Complete genome sequence of Anaerococcus prevotii type strain (PC1T)

Anaerococcus prevotii (Foubert and Douglas 1948) Ezaki et al. 2001 is the type species of the genus, and is of phylogenetic interest because of its arguable assignment to the provisionally arranged family ‘Peptostreptococcaceae’. A. prevotii is an obligate anaerobic coccus, usually arranged in clumps or tetrads. The strain, whose genome is described here, was originally isolated from human plasma; other strains of the species were also isolated from clinical specimen. 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 member of the genus. Next to Finegoldia magna, A. prevotii is only the second species from the family ‘Peptostreptococcaceae’ for which a complete genome sequence is described. The 1,998,633 bp long genome (chromosome and one plasmid) with its 1852 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Anaerococcus prevotii strain PC1 T (= DSM 20548 = ATCC 9321 = JCM 6508) is the type strain of the species and the type species of the genus [1]. Six strains of the species were characterized by Fou-bert and Douglas in 1948, originally designated as 'Micrococcus prevotii', but subsequently placed in the genus Peptococcus [2]. Based on a comparative study published by Ezaki et al. [3], the type strain of P. prevotii was then transferred to the genus 'Peptostreptococcus' and later on assigned to the novel genus Anaerococcus as A. prevotii [1]. The organism is a Gram--positive, anaerobic, indole-negative coccus. The major metabolic end product from metabolism of peptone--yeast--glucose (PYG) is butyric acid. A. prevotii was provisionally as-signed to the arranged family 'Peptostreptococcaceae' within the order Clostridiales, also desig-nated as Family XI Incertae sedis [4]. Here we pre-sent a summary classification and a set of features for A. prevotii strain PC1 T together with the de--scription of the complete genomic sequencing and annotation.

Classification and features
Within the last few years, several changes occurred in the classi`ication of the anaerobic Gram--positive cocci. There are currently `ive genera of anaerobic Gram--positive cocci which may be isolated from humans (Peptostreptococcus, Peptoniphilus, Parvimonas, Finegoldia, and Anaerococcus). Members of the species A. prevotii are frequently recovered from human clinical specimens such as vaginal dis-charges and ovarian, peritoneal, sacral or lung ab-scesses. In particular, A. prevotii was also described as a common isolate of the normal `lora of skin, the oral cavity and the gut [3]. Historically the Gram-positive anaerobic cocci were identi`ied mainly by using phenotypic traits, but as shown by Song et al., this often led to the misidenti`ication of A. vaginalis strains, which were mistakenly assigned to A. prevotii or A. tetradius [5]. Currently Genbank does not contain any16S rRNA sequences from culti-vated strains that can be clearly linked to the spe--cies A. prevotii with over 95% gene sequence simi-larity. Recently, the temporal diversity of the human skin microbiome was analyzed using 16S rRNA gene phylotyping. It is noteworthy that several clones originated from different skin sites (gluteal crease, occiput, umbilicus, popliteal fossa, volar forearm). These isolates were taken from two pa-tients and showed close relationships to A. prevotii [6]. No closely related isolates or uncultivated clones with more than 84% 16S rRNA gene se-quence identity are recorded from global ocean screenings and environmental samples (except for human skin). Figure 1 shows the phylogenetic neighborhood of A. prevotii strain PC1 T in a 16S rRNA based tree. The four 16S rRNA gene copies in the genome of strain PC1 T differ by up to 15 nucleotides from each other, and by up to 9 nucleotides from the previously pub-lished 16S rRNA sequence generated from strain CCUG 41932 (AF542232). 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.

Figure 1.
Phylogenetic tree highlighting the position of A. prevotii PC1 T relative to all type strains of the genus Anaerococcus and the type strains of all other genera within the family 'Peptostreptococcaceae' inferred from 1,302 aligned characters [7,8] of the 16S rRNA sequence under the maximum likelihood criterion [9]. Rooting was done with the type species of the genus Clostridium. 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 [10] are shown in blue, published genomes in bold.
A. prevotii PC1 T cells are Gram--positive and non-motile (Table 1). Cells grown in PYG broth are 0.6--0.9 µm in diameter and occur in pairs, tetrads or ir-regular clumps or short chains ( Figure  2). Colonies range from 0.5 to 2 mm in diameter on Columbia blood agar. Optimum temperature for growth is 37°C. Strain PC1 T metabolizes peptones and amino acids and the major metabolic end product from PYG medium is butyric acid. Most species of the ge-nus Anaerococcus ferment carbohydrates weakly. A.

Chemotaxonomy
Cell wall amino acid analysis of strain PC1 T yielded peptidoglycan type A4αʹ′, composed of L--Lys--D--Glu [22], type A12.2 according to the DSMZ catalogue of strains. Cell wall sugars are glucose, glucosamine and galactose [22]. Major cellular fatty acid compo-sition of the type strain was analyzed by Lambert and Arm`ield in 1979 [23] and by Ezaki et al. in 1983 [3], but the results of these studies are con-tradictory. No other chemotaxonomic data are available at present.

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 pro-ject. The genome project is deposited in the Ge-nomes OnLine Database [10] and the complete ge-nome sequence has been deposited in GenBank. Sequence, inishing and annotation were performed by the DOE Joint Genome Institute (JGI). A sum-mary of the project information is shown in 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 available from the Gene Ontology project [18]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. .

Growth condi tions and DNA isolation
A. prevotii strain PC1 T , DSM 20548, was grown an-aerobically in DSMZ medium 104 [24] at 37°C. DNA was isolated from 1--1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) following the instructions given by the manufac-turer, but with a modi`ied protocol for cell lysis, LALMP, according to Wu et al. [25].

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger, 454 and Illumina sequencing platforms. All general aspects of library construction and se-quencing can be found at the JGI web site. Reads produced by 454 Pyrosequencing were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 2,196 overlapping fragments of 1,000 bp and en-tered into the assembly as pseudo--reads. The se-quences were assigned quality scores based on Newbler consensus q--scores with modi`ications to account for overlap redundancy and to adjust in-lated q--scores. A hybrid 454/Sanger assembly was made using the Arachne assembler. Possible mis-assemblies were corrected and gaps between con-tigs were closed by custom primer walks from sub-clones or PCR products. A total of 66 Sanger inish-ing reads were produced. Illumina reads were used to improve the `inal consensus quality using an in-house developed tool (the Polisher). The `inal as-sembly consisted of 18,576 Sanger and 464,157 Roche/454 reads. The error rate of the completed genome sequence is less than 1 in 100,000. To-gether all sequence types provided 49.1 coverage of the genome.

Genome annotation
Genes were identi`ied using Prodigal [26] as part of the Oak Ridge National Laboratory genome annota-tion pipeline, followed by a round of manual cura-tion using the JGI GenePRIMP pipeline [27]. 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 (IMG--ER) platform [28].

Genome properties
The genome is 1,998,633 bp long (chromosome and one circular plasmid) with a 35.6% GC content (Ta--ble 3). Of the 1,913 genes predicted, 1,852 were protein coding genes, and 61 were RNAs. A total of 46 pseudogenes were also identi`ied, with 73.1% of the genes being assigned a putative function. The remaining genes were annotated as hypothetical proteins. The distribution of genes into COGs func-tional categories is presented in Figure 3 and Table 4.