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Slackia heliotrinireducens (Lanigan 1983) Wade et al. 1999 is of phylogenetic interest because of its location in a genomically yet uncharted section of the family Coriobacteriaceae, within the deep branching Actinobacteria. Strain RHS 1T was originally isolated from the ruminal flora of a sheep. It is a proteolytic anaerobic coccus, able to reductively cleave pyrrolizidine alkaloids. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Slackia, and the 3,165,038 bp long single replicon genome with its 2798 protein-coding and 60 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain RHS 1T (= DSM 20476 = ATCC 29202 = JCM 14554) is the type strain of the species Slackia heliotrinireducens and was originally described by Lanigan in 1976 as Peptococcus heliotrinreducans (sic)  and validly published following an orthographic correction as Peptococcus heliotrinreducens in 1983 [2,3]. The strain was later transferred to the genus Peptostreptococcus on the basis of its G+C content . 16S rRNA gene sequence analysis indicated that it should not be assigned to the genus Peptostreptococcus and therefore the strain was subsequently allocated to the novel genus Slackia as S. heliotrinireducens [5,6]. The three species of the genus Slackia, S. exigua, S. faecicanis, and S. heliotrinireducens form a distinct cluster within the Coriobacteriaceae, located in the neighborhood to the genera Cryptobacterium and Collinsella.
Five additional strains identified as S. heliotrinireducens based on their proteolytic enzyme profiles have been isolated from human polymicrobial abscesses , but these strains were dissimilar from the type strain as shown by pyrolysis mass spectrometry . With 94% sequence identity (16S rRNA gene), S. exigua, the type strain of the closest related species represents the only meaningful (>91%) hit in nucleotide sequence database searches, indicating a complete lack of cultivated and even uncultivated relatives of strain RHS 1T in accessible microbiological diversity. Screening of environmental samples and surveys reported at NCBI BLAST server indicated no closely related phylotypes that can be linked to the species (as of July 2009). Here we present a summary classification and a set of features for S. heliotrinireducens RHS 1T Together with the description of the complete genomic sequencing and annotation.
Figure 1 shows the phylogenetic neighborhood of S. heliotrinireducens strain RHS 1T in a 16S rRNA based tree. The sequence of one of the two 16S rRNA genes differs in two nucleotides from the other copy and from the previously published 16S rRNA sequence generated from ATCC 29202 (AF101241).
S. heliotrinireducens is Gram-positive, nonmotile, obligatly anaerobic, and does not produce endospores (Table 1). Strain RHS 1 forms cocci or coccobacilli (Figure 2) with a diameter of 0.3 to 0.6 µm and 0.8 x 1.2 µm, respectively [5,6]. The strain grows very slowly on blood agar and forms small translucent, glistening colonies, up to 1 mm in diameter after extensive incubation. It does not utilize carbohydrates, but reduces nitrates and pyrrolizidine alkaloids [5,6]. Reductive cleavage of pyrrolizidines (heliotrine, europine, heleurine, supinine and lasiocarpine) occurs by using hydrogen gas or formate as hydrogen donor . Ammonia is formed from tryptone, yeast extract, adenine, uracil and arginine. Nitrates are completely reduced to ammonia if an appropriate electron donor (H2, formate) is present . The strain is bile-sensitive, indole-negative, hydrolyses arginine but not esculin. Does not produce catalase or urease, but is able to dissimilate arginine. Growth is generally stimulated by addition of 0.5% arginine. Metabolic products from S. heliotrinireducens grown in prereduced PYG broth are acetic acid, isovaleric acid, and butyric acid in trace amounts .
Almost nothing is known about the chemotaxonomy of strain RHS 1T, except that its predominant cellular fatty acid is C18:1 .
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 Genome OnLine Database  and the complete genome sequence is 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.
S. heliotrinireducens strain RHS 1T, DSM 20476, was grown anaerobically in DSMZ medium 104 (PYG) ; at 37°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified protocol for cell lysis (FT), as described in Wu et al. .
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 on 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 3,507 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 parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 1,433 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The final assembly consists of 21.045 Sanger and 205,234 pyrosequence (454) reads. Together all sequence types provided 26× coverage of the genome. The error rate of the completed genome sequence is less than 1 in 100,000.
Genes were identified using GeneMark  as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review system (http://img.jgi.doe.ogv/er) , followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org/) . 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. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using the tool RNAmmer . Other non coding RNAs were identified by searching the genome for the Rfam profiles using INFERNAL (v0.81) . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (http://img.jgi.doe.gov/) platform .
The metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 12.5  and MetaCyc version 12.5 , based on annotated EC numbers and a customized enzyme name mapping file. It has undergone no subsequent manual curation and may contain errors, similar to a Tier 3 BioCyc PGDB .
The genome is 3,165,038 bp long and comprises one main circular chromosome with a 60.2% GC content (Table 3 and Figure 3). Of the 2,858 genes predicted, 2,798 were protein coding genes, and 60 RNAs; 33 pseudogenes were also identified. The majority of the protein-coding genes (70.6%) were assigned with a putative function, while those remaining 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, and a cellular overview diagram is presented in Figure 4, followed by a summary of metabolic network statistics shown in Table 5.
We would like to gratefully acknowledge the help of Gabriele Gehrich-Schröter (DSMZ) for growing S. heliotrinireducens cultures. This work was performed under the auspices of the US Department of Energy's 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.