Open Access

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

  • Marcel Huntemann
  • , Hazuki Teshima,
  • , Alla Lapidus
  • , Matt Nolan
  • , Susan Lucas
  • , Nancy Hammon
  • , Shweta Deshpande
  • , Jan-Fang Cheng
  • , Roxanne Tapia,
  • , Lynne A. Goodwin,
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Ioanna Pagani
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Chongle Pan,
  • , Evelyne-Marie Brambilla
  • , Manfred Rohde
  • , Stefan Spring
  • , Markus Göker
  • , John C. Detter,
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz,
  • , Nikos C. Kyrpides
  • , Hans-Peter Klenk
  • and Tanja Woyke
Corresponding author

DOI: 10.4056/sigs.2786069

Received: 04 May 2012

Published: 25 May 2012


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.


facultatively anaerobicnon-motileGram-negativemesophilicmarinechemoheterotrophicFlavobacteriaceaeGEBA


Strain B1T (= 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 B1T 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 B1T 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 B1T, together with the description of the complete genomic sequencing and annotation.

Classification and features

A representative genomic 16S rRNA sequence of M. ruestringensis B1T 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 a 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).

Figure 1

Phylogenetic tree highlighting the position of M. ruestringensis relative to the type strains of the other species within the genus Muricauda. 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 B1T 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 B1T 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].

Figure 2

Scanning electron micrograph of M. ruestringensis B1T

Table 1

Classification and general features of M. ruestringensis B1T according to the MIGS recommendations [13].




     Evidence code

      Current classification

     Domain Bacteria

     TAS [14]

     Phylum Bacteroidetes

     TAS [15,16]

     Class Flavobacteria

     TAS [17,18]

     Order Flavobacteriales

     TAS [19,20]

     Family Flavobacteriaceae

     TAS [21-24]

     Genus Muricauda

     TAS [1,25,26]

     Species Muricauda ruestringensis

     TAS [1]

     Type strain B1

     TAS [1]

      Gram stain


     TAS [1]

      Cell shape


     TAS [1]



     TAS [1]


     not reported

      Temperature range

     mesophile, 20°C–30°C

     TAS [1]

      Optimum temperature


     TAS [1]


     slightly halophilic, optimum 3% NaCl (w/v)

     TAS [1]


      Oxygen requirement

     facultatively anaerobic

     TAS [1]

      Carbon source

     various sugars and amino acids

     TAS [1]

      Energy metabolism


     TAS [1]




     TAS [1]


      Biotic relationship


     TAS [1]





      Biosafety level


     TAS [27]


     seawater sediment suspension

     TAS [1]


      Geographic location

     Jadebusen Bay, coast of North Sea, Germany

     TAS [1]


      Sample collection time

     1998 or earlier












     not reported



     about 0 m, sea level


Evidence codes - 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 [28].


The spectrum of whole-cell fatty acids represents the only chemotaxonomical data published thus far for strain B1T. The spectrum of fatty acids was dominated by branched-chain acids (72%): iso-C17:0 3OH (28.7%), iso-C15:1 (16.3%), iso-C15:0 (15.5%), iso-C15:0 3OH (4.9%), iso-C16:0 3OH (2.9%), iso-C17:0 2OH (2.8%), iso -C15:0 2OH (2.5%), C16:1 ω7c (2.5%), anteiso-C15:0 (2.4%), other acids below 2% [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.

Table 2

Genome sequencing project information





   Finishing quality



   Libraries used

     Four genomic libraries: one 454 pyrosequence standard library,     two 454 PE libraries (4 kb and 8 kb insert size),      one Illumina library


   Sequencing platforms

     Illumina GAii, 454 GS FLX Titanium


   Sequencing coverage

     996.4 × Illumina; 36.4 × pyrosequence



     Newbler version 2.3, Velvet version 0.7.63, phrap version SPS - 4.24


   Gene calling method

     Prodigal 1.4, GenePRIMP



   Genbank Date of Release

     August 19, 2011



   NCBI project ID


   Database: IMG-GEBA



   Source material identifier

     DSM 13258

   Project relevance

     Tree of Life, GEBA

Growth conditions and DNA isolation

M. ruestringensis strain B1T, DSM 13258, was grown in DSMZ medium 917 (Modified Sea Water Agar) [31] at 30°C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the manufacturer’s instructions, with a modified procedure for cell lysis: incubation with 40 μl proteinase K for 40 minutes at 58°C. DNA is available through the DNA Bank Network [32].

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). The initial Newbler assembly consisting of 26 contigs in one scaffold was converted into a phrap [34] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (3,847 Mb) was assembled with Velvet [35] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 268.3 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [34] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [33], Dupfinisher [36], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 46 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. 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 with 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.

Table 3

Genome Statistics



% of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of replicons


Extrachromosomal elements


Total genes



RNA genes



rRNA operons


tRNA genes



Protein-coding genes



Pseudo genes



Genes with function prediction



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


Figure 3

Graphical map of the chromosome. 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.

Table 4

Number of genes associated with the general COG functional categories








    Translation, ribosomal structure and biogenesis




    RNA processing and modification








    Replication, recombination and repair




    Chromatin structure and dynamics




    Cell cycle control, cell division, chromosome partitioning




    Nuclear structure




    Defense mechanisms




    Signal transduction mechanisms




    Cell wall/membrane/envelope biogenesis




    Cell motility








    Extracellular structures




    Intracellular trafficking, secretion, and vesicular transport




    Posttranslational modification, protein turnover, chaperones




    Energy production and conversion




    Carbohydrate transport and metabolism




    Amino acid transport and metabolism




    Nucleotide transport and metabolism




    Coenzyme transport and metabolism




    Lipid transport and metabolism




    Inorganic ion transport and metabolism




    Secondary metabolites biosynthesis, transport and catabolism




    General function prediction only




    Function unknown




    Not in COGs



We would like to gratefully acknowledge the help of Maren Schröder (DSMZ) for growing M. ruestringensis cultures. 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, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2.


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