Identification of Phenol-Degrading Gene Metapyrocatechase in Acinetobacter oleivorans Strain GSN15 -F.Babazadeh, S.Gharavi, Z. Moosavi-Nejad- ATRAN GROUP

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An aerobic bacterial strain GSN 15 was isolated from Tehran oil refinery (Tehran Province, Iran). The strain was found to belong to Acinetobacter oleivorans according to morphological, physiological and its 16S rRNA gene sequence. This strain was able to degrade hydrocarbons such as phenol. Catechol 2,3-dioxygenase (C23O) –encoding gene was detected and amplified with the designed primers from the total DNA of Acinetobacter oleivorans.

Keywords

Bioremediation, petroleum contaminated soil, catechol 2,3dioxygenase-encoding gene

 

  1. Introduction

Phenol is one of the most common toxic environmental pollutants, which mainly originates from industrial processes, such as coking plants, industrial resin manufacturing, oil refineries, pharmaceuticals, plastic, and varnish industries, etc (Whiteley et al. 2000 and Arutchelvan et al. 2003). Phenol and its derivatives like dinitrophenol and pentachlorophenol are widely used as insecticides, but they are very toxic substances. Phenol is a general protoplasmic poison with corrosive local effects that denature proteins. Poisoning with phenol compounds may occur by ingestion, inhalation, and absorption through skin (Meena et al., 2015).

The removal of phenol and its derivatives from a polluted environment by conventional physical and chemical methods was found to be an expensive and not an environment-friendly process (Arora, P et al., 2014). The ability of various microorganisms to metabolize phenolic compounds can be exploited for the efficient degradation of these toxic substances to harmless intermediates and final products. These can be used for the effective bioremediation of environment contaminated with phenolic compounds. Degradation of phenol occurs as a result of the activity of a large number of microorganisms including bacteria, fungi and actinomycetes. Bacterial species include Bacillus sp, Pseudomonas sp, Acinetobacter sp, Achromobacter sp etc. Fusarium sp, Phanerocheate chrysosporium, Corious versicolor, Ralstonia sp, Streptomyces spp (Prieto et al., 2002).

 Bacteria can metabolize phenolic compounds under both aerobic and anaerobic conditions.

The critical step in the metabolism of aromatic compounds is the destruction of the resonance structure by hydroxylation and fission of the benzoid ring which is achieved by dioxygenase-catalyzed reactions in the aerobic systems. Based on the substrate that is attacked

by the ring cleaving enzyme dioxygenase, the aromatic metabolism can be grouped as catechol pathway, gentisate pathway, and proto catechaute pathway. In all

these pathways, the ring activation by the introduction of hydroxyl groups is followed by the enzymatic ring cleavage.

 The ring fission products, then undergoes transformations leading to the general metabolic pathways of the organisms. Most of the aromatic catabolic pathways converge at catechol. Catechols are formed as intermediates from a vast range of substituted and nonsubstituted

mono and poly aromatic compounds.

Aerobically, phenol also is first converted to catechol, and subsequently, the catechol is degraded via ortho or meta fission to intermediates of central metabolism. The initial

ring fission is catalyzed by an ortho cleaving enzyme, catechol 1, 2 dioxygenase or by a meta cleaving enzyme catechol 2,3 dioxygenase, where the product of ring fission is a cis-muconic acid for the former and 2-hydro cis muconic semi aldehyde for the latter (Gurujeyalakshmi and Oriel, 1988),( Nair, C. Indu.,et al., 2008).

In this paper, we report here the identification of phenol-degrading Gene Metapyrocatechase in Acinetobacter oleivorans strain GSN15.

2) Material and method

2-1)Physiological and biochemical characteristics of GSN15

The colony character and bacterium configuration were observed by microscope. The analysis included dying, aerobic test, catalase activity, O/F test.

2-2)16S rRNA amplification and phylogeny analysis of 16S rRNA gene

Strain GSN23 was incubated in LB medium and the genomic DNA was extracted using SET buffer method. The 16S rRNA gene was amplified using primers 27F

(5-AGAGTTTGATCCTGGCTCAG -3)

and 1492R(5-GGCTACCTTGTTACGACTT-3). 25 μL of PCR volume consisted of 1 ng of total DNA, 2.5μL Reaction buffer, 1μL of dNTP, 1μL of  27F, and 1492R, 0.5μL of Taq polymerase and adding ddH2O making the volume up to 25μL. PCR amplification was carried out as  follows: 5 min at 95°C; 30 cycles of 1 min at 94°C, 1 min at 61°C, and 1 min at 72°C, plus an  additional 5 min at 72°C.

2-3) Phenol degradation

The effect of the different substrate concentrations (1000- 2000mg/L-1) on the phenol degradation by GSN 15 was examined. The growth medium contained mineral salts incubated at 35, under a 150 rpm shaking rate and a pH of 7.0.

  1. Results and discussion

3.1 Physiological and biochemical characteristics of GSN15

The physiological, biochemical characteristics of GSN15 are given in Table 1.

3.2 16S rRNA gene phylogeny of GSN15

16S rRNA sequences analysis is a fast and accurate method to identify GSN15 phylogeny. 16S rRNA gene was sequenced and is shown in Fig. 1. (GenBank ID KF815697).

3.3 Detection of catechol 2,3-dioxygenase genes in GSN15

Catechol 2, 3-dioxygenase (C23O) { metapyrocatechase(MPC2)} gene was amplified with primers FC23O and RC23O from Acinetobacter oleivorans gene sequence deposited in the NCBI Genbank. The primer FC23O (5´-GGTTGTGTCATCAAGACC-3´) and

RC23O (5´-GAGTTGAAGATAGAGCCG-3´) were designed by Gene-runner software and primer design server. C23O gene was sequenced and is shown in Fig. 2.

3.4 Effect of substrate concentration

C230 is known as one of key enzymes in benzene ring fission. GSN15 was capable of using phenol as carbon source and its ring cleavage was found at 2, 3-position in follow-up study; two different initial phenol concentrations were used (Table 2).The phenol degradation experienced two phases from the figure. The first was lag phase in which the phenol degradation was slow. In the second logarithmic phase, phenol was rapidly degraded. It was conferred that microorganisms usually need a period of time to adapt the reaction system (Wang, 1993). Figure 3 shows the kinetics of growth in broth containing 500 mg phenol l-1 at 600 nm over 72 h. Cell density and pH were also measured in 1000 and 2000 mg phenol l-1 after 72h as indices of growth capabilities. Significant changes seems to have occurred, when comparison is made to the original medium with pH 7 and initial inoculums with an absorbance greater than 0.05, up to 0.481 (wavelength 600 nm)(Figure3).

  1. Conclusion

The Acinetobacter olivorans isolate GSN15 isolated from the Tehran oil refinery could well degrade phenol while it also had high strength acidic degrading ability. GSN15 belonged to the Acinetobacter genus according to 16S rRNA gene analysis, and it was closely related to A. olivorans. Catechol 2, 3-dioxygenase gene (metapyrocatechase) was found in its genomic DNA. The results from sequencing of the amplified gene shows 94% identity with Acinetobacter oleivorans sequences registered in the NCBI database.

References

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Table 1. The physiological and biochemical characteristics of GSN15.

Characteristics                                        GSN15

Gram stain                                                    –

Aerobic                                                        +

Catalase activity                                           –

O/F test                                                       oxidative non-fermentative

Colony character                                        cream, smooth

Figure1. PCR amplicon with universal primers 27F and 1492R. M,DNA marker(100-3000bp)

Figur2.PCR amplicon by primer FC23O and RC23O.linse 1,2 PCR product C12O and C23O.Line3 DNA marker and line4 negative control.

Figure 2. The sequence of catechol C23O fragment of GSN15

Table2. Final pH value and absorbance of culture broth after 72 h produced by selected and identified isolates

  Strain             The closest species according                Gene bank accession number       1000mgl-1    2000mgl-1

                       to 16S rDNA sequence similarity                                                                A       PH        A         PH

GSN15                 Acinetobacter oleivorans DR1                           KF815697               1.538     4.92     0.091      6

Figure 3. Time course of growth in culture broth for Acinetobacter olivorans GSN15 according to spectrophotometric measurement of absorbency.

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