Genome sequencing and annotation of Geobacillus sp. 1017, a hydrocarbon-oxidizing thermophilic bacterium isolated from a heavy oil reservoir (China).

The draft genome sequence of Geobacillus sp. strain 1017, a thermophilic aerobic oil-oxidizing bacterium isolated from formation water of the Dagang high-temperature oilfield, China, is presented here. The genome comprised 3.6 Mbp, with the G + C content of 51.74%. The strain had a number of genes responsible for numerous metabolic and transport systems, exopolysaccharide biosynthesis, and decomposition of sugars and aromatic compounds, as well as the genes related to resistance to metals and metalloids. The genome sequence is available at DDBJ/EMBL/GenBank under the accession no MQMG00000000. This genome is annotated for elucidation of the genomic and phenotypic diversity of new thermophilic alkane-oxidizing bacteria of the genus Geobacillus.

Direct link to deposited data

https://www.ncbi.nlm.nih.gov/nuccore/MQMG01000000

Experimental design, materials and methods

The genus Geobacillus was originally proposed in 2001 [1]. Members of this genus growing at temperatures between 35 and 75 °C are widespread throughout the world and can be recovered from various natural environments with temperatures ranging from 80 °C to that of glacier ice (such as hot springs, petroleum reservoirs, compost, marine hydrothermal vents, soils, lake and ocean sediments, and arctic waters) [1]. A recent revision of this genus [2] based on analysis of the 16S rRNA gene sequences resulted in a considerable decrease in the number of Geobacillus species. Apart from the 16S rRNA gene, sequence similarity analyses of the recN, gyrB, and parE genes was proposed for differentiation of species within the genus Geobacillus [3], [4].

The strain 1017 (= VKM B-3132) was isolated from a production water sample of the Dagang high-temperature heavy oil reservoir (Hebei Province, China) (38°40′4.7″ N, 117°22′38.0″ E) [4], [5]. Strain 1017 is an aerobic endospore-forming bacterium, motile during the early exponential growth phase, and able to grow within the temperature range from 38 to 68 °C with an optimum at 60 °C. The strain utilizes fatty acids, sugars, amino acids, aromatic compounds, and n-alkanes of crude oil. The 16S rRNA gene sequence of strain 1017 has 99.5% similarity with the respective gene of Geobacillus kaustophilus R-35646 (FN428694) [5]. Analysis of the gyrB and parE genes of the new strain 1017 revealed 96.0% and 97% similarity to the relevant genes of G. kaustophilus NBRC 102445T, (NZ_BBJV01000018, BBJV01000017), which made it possible to suggest this strain as a new Geobacillus species. Analysis of the genome of strain 1017 was carried out in order to specify its taxonomic position and to elucidate the genotypic diversity within the genus Geobacillus.

Genomic DNA was isolated from the biomass using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer's instructions. Geobacillus sp. 1017 genomic DNA was sequenced with a Roche Genome Sequencer (GS FLX), using the Titanium XL + protocol for a shotgun genome library. About 356 Mb of raw sequences with an average read length of 5394 nucleotides were produced providing approximately 100-fold genome coverage. The reads were de novo assembled into contigs using Newbler Assembler version 2.9 (454 Life Sciences, Branford, CT). The draft genome of Geobacillus sp. 1017 consists of 146 contigs longer than 500 bp, with a total length of 3,562,450 bp. The N50 contig size of the genome is 52,882 bp and the G + C content of 51.74%.

Gene search and annotation were performed for all contigs longer than 500 bp using the RAST server [6]. SEED viewer was used for assignment of the predicted genes to functional categories [7]. The genome was predicted to contain 3896 protein-coding sequences, 86 tRNAs and 24 coded rRNAs (5S, 16S, and 23S). Most of the annotated genes determined amino acids and derivatives synthesis (435), carbohydrate metabolism (409), cofactors, vitamins, prosthetic groups and pigment formations (296), protein metabolism (244), and RNA metabolism (149) (Fig. 1).

Fig. 1

Subsystems of Geobacillus sp. 1017 based on SEED database.

The 16S rRNA, gyrB and parE gene sequences revealed in the genome had 100%, 100% and 99.9% similarity, respectively, with those determined earlier for the strain 1017 [5]. The results of phenotypic studies characterizing the strain as an aerobic spore-forming bacterium utilizing a wide range of organic substrates were confirmed by detection of respective genes. Despite utilization of С10,11 and С13–С19,22 n-alkanes by strain 1017, neither alkB nor ladA genes were found in the draft genome. A range of protein-coding sequences involved in the pathways for the oxidation of aromatic compounds was detected in the genome of the strain. Further studies of the genes involved in biodegradation of alkanes and aromatic compounds from crude oil by aerobic thermophilic bacteria are necessary. Overall, in silico results confirmed that the strain 1017 was a member of the genus Geobacillus, though further characterization work is required to determine its species.

Nucleotide sequence accession numbers

This Geobacillus sp. 1017 whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession MQMG00000000. The version described in this paper is version MQMG01000000.

Conflict of interest

The authors declare that there is no conflict of interests on the work published in this paper.

Specifications
Organism/cell line/tissue	Geobacillus sp.
Sequencer or array type	Sequencer; Roche 454
Data format	Processed
Experimental factors	Microbial strains
Experimental features	Draft genome sequence of Geobacillus sp. strain 1017 assembly and annotation
Consent	N/A
Sample source location	A production water sample from the Dagang high-temperature heavy oil reservoir (Hebei Province, China) (38°40′4.7″ N, 117°22′38.0″ E).