Introduction

Strain 11018T (= 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 [24]. 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 [79], 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 [2325]. Here we present a summary classification and a set of features for A. haemolyticum strain 11018T, together with the description of the complete genomic sequencing and annotation.

Classification and features

Strain 11018T 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 11018T (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 11018T 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 MT2.1T (92.3% sequence 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 11018T 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 11018T as described above.

Figure 1 shows the phylogenetic neighborhood of A. haemolyticum strain 11018T 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.

Figure 1.
figure 1

Phylogenetic tree highlighting the position of A. haemolyticum strain 11018T 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.

The cells of strain 11018T are slender or irregular rods (0.3–0.8 × 1.0–5.0 µm) [Table 1 and Figure 2]. The cells are Gram-positive, nonmotile, not acid-fast and without endospores [1]. In young cultures, cells may show clubbed ends sometimes arranged in V formation, but there are no filaments. In older cultures, cells segment into short, irregular rods and cocci [1]. Strain 11018T is facultatively anaerobic. The cells grow slowly on nutrient agar, but grow better on horse blood agar, giving small, convex, translucent colonies surrounded by a zone of complete hemolysis after two days at 37°C [1]. The selective medium for this strain was developed by Coman [39] and contains 5% sheep blood and 3.5% of NaCl. Cell growth is enhanced by the addition of CO2 [1]. The optimum growth temperature is 37°C [1,26]. Cells do not withstand heating at 60°C for 15 min [1,5]. Strain 11018T is chemoorganotrophic and requires nutritionally rich media for growth [1,26]. The fermentative metabolism of this strain produces acid but does not produce gas from glucose and several other carbohydrates on which growth occurs [1,26]. Acid production is mainly acetic, lactic and succinic acids [1,26]. Catalase, nitrate reduction and gelatine hydrolysis reactions are negative [6]. Strain 11018T produces N-acetyl-β-galactosidase, alkaline phosphatase, extracellular DNase, β-galactosidase, α-glucosidase and pyrazinamidase. It does not produce acid phosphatase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), α-fucosidase, α-galactosidase, β-glucosidase, β-glucuronidase, leucine arylamidase, lipase (C14), α-mannosidase, naphthol-AS-BI-phosphohydrolase, trypsin, valine arylamidase and urease [1,6]. Strain 11018T is not able to ferment adonitol, L-arabitol, erythritol, D-fructose, glycerol, glycogen, D-mannitol and D-xylose. It is resistant to oxytetracycline (30µg per disc) but susceptible to nalidixic acid (30µg per disc), sulfamethoxazole trimethoprim (25µg per disc), amikacin (10µg per disc) or cefoxitin (30µg per disc) [1,42].

Figure 2.
figure 2

Scanning electron micrograph of A. haemolyticum strain 11018T

Table 1. Classification and general features of A. haemolyticum strain 11018T according to the MIGS recommendations [34].

Chemotaxonomy

Strain 11018T possesses peptidoglycan type A5α based on L-Lys-L-Lys-D-Glu (unpublished, Norbert Weiss [43]). The predominant menaquinone is MK-9(H4) (85%) complemented by 15% MK-8(H4) [6]. The major cellular fatty acids when grown on blood agar at 35°C are straight-chain unsaturated acids C18:1 ω9c (37.0%), and saturated acids C18:0 (24.7%), C16:0 (22.5%) [6], which is similar to the cellular fatty acids spectrum reported from cells grown on sheep blood agar [31]: C18:1 cis9 (29%), C16:0 (23%), C18:2 (18%), C18:0 (17%), C10:0 (3%) and C14:0 (2%).

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 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

A. haemolyticum strain 11018T, DSM 20595, was grown anaerobically in DSMZ medium 104 (PYG modified medium) [46] at 37°C. DNA was isolated from 1–1.5 g of cell paste using MasterPure Gram Positive DNA Purification Kit (Epicentre MGP04100), with a modified protocol for cell lysis, st/LALM, as described in Wu et al. [45].

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 gapResolution, 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, UniProt, TIGRFam, 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 [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.

Figure 3.
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 3. Genome Statistics
Table 4. Number of genes associated with the general COG functional categories