Kirti Kumari

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

SUMMARY Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.

Polyphasic approach of bacterial classification — An overview of recent advances

Classification of microorganisms on the basis of traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the ‘polyphasic approach’ for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classified on the basis of the polyphasic approach. Also previously classified organisms, as and when required, can be reclassified on this ground in order to obtain information about their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

Proposal of biostimulation for hexachlorocyclohexane (HCH)‐decontamination and characterization of culturable bacterial community from high‐dose point HCH‐contaminated soils

Aims:  To locate a high‐dose point hexachlorocyclohexane (HCH)‐contaminated site, to identify HCH‐degrading bacteria in it and assay HCH‐decontamination by biostimulation.

Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.

Whole Genome Sequence of the Rifamycin B-Producing Strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.

Pseudoxanthomonas indica sp. nov., isolated from a hexachlorocyclohexane dumpsite

A bacterial strain, designated P15(T), was isolated from the soil of an open hexachlorocyclohexane dumpsite. Comparative sequence analysis showed that strain P15(T) displayed high 16S rRNA gene sequence similarities (94.4-97.2 %) with members of the genus Pseudoxanthomonas. The isolate was most closely related to Pseudoxanthomonas mexicana AMX 26B(T) (97.2 % 16S rRNA gene sequence similarity) and Pseudoxanthomonas japonensis 12-3(T) (97.2 %). DNA-DNA relatedness studies showed unambiguously that strain P15(T) represented a novel species that was separate from P. mexicana DSM 17121(T) (7.7 %) and P. japonensis DSM 17109(T) (9.4 %). The predominant cellular fatty acids of strain P15(T) were iso-C₁₆:₀ (21.4 %), iso-C₁₅:₀ (16.1 %), summed feature 9 (comprising iso-C₁₇:₁ω9c and/or 10-methyl C₁₆:₀; 14.9 %), iso-C₁₁:₀ 3-OH (8.3 %) and iso-C₁₄:₀ (7.0 %). The polar lipid profile of strain P15(T) showed the presence of large amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol in addition to unknown glycolipids, phospholipids and an amino-group-containing polar lipid. Ubiquinone 8 was found as the major quinone. The polyamine profile showed the presence of spermidine. The DNA G+C content was 62.9±2 mol%. Strain P15(T) is described as representing a new member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas indica sp. nov. is proposed. The type strain is P15(T) ( = MTCC 8596(T) = CCM 7430(T)).

From bacterial genomics to metagenomics: concept, tools and recent advances

In the last 20 years, the applications of genomics tools have completely transformed the field of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, first describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting findings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world.

Kinetic and sequence-structure-function analysis of known LinA variants with different hexachlorocyclohexane isomers.

Background Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (−)-α -HCH are also discussed. Methodology/Principal Findings Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2B90A A110T, A111C, A110T/A111C and LinA1B90A were constructed using the FoldX computer algorithm. Turnover rates (min−1) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinAs, followed by the γ and then δ isomer. Conclusions/Significance The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.

Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay

Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.

Bacterial diversity and real‐time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real‐time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 105 mg kg−1. The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump‐site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real‐time PCR were 6.2 × 104 and 5.3 × 105, respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation.