Enantioselective Transformation of α-Hexachlorocyclohexane by the Dehydrochlorinases LinA1 and LinA2 from the Soil Bacterium Sphingomonas paucimobilis B90A

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral α-hexachlorocyclohexane (α-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (−) enantiomer. Concurrent formation and subsequent dissipation of β-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral α-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of α-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.     

The first draft of the pigeonpea genome sequence

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety ??Asha??. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550?bp and >10-fold genome coverage, resulting in 510,809,477?bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable ??Arhar?? simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.     

Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation in Sphingobium indicum B90A

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform β- and δ-hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with β- and δ-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including β-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade β- and δ-HCH.     

Cloning and characterization of lin genes responsible for the degradation of Hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.     

Immunotoxicity of soluble and insoluble salts of cadmium instilled intratracheally

Rats were intratracheally (i.t.) exposed to 36.5 or 27.5 microg of cadmium (Cd) as soluble cadmium chloride (CdCl2) and insoluble cadmium oxide (CdO) salts. The retention of metal in lungs, liver and kidney was assessed by atomic adsorption spectrophotometer. The animals were intraperitoneally (i.p.) primed with sheep red blood cells (SRBC) and assessed for the number of antibody forming cells in lung associated lymph nodes (LALN) and spleen. Both the compounds had similar retention of metal in lungs but CdO induced more pulmonary inflammatory and degradative changes than CdCl2. The larger influx of polymorphonuclear cells (PMNs) following CdO exposure appears to be due to the absence of protection afforded by Cd induced metallothionein cytoplasmic protein while the Cd metallothionein complex formed in the case of CdCl2 is more protective. However both forms of Cd had similar local immunosuppressive potential but CdO had more prolonged suppressive effect.