (AJ234059) is 99% identical to six culturable strains that were reported in GenBank (status July 2010). Five strains were isolated from infected horses [23]. Another culturable strain, Tr2-2X-1 (FJ477385), was isolated from gasoline contaminated soil. The 16S rRNA gene of strain 11018 T shares 93.3-97.9% sequence identity with the sequences of the type strains from the other members of the genus Arcanobacterium [27]. The next closest relative outside of the genus Arcanobacterium is Dermacoccus barathri MT2.1 T (92.3% se-quence similarity) [27]. No phylotypes from environmental screening or metagenomic surveys could be linked to A. haemolyticum or even the genus Arcanobacterium, indicating a rare occurrence of these species in the habitats screened thus far (as of July 2010). A representative genomic 16S rRNA sequence of A. haemolyticum 11018 T was compared using BLAST with the most resent release of the Greengenes database [28] and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Arcanobacterium (42.4%), Dermacoccus (12.6%), Actinomyces (10.8%), Terrabacter (9.9%) and Sanguibacter (5.7%). The five most frequent keywords within the labels of environmental samples were 'skin' (6.6%), 'human' (5.0%), 'feedlot' (4.6%), 'elbow' (3.4%) and 'microbiota' (3.3%). The BLAST keywords analysis supports the biological insights into A. haemolyticum strain 11018 T as described above. Figure 1 shows the phylogenetic neighborhood of A. haemolyticum strain 11018 T in a 16S rRNA based tree. The sequences of the four 16S rRNA gene copies in the genome differ from each other by up to two nucleotides, and differ by up to five nucleotides from the previously published sequence generated from CIP 103370 (AJ234059) which contains one ambiguous base call. Phylogenetic tree highlighting the position of A. haemolyticum strain 11018 T relative to the type strains of the other species within the genus Arcanobacterium and to the type strains of the other genera within the family Actinomycetaceae. The trees were inferred from 1,388 aligned characters [29,30] of the 16S rRNA gene sequence under the maximum likelihood criterion [31] and rooted in accordance with the current taxonomy. 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 [32] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [33] are shown in blue, published genomes in bold.

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [44], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [45]. The genome project is deposited in the Genome OnLine Database [33] and the complete genome sequence is deposited in Gen-Bank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. Pyrosequencing reads were assembled using the Newbler assembler version 2.0.0-PostRelease-11/04/2008 (Roche). The initial Newbler assembly consisted of 116 contigs in 28 scaffolds and was converted into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. Illumina GAii sequencing data was assembled with Velvet [47] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 166.4 Mb 454 draft and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment in the following finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolu-tion, Dupfinisher [48], or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [49]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 140 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to improve the final consensus quality using an in-house developed tool -the Polisher [50]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 120.6 ×coverage of the genome. The final assembly contains 2.03 million Illumina reads and 0.52 million pyrosequencing reads.

Genome annotation
Genes were identified using Prodigal [51] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [52]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGRFam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [53].

Genome properties
The genome consists of a 1,986,154 bp long chromosome with a 53.1% GC content (Table 3 and Figure 3). Of the 1,885 genes predicted, 1,821 were protein-coding genes, and 64 RNAs; 90 pseudogenes were also identified. The majority of the protein-coding genes (68.5%) were assigned with 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.