P Somvanshi, V Singh, P Seth
degradation, hexachlorocyclohexane, homologous, lina, linb
P Somvanshi, V Singh, P Seth. Phylogenetic Investigation Of Lin Genes Involved In Degradation Of Hexachlorocyclohexane (Hch). The Internet Journal of Toxicology. 2007 Volume 4 Number 2.
Hexachlorocyclohexane (HCH) is a non-degradable compound extensively used against agricultural pests and various public health programs for controlling mosquitoes. It imbibes into the food chain from crop to animals; biodegradable enzymes present in the genes of some bacteria degrade HCH in soil. Mainly, two genes linA and linB encode dehydrochlorinase and dehalogenase enzymes involved in degradation of HCH. Bioinformatics strategies were used to find other homologous genes and proteins, which can also degrade HCH present in various bacteria for controlling of pollution and offer clue to prevent its entry in the food chain. Total 38 nucleotides and 36 proteins sequences of linA and linB were used for constructing the phylogeny. In addition, analysis on culture dependent and culture independent bacteria were done, which might play a vital role in the degradation of the non-degradable compounds. These bacteria possibly will facilitate in the safety of soil pollution, improving fertility of the soil and prevent entry of HCH in the food chain.
Xenobiotics are chemicals, which did not exist in nature. These are synthetic compounds viz. pesticides, fungicides, herbicides, insecticides, disinfectant and toxic present in the nature. They take action by transferring with microbial reactions in the target organisms. A wide range of halogenated organic compounds were used as solvents, agricultural chemicals, and insulating liquids. Most of them are toxic and recalcitrant in nature, cause serious contamination problems. Generally, these insert in soil and can affect the microorganisms that were important in maintaining fertility of the soil. Some organisms detoxify pesticides in soil gradually. Environmental factors also influence to degrade the compound which includes pH, temperature, bioactivity, nutrient supply and oxygen availability.
In India Hexchlorocyclohexane (HCH) is also known as benzene hexachloride (BHC). It is more toxic than DDT and was used mainly in public health program. HCH was introduced for controlling agricultural pests and vector-borne diseases. It was used extensively around the world and their toxic effect on target and nontarget organisms appeared in the 1980s ( 1 ). These reports finally resulted in prohibition or restrict the use of HCH in various countries. The problem arises in the uptake of HCH residues from soil by crops, from where it enters into the food products through food chain ( 2 ). The decontamination program for HCH polluted soils would diminish the risk posed by HCH residues to human, plant and animal health. The prospect for decontamination is spontaneous or induced microbial degradation of HCH proceeds gradually by number of bacteria ( 3 ). Degradation of HCH was carried out commonly by strains of
Therefore, bacteria which encode linA ( 6 ) and linB ( 7 ) involve in the degradation of HCH also
The pesticide used to control carriers of vector borne disease viz. malaria, sleeping sickness, Dengue fever, yellow fever etc. It has majority of organochlorides as non degradable and accumulates in the environment thus, causes soil pollution, from where it enter the food chain; their concentration heed as they move in the food chain.
This approach is useful in distinguishing other bacteria which comprises the same homologous gene which could biodegrade HCH. Here, emphasis on identification of culture dependent and culture independent bacteria which involved in degrading the non-degradable compounds was done.
Material And Methods
Collection of sequences
The complete nucleotide and protein sequences of linA and LinB from different source of bacteria extracted from biological database, viz. National Centre for Biotechnology Information (NCBI) cited at http://www.ncbi.nlm.nih.gov.The open reading frame of linA and LinB encoding proteins was also analyzed by NCBI.
The relatedness of sequences deposited in databases was evaluated by BLAST (Basic Local Alignment Search Tool) implemented via the NCBI website [www.ncbi.nlm.nih.gov/blast/] against the complete training dataset which is extracted from Genbank database. The BlastP (protein query – protein database comparison) in which conditional composition score adjustment having no filters of BLOSUM 62 matrix with threshold expect value 10 were used.
Construction of phylogenetic tree
The nucleotide sequences of linA and linB were compared with sequences deposited in the Genbank database using the BLAST programme. The sequences were aligned in CLUSTALX 1.83. The computed alignment was then manually checked and corrected. Pairwise evolutionary distances were computed using the Jukes and Cantor equation implemented in the MEGA 3.1 program and a phylogenetic tree was constructed by the neighbour-joining method programs package available online. Protein sequences were also used for construction of phylogenetic tree.
Results And Discussion
In the present study, total twenty nine linA genes having complete open reading frame (ORF) taken from different bacterial source were used. The phylogenetic relationships as demonstrated on the basis of all these structure nucleotide sequences. The homology of these sequences was mainly from three types of bacterial diversity viz
The evolutionary distribution of Lin A gene was present in the whole diversity of bacteria. In spite of nucleotide, we also used Lin A protein sequences for constructing phylogeny. Total nineteen protein sequences of either HCH degrading or almost similar homologous with this protein were used. We observed homology of these sequences mainly from four types belonging to bacterial diversity like
In this study, identification of HCH degrading bacteria on the basis of 16S rDNA gene in the genome was used. Based on 16S rDNA analysis, strain DS3-1 was closely related to
It was reported that
In this study, another gene linB, also involved in the degradation of HCH was used to construct the phylogenetic tree. Total four types of bacteria viz
Bacteria degrade HCH isomers also been isolated from HCH contaminated soils from different geographical locations of the family
The two linA- and linB-like genes coding, respectively, for a gamma-HCH dehydrochlorinase and a dehalogenase were characterized using a PCR strategy based on sequence homologies with previously published sequences from
In conclusion, the investigations of phylogenetic relationship approach on the basis of Lin A and Lin B genes and proteins were useful for other bacteria having the same homologous gene and furthermore used to degrade HCH. We also identified culture dependent and culture independent bacteria that involved in degradation of the non-degradable compounds. In future these bacteria may play a vital role for the safety of soil pollution and thus, improving soil fertility by preventing entry of HCH in food chain for the safety of animal health.