Isolation of Klebsiella pneumoniae from Sungai Skudai and in silico analysis of putative dehalogenase protein

Aims@#The surplus use of herbicide Dalapon® contains 2,2-dichloropropionic acid (2,2-DCP) poses great danger to human and ecosystem due to its toxicity. Hence, this study focused on the isolation and characterization of a dehalogenase producing bacteria from Sungai Skudai, Johor, capable of utilizing 2,2-DCP as a carbon source and in silico analysis of its putative dehalogenase.@*Methodology and results@#Isolation of the target bacteria was done by using 2,2-DCP-enriched culture as the sole carbon source that allows a bacterium to grow in 20 mM of 2,2-DCP at 30 °C with the corresponding doubling time of 8.89 ± 0.03 h. The isolated bacterium was then designated as Klebsiella pneumoniae strain YZ based on biochemical tests and basic morphological examination. The full genome of K. pneumoniae strain KLPN_25 (accession number: RRE04903) which obtained from NCBI database was screened for the presence of dehalogenase gene, assuming both strains YZ and KLPN_25 were the same organisms. A putative dehalogenase gene was then identified as type II dehalogenase from the genome sequence of strain KLPN_25. The protein structure of the type II dehalogenase of KLPN_25 strain was then pairwise aligned with the crystal structure of L-2-haloacid dehalogenase (L-DEX) Pseudomonas sp. strain YL as the template, revealing the existence of conserved amino acids residues, uniquely known to participate in the dehalogenation mechanism. The finding thus implies that the amino acid residues of type II dehalogenase possibly shares similar catalytic functions with the L-DEX.@*Conclusion, significance and impact of the study@#In conclusion, this study confirmed the presence of new dehalogenase from the genus Klebsiella with potential to degrade 2,2-DCP from the river water. The structural information of type II dehalogenase provides insights for future work in designing haloacid dehalogenases.

Dehalogenase producing bacteria from extreme environment: A review

@#Halogenated compounds create the most important class of xenobiotic which commonly lead to pollution. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Many of these toxic chemicals have been shown to occur in various extreme habitats. Pollutant-degrading microorganisms, adapted to grow in various environments, play an important role in the biological treatment of polluted extreme habitats. The presence of dehalogenase producing microorganisms in extreme habitat in particular is necessary since the enzyme can catalyze the removal of a halogen atom from a substrate. Therefore, it can reduce the toxicity of the halogenated compound and some are of interest for study in industrial application. Thermophiles, psychrophiles, acidophiles, alkaliphiles and halophiles are types of extremophiles. Knowledge of the biodegradation of toxic chemicals in extreme environment is limited. Here, examples of dehalogenase producing bacteria isolated from various extreme conditions and its special characteristics/features will be discussed in this review.

In silico analysis of a Rhizobium sp. RC1 putative haloalkanoic permease sequence motif and classification

Aims@#The transport of haloalkanoic acids (haloacids) is important in the metabolism of haloacid pollutants by bacteria. In this study, a computational analysis of Rhizobium sp. RC1 haloacid permease (DehrP) amino acid sequence was conducted to identify its subfamily, sequence motifs and evolutionary position among closely related transporters. @*Conclusion, significance and impact of study@#Blast search in the Pfam and Transmembrane Classification Databases was used to establish the classification and the subfamily of DehrP. Clustal omega sequence alignment approach and MEME Suite motif-based analysis tools were used to locate the transporter motifs of DehrP. Dotplots of DehrP sequence was computed using the EMBOSS Dotmatcher. MEGA7 software was used to analyze the phylogenetic position of DehrP among closely related symporters in the Transmembrane Classification Database. Comparative analysis by Pfam shows that DehrP is a member of the Major Facilitator Superfamily (#2.A.1). PSI-Blast against the Transmembrane Classification Database shows that DehrP is significantly aligned with a subfamily of transporters called the Metabolite: H+ Symporters (#2.A.1.6). DehrP has six similar sequence motifs with the Metabolite: H+ Symporter proteins including the functional motif of GXXXDRXGRR. DehrP is evolutionarily related to Burkholderia caribensis MBA4 Haloacid: H+ Symporters (Dehp2 and Deh4p). @*Methodology and results@#Based on sequence similarity, DehrP is a Major Facilitator Superfamily protein that belongs to the Metabolite: H+ Symporter protein subfamily which might coordinate the transport of a haloacid coupled with a proton (H+). Mutagenesis of DehrP sequence motifs might be useful in the engineering of Rhizobium sp. RC1 for efficient uptake and degradation of haloacids.

Analysis of 2,2-DCP degrading bacteria isolated from a paddy field at a rural area in Malang, Indonesia

Aims@#The use of herbicide effectively controls weeds in agricultural practice. However, its release to the surrounding surface water bodies may lead to environmental issues. The aim of this study was to isolate the bacteria that were able to remove 2,2-dichloropropionic acid (2,2-DCP) from a paddy field located in Malang. @*Methodology and results@#The 2,2-DCP degrading bacteria were isolated and their ability to grow on higher 2,2-DCP concentrations (50 and 80 mM) was tested. Bacterial degradation of 2,2-DCP was examined through measurement of released chloride ions. The potential isolates were identified according to their 16S rDNA sequences. Two potential isolates, BB9.2 and BC14.3 were observed for their growth on 20, 50, and 80 mM 2,2-DCP. Isolate BC14.3 had the shortest cell doubling time of approximately 4.1 h with 100% 2,2-DCP (20 mM) utilization, whereas BB9.2 was only able to degrade 80% of 2,2-DCP at the same concentration. The 16S rDNA gene sequences suggested that BB9.2 and BC14.3 belong to Acinetobacter calcoaceticus and Pseudomonas plecoglossicida, respectively. @*Conclusion, significance and impact of study@#Bacterial strains with 2,2-DCP degrading potentials were successfully isolated from long-term exposed agricultural soil. They demonstrated notable utilization of the organic halide. This is the first time that strains of A. calcoaceticus and P. plecoglossicida were reported to utilize 2,2-DCP.

Purification and characterization of dehalogenase from Bacillus cereus SN1 isolated from cow dung

Aims@#This study was aimed to characterize a dehalogenase derived from Bacillus cereus SN1 isolated from cow dung. @*Methodology and results@#Cell-free extract of Bacillus cereus SN1 was purified using ion exchange and gel filtration chromatography. Fraction B2 of gel filtration gave the highest enzyme specific activity (0.155 μmol CI¯/min/mg). The results of SDS-PAGE showed the enzyme was 25 kDa in size. The enzyme reached its optimum activity at 30 °C at pH 6, and was inhibited by Mercury(II) sulfate (HgSO4). The Km and kcat values were 0.2 mM and 1.22/sec, respectively. The partial dehalogenase gene sequence was amplified using Group I dehalogenase primers. The amplified gene sequence was designated as DehSN1. @*Conclusion, significance and impact of study@#Dehalogenase from Bacillus cereus strain SN1 revealed new characteristics of dehalogenase protein. The findings indicated that the DehSN1 dehalogenase is a promising candidate for further studies as a bioremediation agent for agricultural applications.