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Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well- established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX) often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non- specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting. [Copyright &y& Elsevier]

Abstract: The T4 AsiA is an anti-sigma factor encoded by one of the early genes of Bacteriophage T4. It has been shown that AsiA inhibits transcription from promoters containing −10 and −35 consensus sequence by binding to σ70 of E. coli. Binding of AsiA to σ70 in vivo, in E. coli, leads to inhibition of transcription of essential genes resulting in killing of the organism. By using various in vitro methods, the region of σ70 binding to AsiA have been mapped to domain 4.2. Additionally, mutational analysis of σ70 has also identified amino acid residues in domain 4.1 which are critical for interaction with AsiA. Based on NMR studies it has been suggested that either of these regions can bind to AsiA, a conclusion which was supported by high degree of amino acid homology between domain 4.1 and 4.2. However, it is not clear whether under in vivo conditions, AsiA exerts its transcription inhibitory effect by binding to one of these regions or both the regions together. In order to understand the mechanism of AsiA mediated inhibition of E. coli transcription in vivo, in terms of specific binding requirements to region 4.1 and/or 4.2, we have studied the interaction of these sub-domains with AsiA by Yeast two hybrid system as well as by co-expressing and affinity purification of the interacting partners in vivo in E. coli. It was observed that minimum fragment of σ70 showing observable binding to AsiA, must possess sub-domains 4.1 and 4.2 together. No binding could be detected in σ70 fragments lacking a part of either domain 4.1 or 4.2, in any of the assays. This data was also supported by in vitro binding studies wherein only σ70 fragments carrying both region 4.1 and 4.2 showed binding to AsiA. Co-expression of region 4.1 and 4.2 fragments together also did not show any interaction with AsiA. The results presented here suggest that binding of AsiA to σ70, in vivo, requires the presence of both sub-domains of region 4 of σ70. [Copyright &y& Elsevier]

Mycobacterium tuberculosis is a successful pathogen that overcomes numerous challenges presented by the immune system of the host. This bacterium usually establishes a chronic infection in the host where it may silently persist inside a granuloma until, a failure in host defenses, leads to manifestation of the disease. None of the conventional anti-tuberculosis drugs are able to target these persisting bacilli. Development of drugs against such persisting bacilli is a constant challenge since the physiology of these dormant bacteria is still not understood at the molecular level. Some evidence suggests that the in vivo environment encountered by the persisting bacteria is anoxic and nutritionally starved. Based on these assumptions, anaerobic and starved cultures are used as models to study the molecular basis of dormancy. This review outlines the problem of persistence of M. tuberculosis and the various in vitro models used to study mycobacterial latency. The basis of selecting the nutritional starvation model has been outlined here. Also, the choice of M. smegmatis as a model suitable for studying mycobacterial latency is discussed. Lastly, general issues related to oxidative stress and bacterial responses to it have been elaborated. We have also discussed general control of OxyR-mediated regulation and emphasized the processes which manifest in the absence of functional OxyR in the bacteria. Lastly, a new class of protein called Dps has been reviewed for its important role in protecting DNA under stress. IUBMB Life, 57: 149–159, 2005 [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium smegmatis Dps degrades spontaneously into a species in which 16 C-terminal residues are cleaved away. A second species, in which all 26 residues constituting the tail were deleted, was cloned, expressed and purified. The first did not bind DNA but formed dodecamers like the native protein, while the second did not bind to DNA and failed to assemble into dodecamers, indicating a role in assembly also for the tail. In the crystal structure of the species without the entire C-terminal tail the molecule has an unusual open decameric structure resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. A Dps dodecamer could assemble with a dimer or one of two trimers (trimer-A and trimer-B) as intermediate. Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of the surface area buried on trimerization indicates that association within trimer-B is weak. It weakens further when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail. A species with nine N-terminal residues deleted formed trimers but not dodecamers in solution, unlike wild-type M. smegmatis Dps, under the same conditions. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighboring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants. [Copyright &y& Elsevier]

Abstract: The rel gene is responsible for the maintenance of the level of (p)ppGpp in bacteria under nutrient starvation. This phenomenon known as stringent response plays an important role during survival of the microorganisms in stationary phase. We have cloned 1.6 kb upstream sequence of rel gene of Mycobacterium tuberculosis in a shuttle vector pSD5B containing promoterless lacZ gene and promoter activity was observed in Mycobacterium smegmatis cells by blue/white selection and was measured by β-galactosidase assay. In order to delineate the minimal promoter element of rel gene, a 200 bp fragment from this 1.6 kb upstream sequence was further cloned in promoterless lacZ shuttle vector pSD5B and promoter activity was observed in M. smegmatis cells in similar way. The 200 bp promoter fragment was found to be mycobacterium specific and did not respond when transformed in Escherichia coli. The +1 transcription start site was determined by primer extension method. The −10 promoter region was identified to be TATCCT. The three T bases when mutated, showed a remarkable decrease in the lacZ expression thus confirming the −10 region. The translation start site has also been identified by site directed frame shift mutagenesis. It appears that this rel promoter can be used for expression of proteins in mycobacteria. [Copyright &y& Elsevier]

Abstract: The recognition of DNA is the first and most important condition for biological applications, including transcription and translation regulators and DNA sensors. For this purpose, we have developed few systems where we were able to immobilize long double-stranded DNA (dsDNA) successfully to the surfaces of different solid substrates. To achieve this, we have chosen polystyrene beads and standard Langmuir–Blodgett monolayer of Zn-arachidate. In the first attempt, variant of T7 DNA containing one strong promoter A1 for Escherichia coli RNA polymerase was immobilised on uniform polystyrene microspheres (0.31 μm diameter) by covalent grafting. In the latter case, Zn(II) is bound to arachidic acid through charge neutralization. Since tetrahedral Zn(II) participates in DNA recognition through coordination, we have been able to layer DNA over the Zn-arachidate monolayer. The successful immobilization of DNAs on these different substrates was visualized under fluorescence microscope. These immobilized DNAs were used as a template to study in vitro transcription reaction and thus we introduce a new strategy for the study of transcription in heterogeneous phase. [Copyright &y& Elsevier]