The peptidoglycan of B. cavernae HKI 0122^T contains D- and L-alanine, D- and L-glutamic acid and L-lysine, with the latter widely distributed among actinobacteria [1]. The strain possesses a type A4〈 peptidoglycan with a diagnostic L-Lys←L-Glu interpeptide bridge, type A11.54 according to DSMZ. Glucose, mannose and galactose are the cell wall sugars [1]. The fatty acid profile of strain B. cavernae HKI 0122^T is dominated by 13-methyl tetradecanoic (iso-C_15:0; 43.7%) and 12-methyl tetradecanoic (anteiso-C_15:0; 34.6%) saturated, branched chain acids. Other predominantly saturated fatty acids play a minor role in the cellular fatty acid composition of the strain: iso-C_14:0 (0.9%), C_14:0 (1.9%); C_15:0 (0.9%) isoC_16:0 (2.3%), C_16:0 (6.8%), isoC_17:0 (3.1%), anteiso-C_17:0 (4.9%), und C_18:1 (0.9%) [1]. Mycolic acids are not present [1]. MK-8(H₄) is the major menaquinone, complemented by minor amounts of MK-8(H₂), MK-8 and MK-9(H₄) [1]. The combination of the L-Lys←L-Glu interpeptide bridge and MK-8(H₄) as the dominating menaquinone is shared with the organisms from the neighboring genera Bogoriella and Georgenia. The polar lipids of strain HKI 0122^T consist of phosphatidylinositol and diphosphatidylglycerol together with three yet unidentified phospholipids [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 Genomes OnLine Database [8] and the complete genome sequence in GenBank (CP001618). Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

B. cavernae HKI 0122^T, DSM 12333, was grown in DSMZ medium 736 (Rich Medium) [13] at 28°C. DNA was isolated from 0.5-1 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modification of the standard protocol for cell lysis in first freezing for 20 min. (-70°C), then heating 5 min. (98°C), and cooling 15 min to 37°C; adding 1.5 ml lysozyme (standard: 0.3 ml, only), 1.0 ml achromopeptidase, 0.12 ml lysostaphine, 0.12 ml mutanolysine, 1.5 ml proteinase K (standard: 0.5 ml, only), followed by overnight incubation at 35°C.

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 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 5,256 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 [14]. Gaps between contigs were closed by editing in Consed, custom primer walking or PCR amplification. A total of 1,627 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 19.42x coverage of the genome.

Genes were identified using Prodigal [15] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGIGenePRIMP pipeline [16]. 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 4,669,183 bp long and comprises one main circular chromosome with a 73.1% GC content. (Table 3 and Figure 3). Of the 4,278 genes predicted, 4,225 were protein coding genes, and 53 RNAs. Twenty eight pseudogenes were also identified. The majority of the genes (74.3%) were assigned 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 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