- Original Article
Comparative genome analysis of Oceanimonas sp. GK1, a halotolerant bacterium with considerable xenobiotics degradation potentials
Annals of Microbiology volume 66, pages 703–716 (2016)
The growing pollution by xenobiotic compounds generated through both natural and anthropogenic activities has endangered the environment. The advent of the next generation sequencing has provided fast and cost-effective tools to explore genomes to discover novel xenobiotic-degrading genes. A Gram-negative marine halotolerant Oceanimonas sp. GK1 was analyzed for main physiological and genetically important characteristics at the genome scale while being compared with six other phylogenetically-close sequenced genomes. This exploration revealed high potential of Oceanimonas sp. GK1 for biodegradation of xenobiotics compounds such as phenol. More specifically, the isolate utilizes phenol via the ortho-cleavage pathway as a carbon source in the citrate cycle. This was further confirmed by the significant shortage of carbohydrate active enzymes in Oceanimonas sp. GK1 genome, which has forced this bacterium during the course of evolution to change its metabolism and physiology to benefit unusual carbon and energy sources to survive under harsh conditions.
The increasing level of environmental pollution is among the most catastrophic tragedies of the modern era which in turn has negatively impacted life quality in major parts of the world (Sekoai and Daramola 2015). Oil spills especially into marine ecosystems are regarded by many as the most dreadful disasters which have frequently occurred during the last decades. Apart from through the incidents, xenobiotic compounds, e.g., hydrocarbon aromatic compounds, phenol and their derivates are also the main constituents emitted into the environment by chemicals manufacturers as well as a number of other industries such as paper mills (Bonfá et al. 2013). Among the xenobiotic materials, phenol has been revealed to possess deadly affects on marine microorganisms and aquatic flora even at concentrations as low as parts-per-million (Tišler and Zagorc-Končan 1997; Saha et al. 1999; Kahru et al. 2002; Michałowicz and Duda 2007; Chen et al. 2010).
On the other hand, the enormous growth of contemporary science as well as industrial and technological revolutions have had a massive influence on attempts aimed at finding novel ways to minimize the tragic effects of anthropogenic activities on the biota. Among the solutions offered, naturally-occurring tools and procedures seems to be more advantageous. For instance, Atlas and Philp (2005) and Sinha et al. (2011) in separate studies argued that xenobiotic-degrading microorganisms are the best way to control or reduce the mortal effects of such compounds. Such biodegrading microorganisms could be employed in bioremediation of phenolic effluents of various origins (Bonfá et al. 2013; Le Borgne et al. 2008; McGenity 2010).
In our previous study, Oceanimonas sp. GK1 (IBRC-M 10197), a Gram-negative, rod-shaped, motile, aerobic and marine halotolerant bacterium (up to 12 % NaCl) was isolated from the Gavkhouni wetland [with about 20 % (w/v) total salt; 32°25′N, 52°39′E] in Iran (Yeganeh et al. 2012). This wetland is a grabni hole covered by quaternary sediments and watered by the Zayandehroud river as the only water source of the Gavkhouni basin (Sabzevari et al. 2013). This water basin is severely polluted and contains a high level of phenolic compounds. Since 2001, when Brown et al. proposed to classify a distinct genus as Oceanimonas, the number of physiologically well-characterized and published species has been very limited (Brown et al. 2001; Ivanova et al. 2005). In fact, the closed molecular and physiological relationship between Pseudomonas, Aeromonas and Oceanimonas genera (Brown et al. 2001) make the isolation and registration of new species of this genus very difficult.
One of the most important features of Oceanimonas genus which Brown et al. (2001) stated for Oceanomonas baumannii sp. nov. and Oceanomonas doudoroffii (Baumann et al. 1983) comb. nov. is the phenol degradation capability of this bacteria via the ortho-cleavage pathway, and in the presence of elevated salinity [minimal medium containing 5 % NaCl (w/v) and 4 mM phenol]. On such a basis, the overall aim of this study was to conduct a genome-wide analysis of the halotolerant Oceanimonas sp. GK1 complete sequenced and annotated genome. This was the first complete sequenced genome analysis of this genus in order to characterize the genetic potentials of this bacterium to survive in an extreme environment with respect to phenol concentration. Moreover, other main physiological properties of this isolate were also surveyed.
Materials and methods
Bacterial strain, growth conditions, and genomic DNA extraction
The Oceanimonas sp. GK1 deposited in the Iranian Biological Resource Center (IBRC-M 10197; IBRC, Tehran, Iran) was characterized by using biochemical (i.e., catalase and oxidase reactions, nitrate reduction), physiological (i.e., motility, growth at different salt concentrations, pH and temperatures) and PCR tests. For genomic DNA extraction, cells were grown aerobically for 3–7 days in the DSMZ 10 % MH medium at 34 °C and 220 rpm . DNA extraction was conducted by the “IBRC Gram-negative bacterial genomic DNA extraction kit” (Cat no. MBK0041, Iran) following the manufacturer’s instructions.
Genome sequencing and finishing
Whole genome sequencing of Oceanimonas sp. GK1 was performed on 454 GS-FLX titanium. In total, 93,921,861 bases of 247,884 random reads with an average read length of 378 nucleotides were obtained. The approximate coverage of Oceanimonas sp. GK1 genome was 30-folds. The sequence reads were assembled into 72 contigs using Newbler Assembler software v.2.3. Paired end sequencing resulted in 3 scaffolds with 47 gaps. All gaps were closed using sanger DNA sequencing (Sanger et al. 1977).
Curation and annotation of the genome was carried out in two minimal and enriched levels following International Nucleotide Sequence Database Collaboration (INSDC) guidelines. At minimal level, the Glimmer v.3.02 (Delcher et al. 1999) and GenMark.hmm v. 2.8 (Lukashin and Borodovsky 1998) were used for prediction and finding of prokaryotic coding sequences (CDs), while RNAmmer v.1.2 (Lagesen et al. 2007) and tRNAscan-SE v.1.21 (Lowe and Eddy 1997) were used for identification of 5S, 16S, and 23S ribosomal RNA genes as well as tRNAs and their corresponding genes, respectively. The enriched annotation was performed by means of Rapid Annotation using Subsystem Technology (RAST) v.4.0 (Aziz et al. 2008), Bacterial Annotation System (BASys) v.1.0 (Van Domselaar et al. 2005) and NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP).
Phylogenetic analyses of the completely-sequenced genomes of Oceanimonas sp. GK1 (Acc. No. NC_016745) along with 6 members of the Aeromonadaceae family, including Aeromonas salmonicida ssp. salmonicida A449 (Acc. No. NC_009348), Aeromonas hydrophila ssp. hydrophila ATCC 7966 (Acc. No.: CP000462), Aeromonas caviae Ae398 (Acc. No. CACP01000001 to CACP01000149), Aeromonas veronii B565 (Acc. No. CP002607), Aeromonas salmonicida ssp. salmonicida 01-B526 (Acc. No. AGVO00000000), and Tolumonas auensis DSM 9187 (Acc. No. NC_012691) were performed based on the homology between their concatenated housekeeping genes (adk, argS, gyrA, rpoB, rpoD, metS and dnaE) and core genes of these genomes.
Growth culture preparation and phenol degradation experiments
The isolated bacterium was activated using the same DSMZ growth medium as described earlier. Distilled water was added to make a 1-L solution and the sterilized media were distributed into 250-ml Erlenmeyer flasks under aerobic conditions. The growth was monitored by measuring the turbidity of the cultures.
The isolated strain was first grown aerobically on the same medium containing 1000 mg/l phenol for acclimation and adaption to the environment for 5 days. Then, an acclimated seed culture was prepared and harvested at the mid-exponential phase. For phenol degradation experiments, a 3-ml cell suspension of the acclimated seed culture were transferred to fresh media (100 ml) with a gradual increase in phenol concentration (500–2000 mg/l), while the pH was adjusted to 7.0. The media were incubated at 25 °C on a rotary shaker (150 rpm). The cell growth was measured by the turbidity of the media using a spectrophotometer. A calibration curve was developed based on the cell dry weight with respect to optical density, and the cell dry weight of the samples was measured based on the calibration curve. For measuring the phenol concentration, the culture media was filtered through a filter paper (Whatman No. 42) to remove solid cells and the phenol concentration as indicated by chemical oxygen demand (COD) was monitored every 24 h.
Results and discussion
General genome features
The genome of Oceanimonas sp. GK1 (Acc. No.: NC_016745) as investigated in the present study comprises a single circular chromosome of 3,514,537 bp along with two plasmids of 8462 bp and 4245 bp in length. The average G + C content of the genome was measured at 61.08 %. Genome annotation revealed 3222 protein-coding genes with an average size of 966 bp. These coding regions cover 96.64 % of the genome and 2938 (88.12 %) were functionally assigned. Moreover, 122 rRNA genes were predicted out of which 22 were organized in 7 rRNA operons and the rest (90 of 122) were related to the tRNA genes.
Phylogenetic analysis was performed based on the housekeeping genes (i.e., adk, argS, gyrA, rpoB, rpoD, metS and dnaE) and the core genes in the investigated genomes i.e., that of the present study and six from the literature: Aeromonas salmonicida ssp. salmonicida A449 (Acc. No. NC_009348), Aeromonas hydrophila ssp. hydrophila ATCC 7966 (Acc. No. CP000462), Aeromonas caviae Ae398 (Acc. No. CACP01000001 to CACP01000149), Aeromonas veronii B565 (Acc. No. CP002607), Aeromonas salmonicida ssp. salmonicida 01-B526 (Acc. No. AGVO00000000), and Tolumonas auensis DSM 9187 (Acc. No. NC_012691). The results obtained placed Oceanimonas sp. GK1 and Tolumonas auensis DSM 9187 in the same clade well separated from the Aeromonases (Fig. 1a, b).
Genomic islands, insertion sequence elements and transposases
Four genomic islands (GEIs) were identified on the single chromosome of Oceanimonas sp. GK1. These GEIs cover 1.96 % of genome length with 62 coding sequences. Three of the GEIs identified contained genes related to phage recombination factors and one had insertion sequence (IS) elements. Characteristics of these GEIs are presented in Table 1 and Fig. 2. The GC content range of these islands was measured between 49.31 and 55.81 %.
The results of the blastp similarity analyses obtained for the GEIs’ recombination factors showed that the GEI1 included four coding sequences (i.e., GU3_08700, GU3_08735, GU3_08745 and GU3_08760) which were highly similar to their counterparts in Aeromonas phage phiO18P. GEI2 contained 4 encoding genes for conjugal transfer i.e., TraJ (GU3_09380), TraI (GU3_09385), TrbJ (GU3_09390) and TrbL (GU3_09400) in addition to a single phage integrase-encoding gene (GU3_09355). All these genes were of close proximity to those of Vibrio genus. Among the 13 coding region sequences indentified in GEI3, 3 codings, integrase (GU3_10290), Zonula occludens toxin (Zot)(GU3_10305) and phage replication initiation factor (GU3_10320), were of significance in terms of recombination and pathogenic virulence characteristics. In fact, the protein sequences of these genes suggested high similarities with those of their homologues in Tolumonas auensis DSM 9187, Shewanella baltica OS625 and Vibrio phage VCY-phi, respectively.
Zot protein is a toxin which increases the intestinal permeability by altering the structure of intercellular tight junctions causing diarrhea. This was first reported in an infection caused by Vibrio cholerae (Baudry et al. 1992; Johnson et al. 1993; Rivera et al. 1995). As for the family Aeromonadaceae, within the complete sequenced genomes to date, the presence of Zot protein has only been reported in Aeromonas salmonicida ssp. salmonicida A449 (Reith et al. 2008; Beaz‐Hidalgo and Figueras 2013). However, the protein was found non-functional due to a 1-bp frame shift and an incorporation of an IS element in the coding region (Reith et al. 2008). In the present study, aligning the known Zot proteins reported in Vibrio sp. strains with that of the Oceanimonas sp. GK1 revealed the presence of the catalytic region of Zot protein marking the Oceanimonas sp. GK1 as a potentially pathogenic microorganism.
Finally, the GEI4 was found different from the other 3 GEIs in terms of both nature and components. More specifically, this GEI had no regions coding phage recombination factors while it contained a number of IS630 sequences with high similarity to those of Shewanella oneidensis as determined by NCBI blastn similarity analysis.
Thirty-one IS elements with transposase activity were dispersed throughout the single chromosome. These ISs were classified into 4 main families, IS116/110/902 (7), IS4 (5), IS3/IS911 (16) and IS630 (3). The NCBI blastn similarity search against IS finder database (www-is.biotoul.fr) (Siguier et al. 2006) showed that most of these IS elements were more highly similar to their orthologes in Shewanella sp. than the other microorganisms.
Pan and core genome analysis
The genetical and biochemical characteristics and capabilities of the Aeromonadaceae family members investigated in this study were significantly variable. Pan and core genome analysis of these bacterial genomes assisted in making orthology comparisons in order to identify core, dispensable and unique genes. The gene content analyses conducted among the 7 genera of the family of Aeromonadaceae showed enormous variability i.e., 3222 CDS in Oceanimoans sp. GK1 to 4724 CDS in Aeromonas salmonicida ssp. salmonicida 01-B526 . A high similarity was observed in terms of CDS number between Oceanimoans sp. GK1 and Tolumonas auensis DSM 9187 while these two genomes were significant apart from the other five members of the Aeromonadaceae family.
In order to compute pan genome, core genome and singletons, Edgar was used (Blom et al. 2009). In total, there were 9481 CDSs as the pan genome of the mentioned 7 bacterial genomes with the average genome of the Oceanimoans sp. GK1 selected as the reference genome. This value was 2–3 times larger than the CDS numbers for each of the investigated bacteria. This great variability can be attributed to the phylogenetical distance and differences in physiological characteristics. The core genome analysis of these bacteria as compared with iterative pairwise comparison resulted in 1137 CDSs with Oceanimonas sp. GK1 used as the reference. Also, there were 1130 unique singleton genes in Oceanimonas sp. GK1 genome, which were not present in the other investigated genomes. Oof those unique singleton genes, 365 CDSs were annotated as hypothetical proteins which could be the origin of the signature characteristics of Oceanimonas sp. GK1. Due to EDGAR’s limitation of a maximum 5 genomes for the generation of Venn diagrams, only the genome sequences of Aeromonas caviae Ae398, Aeromonas hydrophila ssp. hydrophila ATCC 7966, Aeromonas salmonicida ssp. Salmonicida A449, Oceanimonas sp. GK1, and Tolumonas auensis DSM 9187 were used to construct the diagram (Fig. 3). For the selected genomes and based on the resulting diagram, 8189 and 1169 CDSs were detected as pan and core genome, respectively.
In addition, the Tolumonas auensis DSM 9187 and Oceanimonas sp. GK1 with 1343 and 1265 CDSs, respectively, were found to possess the most strain-specific genes among these 5 genomes. Also, the binary comparison to assess the shared genes for the paired genomes revealed that Oceanimonas sp. GK1 had 1770, 1751, 1741 and 1277 homologues with Aeromonas caviae Ae398, Aeromonas salmonicida ssp. salmonicida A449, Aeromonas hydrophila ssp. hydrophila ATCC 7966 and Tolumonas auensis DSM 9187, respectively (Fig. 3).
General adaptive features
Membrane transport systems are crucial for the translocation of proteins and acquisition of organic nutrients and play vital roles in primary cellular processes such as preservation of ion homeostasis, cell signaling and environmental sensing (Ren and Paulsen 2005). Whole-genome transporter analyses were carried out on 5 of the 7 studied genomes whose protein sequences in FASTA format were available. For each of the investigated organisms, Transporter Automatic Annotation Pipeline (TransAAP) (Ren et al. 2007) analysis characterized the complete set of membrane transport systems, predicted their functions, and categorized them into protein families based on the transporter classification system. The results obtained are tabulated in Table 2. In general, larger genomes possessed a relatively greater number of transport systems while the number of transporters per Mb genome was approximately identical. In all genomes, ATP-dependent transporters and secondary transporters occurred in the most frequencies (collectively 83.2–92.2 %). In contrast to the other genomes, the secondary transporters of Oceanimonas sp. GK1 contributed a greater portion (47.6 %) to its whole transporters.
ABC type transporters are the extensive part of translocators in all organisms (Tomii and Kanehisa 1998). They are typically composed of two functional parts to bind and hydrolyze ATP (Nucleotide Binding Domain, NBD) and translocate via the multiple transmembrane segments (Membrane Spaning Domain, MSD) (Davidson and Chen 2004). The MSD domains of ABC transporters are involved in signal transduction processes using the extracytoplasmic loop. Thus, ABC-type transporters could play a role in the adaptive bacterial response to environmental changes (Seo et al. 2002; Coumes-Florens et al. 2011). Comparative analysis of ABC translocation capabilities as realized by using the complete sequenced genomes of the Aeromonadaceae family revealed various adaptation systems depending on environmental differences and physiological characteristics.
In the present study, the ABC transporters of all 7 genomes were assigned based on substrate specificity. The assignment is summarized in Table 3. In short, based on in silico analysis, the Oceanimonas sp. GK1 revealed the potential for ABC transpotation of iron (II)/manganese and disability for ABC transportation of maltose/maltodextrin and methyl-galactoside in contrast to other genomes.
Secretion of extracellular proteins is one of the most important mechanisms in bacteria in order to adjust their interactions with the surrounding environments for adaptation, survival and pathogenecity (Glenn 1976; Tseng et al. 2009). In fact, bacterial species develop different strategies to secret their extracellular enzymes and toxins. Six highly specialized pathways are so far known including: type I: signal sequence-independent pathway; type II: the main terminal branch of general secretion pathway; type III: the contact-dependent pathway; type IV: filamentous hemagglutinin secretion pathway; type V: autotransporter pathway (Binet et al. 1997; Collazo and Galán 1997; Burns 1999; Cornelis and Van Gijsegem 2000; Sandkvist 2001; Wooldridge et al. 2005; Deane et al. 2010; Silverman et al. 2011; Büttner 2012; Korotkov et al. 2012; and finally; type VI whose structure is like the bacteriophage cell-puncturing machine and is widely distributed in pathogenicGgram-negative bacteria such as Vibrio cholerae and Pseudomonas aeruginosa (Pukatzki et al. 2007, 2009; Bingle et al. 2008; Boyer et al. 2009; MacIntyre et al. 2010).
The type II secretion system (T2SS) is just present in the Gram-negative proteobacteria phylum in order to secret proteases, cellulases, phospholipases, pectinases, lipases as well as toxins (Filloux 2004; Cianciotto 2005). The expression and secretion of most of these extracellular enzymes, with the cell wall degrading and virulence effects, are firmly controlled by the environmental needs (Sandkvist 2001).
The Oceanimonas sp. GK1 also secrets its extracellular enzymes through T2SS composed of 10 T2SS specific proteins [Secretin (GspD), Inner Membrane Proteins (GspF, GspG, GspH, GspI, GspJ, GspK, GspL and GspM) and ATPase (GspE)], 10 s-SRP [Inner Membrane Proteins (SecD/F, SecE, SecG, SecY, YajC and YidC), ATPase (SecA), SRP receptor (FtsY) and Targeting protein (SecB and ffh)] and 3 Twin arginine targeting (Tat) proteins [Inner Membrane Proteins (TatA, TatB and TatC)].
Resistance to toxic compounds
A wide-range in-silico analysis conducted on the 7 studied genome sequences provided their profiles with respect to resistance to toxic compounds such as antibiotics and heavy metals. The detoxifying mechanism of the heavy metal resistant bacteria involves the utilization of an inducible ion efflux system for reduction of intracellular concentration of a specified ion via active export. This mechanism is contrary to that of the thermophilic or psycrophilic organisms only produce xenobiotic-degrading enzymes under extreme circumstances (Nies 2000). Figure 4 represents each organism prospective to degrade, survive, and adapt to toxic compounds.
The growing pollution by xenobiotic compounds generated through both natural and anthropogenic activities has endangered the environment, especially the aquatic ecosystems and consequently has posed serious threats to the public health. Xenobiotics including phenols, phenyl carbonates, benzoate, biphenyl and the other aromatic compounds make up a major part of pesticides and herbicides, petroleum (oil), solvents, alkanes, polycyclic hydrocarbons (PAHs), antibiotics, and synthetic azo dyes which are increasingly being introduced to the environment (Ojo 2007; Sinha et al. 2011). These substances are toxic and carcinogenic even at trace concentrations. Among the organisms with detoxifying capabilities, microorganisms, particularly bacteria, due to their wide range of diversity and flexibility for adaptation to xenobiotics, are the best candidates to recycle xenobiotic compounds back into natural biogeochemical cycles (Bonfá et al. 2013). Our analysis of Oceanimonas sp. GK1 genome revealed that the strain had high potentials to degrade xenobiotic compounds contained in wastewaters such as high-phenol content and oil wastewaters.
Phenol degradation: in silico analysis
Phenols and its derivatives as troublesome environmental contaminants are present in the commonly hypersaline wastewater of many industries such as petroleum industry and the chemical manufacturing plants (Lefebvre and Moletta 2006; Agarry et al. 2008; Nagamani et al. 2009; Bonfá et al. 2013). Such phenolic hypersaline effluents are commonly treated by physico-chemical means or biological methods (Dubey et al. 2013; Haddadi and Shavandi 2013). While physico-chemical techniques are usually energy-consuming and expensive, the biological removal of phenol and other dissolved organics from such effluents has been shown to be reasonably more advantageous (Lefebvre and Moletta 2006). However, biological treatment of hypersaline wastewaters requires prior desalinization for most microorganisms with the xenobiotic capabilities which cannot cope with hypersaline conditions. This pretreatment step involves expensive procedures or dilution with fresh water (Bonfá et al. 2013). The latter although less costly increases the volume of wastewater and also puts extra pressure on fresh water resources. As a result, halophilic microorganisms with xenobiotic degradation capabilities are the best choice to overcome these challenges. Unluckily, the known or characterized halophilic or halotolerant phenol-degrading bacteria are very limited until now (Bonfá et al. 2013).
Recently, Fathepure (2014) in an excellent review listed the main organisms of biodegradation capability for petroleum hydrocarbons in hypersaline environments. Fathepure also categorized 10 phenol-degrading species under moderate to highly saline conditions (0–30 %), i.e., the halophilic isolate (1–15 %) (Woolard and Irvine 1995), Halomonas sp. (1–14 %) (Hinteregger and Streichsbier 1997), Candida tropicalis (15 %) (Bastos et al. 2000), Halomonas campisalis (0–15 %) (Alva and Peyton 2003), Halomonas organivorans (1.5–30 %) (García et al. 2004, 2005b), Thelassobacillus devorans (7.5–10 %) (García et al. 2005a), Arthrobacter sp. (6–9 %) (Plotnikova et al. 2011), Halomonas organivorans, Arhodomonas aquaeolei (10 %) (Bonfá et al. 2013), and Modicisalibacter tunisiensis Strain C5 (12 %) (Chamkha et al. 2008).
The comprehensive comparison of the 7 studied genomes in terms of their phenolic and other aromatic compounds degradation potentials well segregated Oceanimonas sp. GK1 from the others (Fig. 5). Comparative pathway analysis of Oceanimonas sp. GK1, Aeromonas hydrophila ssp. hydrophila ATCC 7966, Aeromonas salmonicida ssp. salmonicida A449, Aeromonas veronii B565 and Tolumonas auensis DSM 9187 using the Metacyc pathway tool v.18.0 (Caspi et al. 2008; Karp and Caspi 2011), revealed the significant superiority of Oceanimonas sp. GK1 compared to the others in terms of biodegradation of aromatic compounds (Table 4 and Fig. 5).
This wide genome analysis has shown high potentials of the Oceanimonas sp. GK1 isolate for phenol biodegradation through its utilization as carbon source via the ortho-cleavage pathway. More specifically, in the present study, and based on the “degradation of aromatic compounds pathway” in KEGG and genome analysis, the Oceanimonas sp. GK1 was found capable of converting benzene to phenol, followed by its oxidization to catechol. Catechol then undergoes ring cleavage by catechol 1, 2-dioxygenase (EC 22.214.171.124) (GU3_01755) via the intradiol pathway. After several subsequent reactions, the pathway leads to acetyl-CoA and the citrate cycle, which could be up-taken as an energy source (Fig. 5).
Phenol degradation: experimental analysis
The effect of initial phenol concentration ranging from 500 to 2100 mg/l (corresponding to 1200–5092 mg COD/l, respectively) on biodegradation capabilities of Oceanimonas sp. GK1 in terms of COD removal efficiency was investigated. Increasing the initial phenol concentration from 500 to 1000 mg/l (1200–2540 mg COD/l, respectively) resulted in decreasing COD removal efficiency from 40.2 to 36.8 %. By further increasing the phenol concentration to 1500, 1750 and 2100 mg/l (corresponding to 5092, 4175 and 3473 mg COD/l), the decreasing trend continued and the COD removal efficiency further decreased to 24.3, 26.8 and 32.2 %, respectively. On the other hand, the cell dry weight (CDW) was found to react similarly to increasing phenol concentrations as the COD removal efficiency. Maximum CDW of 67, 51, 44, 32 and 26 g/l were achieved for initial COD concentrations of 1200, 2540, 3473, 4175 and 5092 mg COD/l, respectively. In an investigation performed by González et al., the ability of Pseudomonas putida ATCC 17484 to degrade phenol at concentrations ranging from 200 t 1000 mg/l in batch mode was evaluated. A maximum removal efficiency of 90 % was achieved for phenol concentration of 1000 mg/l (González et al. 2001). These results as well as the presence of multiple copies of the key genes involved in the degradation of xenobiotics compounds such as phenol would suggest Oceanimonas sp. GK1 as a potential platform to develop genetically-engineered strains of superior degrading capabilities.
Another feature of Oceanimonas sp. GK1 is its abilities and disabilities in metabolizing carbohydrates. According to the carbohydrate-active enzyme (CAZy) analysis, Oceanimonas sp. GK1 is predicted to possess 39 genes (1.36 % of all genes) encoding carbohydrate-degrading enzymes. These enzymes include glycosyl hydrolases (GHs)(5 families), carbohydrate binding modules (CBMs) (1 family), carbohydrate esterases (CEs) (4 families), and glycosyl transferase (GTs) (9 families) (Table 5). Some of these enzymes fall into more than one CAZy type category (i.e., both GHs and CBMs). Therefore, a total of 44 CAZymes were identified using this ontology.
As a case in point, in comparison with the other studied genomes, the enzymes involved in the starch metabolism pathways are very limited in Oceanimonas sp. GK1. The resulting heatmap obtained by the PATRIC comparative pathway tool (Wattam et al. 2014) for this comparison showed that Oceanimonas sp. GK1 possesses just 7 enzymes of the 24 enzymes generally existing in the starch metabolism pathways (Fig. 6). Interestingly, the Oceanimonas sp. GK1 retains the cellulose synthase (EC 126.96.36.199) and cellulase (EC 188.8.131.52)-encoding genes. Nevertheless, with the exception of Aeromonas caviae Ae398, the other genomes lacked both enzymes and such a capability would make Oceanimonas sp. GK1 a good candidate to be considered for further cellulolytic activity analyses for applications in biotechnology and industry.
In the present study, the biochemical and physiological properties of a hypersaline Oceanimonas sp. GK1 were investigated both experimentally and in silico at the genome scale. The microbe’s capabilities in degrading aromatic compounds, specially phenol, and utilizing its degradation products in the citrate cycle (despite its enzymatic limitations in the carbohydrate metabolism pathway) represents the high level of adaptation exhibiting by this bacterium to the extreme life style and utilization of unusual carbon and energy sources. Finally, the investigation and analysis of the other adaptation features and genetical potentials of Oceanimonas sp. GK1 make it highly recommended.
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The authors would like to thank the Iranian Biological Resource Center for financially supporting this study through the grant MO-1388-02.
Reza Azarbaijani and Laleh Parsa Yeganeh contributed equally to this work.
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Azarbaijani, R., Yeganeh, L.P., Blom, J. et al. Comparative genome analysis of Oceanimonas sp. GK1, a halotolerant bacterium with considerable xenobiotics degradation potentials. Ann Microbiol 66, 703–716 (2016). https://doi.org/10.1007/s13213-015-1156-4
- Xenobiotic pollution
- Next generation sequencing
- Genome annotation
- Comparative study