Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains 一株分离自地层水的石油降解菌的特性研究：Ochrobactrum intermedium环境分离菌株与临床分离菌株的比较基因组分析 1 first-author 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhengbw@zju.edu.cnsheyuehui@163.com †E-mail:zhengbw@zju.edu.cnsheyuehui@163.com zhengbw@zju.edu.cn 3 3 4 4 5 1 3 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhengbw@zju.edu.cnsheyuehui@163.com †E-mail:zhengbw@zju.edu.cnsheyuehui@163.com zhengbw@zju.edu.cn Key Laboratory of Marine Reservoir Evolution and Hydrocarbon Accumulation Mechanism,School of Energy Resources, China University of Geosciences, Beijing 100083, China Key Laboratory of Marine Reservoir Evolution and Hydrocarbon Accumulation Mechanism,School of Energy Resources, China University of Geosciences, Beijing 100083, China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital,School of Medicine, Zhejiang University, Hangzhou 310003, China State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital,School of Medicine, Zhejiang University, Hangzhou 310003, China College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, China College of Chemistry and Environmental Engineering, Yangtze University, Jingzhou 434023, China Institute of Porous Flow & Fluid Mechanics, Chinese Academy of Sciences, Langfang 065007, China Institute of Porous Flow & Fluid Mechanics, Chinese Academy of Sciences, Langfang 065007, China State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 257061, China State Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing 257061, China In this study, we isolated an environmental clone of Ochrobactrum intermedium , strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301 T ) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2. 目的 2 对一株地层水分离的石油降解菌 Ochrobactrum intermedium 2745-2进行生理生化特性的研究、全基因组测序以及比较基因组研究。 创新点 2 首次对一株分离自地层水的石油降解菌 O. intermedium 2745-2进行了生理生化特性研究以及基因组测序，从基因组角度解释菌株2745-2对石油的降解能力。通过菌株2745-2与同种其他临床分离菌株的比较基因组学分析，表明2745-2仍具有多种与致病性相关的基因。 方法 2 通过微生物富集培养的方法从油井的地层水中分离石油降解微生物，通过聚合酶链反应（PCR）扩增16S核糖体RNA（rRNA）序列进行比较和分析确定菌株的分类地位属于 O. intermedium （图1）。采用Illumina HiSeq2000对菌株2745-2进行高通量测序，采用Velvet 1.2.07和RAST server分别进行数据组装和注释（表1）。PHAST寻找基因组中的噬菌体相关序列（图4和表2）。通过BLAST+和BRIG对环境分离菌株（2745-2）和临床分离菌株（M86、229E和LMG3301 T ）进行基因组比较（表3、表4和图5）。 结论 2 首次对一株环境分离的 O. intermedium （2745-2）进行全基因组测序，揭示具有多种与石油降解相关的基因。通过环境分离菌株（2745-2）与临床分离菌株（M86, 229E和LMG3301 T ）的基因组比较分析，表明2745-2仍具有多种致病性相关的基因。 Comparative genome Ochrobactrum intermedium Oil degradation Pathogen Ochrobactrum intermedium 石油降解 比较基因组 病原菌 Introduction 1 Oil contamination is a worldwide problem, which is growing more serious with economic development; its effects are long lasting and remediation is difficult. Several methods of oil degradation have been developed, the method with the longest history being land farming, which is “low-tech” but time consuming (Genouw et al., 1994 1994 ). Physical methods such as surface heating are more efficient but energy consuming (Edelstein et al., 1994 1994 ). Microbial oil degradation shows promise of being sustainable and environmentally friendly, and the screening of potential oil degrading microorganisms is becoming increasingly important. Bacteria from different habitats, such as soil (Jesubunmi, 2014 2014 ; Kumar et al., 2014 2014 ; Pham et al., 2014 2014 ) and the ocean (Hazen et al., 2010 2010 ; Hassanshahian et al., 2014 2014 ), are screened for their oil degrading properties. These bacteria are then used individually or in a mixture (Creencia et al., 2014 2014 ; Silva et al., 2015 2015 ). In our previous studies, several strains of bacteria, which have the ability to degrade crude oil, were isolated from the formation water of Chinese oilfields (She et al., 2011 2011 ; 2014 2014 ; Zhang et al., 2012 2012 ; 2014 2014 ; Zheng et al., 2014 2014 ). In this study, we isolated from the Changqing oilfield a strain which has rarely been isolated from an oilfield, Ochrobactrum intermedium strain 2745-2. O. intermedium was first described in 1998 with five strains formerly known as members of Ochrobactrum anthropi (Holmes et al., 1988 1988 ; Velasco et al., 1998 1998 ). The name, O. intermedium , indicates an intermediate position between O. anthropi and Brucella spp. (Velasco et al., 1998 1998 ). O. anthropi is an emerging opportunistic pathogen in immunocompromised patients (Mudshingkar et al., 2013 2013 ) and members of Brucella are pathogens causing brucellosis which is a common zoonotic infection globally (Dean et al., 2012 2012 ). Strains of O. intermedium are associated with both human beings and the environment. Some strains are pathogens which cause infection (Möller et al., 1999 1999 ; Apisarnthanarak et al., 2005 2005 ); some live in environments polluted by chromium (Kavita and Keharia, 2012 2012 ), lead (Waranusantigul et al., 2011 2011 ), and tobacco waste (Yuan et al., 2007 2007 ), etc. As a human pathogen and environmental bacterium, O. intermedium attracts a lot of interest. From a database survey, we found three draft genome sequences within O. intermedium , two of which (strains M86 and 229E) have been published (Kulkarni et al., 2013 2013 ; 2014 2014 ). Strains M86, 229E, and LMG3301 T were isolated from a stomach biopsy and blood taken from a non-ulcer dyspeptic individual from India. Thus, all of these three strains are associated with humans; no environmental strain had been sequenced before our study. Comparative genomic analysis is needed between human and environmental isolates of O. intermedium to give us a better understanding of the mechanisms by which it adapts to its environment. Here, we describe the classification and features of O. intermedium strain 2745-2, together with its genome sequence and the comparative genomic study we conducted with its clinical relatives, strains LMG3301 T , 229E, and M86. The aims of this work are to investigate the oil-degrading genes of strain 2745-2 and to find the distinction and similarities among the genomes and genes that reflect adaptation to specific environments. Materials and methods 1 Sampling and isolation of oil degrading bacteria 2 A water sample was collected from an oil producing well in Changqing oilfield, Shanxi Province, China, in 2012. The sample was stored immediately at 4 °C. Oil degrading bacteria were isolated using sterile crude oil as the medium. After incubation, the culture was spread on LB agar plates containing 5.0 g/L yeast extract (Difco, USA), 10.0 g/L NaCl, 10.0 g/L tryptone, and 20.0 g/L agar (Difco, USA) to select the single clones. One strain (2745-2) was further characterized. It was cultured in LB medium and genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. 16S ribosomal RNA (rRNA) was amplified by polymerase chain reaction (PCR) using the primers as follows: 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). Phylogenetic tree construction 2 16S rRNA nucleotide sequence analysis was conducted using the BLASTN program against the national center for biotechnology information (NCBI)-nucleotide collection (nr/nt) database. Sequences were aligned by the CLUSTALW (Larkin et al., 2007 2007 ). A Neighbor-Joining phylogenetic tree based on the Tamura-Nei model was constructed using MEGA6 software (Tamura et al., 2013 2013 ). Characterization of strain 2745-2 2 Cell morphology of strain 2745-2 was examined using a scanning electron micrograph (Quanta 200, FEI Co., USA). The temperature range, pH range, and NaCl range for growth were determined using methods described before (Cheng et al., 2015 2015 ). Gram-reaction was carried out according to Bergey’s manual (Holt et al., 1994 1994 ). Tests for H 2 S production and indole production were conducted using the method described by Mata et al. ( 2002 2002 ). Hydrolase of starch, gelatin, and casein were tested. Single carbon source utilization tests were performed using D -glucose, maltose, lactose, D -galactose, rhamnose, raffinose, sorbitol, glycerol, cellobiose, sucrose, tetradecane, and hexadecane. Resistance to ampicillin, erythromycin, tetracycline, kanamycin, and gentamicin were tested. Whole genome sequencing 2 Strain 2745-2 was cultivated aerobically in LB medium, pH 5.5 at 42 °C overnight. Genomic DNA was extracted using the method described by Marmur and Doty ( 1962 1962 ). The resulting genomic DNA was then measured using gel electrophoresis 0.7% (7 g/L) agarose with λ-Hind III digest DNA as the marker (TaKaRa, Dalian, China). The concentration of the genomic DNA was measured by NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific Inc., USA). Genomic DNA sequencing was performed using Illumina HiSeq2000 with Solexa paired-end sequencing strategy. One DNA library (500 bp insert size with Illumina adapter at both ends) was generated and detected by Agilent DNA analyzer 2100 (Agilent Technologies, USA). Sequence assembly and annotation 2 Clean reads were assembled into scaffolds using the Velvet version 1.2.07 (Zerbino and Birney, 2008 2008 ). We then used PAGIT flow (Swain et al., 2012 2012 ) to prolong the initial contigs and correct sequencing errors. The transfer RNAs (tRNAs) and rRNAs were identified using tRNAscan-SE (Lowe and Eddy, 1997 1997 ), RNAmmer (Lagesen et al., 2007 2007 ), and Rfam database (Griffiths-Jones et al., 2003 2003 ; Burge et al., 2012 2012 ). The genome annotation was predicted using the RAST server online (Aziz et al., 2008 2008 ). Predicted genes were blast against the Clusters of Orthologous Groups (COGs) database (Tatusov et al., 2000 2000 ; 2001 2001 ). We applied the PHAST program to predict the prophages and putative phage-like elements in the genome (Zhou et al., 2011 2011 ). Comparative genomic analysis 2 The genome sequences of O. intermedium M86, 229E, and LMG3301 T were downloaded from the NCBI database under the accession numbers AOGE00000000.1, ASXJ00000000.1, and ACQA 00000000.1, respectively (Kulkarni et al., 2013 2013 ; 2014 2014 ). All these genomes were annotated by the RAST on-line server, which was also used for subsystem annotations (Aziz et al., 2008 2008 ). Contigs were re-ordered using the Mauve program (Darling et al., 2010 2010 ). Blasts of the three genomes together with strain 2745-2 were performed using the BLAST+program (Camacho et al., 2009 2009 ). The BLAST Ring Image Generator (BRIG) was used for genome alignment visualization (Alikhan et al., 2011 2011 ). Nucleotide sequence accession number 2 The genome sequence of O. intermedium 2745-2 has been deposited in GenBank with the accession number JFHY00000000.1. Results 1 Phylogenetic analysis and characterization of strain 2745-2 2 Neighbor-Joining phylogenetic analysis indicated the taxonomic status of 2745-2, which is clearly classified into the same branch as O. intermedium LMG3301 T . Rhizobium leguminosarum IAM 12609 T was used as an out group (Fig. 1 1 ). GenBank accession numbers for 16S rRNA genes of the strains used in this phylogenetic tree are shown following the organism names. The bootstrap values on the branching nodes were calculated on 1000 replications. Rhizobium leguminosarum IAM 12609 T was used as an out group. The scale bar indicated 0.005 substitutions per nucleotide position Strain 2745-2 was capable of growing at 15–45 °C and pH 5.5–9.0 with optimum conditions being 42 °C and pH 5.5. Cells are straight rods, 0.6–0.9 μm in diameter and 1.7–5.3 μm long (Fig. 2 2 ). Colonies grown at 42 °C on LB agar plate are gray, circular, and convex. H 2 S and indole are produced. Gelatin and casein are hydrolyzed, but not starch. Lactose, rhamnose, tetradecane, and hexadecane are used as the carbon source, while D -glucose, maltose, D -galactose, raffinose, sorbitol, glycerol, cellobiose, and sucrose are not used. An antimicrobial susceptibility test showed that strain 2745-2 is resistant to ampicillin, erythromycin, tetracycline, kanamycin, and gentamicin. Cells were grown at 42 °C in LB broth for 16 h (about early stationary phase) Genome features 2 The draft genome size of O. intermedium 2745-2 was 4 800 175 bp with a G+C content of 57.62%. The draft genome contains 4737 coding sequences (CDSs) and 60 RNAs including two complete rRNA operons. Detailed information on the genome is summarized in Table 1 1 . A total of 4285 genes were categorized into COGs functional groups (Fig. 3 3 ). Five prophage regions have been identified (Fig. 4 4 ), including one intact, two incomplete, and two questionable regions (Table 2 2 ). 2 Size (bp) 2 G+C content (bp) 2 Coding region (bp) 4 Gene number Total RNA Protein-coding COGs 4 800 175 2 766 132 4 145 190 4797 60 4737 4285 Region Region length (kb) Completeness CDS Specific keyword 1 51.0 Intact 47 Terminase, portal, plate, tail 2 10.3 Incomplete 16 Tail 3 35.3 Questionable 32 Integrase, terminase, portal, head, capsid 4 40.1 Questionable 54 Terminase, portal, head, capsid, tail 5 15.5 Incomplete 20 Terminase, capsid, head, tail Comparative genomic analysis 2 Comparative genomic analyses of O. intermedium M86, 229E, LMG3301 T , and 2745-2 were conducted. The isolation source and genomic statistics are shown in Table 3 3 . Comparisons of subsystem features between the four genomes revealed some distinctions between 2745-2 and the other three strains (Table 4 4 ). The numbers of genes involved in virulence, disease, and defense were significantly lower in 2745-2 than in the other strains. Genes involved in secondary metabolism were higher in 2745-2. This difference in gene numbers may be due to the different habitat of 2745-2. BLAST of nucleotide sequence between 2745-2 and the other three strains was performed and the identities were visualized (Fig. 5 5 ). 2 Strain 2 Isolation source 2 Accession No. 2 Size (Mb) 2 GC (%) 4 Number Contig Subsystem CDS RNA 2745-2 Formation water JFHY00000000.1 4.80 57.6 95 440 4737 60 M86 Gastric biopsy AOGE00000000.1 5.19 57.9 148 480 5473 66 229E Stomach biopsy ASXJ00000000.1 4.81 57.9 378 468 5610 58 LMG3301 T Blood ACQA00000000.1 4.73 57.7 4 474 4723 70 2 Subsystem feature 4 Number of genes 2745-2 M86 229E LMG3301 T  Cofactors, vitamins, prosthetic groups, pigments 224 276 305 275  Cell wall and capsule 111 107 134 111  Virulence, disease, and defense 77 102 112 94  Potassium metabolism 15 18 24 17  Photosynthesis 0 0 0 0  Miscellaneous 55 63 76 63  Phages, prophages, transposable elements, plasmids 19 38 0 15  Membrane transport 180 254 281 226  Iron acquisition and metabolism 41 55 80 56  RNA metabolism 127 145 189 157  Nucleosides and nucleotides 100 109 121 110  Protein metabolism 207 273 305 265  Cell division and cell cycle 16 30 30 28  Motility and chemotaxis 77 84 78 88  Regulation and cell signaling 70 82 84 75  Secondary metabolism 9 4 4 4  DNA metabolism 98 123 124 107  Fatty acids, lipids, and isoprenoids 113 153 169 153  Nitrogen metabolism 35 38 51 38  Dormancy and sporulation 2 2 3 1  Respiration 126 134 157 134  Stress response 119 131 146 128  Metabolism of aromatic compounds 30 32 42 32  Amino acids and derivatives 409 522 618 503  Sulfur metabolism 11 54 65 54  Phosphorus metabolism 43 53 57 49  Carbohydrates 440 496 580 484 The rings illustrate a shared identity with the four strains Discussion 1 Crude oil degradation related genes 2 Crude oil is a mixture of hydrocarbons of various molecular weights. Many bacteria in nature have been found to be capable of degrading crude oil (Dawar and Aggarwal, 2015 2015 ; Lincoln et al., 2015 2015 ), using it as their sole carbon source. Strain 2745-2 is one of them. Experiments showed that this strain can use tetradecane and hexadecane, the main compounds in crude oil, as its carbon source. Tetradecane and hexadecane are alkanes which can be oxidized by alkane hydroxylases, such as AlkB and P450. Two alkB genes and one P450 gene were found in the genome of 2745-2. alkB encodes a protein named alkane 1-monooxygenase, which is the key enzyme in the degradation of alkanes. Genes encoding for 2-polyprenylphenol hydroxylase and alkaline phosphatase, which are responsible for degrading aminobenzoate, were found in the genome. Genes of 2-haloalkanoic acid dehalogenase and alcohol dehydrogenase for chloroalkane and chloroalkene degrading exist in the genome. Furthermore, 2745-2 also contains all genes involved in the assembly of flagella which allows the bacterium to move to the oil-water interface the degradation process. Many other oil-degrading bacteria also have suits of flagella assembly-related genes and it is believed that these genes can also benefit emulsification of the hydrocarbon in crude oil (Das et al., 2015 2015 ). Strain 2745-2 also contains genes encoding for phosphomannomutase, acyl transferase, glycosyl transferase, rhamnosyltransferase, glucose-1-phosphate thymidylyltransferase, dTDP-glucose 4,6-dehydratase, dTDP-4-dehydrorhamnose 3,5-epimerase, dTDP-4-dehydrorhamnose reductase, and N -acyl- L -homoserine lactone synthase. These enzymes are involved in the synthesis of rhamnolipids, a class of glycolipid, which work as bacterial surfactants by reducing the surface tension, critical micelle concentration, and interfacial tension, and increasing the emulsification and solubility of hydrocarbons in mixtures such as crude oil (Das et al., 2015 2015 ). All these genes reflect the ability of strain 2745-2 to degrade crude oil. Pathogen potential of strain 2745-2 2 Previous studies on the genome of two strains, O. intermedium M86 and 229E, identified many gene clusters related to virulence (Kulkarni et al., 2013 2013 ; 2014 2014 ). In our study, the genomes of three strains of O. intermedium , which are available in the public database (M86, 229E, and LMG3301 T ), were compared with the genome of strain 2745-2. All the strains have an average genomic size of 4.8 Mb except M86 (Table 3 3 ). 2745-2 is an environmental strain and the other three are clinical. There are fewer genes involved in virulence, disease, and defense in strain 2745-2 compared with the others (Table 4 4 ). However, 2745-2 contains several genes that are related to phages, prophages, transposable elements, and plasmids. Furthermore, we identified five prophage regions in 2745-2. One region is an intact phage with a length of 51 kb, which encodes for phage-like and hypothetical proteins (Fig. 4 4 ). It is believed that the phage-like sequences improve the cell adhesion and the ability to acquire antibiotic resistance, properties that can enable bacteria to survive in new environments and become pathogens (Casjens, 2003 2003 ; Zhou et al., 2011 2011 ). The clinical isolates of O. intermedium , which are related to pathogens such as O. anthropi and Brucella spp., display a high level of resistance to forms of β-lactam antibiotics (Teyssier et al., 2005 2005 ) and are considered to be pathogens. Although 2745-2 was isolated from a non-clinical environment, it was found to be resistant to ampicillin and the genomic annotation results showed the presence of several β-lactamase genes that provide resistance to β-lactam antibiotics. With all these properties, strain 2745-2 may have the potential to be a pathogen. Conclusions 1 As the first environmentally-derived strain of O. intermedium whose genome has been sequenced, strain 2745-2 is giving us a new perspective on its adaption to the environment. Genes involved in crude oil degradation are annotated in its genome, reflecting its ability to degrade crude oil. Further comparative genomic studies between 2745-2 and strains isolated from clinical samples will give us a better understanding of the adaption and evolution of environmental bacteria into host pathogens. References NF Alikhan , NK Petty , NLB Zakour , 等 . BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons . 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DOI: 10.1093/nar/gkr485 http://doi.org/10.1093/nar/gkr485 . PHAST: a fast phage search tool Compliance with ethics guidelines Lu-jun CHAI, Xia-wei JIANG, Fan ZHANG, Bei-wen ZHENG, Fu-chang SHU, Zheng-liang WANG, Qing-feng CUI, Han-ping DONG, Zhong-zhi ZHANG, Du-jie HOU, and Yue-hui SHE declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500029 Project supported by the National High-Tech R & D Program (863) of China (No. 2013AA064402), the National Natural Science Foundation of China (Nos. 81301461 and 51474034), the Zhejiang Provincial Natural Science Foundation of China (No. LQ13H190002), and the Scientific Research Foundation of Zhejiang Provincial Health Bureau (No. 2012KYB083), China 2015-01-26 2015-09-17 2015-07-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 16