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.

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.

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.

Determination of Ergosterol as a Potential Biomarker in Pathogenic Medically Important Fungal Isolates / Jurnal Sains Kesihatan Malaysia

Ergosterol, a component of fungal cell membrane, has been frequently detected as an indicator of fungal presence and massin environmental samples like soil. However, its detection in major pathogenic fungal species has not been investigated.In this study, the ergosterol contents of ten pathogenic fungal species were determined. Liquid chromatography was usedfor the detection and quantification of ergosterol extracted from fungal broth cultures. Results showed that ergosteroleluted as a single, well resolved peak in the chromatogram profiles of all tested fungi. Based upon relative amounts ofergosterol produced per fungal mycelial dry weight, three groups of fungal pathogens were identified, namely low ergosterol(Aspergillus niger, Candida albicans and Cryptococcus neoformans at 4.62, 6.29 and 7.08 μg/mg, respectively), mediumergosterol (Fusarium solani, Aspergillus fumigatus, Mucor sp., Penicillium sp., Cryptococcus gattii and Rhizopus sp.at 9.40, 10.79, 10.82, 11.38, 12.60 and 13.40 μg/mg, respectively), and high ergosterol (Candida tropicalis at 22.84 μg/mg), producers. Ergosterol was not detectable in bacterial samples, which were included as controls. This first report onergosterol detection in major pathogenic fungal species indicates that ergosterol may be used as a biomarker to diagnoseinvasive fungal infections in clinical sampl

The potential of a novel β-specific dehalogenase from Bacillus cereus WH2 as a bioremediation agent for the removal of β-haloalkanoic acids

Aims: This study aims to describe the biochemical and kinetic properties of a dehalogenase produced by a bacterium, Bacillus cereus WH2 (KU721999), that is uniquely adept at degrading a β-haloalkanoic acid, i.e., 3-chloropropionic acid (3-CP), and using it as the bacterium’s sole carbon source. The bacterium was isolated from abandoned agricultural land in Universiti Teknologi Malaysia that was previously exposed to herbicides and pesticides. Methodology and results: The B. cereus impressively removed 97% of 3-CP after 36 h of culturing. The intracellular WH2 dehalogenase of the bacterium was purified 2.5-fold and has an estimated molecular mass of 37 kDa. The highest activity of the dehalogenase was achieved under conditions of 30 °C and pH 7. The metal ions Hg2+ and Ag2+ substantially repressed the enzyme’s activity, but the enzyme’s activity was uninhibited by dithiothreitol (DTT) and EDTA. The WH2 dehalogenase showed a higher affinity for 3-CP (Km = 0.32 mM, kcat = 5.74 s-1 ) than for 3-chlorobutyric acid (3-CB) (Km = 0.52 mM; kcat = 5.60 s-1 ). The enzyme was ~1.6-fold more catalytically efficient (kcat/Km) in dehalogenating the three-carbon substrate 3-CP (17.8 mM-1 s -1 ) than the four-carbon 3-CB (11.2 mM-1 s -1 ). Conclusion, significance and impact of study: The novel B. cereus bacterium isolated in this study may prove applicable as a bioremediation agent to cleaning environments that are polluted with β-halogenated compounds. Furthermore, such an approach to treat polluted environments is more sustainable and potentially safer than chemical treatments.

Microbial isolation and degradation of selected haloalkanoic aliphatic acids by locally isolated bacteria: A review

The liberation of halogenated compounds by both natural processes and man-made activities has led to extensive contamination of the biosphere. Bioremediation via the dehalogenation process offers a sustainable way to eliminate such hazardous contaminants. Whereas, a large number of natural soil microorganisms (i.e., bacteria and fungi) that have been isolated are capable of degrading and detoxifying such contaminants, information on the preferred types of halogenated compounds that they catalyze is lacking. In this review, we discuss those microorganisms that have the potential to perform bioremediation of such environmental contaminants. We also present a method for isolating novel dehalogenase-producing microorganisms from cow dung.

In silico molecular analysis of novel L-specific dehalogenase from Rhizobium sp. RC1

Aims: This study presents the first structural model and proposed the identity of four important key amino acid residues, Asp13, Arg51, Ser131 and Asp207 for the stereospecific haloalkanoic acid dehalogenase from Rhizobium sp. RC1. Methodology and results: The enzyme was built using a homology modeling technique; the structure of crystallized LDEX YL from Pseudomonas sp. strain YL as a template. Model validation was performed using PROCHECK to generate the Ramachandran plot. The results showed 80.4% of its residues were located in the most favoured regions suggested that the model is acceptable. Molecular dynamics simulation of the model protein was performed in water for 10 nanoseconds in which Na+ was added to neutralize the negative charge and achieved energy minimization. The energy value and RMSD fluctuation of Cα backbone of the model were computed and confirmed the stability of the model protein. Conclusion, significance and impact of study: In silico or computationally based function prediction is important to complement with future empirical approaches. L-haloacid dehalogenase (DehL), previously isolated from Rhizobium sp. RC1 was known to degrade halogenated environmental pollutants. However, its structure and functions are still unknown. This structural information of DehL provides insights for future work in the rational design of stereospecific haloalkanoic acid dehalogenases.

Isolation and identification of bacteria isolated from ruminant animal waste that able to degrade 2,2-dichloropropionic acid (2,2-DCP)

Aims: A 2,2-dichloropropionic acid (2,2-DCP) naturally degrading bacterial species, strain SN1 was successfully isolated from cow dung capable of utilizing the substance as the sole carbon source and energy. Methodology and results: Strain SN1 was preferred over other strains (SN2, SN3 and SN4) following observations on its rapid growth in 20 mM 2,2-DCP liquid minimal media. Since strain SN1 clearly exhibited tolerance towards 2,2-DCP, its growth in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2-DCP with the highest chloride release of 39.5 µmole Cl- /mL while exhibiting a remarkably short doubling time of 3.85 h. In view of such notable characteristics, species identification via Biolog GEN III system and 16S rRNA analysis was performed and established strain SN1 as Bacillus cereus. Conclusion, significance and impact of study: Considering the rapid growth of B. cereus strain SN1 in such medium, its employment as a bioremediation agent to treat 2,2-DCP contaminated soils may prove beneficial. Moreover, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2-DCP. Therefore, further assessment into its ability to degrade other types of haloalkanoic acids merit special consideration.

A novel putative non-ligninolytic dehalogenase activity for 3-chloropropionic acid (3CP) utilization by Trichoderma asperellum strain SD1

Aims: Trichoderma asperellum strain SD1 grows on 3-chloropropionic acid (3CP), a β-haloalkanoic acid, and produces a putative extracellular dehalogenase that can degrade this acid. Here we further characterized the fungal enzyme system responsible for biodegradation of 3CP. Methodology and results: The primary qualification of the ligninolytic potential in T. asperellum strain SD1 was performed using guaiacol oxidation. When strain SD1 was grown in liquid minimal medium with the presence of 3CP as the sole carbon source, no lignin peroxidase, manganese peroxidase, or laccase activity was detected. The ligninolytic condition was achieved only in the presence of glucose or when guaiacol was present as an inducer. Under nonligninolytic conditions, 3CP was utilized by strain SD1. Therefore, 3CP was utilized under ligninolytic conditions as well as under non-ligninolytic conditions, suggesting that extracellular peroxidases and laccase are not involved in the degradation of 3CP by T. asperellum strain SD1. Conclusion, significance, and Impact of study: Very few studies have explained the degradation of β-chloro– substituted haloalkanoic acids such as 3CP by dehalogenases. This is the first report to identify a novel putative β- haloacid dehalogenase that degrades 3CP under ligninolytic and non-ligninolytic conditions. T. asperellum strain SD1, thus has the potential in the development of dehalogenating enzymes for industrial biocatalytic processes, in future.

Molecular identification and biodegradation of 3-chloropropionic acid (3CP) by filamentous fungi-Mucor and Trichoderma species isolated from UTM agricultural land

Aims: This study was carried out to further characterize fungal species that could degrade 3-chloropropionic acid (3CP) as sole source of carbon and energy. Methodology and Results: Both fungi were able to grow on 3CP after 10 days on solid minimal media. Based on sequencing of its segment of 18S rRNA these isolates were identified as Mucor sp. SP1 and Trichoderma sp. SP2. The isolated strains were not able to grow on media plates containing 10 mM of 2,2-dichloropropionate (2,2DCP) as sole source of carbon. 3CP degradation was observed in liquid minimal medium containing 10 mM 3CP after 18 days culture period. The chloride ion released was detected in both growth medium containing Mucor sp. SP1 and Trichoderma sp. SP2. At least 80% of 10 mM 3CP was utilized in the growth medium. Conclusion, significance and impact of study: Dehalogenase enzyme that can degrade α-chloro-substituted haloalkanoic acids for example 2,2DCP is well studied up to protein crystallization. Very few reports on the degradation of β-chloro-substituted haloalkanoic acids such as 3CP and none from fungi. This study is considered important because it can be compared to that of well-documented α-chloro-substituted haloalkanoic acids degradation. This is the first study to indicate fungal growth on 3CP as sole carbon and energy sources.

Identification of Serratia marcescens SE1 and determination of its herbicide 2,2- dichloropropionate (2,2-DCP) degradation potential

Aims: The goal of the study is to isolate species of bacteria that capable of utilizing 2,2-dichloropropionic acid (2,2-DCP) as sole carbon source from soil sample collected from surrounding lake water located in Universiti Teknologi Malaysia, Skudai, Johor. Methodology and Results: Genomic DNA from bacterium SE1 was extracted and PCR amplification was carried out using universal primers, Fd1 (5’ - AGA GTT TGA TCC TGGCTC AG - 3’) and rP1 (5’- ACG GTC ATA CCT TGT TAC GAC TT - 3’) before sending for sequencing. The 16S rDNA nucleotide sequences were compared with Basic Local Alignment Search Tool nucleotide (BLASTn) and further analyzed using phylogenetic tree of Neighbour-Joining method (MEGA 5). Phylogenetic analysis indicated that SE1 strain clearly shared 97% homology to the genus of Serratia marcescens and therefore designated as Serratia marcescens sp. SE1. SE1 exhibited the ability to utilize 2,2-DCP as sole carbon source at 20 mM concentration with cell doubling time of 5 h and maximum chloride ion release of 38 μmolCl-/mL. This result suggests that the dehalogenase enzyme present in the bacteria has high affinity towards the substrate. Based on morphological and partial biochemical characteristics, strain SE1 was a non-motile Gram negative bacterium with red colonies, that gave a catalase positive reaction. Conclusion, significance and impact of study: A better understanding of dehalogenases enzyme produce by this S. marcescens sp. SE1 in general will be useful to be used as bioremediation tools for environmental management. This is the first reported case that Serratia sp. has the ability to degrade halogenated compound.

Characterization of Exopolysaccharides Produced by Bacillus cereus and Brachybacterium sp. Isolated from Asian Sea Bass (Lates calcarifer)

Aims: EPS extracted from marine bacteria, which associated with Asian sea bass has potential antimicrobial activities. Methodology and Results: Two marine Bacteria were isolated from Asian sea bass (Lates calcarifer) obtained from aquaculture farm, located at Johor bahru Malaysia. 16S rRNA analysis for bacteria identity revealed that bacteria ors1 had 99 % identity to Bacillus cereus and ors2 had 96 % identity with Brachybacterium sp. All bacteria shared many similarities and variation in terms of biochemical reactions and microscopic observation. Exopolysaccharides (EPSs) were extracted and purified from bacteria as they produced mucous colonies. Average analysis of EPS components showed 50 % carbohydrates, 26 % protein and 24 % fatty acids. The FTIR analysis confirmed the functional groups of the EPS. Screening for antimicrobial activities assays using Kirby-Bauer methods against both grams positive and negative had shown presence of inhibition zones. Conclusion, significance and impact of study: This study recommends that bacteria isolated from Asian sea bass are having antimicrobial activities and could be used as a potential source for the development of marine drugs.