Complete genome sequence of Stackebrandtia nassauensis type strain (LLR-40K-21T)

Stackebrandtia nassauensis Labeda and Kroppenstedt (2005) is the type species of the genus Stackebrandtia, and a member of the actinobacterial family Glycomycetaceae. Stackebrandtia currently contains two species, which are differentiated from Glycomyces spp. by cellular fatty acid and menaquinone composition. Strain LLR-40K-21T is Gram-positive, aerobic, and nonmotile, with a branched substrate mycelium and on some media an aerial mycelium. The strain was originally isolated from a soil sample collected from a road side in Nassau, Bahamas. 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 actinobacterial suborder Glycomycineae. The 6,841,557 bp long single replicon genome with its 6487 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain LLR-40K-21 T (=DSM 44728 = NRRL B-16338 = JCM 14905) is the type strain of Stackebrandtia nassauensis, which is the type species of the genus Stackebrandtia [1]. S. nassauensis was originally isolated by M. P. Lechevalier and subsequently described by Labeda and Kroppenstedt [1] during the course of a 16S rRNA survey of putative Glycomyces strains. The genus was named after Erko Stackebrandt, a German microbiologist of note, who has contributed significantly to the molecular systematics the Actinobacteria. At present the genus Stackebrandtia contains only one addi-tional species: S. albiflava, isolated from a soil sample collected from a tropical rainforest in China [2] .Here we present a summary classification and a set of features for S. nassauensis strain LLR-40K-21 T together with the description of the complete genomic sequencing and annotation.

Classification and features
A search of GenBank revealed no 16S rRNA reference sequences that were closely related to S. nassauensis. With 95% sequence similarity, the type strain S. albiflava, YIM 45751 [2], is the only cultivated strain with a sequence similarity above 91%, whereas a 16S rRNA gene sequence derived from a sample isolated from pig slurry (pig saw dust spent bedding in France, M982657, Snell-Castro et al., unpublished), represents the only related phylotype (with the same degree of sequence similarity as YIM 45751). Curiously, the type strains of the neighboring genus Glycomyces [3] were not within the 250 top hits in BLAST searches, with the 16S rRNA of type species G. harbinensis [3] sharing only 89% sequence similarity. Screening of envi-ronmental genomic samples and surveys reported at the NCBI BLAST server also showed no closely related phylotypes (with 93% sequence identity at the maximum), indicating a rather limited environmental occurrence of the species S. nassauensis (as of July 2009). Figure 1 shows the phylogenetic neighborhood of S. nassauensis in a 16S rRNA based tree. The two 16S rRNA gene sequences in the genome of strain LLR-40K-21 T are identical and do not differ from the previously published 16S rRNA sequence generated from NRRL B-16338 (AY650268).

Figure 1.
Phylogenetic tree of S. nassauensis strain LLR-40K-21 T and all type strains of the family Glycomycetaceae, inferred from 1,390 aligned characters [4] of the 16S rRNA sequence under the maximum likelihood criterion [5,6]. The tree was rooted with Actinomyces bovis, the type strain of the order Actinomycetales. 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 [7] are shown in blue, published genomes in bold.

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 [7] and the complete genome sequence 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. 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 from the Gene Ontology project [14]. If the evidence code is IDA, then the property was observed for a living isolate by one of the authors or an expert mentioned in the acknowledgments.

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 7,157 overlap-ping fragments of 1,000 bp and entered into 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 phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [17]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 308 Sanger finishing reads were produced to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. The final assembly consists of 81,931 Sanger and 851,638 pyrosequence reads. Together all sequence types provided 40.0× coverage of the genome.

Genome annotation
Genes were identified using Prodigal [18] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [19]. 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 manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [20].

Genome properties
The genome is 6,841,557 bp long and comprises one circular chromosome with a 68.1% GC content ( Table 3 and Figure 3). Of the 6,450 genes predicted, 6,487 were protein coding genes, and 53 RNAs; One hundred eight pseudogenes were also identified. The majority of the protein-coding genes (66.8%) were assigned 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.