Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds b... more Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
Isolation and characterization of rice (Oryza sativa L. ssp. indica) OsPHOT1 cDNA, encoding a blu... more Isolation and characterization of rice (Oryza sativa L. ssp. indica) OsPHOT1 cDNA, encoding a blue light photoreceptor phototropin 1, is reported. The predicted amino acid sequence of OsPHOT1 corresponds to a protein of 103.6 kDa harboring two LOV domains (LOV1 and LOV2) and a C-terminal protein kinase domain conserved in other phototropins. The structural part of the gene is interrupted by 22 introns. The OsPHOT1 gene is represented by two copies in the rice genome (designated as OsPHOT1a and OsPHOT1b), one each on the duplicated segments of chromosomes 11 and 12. BLASTN and TBLASTN searches also led to the identification of another phototropin homologue, OsPHOT2, present on chromosome 4. The real-time PCR analysis showed that both the OsPHOT1 and OsPHOT2 are expressed at significantly higher levels in mature leaves. The mRNA levels of OsPHOT1 were higher in the coleoptiles and that too in the apical portion, whereas OsPHOT2 was strongly expressed in leaves of etiolated rice seedlings. The transcript levels of OsPHOT1 and OsPHOT2 were differentially regulated by a broad spectrum of light, including white, blue, red, and far-red light; the transcript levels of OsPHOT1 declined, whereas those of OsPHOT2 were upregulated. These results indicate the participation of red/far-red sensing phytochromes and blue light (perceived through either cryptochromes or phototropins) in differential regulation of transcript abundance of phototropins, OsPHOT1 and OsPHOT2.
Environmental Science and Pollution Research, 2011
Purpose α-Hexachlorocyclohexane (HCH), β-HCH, and lindane (γ-HCH) were listed as persistent organ... more Purpose α-Hexachlorocyclohexane (HCH), β-HCH, and lindane (γ-HCH) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and hence must be phased out and their wastes/stockpiles eliminated. At the last operating lindane manufacturing unit, we conducted a preliminary evaluation of HCH contamination levels in soil and water samples collected around the production area and the vicinity of a major dumpsite to inform the design of processes for an appropriate implementation of the Convention. Methods Soil and water samples on and around the production site and a major waste dumpsite were measured for HCH levels. Results All soil samples taken at the lindane production facility and dumpsite and in their vicinity were contaminated with an isomer pattern characteristic of HCH production waste. At the dumpsite surface samples contained up to 450 g kg−1 Σ HCH suggesting that the waste HCH isomers were simply dumped at this location. Ground water in the vicinity and river water was found to be contaminated with 0.2 to 0.4 mg l−1 of HCH waste isomers. The total quantity of deposited HCH wastes from the lindane production unit was estimated at between 36,000 and 54,000 t. Conclusions The contamination levels in ground and river water suggest significant run-off from the dumped HCH wastes and contamination of drinking water resources. The extent of dumping urgently needs to be assessed regarding the risks to human and ecosystem health. A plan for securing the waste isomers needs to be developed and implemented together with a plan for their final elimination. As part of the assessment, any polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) generated during HCH recycling operations need to be monitored.
In the last 20 years, the applications of genomics tools have completely transformed the field of... more 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.
The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical H... more 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.
Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds b... more Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.
Isolation and characterization of rice (Oryza sativa L. ssp. indica) OsPHOT1 cDNA, encoding a blu... more Isolation and characterization of rice (Oryza sativa L. ssp. indica) OsPHOT1 cDNA, encoding a blue light photoreceptor phototropin 1, is reported. The predicted amino acid sequence of OsPHOT1 corresponds to a protein of 103.6 kDa harboring two LOV domains (LOV1 and LOV2) and a C-terminal protein kinase domain conserved in other phototropins. The structural part of the gene is interrupted by 22 introns. The OsPHOT1 gene is represented by two copies in the rice genome (designated as OsPHOT1a and OsPHOT1b), one each on the duplicated segments of chromosomes 11 and 12. BLASTN and TBLASTN searches also led to the identification of another phototropin homologue, OsPHOT2, present on chromosome 4. The real-time PCR analysis showed that both the OsPHOT1 and OsPHOT2 are expressed at significantly higher levels in mature leaves. The mRNA levels of OsPHOT1 were higher in the coleoptiles and that too in the apical portion, whereas OsPHOT2 was strongly expressed in leaves of etiolated rice seedlings. The transcript levels of OsPHOT1 and OsPHOT2 were differentially regulated by a broad spectrum of light, including white, blue, red, and far-red light; the transcript levels of OsPHOT1 declined, whereas those of OsPHOT2 were upregulated. These results indicate the participation of red/far-red sensing phytochromes and blue light (perceived through either cryptochromes or phototropins) in differential regulation of transcript abundance of phototropins, OsPHOT1 and OsPHOT2.
Environmental Science and Pollution Research, 2011
Purpose α-Hexachlorocyclohexane (HCH), β-HCH, and lindane (γ-HCH) were listed as persistent organ... more Purpose α-Hexachlorocyclohexane (HCH), β-HCH, and lindane (γ-HCH) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and hence must be phased out and their wastes/stockpiles eliminated. At the last operating lindane manufacturing unit, we conducted a preliminary evaluation of HCH contamination levels in soil and water samples collected around the production area and the vicinity of a major dumpsite to inform the design of processes for an appropriate implementation of the Convention. Methods Soil and water samples on and around the production site and a major waste dumpsite were measured for HCH levels. Results All soil samples taken at the lindane production facility and dumpsite and in their vicinity were contaminated with an isomer pattern characteristic of HCH production waste. At the dumpsite surface samples contained up to 450 g kg−1 Σ HCH suggesting that the waste HCH isomers were simply dumped at this location. Ground water in the vicinity and river water was found to be contaminated with 0.2 to 0.4 mg l−1 of HCH waste isomers. The total quantity of deposited HCH wastes from the lindane production unit was estimated at between 36,000 and 54,000 t. Conclusions The contamination levels in ground and river water suggest significant run-off from the dumped HCH wastes and contamination of drinking water resources. The extent of dumping urgently needs to be assessed regarding the risks to human and ecosystem health. A plan for securing the waste isomers needs to be developed and implemented together with a plan for their final elimination. As part of the assessment, any polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) generated during HCH recycling operations need to be monitored.
In the last 20 years, the applications of genomics tools have completely transformed the field of... more 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.
The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical H... more 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.
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