Unfolding of in planta activity of anti-rep ribozyme in presence of a RNA silencing suppressor.

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double-stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep-mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA-silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA-silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down-regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.

Physiological and biochemical consequences of host-plasmid interaction--a case study with Corynebacterium renale, a multiple cryptic plasmid containing strain.

Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, µ, and respiratory rate, r. Both µ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in µ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.

Purification of dibenzothiophene monooxygenase from a recombinant Escherichia coli.

Dibenzothiophene monooxygenase is the first enzyme involved in the degradation of dibenzothiophene. This gene was expressed via the pET28a vector in E. coli and was purified in a single step using affinity chromatography. The protein was purified 39-fold with a specific activity of 38 U/mg.

Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein.

Geminiviruses pose serious threat to many economically important crops such as mungbean, tomato, cotton, etc. To devise a specific antiviral strategy at the viral DNA replication level, a hammerhead ribozyme was directed against the mRNA of the replication initiator protein (Rep). Rep is the most important viral protein for the DNA replication of the Mungbean yellow mosaic India virus (MYMIV), a member of the Geminiviridae family. The ribozyme showed approximately 33% cleavage activity on synthetic rep transcript within 1h under in vitro conditions, whereas the mutant ribozyme, designed to lack the catalytic activity but target the same site, showed no cleavage. The in vivo efficiency of ribozyme was evaluated in Saccharomyces cerevisiae as it can act as a surrogate host for replication of the MYMIV-DNA and lacks RNAi machinery. In the presence of the ribozyme, growth of the yeast cells that are dependent on geminiviral replication was inhibited by 30% and cellular generation time was increased by 2h. The RT-PCR analysis showed a maximum of about 50% reduction in the rep mRNA level in presence of the ribozyme compared to its noncatalytic mutant control. About 65% decrease in geminiviral DNA replication was observed due to the downregulation of replication initiator protein by the ribozyme. These results raise the possibility of engineering resistance to geminiviruses employing the ribozyme approach.

Development of expression vectors for Escherichia coli based on the pCR2 replicon.

BACKGROUND: Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. RESULTS: We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E. coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNgamma was obtained by cloning the h-IFNgamma under a T7 promoter in the expression plasmid pCR2-IFNgamma and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNgamma, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. CONCLUSION: We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy.

Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of lambda(max) point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (approximately 5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment.

Characterization of broad host range cryptic plasmid pCR1 from Corynebacterium renale.

Plasmid pCR1 is a cryptic plasmid harboured by Corynebacterium renale. It is the smallest corynebacterial plasmid known to date. Although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. It can also replicate in Escherichia coli, in which it is stably maintained. The copy number of pCR1 in this host is higher than that of pUC19, with which it shows unidirectional incompatibility. It is also incompatible with pBK2, a plasmid bearing the common corynebacterial replicon pBL1. Its size is 1488bp, as revealed by DNA sequencing. A total of eight open reading frames (ORF) were detected in this plasmid, the largest of which codes for a putative Rep protein of predicted molecular mass of 21kDa. The plasmid pCR1 can be mobilized by the plasmid R6K from E. coli to other corynebacteria. Sequence analysis revealed the presence of an oriT homologous to that of R64. An E. coli plasmid pKL1 shows more than 90% identity with pCR1. Like many coryenbacterial plasmids, pCR1 also replicates by rolling circle mode.

Molecular understanding of aminoglycoside action and resistance.

Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. They are particularly active against aerobic, Gram-negative bacteria and act synergistically against certain Gram-positive organisms. Aminoglycosides are used in the treatment of severe infections of the abdomen and urinary tract, bacteremia, and endocarditis. They are also used for prophylaxis, especially against endocarditis. Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat. This might be the reason for the interest in understanding the mechanisms of resistance. It is now clear that the resistance occurs by different mechanisms such as prevention of drug entry, active extrusion of drugs, alteration of the drug target (mutational modification of 16S rRNA and mutational modification of ribosomal proteins), and enzymatic inactivation through the expression of enzymes, which covalently modify these antibiotics. Enzymatic inactivation is normally due to acetyltransferases, nucleotidyltransferases, and phosphotransferases. In this review, we focus on the recent concept of molecular understanding of aminoglycoside action and resistance.

Kinetic mechanism of streptomycin adenylyltransferase from a recombinant Escherichia coli.

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily by enzymic modification of these drugs. One important mechanism of streptomycin modification is through ATP-dependent O-adenylation, catalyzed by streptomycin adenylyltransferase. Initial velocity patterns deduced from steady state kinetics indicate a sequential mechanism. Dead-end inhibition by tobramycin and neomycin is non-competitive versus streptomycin and uncompetitive versus ATP, indicative of ordered substrate binding where ATP binds first and then streptomycin. These results surmise that streptomycin adenylyltransferase follows an ordered, sequential kinetic mechanism in which one substrate (ATP) binds prior to the antibiotic and pyrophosphate is released prior to formation of AMP-streptomycin.

Molecular targets for design of novel inhibitors to circumvent aminoglycoside resistance.

Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and Gram-negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Antibiotic resistance is a growing problem for all classes of anti-infective agents. One of the first groups of antibiotics to encounter the challenge of resistance was the aminoglycoside -aminocyclitol family. Initially, the resistance that emerged in organisms such as Mycobacterium tuberculosis was restricted to modification of the antibiotic targets, which we now know to be the bacterial ribosomal rRNA and proteins. As new aminoglycosides came to the clinic, however, the prevalence of chemical modification mechanisms of resistance became dominant. Enzymatic modification of aminoglycosides through kinases (O-phosphotransferases, APHs), O-adenyltransferases (ANTs) and N-acetyltransferases (AACs) has emerged in virtually all clinically relevant bacteria of both Gram-positive and Gram-negative origin. Although their clinical use has been extensive, their toxicity and the prevalence of resistance in clinical strains have prompted the pharmaceutical industry to look for alternatives. Whereas the search for novel targets for antibiotics from the genomic information is ongoing, no antibacterial agent based on these efforts has so far entered clinical trials. Meanwhile, structural knowledge of the ribosome, the target for aminoglycosides, has invigorated the field of antibiotic development. It is expected that knowledge of the binding interactions of aminoglycosides and the ribosome would lead to concepts in drug design that would take us away from the parental structures of aminoglycosides in the direction of different structural classes that bind to the same ribosomal target sites as aminoglycosides. The challenge to ensure the continued use of these highly potent antibacterial agents will require the effective management of resistance at several levels. One potential mechanism of circumventing resistance is the development of inhibitors of modification enzymes, a methodology that is now well established in the beta-lactam field. This approach requires knowledge of resistance at the molecular and atomic levels for the rational design of inhibitory molecules. The understanding of the molecular basis for aminoglycoside resistance modification has been greatly enhanced by the recent availability of representative 3D-structures from the three classes of modifying enzymes: kinases, acetyltransferases and adenyltransferases. The challenge is now to firmly establish the mechanisms of enzyme action and to use this information to prepare effective and potent inhibitors that will reverse antibiotic resistance. In this review, we discuss the molecular mechanisms of resistance of aminoglycosides specifically on aminoglycoside-modifying enzymes and newly developed strategies to circumvent resistance including antisense technology, which is an example of new strategy to deal with antibiotic resistance.

Gene expression systems in corynebacteria.

Corynebacterium belongs to a group of gram-positive bacteria having moderate to high G+C content, the other members being Mycobacterium, Nocardia, and Rhodococcus. Considerable information is now available on the plasmids, gene regulatory elements, and gene expression in corynebacteria, especially in soil corynebacteria such as Corynebacterium glutamicum. These bacteria are non-pathogenic and, unlike Bacillus and Streptomyces, are low in proteolytic activity and thus have the potential of becoming attractive systems for expression of heterologous proteins. This review discusses recent advances in our understanding of the organization of various regulatory elements, such as promoters, transcription terminators, and development of vectors for cloning and gene expression.

Purification of streptomycin adenylyltransferase from a recombinant Escherichia coli.

Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modification is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni(2+)-IDA-His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The purified fusion protein showed a single band on SDS-PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold purification. The fusion system provided a single step, easy and very rapid purification of SMATase and is suitable for obtaining a highly purified functional protein of interest. The fusion does not affect the functionality of the protein.

Strategies for efficient production of heterologous proteins in Escherichia coli.

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost.

Biotechnology of desulfurization of diesel: prospects and challenges.

To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to make a huge investment to bring down the sulfur content in diesel to the desired level, using conventional hydrodesulfurization (HDS) technology, by which sulfur is catalytically converted to hydrogen sulfide in the presence of hydrogen. These reactions proceed rapidly only at high temperature and pressure and therefore the capital cost as well as the operating cost associated with HDS very high. Biological desulfurization has the potential of being developed as a viable technology downstream of classical HDS. Various attempts have been made to develop biotechnological processes based on microbiological desulfurization employing aerobic and anaerobic bacteria. However, there are several bottlenecks limiting commercialization of the process. This review discusses various aspects of microbial desulfurization and the progress made towards its commercialization.

RNA interference: potential therapeutic targets.

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi methodologies hold the promise to selectively inhibit gene expression in mammals. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cell, causing the degradation of not only the invading dsRNA molecule, but also single-stranded (ssRNAs) RNAs of identical sequences, including endogenous mRNAs. The use of RNAi for genetic-based therapies has been widely studied, especially in viral infections, cancers, and inherited genetic disorders. As such, RNAi technology is a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics.

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Lithium toxicity - a descriptive study.

Lithium is the treatment for acute mania and bipolar disorders. Ever since its introduction in the psychiatric arsenal, case reports of toxicity have been appearing in the literature at regular intervals. This study was thus carried out to study the presentation and associated features of lithium toxicity. In this retrospective study, case record files of all patients suspected to have developed lithium toxicity during a five year period were retrieved. It was found that toxicity presented most commonly with cerebellar symptoms and appeared at lower serum levels. Lithium could be restarted albeit at a lower dose and with a gradual titration in a number of cases.

Plasmids of corynebacteria.

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.

Lithium neurotoxicity at therapeutic level--a case report.

A 30 years old Hindu male presenting with symptoms of lithium toxicity. On investigation, serum lithium level was found to be 0.5 meq/l. Though toxicity at this level of lithium is unusual, still neurotoxicity happened to be the cause of his hospital admission. He was debarred from taking lithium further and carbamazepine was started as mood elevator. He responded favourably.

Partial characterization of small plasmids from Corynebacterium renale.

A naphthalene-degrading strain of corynebacteria, Corynebacterium renale, harbors multiple small plasmids designated pCR1, pCR2, pCR3, and pCR4 with sizes of 1.4, 3.2, 4.4, and 5.7 kb, respectively. Plasmid pCR1 of 1.4 kb is the smallest plasmid reported in this group of bacteria and is present in high copy number. Attempts to clone whole pCR1 in Escherichia coli were unsuccessful but two of its fragments (750 and 650 bp) could be separately cloned in it. The 4.4-kb plasmid, pCR3, bears considerable restriction pattern similarity to a 4.4-kb plasmid belonging to the pBL1 group of cryptic plasmid of corynebacteria but has no sequence homology, suggesting that pCR3 represents a new member of the 4.4-kb group of corynebacterial plasmids.