Mrutyunjay Suar

Cloning and Characterization of lin Genes Responsible for the Degradation of Hexachlorocyclohexane Isomers by Sphingomonas paucimobilis Strain B90

ABSTRACT 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, α, β, γ, and δ. While all these isomers pose serious environmental problems, β-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 α-, γ-, and δ-HCH but not of β-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of β-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 ∼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.

Organization of lin Genes and IS6100 among Different Strains of Hexachlorocyclohexane-Degrading Sphingomonas paucimobilis: Evidence for Horizontal Gene Transfer

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.

Enantioselective transformation of α-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A

ABSTRACT 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.

Dynamics of multiple lin gene expression in Sphingomonas paucimobilis B90A in response to different hexachlorocyclohexane isomers

ABSTRACT Sphingomonas paucimobilis B90A is able to degrade the α-, β-, γ-, and δ-isomers of hexachlorocyclohexane (HCH). It contains the genes linA, linB, linC, linD, linE, and linR, which have been implicated in HCH degradation. In this study, dynamic expression of the lin genes was measured in chemostat-grown S. paucimobilis B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH, linA, linB, and linC were all expressed constitutively. In contrast, linD and linE were induced with α-HCH (2 mg/liter) and γ-HCH (7 mg/liter). A sharp increase in mRNA levels for linD and linE was observed from 10 to 45 min after the addition of α- or γ-HCH. Induction of linD and linE was not detectable upon the addition of 0.7 mg of γ-HCH per liter, although the compound was degraded by the cells. The addition of β-HCH (5 mg/liter) or δ-HCH (20 mg/liter) did not lead to linE and linD induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of β- and δ-HCH proceeds by a different pathway than that of α- and γ-HCH.

Microbial Biodiversity Study of a Brackish Water Ecosystem in Eastern India

Abstract Brackish aquatic ecosystems are replete with microbes which are the most diverse and abundant organisms of these natural surroundings. The significance of microbial activities in rivers, lakes, and oceans has been emphasized in the current global climate change scenario. The magnitude of participation of microorganisms to impact worldwide flux of nutrients and greenhouse gas emissions is enormous. Estuarine water and sediments are dominated by bacteria which mineralize or decompose a wide variety of biopolymeric substrates with the aid of hydrolytic extracellular enzymes, produce bioactive compounds, secondary metabolites, antibiotics. Moreover, microbes in estuarine conditions constantly undergo tidal flushing which helps them to adapt to changing salt concentration and this helps to combat osmotic stress and tolerance to broad range of environmental factors like pH, temperature, and salinity in nature. However, in terms of microbial community composition, brackish water ecosystems are poorly understood. The interactions of microbes with biotic and abiotic factors give insights into the larger picture of ecosystem functioning. By using classical culture-dependent and modern culture-independent methods, the microbial diversity of Chilika Lake was evaluated to give insights into their functional and biological role in maintenance of health of estuarine ecosystem for exploiting their future potential.

High Prevalence of Bacterial Spore-Formers Active Against Mosquito Larvae in Temporary Monsoon Flooded Sites in Orissa, India

Abstract Different ecosystems were probed in the vicinity of the city of Bhubaneswar in the Indian state of Orissa for the presence of bacterial spore-formers with activity against mosquito larvae. The most productive sites were places that were flooded during the monsoon season, including roadside ditches and shorelines of ponds. Among 630 isolates screened, 44 (7%) showed larvicidal activity against larvae of Aedes aegypti. The specific activity of the bacterial spore-formers varied greatly. Isolates were found with specific activities superior to the Bacillus thuringiensis israelensis reference strain of the Pasteur Institute. All mosquitocidal strains produced crystal proteins, and based on the biochemical analyses could be classified into the species B. thuringiensis. Such strains possess the potential for the development of new microbial products for mosquito control in India.