Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Optimum DNA Curvature Using a Hybrid Approach Involving an Artificial Neural Network and Genetic Algorithm

Abstract

In the present paper, a hybrid technique involving artificial neural network (ANN) and genetic algorithm (GA) has been proposed for performing modeling and optimization of complex biological systems. In this approach, first an ANN approximates (models) the nonlinear relationship(s) existing between its input and output example data sets. Next, the GA, which is a stochastic optimization technique, searches the input space of the ANN with a view to optimize the ANN output. The efficacy of this formalism has been tested by conducting a case study involving optimization of DNA curvature characterized in terms of the R_L value. Using the ANN-GA methodology, a number of sequences possessing high R_L values have been obtained and analyzed to verify the existence of features known to be responsible for the occurrence of curvature. A couple of sequences have also been tested experimentally. The experimental results validate qualitatively and also near-quantitatively, the solutions obtained using the hybrid formalism. The ANN-GA technique is a useful tool to obtain, ahead of experimentation, sequences that yield high R_L values. The methodology is a general one and can be suitably employed for optimizing any other biological feature.     

Woody plant diversity in relation to environmental factors in a seasonally dry tropical forest landscape

Questions

Water availability is known to be a first‐order driver of plant diversity; yet water also affects fire regimes and soil fertility, which, in turn, affect plant diversity. We examined how precipitation, fire and soil properties jointly determine woody plant diversity. Specifically, we asked how woody plant diversity varies along a sharp precipitation gradient (about 600–1,800 mm mean annual precipitation [MAP ]within a ~45‐km distance) exhibiting considerable variation in long‐term fire burn frequency and soil fertility, in a southern Indian seasonally dry tropical forest (SDTF ) landscape.

Location

Mudumalai, Western Ghats, India.

Methods

Woody plants ≥1‐cm DBH were enumerated in 19 1‐ha permanent plots spanning a range of tropical vegetation types from dry thorn forest, through dry and moist deciduous forest to semi‐evergreen forest. Burn frequencies were derived from annual fire maps. Six measures of surface soil properties – total exchangeable bases (Ca + Mg + K), organic carbon (OC ), total N, pH , plant available P and micronutrients (Fe + Cu + Zn + Mn) were used in the analyses. Five measures of diversity – species richness, Shannon diversity, the rarefied/extrapolated versions of these two measures, and Fisher's α – were modelled as functions of MAP , annual fire burn frequency and the principal components of soil properties.

Results

Most soil nutrients and OC increased with MAP , except in the wettest sites. Woody productivity increased with MAP , while fire frequency was highest at intermediate values of MAP . Woody plant diversity increased with MAP but decreased with increasing fire frequency, resulting in two local diversity maxima along the MAP gradient – in the semi‐evergreen and dry thorn forest – separated by a low‐diversity central region in dry deciduous forest where fire frequency was highest. Soil variables were, on the whole, less strongly correlated with diversity than MAP .

Conclusions

Although woody plant diversity in this landscape, representative of regional SDTF s, is primarily limited by water availability, our study emphasizes the role of fire as a potentially important second‐order driver that acts to reduce diversity in this landscape.

     

Structure of a Transducing Mycobacteriophage

Electron micrographs of the transducing phage I3 for Mycobacterium smegmatis strain SN2 revealed a phage with a contractile tail and a head with isometric symmetry and visible capsomeres.     

Salmonella typhimurium mutants affecting establishment of lysogeny

Summary Salmonella typhimurium mutants have been isolated in which phage P22 fails to establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c ₁ and gene 12.     

Prevalence of Escherichia coli O157:H7 in Gallbladders of Beef Cattle

Gallbladders and rectal contents were collected from cattle (n = 933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.     

Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.