Complete genome sequence of the facultatively anaerobic, appendaged bacterium Muricauda ruestringensis type strain (B1T)

Muricauda ruestringensis Bruns et al. 2001 is the type species of the genus Muricauda, which belongs to the family Flavobacteriaceae in the phylum Bacteroidetes. The species is of interest because of its isolated position in the genomically unexplored genus Muricauda, which is located in a part of the tree of life containing not many organisms with sequenced genomes. The genome, which consists of a circular chromosome of 3,842,422 bp length with a total of 3,478 protein-coding and 47 RNA genes, is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain B1 T (= DSM 13258 = LMG 19739 = KCTC 12928) is the type strain of the species Muricauda ruestringensis, which is the type species of the currently six species containing genus Muricauda [1,2]. The genus name was derived from the Latin words muris, of the mouse, and cauda, the tail; Muricauda, tail of the mouse, referring to the cellular appendages observed on some cells [1]. The species epithet is derived from the Neo-Latin word ruestringensis, pertaining to the former village of Rüstringen, which was destroyed by a tidal wave in 1362 [1]. Stain B1 T was isolated from a seawater sediment suspension from intertidal sediment at the German North Sea coast, which contained hexadecane as the sole carbon source during the initial cultivation. Later, the organism either turned out to be unable to degrade hexadecane or lost its ability to do so [1]. Other isolates belonging to the species are not known, nor was strain B1 T used for scientific work other than the description of the species M. ruestringensis. Here we present a summary classification and a set of features for M. ruestringensis strain B1 T , together with the description of the complete genomic sequencing and annotation.

Classification and features
A representative genomic 16S rRNA sequence of M. ruestringensis B1 T was compared using NCBI BLAST [3,4] under default settings (e.g., considering only the high-scoring segment pairs (HSPs) from the best 250 hits) with the most recent release of the Greengenes database [5] and the relative frequencies of taxa and keywords (reduced to their stem [6]) were determined, weighted by BLAST scores. The most frequently occurring genera were Muricauda (24.7%), Maribacter (24.0%), Cytophaga (12.3%), Zobellia (9.6%) and Flavobacterium (7.1%) (118 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.7%, whereas the average coverage by HSPs was 93.8%. Regarding the six hits to sequences from other members of the genus, the average identity within HSPs was 97.9%, whereas the average coverage by HSPs was 97.9%. Among all other species, the one yielding the highest score was Muricauda aquimarina (EU440979), which corresponded to an identity of 98.7% and an HSP coverage of 98.4%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was HQ326265 ('Microbial structure biofilm on SWRO membranes clone SBS-FW-047'), which showed an identity of 98.5% and an HSP coverage of 98.0%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'microbi' (4.7%), 'sediment' (4.1%), 'sea' (2.9%), 'marin' (2.4%) and 'biofilm' (2.4%), (132 hits in total). Environmental samples which yielded hits of a higher score than the highest scoring species were not found. Figure 1 shows the phylogenetic neighborhood of M. ruestringensis in a 16S rRNA based tree. The sequences of the two identical 16S rRNA gene copies in the genome differ by one nucleotide from the previously published 16S rRNA sequence (AF218782). The tree was inferred from 1,481 aligned characters [7,8] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [9]. Flavobacterium aquatile was included in the dataset for use as outgroup taxa. The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 850 ML bootstrap replicates [10] (left) and from 1,000 maximum-parsimony bootstrap replicates [11] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [12] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks.
Cells of strain B1 T are rod-shaped with rounded ends, 0.3 -0.6 µm wide and 1.1 -2.7 µm long (Figure 2 and Table 1) [1]. Cells of older cultures are characterized by mainly polar appendages with vesicle-like structures (blebs) at the end ( Figure  2), which were discussed in detail by Bruns et al. in [1] and probably serve to contact cells to each other or for colonization of a substratum [1]. The non-motile cells (see missing genes in the motility category in COGs table) stain Gram-negative and grow as facultative anaerobes in seawater. The temperature range for growth is between 8°C and 40°C, with an optimum between 20 and 30°C [1]. The pH range for growth is 6.0-8.0, with an optimum at pH 6.5-7.5 [1]. Physiology and metabolism are discussed in detail in [1], with the surprising discovery that although strain B1 T was isolated from a continuous-flow culture containing hexadecane as a sole carbon source, the strain was unable to degrade hexadecane (even if it was offered as cosubstrate along with other carbon sources); nor could it use acetate or pyruvate as sole carbon sources, but required a wide spectrum of amino acids as carbon and energy sources in addition to some carbohydrates [1].

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [29], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [30]. The genome project is deposited in the Genomes On Line Database [12] and the complete genome sequence is deposited 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.

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [33].
Pyrosequencing reads were assembled using the Newbler assembler (Roche Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [37]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 1,032.9 × coverage of the genome. The final assembly contained 422,407 pyrosequence and 49,819,141 Illumina reads.

Genome annotation
Genes were identified using Prodigal [38] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [39]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [40].

Genome properties
The genome consists of a 3,842,422 bp long circular chromosome with a G+C content of 41.4% (Table 3 and Figure 3). Of the 3,525 genes predicted, 3,478 were protein-coding genes, and 47 RNAs; 46 pseudogenes were also identified. The majority of the protein-coding genes (66.6%) 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.