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, ␣, ␤, ␥, and ␦. While all these isomers pose serious environmental problems, ␤-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 ␣-, ␥-, and ␦-HCH but not of ␤-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of ␤-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 ϳ96 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.

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp؉ showed that they were able to transform ␤-and ␦-hexachlorocyclohexane (␤-and ␦-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 ␤-and ␦-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 ␤-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade ␤-and ␦-HCH.Hexachlorocyclohexane (HCH), a broad-spectrum insecticide, was one of the most extensively used organochlorine pesticides for the control of agricultural pests and the control of mosquitoes in malaria health programs during the 1940s. Technical HCH is prepared by chlorination of benzene in the presence of UV, resulting in the formation of a mixture primarily containing the isomers ␥-HCH (10 to 12%), ␣-HCH (60 to 70%), ␤-HCH (5 to 12%), and ␦-HCH (6 to 10%) (20). Of these isomers, only ␥-HCH (also known as lindane) has insecticidal properties. For purely economic reasons, technical HCH was used indiscriminately instead of lindane in many countries, and its use continued unabated until the 1990s, when the persistent and toxic nature of the components of technical mixtures was realized. Today, the use of technical HCH is banned in most countries, and the use of lindane is either banned or severely restricted. The extensive and widespread use, unregulated disposal, and persistent nature of HCH isomers have created the following two types of contamination problems: (i) low levels of contamination of agricultural soils and groundwater (3,14,25,32,39,45), mainly caused by the intended usage, and (ii) high levels of contamination at the production sites caused by inappropriate disposal of the noninsecticidal isomers ␣-, ␤-, and ␦-HCH. A large number of open or sealed dumping sites exist in The Netherlands (53), Brazil (36), Spain (24), Germany (13), India (39), and Eastern and Central Europe (53), which still pose serious risks for soils and groundwater.␣-and ␥-HC...

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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.

A Gram-negative, motile, rod-shaped, non-sporulating, aerobic bacterial strain (Esp-1 T ) was isolated from oil-contaminated soil of Panipat Oil Refinery, India, and its taxonomic position was determined using a polyphasic approach. Strain Esp-1 T grew in the presence of 2 % NaCl at 30 6C and was characterized chemotaxonomically by having C 16 : 0 as the major fatty acid followed by C 17 : 0 cyclo and C 18 : 1 v7c. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Esp-1 T formed a cluster together with Pseudomonas knackmussii DSM 6978 T (98.9 % sequence similarity), Pseudomonas delhiensis MTCC 7601 T (98.5 %), Pseudomonas nitroreducens DSM 14399 T (98.5 %), Pseudomonas citronellolis DSM 50332 T (98.7 %), Pseudomonas multiresinivorans ATCC 700690 T (98.9 %) and Pseudomonas jinjuensis DSM 16612 T (97.8 %). DNA-DNA hybridization values of strain Esp-1 T with P. knackmussii DSM 6978 T , P. delhiensis MTCC 7601 T , P. jinjuensis DSM 16612 T , P. citronellolis DSM 50332 T , P. multiresinivorans ATCC 700690 T and P. nitroreducens DSM 14399 T were 32. 9, 30.2, 20.6, 23.4, 23.4 and 20.0 %, respectively. Low levels of DNA-DNA hybridization and phenotypic and chemotaxonomic results are sufficient to delineate strain Esp-1 T from other closely related species of Pseudomonas. Phenotypic and chemotaxonomic data confirm that strain Esp-1 T represents a novel species, for which the name Pseudomonas panipatensis sp. nov. is proposed. The type strain of Pseudomonas panipatensis sp. nov. is Esp-1 T (5MTCC 8990 T 5CCM 7469 T ).The genus Pseudomonas is an extremely heterogeneous group that has been reclassified several times on the basis of phenotypic features (Sneath et al., 1981), DNA-DNA hybridization (Palleroni, 1984), 16S rRNA gene sequence similarity (Anzai et al., 2000) and chemotaxonomic data (Oyaizu & Komagata, 1983;Vancanneyt et al., 1996). Its members grouped previously into the a, b and c subclasses of the proteobacteria (Palleroni, 1984), but are now restricted to the c subclass, with Pseudomonas aeruginosa as the type species; members of the a and b subclasses have been transferred to other genera (Kersters et al., 1996;Anzai et al., 2000).Members of the genus Pseudomonas are ubiquitous in nature due to their metabolic versatility (Palleroni, 1993;Elkin & Geddes, 2003; Ló pez-Romalde et al., 2003;Levitski-Heikkila & Ullian, 2005). A number of pseudomonads are known to utilize a variety of xenobiotics as sources of carbon and energy (Kiyohara et al., 1992;Johnsen et al., 1996;Stolz et al., 2007) and are thus exploited for the bioremediation of such compounds (O'Mahony et al., 2006;Onaca et al., 2007). Owing to their success in the bioremediation of soils with oily sludge (Lal & Khanna, 1996;Mishra et al., 1999Mishra et al., , 2001Whyte et al., 2001), there have been quite a large number of attempts to isolate Pseudomonas from such sites (Bhattacharya et al., 2003;Prakash et al., 2007a).In an ongoing study, a bacterial strain (Esp-1 T ) was isolated from an oil refinery (Panipat, India) that degra...

SummaryLong-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect longchain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

A Gram negative, yellow pigmented, rod shaped bacterium designated as RL(T) was isolated from a hot water spring (90-98 °C) located at Manikaran in Northern India. The isolate grows at 60-80 °C (optimum, 70 °C) and at pH 7.0-9.0 (optimum pH 7.2). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicate that the new isolate represents a novel species of the genus Thermus with closest affinity to Thermus thermophilus HB8(T) (99.5 %) followed by Thermus arciformis (96.4 %). A comparative analysis of partial sequences of housekeeping genes (HKG) further revealed that strain RL(T) is a novel species belonging to the genus Thermus. The melting G+C content of strain RL(T) was calculated as 68.7 mol%. The DNA-DNA relatedness value of strain RL(T) with its nearest neighbours (>97 %) was found to be less than 70 % indicating that strain RL(T) represents a novel species of the genus Thermus. MK-8 was the predominant respiratory quinone. The presence of characteristic phospholipid and glycolipid further confirmed that strain RL(T) belongs to the genus Thermus. The predominant fatty acids of strain RL(T) were iso-C17:0 (23.67 %) and iso-C15:0 (24.50 %). The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished strain RL(T) from its closely related species. Thus, strain RL(T) represents a novel species of the genus Thermus for which the name Thermus parvatiensis is proposed (=DSM 21745(T)= MTCC 8932(T)).