Genome sequence of the orange-pigmented seawater bacterium Owenweeksia hongkongensis type strain (UST20020801T)

Owenweeksia hongkongensis Lau et al. 2005 is the sole member of the monospecific genus Owenweeksia in the family Cryomorphaceae, a poorly characterized family at the genome level thus far. This family comprises seven genera within the class Flavobacteria. Family members are known to be psychrotolerant, rod-shaped and orange pigmented (β-carotene), typical for Flavobacteria. For growth, seawater and complex organic nutrients are necessary. The genome of O. hongkongensis UST20020801T is only the second genome of a member of the family Cryomorphaceae whose sequence has been deciphered. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,000,057 bp long chromosome with its 3,518 protein-coding and 45 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain UST20020801 T (= DSM 17368 = NRRL B-23963 = JCM 12287) is the type strain of the species Owenweeksia hongkongensis [1] in the monotypic genus Owenweeksia [1]. The genus was named after Owen B. Weeks for his work on Flavobacterium and Cytophaga during the 1950s to 1970s [1]. The species epithet points to Hong Kong, P. R. China, the place where the stain was isolated [1]. Strain UST20020801 T was first isolated in August 2002 from seawater samples collected from Port Shelter in Hong Kong during a study of the bacterial diversity in Hong Kong coastal sea water. Members of the phylum Bacteroidetes are widely distributed in marine and freshwater ecosystems. Compared to free-living bacteria, they were more frequently attached to aggregates [2,3] and occurred during algae blooms [4,5]. Representatives of the phylum Bacteroidetes, especially of the class Flavobacteria, are well-known to efficiently degrade and consume high-molecular-mass organic matter [6][7][8][9][10][11]. Recently, the family Cryomorphaceae was proposed to constitute a branch within the phylum Bacteroidetes [12]. This family encompasses the marine genera Brumimicrobium, Cryomorpha, Crocinitomix [12], Owenweeksia [1], Lishizhenia [13], Wandonia [14], and "Phaeocystidibacter" [15] as well as the freshwaterliving genus Fluviicola [16]. Here we present a summary classification and a set of features for O. hongkongensis UST20020801 T , together with the description of the genomic sequencing and annotation.

Genome sequencing and annotation Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [41], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [42]. The genome project is deposited in the Genomes On Line Database [27] 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. The tree was inferred from 1,409 aligned characters [21,22] of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion [23]. Rooting was done initially using the midpoint method [24] and then checked for its agreement with the current classification ( Table 1). The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 400 ML bootstrap replicates [25] (left) and from 1,000 maximum-parsimony bootstrap replicates [26] (right) if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [27] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [28].   [29] and NamesforLife [30]. Evidence codes -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). Evidence codes are from the Gene Ontology project [40]. . DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification kit (GENOMED 600100) following the standard protocol as recommended by the manufacturer with an extended cell-lysis procedure, i.e. incubation with additional 80 µl protease K for one hour at 58°C. DNA is available through the DNA Bank Network [44].

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 [45]. Pyrosequencing reads were assembled using the Newbler assembler (Roche Possible mis-assemblies were corrected with gapResolution [45], Dupfinisher [48], 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 58 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 the software Polisher developed at JGI [49]. 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 308.6 x coverage of the genome. The final assembly contained 291,505 pyrosequence and 75,503,620 Illumina reads.

Genome annotation
Genes were identified using Prodigal [50] as part of the Oak Ridge National Laboratory genomeannotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [51]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [52].

Genome properties
The genome consists of a 4,000,057 bp long circular chromosome with a G+C content of 40.2% ( Figure 3 and Table 3). Of the 3,563 genes predicted, 3,518 were protein-coding genes, and 45 RNAs; 33 pseudogenes were also identified. The majority of the protein-coding genes (67.9%) 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.

Insights into the genome sequence
Genome analysis of strain UST20020801 T revealed the presence of genes encoding an arylsulfatase A family protein (Oweho_0043), a bacteriophytochrome (light-regulated signal transduction histidine kinase (Oweho_0350), a cytochrome c2 and a cytochrome c oxidase cbb3 type (Oweho_2085)). Additional gene sequences of interest encode a homogenisate 1,2-dioxigenase (Oweho_2010), a haloacid dehalogenase superfamily protein (Oweho_2094) as well as a 2-haloalkanoic acid dehalogenase type II (Oweho_2503). The presence of these genes could indicate that strain UST20020801 T plays a role in the respiratory degradation of recalcitrant compounds in its ecological niche. Further, a light-dependent regulation of metabolic activities using bacteriophytochrome as a sensor seems to be possible.