Sukanya Lal

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.

Engineering antibiotic producers to overcome the limitations of classical strain improvement programs.

Improvement of the antibiotic yield of industrial strains is invariably the main target of industry-oriented research. The approaches used in the past were rational selection, extensive mutagenesis, and biochemical screening. These approaches have their limitations, which are likely to be overcome by the judicious application of recombinant DNA techniques. Efficient cloning vectors and transformation systems have now become available even for antibiotic producers that were previously difficult to manipulate genetically. The genes responsible for antibiotic biosynthesis can now be easily isolated and manipulated. In the first half of this review article, the limitations of classical strain improvement programs and the development of recombinant DNA techniques for cloning and analyzing genes responsible for antibiotic biosynthesis are discussed. The second half of this article addresses some of the major achievements, including the development of genetically engineered microbes, especially with reference to beta-lactams, anthracyclines, and rifamycins.

Rifamycins: Strain Improvement Program

Rifamycins are primarily produced by Gram-positive bacterium Amycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens including Mycobacterium avium and Pneumococcus. Because of the importance of rifamycin, the producer strain A. mediterranei has been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations of A mediterranei are discussed.

Bioconcentration and metabolism of DDT, fenitrothion and chlorpyrifos by the blue-green algae Anabaena sp. and Aulosira fertilissima.

Anabaena and Aulosira fertilissima showed a marked ability to accumulate DDT, fenitrothion and chlorpyrifos. Although the maximum accumulation of DDT was almost the same in both organisms, there were significant differences in their abilities to accumulate fenitrothion and chlorpyrifos. Patterns of uptake of DDT under different treatments were also similar in both Anabaena and Aulosira, but there were significant differences in the patterns of accumulation of fenitrothion between these two organisms. In Aulosira the maximum accumulation of fenitrothion was observed on the second day, whereas, in Anabaena, maximum accumulation was noticed on the first day. A completely different pattern of accumulation of chlorpyrifos was observed in Aulosira, which continued to accumulate chlorpyrifos throughout the experimental period. Bioconcentration of DDT in Anabaena and Aulosira ranged from 3 to 1568 ppm (microg g(-1)) and 6 to 1429 ppm, respectively. Bioconcentration of fenitrothion and chlorpyrifos in Anabaena varied from 53 to 3467 ppm and 7 to 6779 ppm, respectively. In Aulosira the bioconcentration varied from 100 to 6651 ppm and 53 to 3971 ppm for fenitrothion and chlorpyrifos, respectively. Anabaena and Aulosira metabolised DDT to DDD and DDE. Amounts of these DDT metabolites detected in the organisms were dependent on the concentration of treatment. DDD was the major, and DDE the minor, metabolite. These organisms were not able to metabolise the organophosphorus insecticides, fenitrothion and chlorpyrifos.

Effects of DDT, fenitrothion and chlorpyrifos on growth, photosynthesis and nitrogen fixation in Anabaena (Arm 310) and Aulosira fertilissima

Abstract The response of blue-green algae to DDT, fenitrothion and chlorpyrifos revealed that algae are quite sensitive to insecticides and the effects depend on the type and nature of the insecticide, the organisms and the experimental conditions. DDT inhibited the growth of Anabaena whereas it was stimulatory to Aulosira . Fenitrothion and chlorpyrifos were, however, quite toxic even at concentrations of 100 times less than DDT. Organisms recovered from the toxic effect if the treatment continued for 35 days. DDT at all concentrations inhibited photosynthesis in Anabaena and Aulosira . Fenitrothion was extremely toxic to both these organisms as it inhibited photosynthesis by more than 75% at the highest concentration. Chlorpyrifos was comparatively less toxic to Aulosira than Anabaena as it inhibited 14 CO 2 -uptake at 10 ppm by 76.0 and 69.4%, respectively. Nitrogenase activity was stimulated by DDT in Anabaena but inhibited by it in Aulosira , whereas fenitrothion and chlorpyrifos inhibited nitrogenase activity in both the organisms.

Bioaccumulation, metabolism, and effects of DDT, fenitrothion, and chlorpyrifos on Saccharomyces cerevisiae.

Saccharomyces cerevisiae accumulated DDT, fenitrothion, and chlorpyrifos rapidly from yeast glucose medium. The maximum concentrations of DDT, fenitrothion, and chlorpyrifos accumulated were 8,253, 18,960 and 11,579 μg/g (dry wt), respectively. The pattern of accumulation was similar for all insecticides. The bioconcentration factor was inversely proportional to insecticide solubilities.Saccharomyces metabolized the three insecticides, but only two metabolites of DDT (DDD and DDE) were identified. Protoplast cultures were more sensitive to DDT and fenitrothion compared to normal cultures but were less sensitive to chlorpyrifos. Both the normal and protoplast cultures recovered from the toxic effect after 24 hr.

Development of cloning vectors and transformation methods for Amycolatopsis

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA-rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.

Manipulations of Catabolic Genes for the Degradation and Detoxification of Xenobiotics

Publisher Summary This chapter describes the manipulations of catabolic genes for the degradation and detoxification of xenobiotics. In vitro strain construction requires detailed genetic and biochemical information on the degradation pathways of xenobiotics. The control of catabolic pathways can also be modified by placing key biodegradation enzymes that require inducers, some of which are pollutants themselves, under the control of new regulatory systems. For example, genetic engineering has been used to uncouple the Pseudomonas mendocina toluene monoxygenase from toluene induction to derive Pseudomoms transconjugants that constitutively express the 2,4-D degradation pathway and to derive E. coli recombinant strains to enhance PCBs degradative activity in the presence of exogenous catabolite repressor substance. The cloning of genes for modified enzymes that have useful catabolic properties (such as relaxed substrate specificities or enhanced induction) provides an important repository of genetic diversity for future research.