Draft genome sequence of Paenibacillus sp. strain A2

Paenibacillus sp. strain A2 is a Gram-negative rod-shaped bacterium isolated from a mixture of formation water and petroleum in Daqing oilfield, China. This facultative aerobic bacterium was found to have a broad capacity for metabolizing hydrocarbon and organosulfur compounds, which are the main reasons for the interest in sequencing its genome. Here we describe the features of Paenibacillus sp. strain A2, together with the genome sequence and its annotation. The 7,650,246 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 54.2 % and contains 7575 protein-coding and 49 RNA genes, including 3 rRNA genes. One putative alkane monooxygenase, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter and four putative sulfate transporters were found in the draft genome.


Introduction
Paenibacillus is a genus of aerobic, Gram-positive, rodshaped, and endospore forming bacteria, formerly included within the genus Bacillus, but was proposed as a separate genus in 1993 on the basis of its unique distrinctive phenotypic and genotypic features [1]. Strains in this genus have been detected in a variety of environments including soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples [2][3][4][5][6]. One hundred and forty nine species and four subspecies have previously been recorded in the genus Paenibacillus. These bacteria produce various metabolites, which can catalyze a wide variety of synthetic reactions in fields ranging from cosmetics to biofuel production and have gained importance in agriculture, industrial and medical applications [7].
Surfactant flooding is an important form of EOR to reduce the interfacial tension between oil and water to an ultra-low value [8]. Until now, sulfonate surfactants have been widely adopted as flooding agents in EOR in some oilfields under different geological conditions [9].
Surfactant flooding technology has been widely applied in the Daqing oilfield (China), and in our previous work three indigenous bacteria were isolated as crude-oil degrading species that enhance oil recovery [10]. While screening hydrocarbon-degrading bacteria previously, we isolated a Paenibacillus sp. strain A2 from a mixture of formation water and petroleum in Daqing oilfield. Strain A2 grows aerobically with tetradecane and hexadecane as the sole carbon and energy source, and was also found to have a capacity to metabolize organosulfur compounds. To date, data on the genetic basis of metabolizing hydrocarbon and sulfur compounds in genus Paenibacillus are only sparsely available. To gain insight into the nature and genomic plasticity of this strain from a unique niche its genome was sequenced and here we report a summary classification and genome annotations for Paenibacillus sp. strain A2.
predominantly singly (Fig. 1). Growth occurs under aerobic condition. The optimum temperature for growth is 37°C, with a temperature range of 15-45°C (Table 1). Cell morphology, motility and sporulation were examined by using scanning electron microscopy (Quanta 200, FEI Co., USA).
Biochemical features were tested by using two automated systems, the Vitek2 Compact (bioMérieux, Marcy l'Etoile,  Evidence codes -IDA inferred from direct assay, 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). These evidence codes are the Gene Ontology project [43] France) and Phoenix 100 ID/AST system (Becton Dickinson Company, Sparks, MD. USA). Positive reactions were obtained for glucose, xylose, mannitol and arabinose. Negative reactions were observed for fructose, trehalose, gluconic acid, sucrose, maltose, urea, cellobiose, glucoside, tagatose and maltotriose. This strain was susceptible to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, tri-methoprim/sulfamethoxazole, amoxicillin, imipenem, meropenem, ciprofloxacin, tigecycline and rifampicin, but resistant to metronidazole.

Genome sequencing information
Genome project history Paenibacillus sp. strain A2 was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the genus Paenibacillus, and is part of a microbial diversity study of the oilfield aiming at isolating all bacterial species degrading crude-oil. This whole genome shotgun project of Paenibacillus sp. strain A2 is deposited in the Genome On Line Database and the draft genome sequence is deposited at DDBJ/EMBL/GenBank under the accession  Table 2 [11].

Growth conditions and genomic DNA preparation
Paenibacillus sp. strain A2 was grown aerobically on LB broth, at 37°C for 16 h. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Germany), according to the manufacturer's recommended protocol. The quantity of DNA was measured by the NanoDrop Spectrophotometer and Cubit. Then 10 μg of DNA was sent to BGI (Shenzhen, China) for sequencing on a Hiseq2000 system.

Genome sequencing and assembly
One DNA library was generated (450 bp insert size, with the Illumina adapter at both ends detected by Agilent DNA analyzer 2100), then sequenced using an Illumina Hieseq 2000 genomic sequencer, with a 2 × 100 pair end sequencing strategy. Finally, we obtained a total of 5,728,134 M bp and performed the following quality control steps: 1) Reads linked to adapters at both end were considered as sequencing artifacts and removed. 2) Bases with quality index lower than Q20 at both ends were trimmed. 3) Reads with ambiguous bases (N) were removed. 4) Single qualified reads were discarded (In this situation, one read is qualified but its mate is not). Filtered 1378 M clean data were assembled into scaffolds using the Velvet version 1.2.07 with parameters "-scaffolds no" [12], then we use a PAGIT flow [13] to prolong the initial contigs and correct sequencing errors to arrive at a set of improved scaffolds.  The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome The total is based on the total number of protein coding genes in the annotated genome

Genome annotation
Predicted genes were identified using Glimmer version 3.0 [14]. tRNAscan-SE version 1.21 [15] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer version 1.2 [16]. To annotate predicted genes, we used HMMER version 3.0 [17] to align genes against Pfam version 27.0 [18] (only pfam-A was used) to find genes with conserved domains. KAAS server [19] was used to assign translated amino acids into KEGG Orthology [20] with single-directional best hit method. Translated genes were aligned with the COG database [21,22] using NCBI blastp (hits should have scores no less than 60, e value is no more than 1e-6). To find genes with hypothetical or putative functions, we aligned genes against the NCBI nucleotide sequence database (nt database was downloaded at Sep 20, 2013) by using NCBI blastn, only if hits have identity no less than 0.95, coverage no less than 0.9, and reference genes were annotated as putative or hypothetical. To define genes with a signal peptide, we use SignalP version 4.1 [23] to identify genes using default parameters. TMHMM 2.0 [24] was used to identify genes with transmembrane helices. Prophages and putative phage like elements in the genome were identified using prophage-predicting PHAST [25]. Blast of the three genomes together with strain 2745-2 were performed using blast+program [26]. BLAST Ring Image Generator (BRIG) was used for genome alignment visualization [27].

Genome properties
The draft genome sequence of Paenibacillus sp. strain A2 revealed a genome size of 7,650,246 bp and a G+C content of 54.2 % ( Table 3). The genome contain 7575 coding sequences, 46 tRNAs (excluding 1 pseudo tRNAs) and incomplete rRNA operons (one small subunit rRNA and two large subunit rRNAs). A total of 3112 proteincoding genes were assigned as putative function or hypothetical proteins. Four thousand seven hundred ten genes were categorized into COGs functional groups (including putative or hypothetical genes). The properties and the statistics of the genome are summarized in Tables 3 and 4. Nine prophage regions have been identified in the genome of strain A2 (Fig. 3), including one intact, six incomplete and two questionable regions (Table 5).

Insights from the genome sequence
Paenibacillus sp. strain A2 grows aerobically with tetradecane and hexadecane as the sole carbon and energy source, and has capability of degrading alkanesulfonate suggesting that it has developed a number of evolutionary  strategies that allow for habitat adaptation. To identify pathways associated with niche adaptation to a petroleum reservoir, we explored the genome content for genes associated with hydrocarbon and sulfur metabolism (Table 6). Alkane monooxygenases have been proposed as one of the two unrelated classes of enzymes responsible for the aerobic transformation of midchain-length n-alkanes (C5 to C16) and in some cases even longer alkanes [28]. Sulfate transporters and alkanesulfonate transporter have been shown to play an essential role in metabolizing organosulfur compounds [29,30]. Based on this knowledge, the genome sequence of strain A2 provides the basis to elucidate its genetic basis for crude oil degradation and adaptation to the petroleum reservoir. BLAST search of nucleotide sequence between strain A2 and other seven Paenibacillus species showed that A2 has highest similarity with Paenibacillus elgii B69, which is consistent with the 16 s rRNA sequence alignment (Fig. 4).

Conclusions
Paenibacillus sp. strain A2, was isolated from a mixture of formation water and petroleum and has a broad capacity for metabolizing hydrocarbon and organosulfur compounds. To date, no metabolc pathways involved in petroleum degradation or sulfur compounds have been characterized in genus Paenibacillus. The genome sequence of the A2 will hopefully provide new insights into the mechanism of degradation and microorganisms adapt to the petroleum reservoir after surfactant flooding. Furthermore, our data takes a step toward a comprehensive genomic catalog of the metabolic diversity of genus Paenibacillus.