Genome sequence and overview of Oligoflexus tunisiensis Shr3T in the eighth class Oligoflexia of the phylum Proteobacteria

Oligoflexus tunisiensis Shr3T is the first strain described in the newest (eighth) class Oligoflexia of the phylum Proteobacteria. This strain was isolated from the 0.2-μm filtrate of a suspension of sand gravels collected in the Sahara Desert in the Republic of Tunisia. The genome of O. tunisiensis Shr3T is 7,569,109 bp long and consists of one scaffold with a 54.3% G + C content. A total of 6,463 genes were predicted, comprising 6,406 protein-coding and 57 RNA genes. Genome sequence analysis suggested that strain Shr3T had multiple terminal oxidases for aerobic respiration and various transporters, including the resistance-nodulation-cell division-type efflux pumps. Additionally, gene sequences related to the incomplete denitrification pathway lacking the final step to reduce nitrous oxide (N2O) to nitrogen gas (N2) were found in the O. tunisiensis Shr3T genome. The results presented herein provide insight into the metabolic versatility and N2O-producing activity of Oligoflexus species.


Introductions
The phylum Proteobacteria traditionally comprises five classes of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria [1,2], with two additional classes 'Zetaproteobacteria' and Acidithiobacillia proposed by Emerson et al. [3] and Williams and Kelly [4], respectively. Proteobacteria hosts the greatest number of isolates and sequenced genomes among the prokaryotic phyla [5] and contains members exhibiting extremely diversified metabolisms relevant to global carbon, nitrogen, and sulfur cycles [2]. This phylum recently gained the eighth (or seventh if yet-to-be-validated 'Zetaproteobacteria' is excluded) class Oligoflexia with the cultured species Oligoflexus tunisiensis type strain Shr3 T [6]. The class Oligoflexia includes environmentally-derived 16S rRNA gene sequences, otherwise known as environmental clones or phylotypes, recovered from a variety of habitats including soils, the Taklamakan Desert, glacial ice, lake water, seawater, human skin, and the guts of earthworms [6]. In contrast to their wide distribution, Oligoflexia-affiliated clones have rarely been found in clone libraries [7]; accordingly, it has been suggested that the Oligoflexia members show a small population size, belonging to the so-called rare biosphere [8].
At the time of writing, O. tunisiensis Shr3 T was the only cultured species within the class Oligoflexia. Physiological and biochemical features of strain Shr3 T could not be fully characterized because of restrictive culture conditions owing to the slow-growing nature of this strain [6]. The phenotypic information is essential for understanding its ecological role and biotechnological potentials. Here, we compensated for the limited knowledge regarding Oligoflexia members by conducting genomic analysis of strain Shr3 T .

Classification and features
During a study of ultramicro-sized bacteria that could pass through 0.2-μm pore-size filters, which are generally used for sterile filtration to remove microorganisms, we isolated the bacterium designated isolate Shr3 [9]. The isolation source of this bacterium was a 0.2-μm filtrate of the suspension of sand gravels collected in December 2008 in Matmata (33°3 1' N 9°57' E) on the eastern margin of the Sahara Desert in the Republic of Tunisia. Isolate Shr3 was thereafter described as the type strain of Oligoflexus tunisiensis, the first cultured representative of the novel class Oligoflexia [6]. Figure 1 shows the phylogenetic position of O. tunisiensis and related environmental clones in a 16S rRNA-based evolutionary tree. The sequence of the three 16S rRNA gene copies in the genome was 100% identical to the previously published 16S rRNA gene sequence (DDBJ/EMBL/ GenBank accession no. AB540021 [6]). The database search showed that seven environmental clones had a >97% high similarity with the O. tunisiensis 16S rRNA gene sequence [7]. The seven clones were from rice paddy soil, cyanobacterial blooms in a hypereutrophic lake, a microalgal photobioreactor, a bio-filter, and human skin [7]. Strain Shr3 T has been deposited in the Japan  Table 1.
O. tunisiensis Shr3 T is a Gram-negative, aerobic, nonmotile, filamentous bacterium of 0.4-0.8 μm in width when cultivated under the experimental culture conditions [6]. Some cells exhibited a spiral, spherical (or curled), or curved rod morphology [7]. Although the factors controlling the cell shapes are still unclear, the morphological flexibility is likely associated with their ability to pass through 0.2-μm filters. Strain Shr3 T grows in the R2A medium [6]. The cells showed slow growth, with 3-5 days required before colonies could be seen by the naked eye [6]. The growth occurs at NaCl concentrations <1.0% (w/v), 20-37°C (optimum 25-30°C), and pH 7.0-9.5 (optimum pH 7.0-8.0) [6]. Enzyme activities of esterase lipase, leucine arylamidase, trypsin, naphthol-AS-BI-phosphohydrolase and α-mannosidase are positive [6]. Transmission electron microscopy revealed that cells contained many low electron-dense particles (Fig. 2). Some, but not all, particles were stained by Sudan black B upon staining PHB or lipophilic particles. Because cells swollen by accumulated PHB were not observed when grown on PHB-containing medium [6], the particles stained with Sudan black B are likely lipophilic granules.

Genome project history
Phenotypic features of strain Shr3 T are described above, but could not be fully tested because of restrictive culture conditions [6]. Therefore, this organism was selected for genome sequencing to investigate the basis of its ecological role and biotechnological potentials. The genome project is deposited in the Genomes OnLine Database [11] under the accession number Gp0139475. The information genome sequence is available from the DDBJ/EMBL/GenBank database. A summary of this genome project is shown in Table 2.

Growth conditions and genomic DNA preparation
A culture of O. tunisiensis Shr3 T grown aerobically in R2A broth (DAIGO; Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) at 30°C was used to prepare genomic DNA. The genomic DNA was extracted using Qiagen Genomic-Tip 500/G columns according to the manufacturer's instructions. The quantity and purity of the extracted DNA was checked by spectrophotometric measurement at 260 nm and agarose gel electrophoresis.

Genome sequencing and assembly
The genome sequence was generated using paired-end sequencing (2 × 90 bp) on an Illumina HiSeq 2000 platform at the BGI with the pair-end library and mate-pair library of two different insert sizes, 456 to 496 bp and 6310 to 6350 bp. After trimming of low quality reads, 1130 Mb was obtained and assembled into 19 contigs in one scaffold using SOAPdenovo version 2.04 [12]. The assembly result was locally optimized according to the paired-end and overlap relationship via mapping reads to obtained contigs. A summary of this genome sequence is shown in Table 3.

Genome annotation
Gene sequences were identified via the Prodigal V2.6.3 [13] as part of the DOE-JGI genome annotation pipeline in the Integrated Microbial Genomes-Expert Review (IMG-ER) system [14]. Gene functional annotation as well as data visualization was conducted within the IMG-ER [15]. The predicted coding sequences were translated and used to search the National Center for Biotechnology Information nonredundant, UniProt, TIGR-Fam, Pfam, KEGG, COG, and InterPro databases. Identification of RNA gene sequences and miscellaneous features were carried out using HMMER 3.1b2 [16] and INFERNAL 1.0.2 and 1.1.1 [17]. Additional functional prediction was performed with the RAST server [18] under accession   [19].

Genome Properties
The genome of O. tunisiensis Shr3 T consists of a 7,569,109 bp long chromosome with a 54.3% G + C content (Table 3). Of the 6463 predicted genes, 6406 were protein-coding genes and 57 were RNA genes (three rRNA operons, 46 tRNAs, and two miscRNAs). The majority of the protein-coding genes (62.7%) were assigned to a putative function. The remaining ones were annotated as hypothetical proteins. The distribution of genes classified into COGs functional categories is shown in Table 4 and Fig. 3.

Insights from the genome sequence
The genome of O. tunisiensis Shr3 T encoded genes for ABC transporters of amino acid, oligopeptide/dipeptide, and phosphonate, ammonium and nitrate/nitrite transporters, as well as RND-type efflux pumps. One of the amino acid sequences (Ga0118670_114686) classified as an RND pump showed a high similarity (67% identity and 99% coverage) to sequences of the pathogenic  The total is based on the total number of protein coding genes in the genome bacteria Achromobacter xylosoxidans and Pseudomonas aeruginosa. The RND-type efflux system is widely distributed in Gram-negative bacteria and known to promote resistance to various kinds of antimicrobial substances, termed as multidrug resistance [20].
In support of its aerobic growth, gene sequences assigned to different terminal oxidases including aa 3 -and cbb 3 -type cytochrome c oxidases (COG0843 and COG3278) and cytochrome bd-type quinol oxidase (COG1271 and COG1294) were found in the Shr3 T genome.
The Shr3 T genome contained a nirK gene coding for a copper-dependent nitrite reductase (Nir) (Ga0118670_114712) involved in denitrification, a major component of the nitrogen cycle [21]. Denitrification is the dissimilatory reduction of nitrate or nitrite to nitrogen gas (NO 3 − → NO 2 − → NO → N 2 O → N 2 ) [22] that usually occurs under oxygen-limiting conditions [21]. The key steps releasing gaseous products NO, N 2 O, and N 2 are catalyzed by Nir, nitric-oxide reductase (Nor) and nitrous oxide reductase (Nos), respectively [23,24]. There are two structurally different nitrite reductases among denitrifiers: a copper-containing type (Cu-Nir) encoded by the nirK gene and a cytochrome cd 1 -containg one (cd 1 -Nir) encoded by the nirS gene [24]. The nirS gene was absent from the O. tunisiensis Shr3 T genome.
The NirK deduced amino acid sequence of O. tunisiensis Shr3 T was most closely related to that of Bdellovibrio bacteriovorus of the class Deltaproteobacteria, with 70% identity and 96% coverage. B. bacteriovorus has an incomplete denitrifying pathway with a Cu-Nir, a cytochrome cdependent Nor (cNor), and no Nos [25,26]. O. tunisiensis Shr3 T also had a partial pathway containing the Cu-Nir described above, a quinol-dependent Nor (qNor), and no Nos inferred from the genome data. Strain Shr3 T has two copies of the gene encoding qNor (Ga0118670_112818 and Ga0118670_114769). NorR protein is known to regulate Nor expression in response to NO [27,28]. The transcription regulator norR gene (Ga0118670_114771) was nearly adjacent to one of two copies of the qNor-encoding gene in the genome.
Our results suggest that the Oligoflexus species has the capability to produce N 2 O as a final product of the incomplete denitrification lacking the last step (reduction of N 2 O to N 2 ). N 2 O is known as a strong greenhouse gas, as well as an ozone-depleting substance [29]. Accordingly, future studies should examine the N 2 Oproducing phenotype of strain Shr3 T .

Conclusions
In this study, we characterized the genome of O. tunisiensis Shr3 T , the first cultured representative of the novel proteobacterial class Oligoflexia. The genome sequence gives us insight into the metabolic versatility and incomplete denitrification pathway of Oligoflexus species. The genome information will facilitate future systematics and comparative genomics studies within the phylum Proteobacteria.