Asis Das
University of Connecticut Health Center
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Featured researches published by Asis Das.
Cell | 1989
David Lazinski; Elizabeth Grzadzielska; Asis Das
We have dissected the protein and nucleic acid determinants that direct a group of transcriptional antiterminators to their specific target operons. These antiterminators, the N gene products of phages lambda, 21, and P22, function solely with their respective recognition sites, nut, to modify RNA polymerase to a termination-resistant form. We demonstrate that a unique hairpin sequence within each nut site, called boxB, confers genome specificity by interacting with a small amino-terminal domain of the cognate N protein. This interaction is dependent upon an arginine-rich subdomain, which is conserved not only among the N proteins but also in many RNA binding proteins from ribosomes and RNA virus capsids. Notably, this motif constitutes an essential domain of the HIV protein Tat whose function as a trans-activator requires a specific hairpin sequence.
Journal of Molecular Biology | 1980
Max Gottesman; Sankar Adhya; Asis Das
Abstract In the presence of the lambda N gene product, transcription originating at the prophage p L promoter can extend beyond termination signals and into the neighboring bacterial gal operon. In Escherichia coli rho mutants, N function is still required for p L transcription to enter gal . The requirement for N function in rho mutants is abolished by deletion of the prophage genome between 57.4 and 70.9 lambda fractional lengths. These results suggest: 1. (1) Rho-independent termination sites lie in the p L operon of lambda. 2. (2) Both Rho-independent and Rho-dependent terminators are suppressed by N function. N product must, therefore, be considered as a transcription antitermination factor, rather than as an antagonist of Rho, as previously suggested. Various DNA segments have been interposed between p L and gal and their effect on gal escape synthesis studied. Lambda DNA encoding the 6 S transcript, which ends at a Rho-independent terminator, does not reduce gal escape synthesis. The J-b2 region of the phage chromosome, however, completely blocks p L -promoted gal enzyme synthesis; the block is partially relieved by the rho15 mutation. The introduction of large DNA segments lowers gal escape in both rho + and rho15 mutants. The extent of gal escape is directly proportional to the efficiency of suppression of a prophage Nam mutation, suggesting that N product is not made in excess.
Cell | 1982
M.Andrew Hoyt; David M. Knight; Asis Das; Harvey I. Miller; Harrison Echols
The cII protein of bacteriophage lambda has a decisive role in the regulatory switch between the lysogenic and lytic pathways of viral development. Recent work has indicated that cII may be the primary control function providing for the initial partition between the two pathways, with other host and viral regulatory genes acting to determine the levels of cII in an infected cell. We have studied the synthesis and stability of cII protein with two experimental systems, phage infection and a cII-producing plasmid. We have found that the stability of cII is controlled by the host hflA and viral cIII genes; hflA protein facilitates degradation of cII, whereas cIII protects cII. The synthesis of cII appears to be under the positive control of the host himA and himD genes. We conclude that posttranscriptional regulation of cII by host and viral genes is critical for the choice of a developmental pathway.
Molecular Microbiology | 2007
Anjali Mandlik; Arlene Swierczynski; Asis Das; Hung Ton-That
Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA‐type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.
Molecular Microbiology | 2007
Anu Swaminathan; Anjali Mandlik; Arlene Swierczynski; Andrew H. Gaspar; Asis Das; Hung Ton-That
Many surface proteins in Gram‐positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres – SrtA for the SpaA‐type pilus, SrtB/SrtC for the SpaD‐type pilus, and SrtD/SrtE for the SpaH‐type pilus. We demonstrate here the function of SrtF, the so‐called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall‐linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface‐linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild‐type levels of the SpaA‐type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two‐stage model of pilus assembly: pilins are first polymerized by a pilus‐specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.
Proceedings of the National Academy of Sciences of the United States of America | 2008
Anjali Mandlik; Asis Das; Hung Ton-That
Cell surface pili in Gram-positive bacteria orchestrate the colonization of host tissues, evasion of immunity, and the development of biofilms. Recent work revealed that pilus assembly is a biphasic process wherein pilus polymerization is catalyzed by a pilus-specific sortase followed by cell wall anchoring of the pilus that is promoted by the housekeeping sortase. Here, we present molecular genetic and biochemical studies of a heterotrimeric pilus in Corynebacterium diphtheriae, uncovering the molecular switch that terminates pilus polymerization in favor of cell wall anchoring. The prototype pilus contains a major pilin (SpaA) forming the shaft, a tip pilin (SpaC), and another minor pilin (SpaB). Cells lacking SpaB form pilus fibers, but they are largely secreted in the medium, a phenotype also observed when cells lack the housekeeping sortase. Furthermore, the average pilus length is greatly increased in the absence of SpaB. Remarkably, a SpaB mutant that lacks the cell wall sorting signal but contains a critical lysine residue is incorporated in the pilus. However, the resulting pili fail to anchor to the cell wall. We propose that a specific minor pilin acts as the terminal subunit in pilus assembly. Cell wall anchoring ensues when the pilus polymer assembled on the pilus-specific sortase is transferred to the minor pilin presented by the housekeeping sortase via lysine-mediated transpeptidation.
The EMBO Journal | 2000
Francine Toulme; Christine Mosrin-Huaman; Jason Sparkowski; Asis Das; Marc Leng; A. Rachid Rahmouni
The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)n sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3′ terminus and its re‐synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB‐induced ‘cleavage‐and‐restart’ process to become catalytically competent. Clearly, by promoting transcript shortening and re‐alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.
Cell | 1987
Sailen Barik; Balaram Ghosh; William Whalen; David Lazinski; Asis Das
As a transcriptional activator, the N protein of phage lambda acts to suppress transcription termination by recognizing a promoter-proximal site, nut, which is separated from the terminators by thousands of base pairs. We demonstrate here that N interacts with the elongating RNA polymerase in transit through the boxB domain of nut. This interaction leads to the stable association of N as an integral component of the transcription apparatus. During subsequent elongation, N translocates along with polymerase through several defined terminators positioned beyond nut. Therefore, by being an operon-specific subunit of the transcription apparatus, N presumably prevents the interaction of polymerase with termination signals.
Journal of Bacteriology | 2007
Arunima Mishra; Asis Das; John O. Cisar; Hung Ton-That
Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.
Cell | 1984
Asis Das; Krystyna Wolska
Employing specifically engineered plasmids in which the expression of E. coli galK cistron is regulated by transcription termination, we have analyzed the antitermination function of phage lambda N gene product in S30 extracts. Antitermination by N, dependent on its site of action, nutL, is defective in the extracts prepared from nusA, nusB, and nusE mutants. By complementation analysis, we demonstrate that none of the these nus mutations affects the synthesis of N or the other nus gene products to cause a defect in antitermination. Rather, these mutations have inactivated a set of specific host components, the Nus factors, which are essential for N activity. Curiously, an appreciable portion of N and Nus complementation activities of an S30 extract is ribosome-associated. The significance of this finding remains to be uncovered.