MATERIALS AND METHODS

Bacteria cultivation: The soil sample was taken from volcanic area of Gunung Sibayak, Indonesia. All strains were cultivated aerobically at 37°C on solid minimal media containing 20 mM of 2,2-DCP using streak plate method. The sample was repeatedly streaked on the same type of medium to obtain a pure colony.

The liquid minimal media was prepared as 10X concentrated basal salts containing K₂HPO₄. 3H₂O (42.5 g L^-1), NaH₂PO₄.2H₂O (10.0 g L^-1) and (NH₄)2SO₄ (25.0 g L^-1). The trace metal salts solution was a 10X concentrate that contained nitriloacetic acid (NTA) (1.0 g L^-1), MgSO₄ (2.0 g L^-1), FeSO₄. 7H₂O (120.0 mg L^-1), MnSO₄. 4H₂O (30.0 mg L^-1), ZnSO₄.H₂O (30 mg L^-1) and COCl₂ (10.0 mg L^-1) in distilled water (Hareland et al., 1975). Minimal media for growing bacteria contained 10 mL of 10X basal salts and 10 mL of 10X trace metal salts per 100 mL of distilled water and were autoclaved. The sterilized 2,2-DCP carbon source was added to the autoclaved salts medium to a final concentration of 20 mM. The extent of growth determined by measuring the absorbance at A_680nm.

Molecular analysis
PCR and dehalogenase gene identification: The PCR primers were described according to Fortin et al., (1998), from Xanthobacter autotrophicus dhlB 314 5’-TCT GGC GGC AGA AGC AGC TGG- 3’ dhlB 637 5’- CGC GCT TGG CAT CGA CGC TGA TG- 3’ (Van Der Ploeg et al., 1991). The PCR conditions were set at 30 cycles of the following parameters: Denaturation, 95°C for 1 min; annealing, 55°C for 1 min; extension, 72°C for 2 min. The reaction mixture was electrophoresed on a 1% agarose gel and purified using ExoSAP-IT PCR Clean-up Kit (GE Healthcare Bio-Sciences Corp. USA) for sequencing at 1st Base Laboratory, Malaysia. The DNA sequencing results were converted into amino acids. The amino acid sequence was analysed by sequence comparison in the public databases using BLASTp search program on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/).

RESULTS

Growth experiment of strain GS1 on 2,2DCP: A pure colony of GS1 grew well on liquid and solid minimal media supplied with various concentrations of 2,2-DCP up to 40 mM at 37°C. However, no growth was detected at 50 mM 2,2-DCP suggesting 50 mM of 2,2-DCP is toxic to the cells. A control plate without 2,2-DCP showed no growth at all.

Molecular analysis of strain GS1 and the presence of putative dehalogeanse gene: Oligonucleotide primers (Van Der Ploeg et al., 1991) were used to perform PCR amplification using genomic DNA extracted from strain GS1 grown in 2,2DCP. PCR fragment of the expected size of 500-600 bp (Fig. 1) was generated and the negative control using E. coli genomic DNA did not show any amplification. The PCR fragment was sent for sequencing. The nucleotide and amino acid sequences were analyzed against EMBL and SWISSPROT databases, respectively. A FASTA search revealed that the PCR fragment was 69% homologous to the known coding region of the haloacid dehalogenase from X. autotrophicus (Fig. 2).

Fig. 1:	PCR analysis of genomic DNA from strain GS1 using nucleotide primers derived from dhlB. Lane 1: GS1 PCR product (Approximately 500 bp); Lane 2. 21 kb DNA ladder; Lane 3; Negative control

Fig. 2:	Amino acid sequence comparison of dehGS1 and the dehalogenase gene dhlB. The dehGS1 PCR product is 69% homologous to dhlB, encoding the haloalkanoic acid dehalogenase from X. autotrophicus. Identical amino acid residues are asterisks. The GeneBank accession number for dhlB is M81691

Fig. 3:	Amino acid sequence comparison between DehGS1 and HadL, DehH109 and HdlIV. Identical amino acid residues are asterisks. Two dots indicate amino acid similarity

This PCR product was named DehGS1 and has some homology with a variety of other haloalkanoic acid dehalogenases as shown in Fig. 3. The overall protein sequence identity was 25%. Examples are: The HadL from Pseudomonas putida AJ1- 25% (Jones et al., 1992); DehH109 dehalogenase from P. putida H109-26% (Kawasaki et al., 1994) and the HdlIV dehalogenase from Pseudomonas cepacia MBA4- 25% (Murdiyatmo et al., 1992). However, there was less than 5% homology with haloacetate dehalogenase and most of group 1 αHA. These results suggest current DehGS1 might be a novel group II αHA.

DISCUSSION

In previous study, a group of bacteria that can grow on halogenated compound as sole source of carbon were identified (Jing and Huyop, 2008; Ismail et al., 2008; Jing et al., 2008; Mesri et al., 2009; Thasif et al., 2009; Zulkifly et al., 2010). In addition, some of the dehalogenase producing microorganisms were intensively studied and their corresponding genes were identified (Thomas et al., 1992; Cairns et al., 1996; Stringfellow et al., 1997; Yusn and Huyop, 2009).

To the best of our knowledge, this is the first reported Bacillus sp. that can degrade 2,2DCP as sole source of carbon. There are very limited studies focused on chlorinated compounds degradation by Bacillus species. Other reported strains of the same genus are dichloromethane degrading bacteria (Wu et al., 2009) and degradation of low concentration of monochloroacetic acid by Bacillus sp. TW1 (Zulkifly et al., 2010).

Oligonucleotide primers for the dhlB gene were capable of detecting the presence of dehalogenase gene in a bacterium that could grow on 2,2DCP. In future using similar technique might be useful in detecting the presence of bacteria with the capacity to degrade halogenated compounds by hydrolytic dehalogenation. In this study, the PCR product was identified to be a dehalogenase gene associated with L-haloacid dehalogenases.

DehL dehalogenase in Rhizobium sp. could not act on 2,2DCP (Leigh, 1986). However, growth of Rhizobium sp. on 2,2DCP could trigger production of DehL dehalogenase suggesting 2,2DCP is substrate-inducer. In current study might suggest that the identified gene might or might not be responsible for growth on 2,2DCP.

CONCLUSION

In conclusion, using molecular approach can be used in finding a novel gene of interest. This is the first report demonstrating the dehalogenase gene is present in Bacillus sp. associated with growth on halogenated substrate as sole source of carbon. This technique could also apply to monitor for microorganisms possessing specific dehalogenase gene(s) in soil community or in water systems. In future, it was hoped that such studies would be possible to identify new kind of dehalogenase from newly isolated microorganism. Therefore, it provides a better understanding of the microbial populations as a whole.

ACKNOWLEDGMENTS

The authors would like to express their gratitude to the Faculty of Biosciences and Bioengineering UTM/GUP Q.J130000.7135.00H34&FRGS 4F008 for supporting this research.