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Complete genome sequence of Anaerococcus prevotii type strain (PC1T)
Standards in Genomic Sciencesvolume 1, pages159–165 (2009)
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.
Anaerococcus prevotii strain PC1T (= DSM 20548 = ATCC 9321 = JCM 6508) is the type strain of the species and the type species of the genus . Six strains of the species were characterized by Foubert and Douglas in 1948, originally designated as ‘Micrococcus prevotii’, but subsequently placed in the genus Peptococcus . Based on a comparative study published by Ezaki et al. , 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 . 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 assigned to the arranged family ‘Peptostreptococcaceae’ within the order Clostridiales, also designated as Family XI Incertae sedis . Here we present a summary classification and a set of features for A. prevotii strain PC1T together with the description of the complete genomic sequencing and annotation.
Classification and features
Within the last few years, several changes occurred in the classification of the anaerobic Gram-positive cocci. There are currently five 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 discharges and ovarian, peritoneal, sacral or lung abscesses. In particular, A. prevotii was also described as a common isolate of the normal flora of skin, the oral cavity and the gut . Historically the Gram-positive anaerobic cocci were identified mainly by using phenotypic traits, but as shown by Song et al., this often led to the misidentification of A. vaginalis strains, which were mistakenly assigned to A. prevotii or A. tetradius . Currently Genbank does not contain any16S rRNA sequences from cultivated strains that can be clearly linked to the species A. prevotii with over 95% gene sequence similarity. 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 patients and showed close relationships to A. prevotii . No closely related isolates or uncultivated clones with more than 84% 16S rRNA gene sequence identity are recorded from global ocean screenings and environmental samples (except for human skin).
Figure 1 shows the phylogenetic neighborhood of A. prevotii strain PC1T in a 16S rRNA based tree. The four 16S rRNA gene copies in the genome of strain PC1T differ by up to 15 nucleotides from each other, and by up to 9 nucleotides from the previously published 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.
A. prevotii PC1T 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 irregular 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 PC1T metabolizes peptones and amino acids and the major metabolic end product from PYG medium is butyric acid. Most species of the genus Anaerococcus ferment carbohydrates weakly. A. prevotii is proteolytic. α-Glucosidase, α-galactosidase, ß-glucuronidase and pyroglutamyl arylamidase activities are detectable [19,20]. Production of urease may vary among strains of the species. Most strains produce ammonia from threonine and serine  by deamination of the amino acids to pyruvate. A. prevotii is resistant to sodium polyanethol sulfonate , but susceptible to the penicillins .
Cell wall amino acid analysis of strain PC1T yielded peptidoglycan type A4α′, composed of L-Lys-D-Glu , type A12.2 according to the DSMZ catalogue of strains. Cell wall sugars are glucose, glucosamine and galactose . Major cellular fatty acid composition of the type strain was analyzed by Lambert and Armfield in 1979  and by Ezaki et al. in 1983 , but the results of these studies are contradictory. 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 project. The genome project is deposited in the Genomes OnLine Database  and the complete genome sequence has been deposited in GenBank. Sequence, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
A. prevotii strain PC1T, DSM 20548, was grown anaerobically in DSMZ medium 104  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 manufacturer, but with a modified protocol for cell lysis, LALMP, according to Wu et al. .
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 sequencing 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 entered into the 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 Arachne assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. A total of 66 Sanger finishing reads were produced. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher). The final assembly 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. Together all sequence types provided 49.1 coverage of the genome.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . 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 .
The genome is 1,998,633 bp long (chromosome and one circular plasmid) with a 35.6% GC content (Table 3). Of the 1,913 genes predicted, 1,852 were protein coding genes, and 61 were RNAs. A total of 46 pseudogenes were also identified, 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 functional categories is presented in Figure 3 and Table 4.
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We would like to gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. 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, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.