Poonam Sharma

Polyphasic approach of bacterial classification — An overview of recent advances

Classification of microorganisms on the basis of traditional microbiological methods (morphological, physiological and biochemical) creates a blurred image about their taxonomic status and thus needs further clarification. It should be based on a more pragmatic approach of deploying a number of methods for the complete characterization of microbes. Hence, the methods now employed for bacterial systematics include, the complete 16S rRNA gene sequencing and its comparative analysis by phylogenetic trees, DNA-DNA hybridization studies with related organisms, analyses of molecular markers and signature pattern(s), biochemical assays, physiological and morphological tests. Collectively these genotypic, chemotaxonomic and phenotypic methods for determining taxonomic position of microbes constitute what is known as the ‘polyphasic approach’ for bacterial systematics. This approach is currently the most popular choice for classifying bacteria and several microbes, which were previously placed under invalid taxa have now been resolved into new genera and species. This has been possible owing to rapid development in molecular biological techniques, automation of DNA sequencing coupled with advances in bioinformatic tools and access to sequence databases. Several DNA-based typing methods are known; these provide information for delineating bacteria into different genera and species and have the potential to resolve differences among the strains of a species. Therefore, newly isolated strains must be classified on the basis of the polyphasic approach. Also previously classified organisms, as and when required, can be reclassified on this ground in order to obtain information about their accurate position in the microbial world. Thus, current techniques enable microbiologists to decipher the natural phylogenetic relationships between microbes.

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

ABSTRACT 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.

Spatial and Temporal Changes in Endogenous Polyamine Levels Associated with Somatic Embryogenesis from Different Hypocotyl Segments of Eggplant (Solanum melongena L.)

Summary In Solanum Melongena L. Cv. Pusa Purple Long, Significant Differences Were Observed For Embryogenic Potential Within A Single Explant (Hypocotyl System). Terminal Hypocotyl Segments (Apical and Basal) Yielded A Higher Number Of Somatic Embryos Than The Medial (Sub-Apical and Sub-Basal) Segments. High Levels Of Conjugated Spermidine Along With High Levels Of Total Polyamines (Primarily Free Fraction) Could Be Correlated With The Formation Of Somatic Embryos In Terminal Segments. Temporal Changes In Endogenous Levels Of Free, Conjugated and Bound Putrescine, Spermidine and Spermine Were Analyzed At Critical Stages Of Somatic Embryogenesis From Four Different Hypocotyl Segments. Within 3 Days Of Culture, There Was A Sharp Decline In Free (and Total) Putrescine and An Increase In Conjugated Spermidine Levels. All Hypocotyl Segments Attained Similar Levels Of Free Spermidine, Irrespective Of Their Subsequent Embryogenic Response. As The Tissues Become Committed For Embryogenesis, Free, Conjugated and Total Putrescine and Spermidine Reach Uniformly Minimal Levels For All Segments. Just Before The Onset Of Embryogenesis, There Was A Dramatic Increase In All Fractions Of Putrescine, and In Free and Conjugated (Along With Total) Spermidine Levels. Intermediate Levels Of Conjugated and Total Putrescine and Spermidine Were Reached In Tissues With Good Embryogenic Potential: tissues with poor embryogenic potential attained lower or higher levels. After the embryos were formed, their levels fell sharply, and continued to decline. Free putrescine, however, reached another peak when embryos had fully matured. The changes in bound putrescine and spermidine, and in spermine (all forms) could not be correlated with somatic embryogenesis.

Proposal of biostimulation for hexachlorocyclohexane (HCH)‐decontamination and characterization of culturable bacterial community from high‐dose point HCH‐contaminated soils

Aims:  To locate a high‐dose point hexachlorocyclohexane (HCH)‐contaminated site, to identify HCH‐degrading bacteria in it and assay HCH‐decontamination by biostimulation.

New Metabolites in the Degradation of α- and γ-Hexachlorocyclohexane (HCH): Pentachlorocyclohexenes Are Hydroxylated to Cyclohexenols and Cyclohexenediols by the Haloalkane Dehalogenase LinB from Sphingobium indicum B90A

Technical hexachlorocyclohexane (HCH) and lindane are obsolete pesticides whose former production and use led to widespread contaminations posing serious and lasting health and environmental risks. Out of nine possible stereoisomers, alpha-, beta-, gamma-, and delta-HCH are usually present at contaminated sites, and research for a better understanding of their biodegradation has become essential for the development of appropriate remediation technologies. Because haloalkane dehalogenase LinB was recently found responsible for the hydroxylation of beta-HCH, delta-HCH, and delta-pentachlorocyclohexene (delta-PCCH), we decided to examine whether beta- and gamma-PCCH, which can be formed by LinA from alpha- and gamma-HCH, respectively, were also converted by LinB. Incubation of such substrates with Escherichia coli BL21 expressing functional LinB originating from Sphingobium indicum B90A showed that both beta-PCCH and gamma-PCCH were direct substrates of LinB. Furthermore, we identified the main metabolites as 3,4,5,6-tetrachloro-2-cyclohexene-1-ols and 2,5,6-trichloro-2-cyclohexene-1,4-diols by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. In contrast to alpha-HCH, gamma-HCH was not a substrate for LinB. On the basis of our data, we propose a modified gamma-HCH degradation pathway in which gamma-PCCH is converted to 2,5-cyclohexadiene-1,4-diol via 3,4,5,6-tetrachloro-2-cyclohexene-1-ol and 2,5,6-trichloro-2-cyclohexene-1,4-diol.

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.

Effect of cyclic 3',5'-adenosine monophosphate and kinetin on growth and fertility in the male clones of the moss Microdus brasiliensis (Dub.) Ther.

On basic medium the male clones of Microdus brasiliensis form normal gametophores, of which 18 % bear antheridia. Kinetin enhances the number of buds but inhibits the formation of normal gametophores. With cAMP the number of buds is reduced and the gametophores are also stunted. Co-addition of kinetin and cAMP increases the number of buds, improves the morphology of gametophores, and in some combinations bring about considerable enhancement in antheridial produ ction.

Sustainable Smart Cities in India

Themodern form of urbanization began around early nineteenth century in developed countries. Early cities of world New York, Boston and Philadelphia were the largest in 1790. At this stage only 3% of global population lived in cities. By 2000 the mega cities (cities with 10 million Population) rose to sixteen and the number is expected to reach around 27 by 2025. Massive urbanization creates opportunities on one hand and poses challenges on the other hand. The cities which are magnets for small and medium town population slowly steadily face resource and infrastructure crunch. The scarcity of resources coupled with environmental pollution chokes the urban environment and makes the cities unhealthy and unsustainable. According to Word Urbanisation Prospects, United Nations by 2050, nearly 70% of the world’s population would live in cities and there would be many cities with more than 10 million population. India too is experiencing the upsurge of population especially in urban areas. To accommodate and resolve the problems associated with rapid urbanization, the Government of India has planned to build hundred new Smart cities. Building Smart cities in India is challenging yet imperative solution for managing massive urbanization and optimizing resource availability and distribution processes.

Perspectives of Smart Cities: Introduction and Overview

The modern form of urbanization began around early nineteenth century in developed countries. Early cities of world New York, Boston and Philadelphia were the largest in 1790. At this stage only 3% of global population lived in cities. By 2000 the mega cities (cities with 10 million Population) rose to sixteen and the number is expected to reach around 27 by 2025. Massive urbanization creates opportunities on one hand and poses challenges on the other hand. The cities which are magnets for small and medium town population slowly steadily face resource and infrastructure crunch. The scarcity of resources coupled with environmental pollution chokes the urban environment and makes the cities unhealthy and unsustainable. According to Word Urbanisation Prospects, United Nations by 2050, nearly 70% of the world’s population would live in cities and there would be many cities with more than 10 million population. India too is experiencing the upsurge of population especially in urban areas. To accommodate and resolve the problems associated with rapid urbanization, the Government of India has planned to build hundred new Smart cities. Building Smart cities in India is challenging yet imperative solution for managing massive urbanization and optimizing resource availability and distribution processes.

The Energy Scenario of India: An Assessment

Energy as one of the most remarkable invention has come to be known as a strategic commodity and any uncertainty about its demand and supply can threaten the smooth functioning of the entire economy that too in developing economies. In case of the Indian power sector which is characterized by large demand-supply gap, is facing challenge despite significant growth in generation over the years, it has been suffering from shortages and supply constraints. Energy consumption rose by half between 2001 and 2010, and the trajectory remains one of steep ascent. India imports a substantial portion of its energy 80% of its oil, 18% of its gas, and now even 23% of its coal. As GDP growth accelerates to an ambitious 8–10% in recent years the shortage of power will become more severe for future scenario. As India’s substantial and sustained economic growth is placing enormous demand on its energy resources there is a need for giving emphasis on alternate energy supplies. Energy requirement is increasing at a rapid rate as current urbanization pattern is transforming cities into smart cities thereby increasing their energy demand. This paper provides a comprehensive overview of India’s energy sector and identifies challenges towards achieving the country’s energy objectives for meeting future goals.