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Cell Survival Strategies : Role Of Gyrase Modulatory Proteins
No full textA steady state level of negative supercoiling is essential for chromosome condensation, initiation of replication and subsequent elongation step. DNA gyrase, found in every eubacteria, serves the essential housekeeping function of maintenance of the negative supercoiling status of the genome. The functional holoenzyme is a heterotetramer, comprising of two GyrA and two GyrB subunits. DNA gyrase is an indispensable enzyme and serves as a readily susceptible target for natural antibacterial agents. The enzymatic steps of topoisomerisation by gyrase involve transient double strand break and rejoining of the strands after intact duplex transfer. Corruption of its catalytic cycle can lead to the generation of cytotoxic double-strand DNA breaks. Most of the anti-gyrase agents achieve their objective by targeting the vulnerable step of the reaction cycle i.e. DNA cleavage step. Bacteria on their part must have evolved and adopted strategies to counter the action of external agents and prevent the generation of double strand breaks thereby safeguarding their genome.
In the present thesis, attempts have been made to understand the role of three endogenous gyrase interacting proteins in gyrase modulation and cellular defense against anti-gyrase agents. The thesis is divided into six chapters. Chapter 1 introduces the wonder enzymes “DNA topoisomerases” starting with a brief classification of these enzymes and their physiological functions. In the next section, DNA gyrase has been discussed in greater detail. The structural aspects as well as the mechanism of the topoisomerisation reaction catalyzed by gyrase have been discussed. Final section gives an overview of different gyrase modulators known till date focusing on their source, structure and mode of action. The scope and objectives of the present study is presented at the end of this chapter.
In Chapter 2 is aimed at understanding the physiological role of GyrI. GyrI, originally identified in Escherichia coli as an inhibitor of DNA gyrase, has been previously shown in the laboratory to render protection against gyrase poisons and also various other DNA damaging agents (mitomycin C, MNNG). Abolishing GyrI expression renders the cell hypersensitive to these cytotoxic agents. Interestingly, GyrI exhibits contrasting behavior towards two plasmid encoded proteinaceous poisons of DNA gyrase. It reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin. However, a positive cooperation between GyrI and F plasmid encoded toxin CcdB, results in enhanced DNA damage and cell death. These results suggest a more complex functional interplay and physiological role for GyrI.
Search for other chromosomally encoded gyrase inhibitors led to YacG, a small zinc finger protein (7.3kDa) from E. coli, shown to be a member of DNA gyrase interactome, in a protein-protein interaction network described recently. Chapter 3 deals with the detailed characterization of YacG. It is shown that YacG inhibits DNA gyrase by binding to GyrB subunit and preventing DNA binding activity of the enzyme. More importantly, it protects against the cytotoxic effects of other gyrase inhibitors like ciprofloxacin, novobiocin, microcin B17 and CcdB. Further investigations revealed that YacG and its homologues are found only in proteobacteria. Hence, it appears to be a defense strategy developed by gram-negative bacteria to fight against the gyrase targeting cytotoxic agents. Inhibition by YacG appears to be specific to E. coli gyrase as mycobacterial enzyme is refractile to YacG action. GyrB, only in gram-negative organisms, possesses extra stretch of 165 amino acids, indispensable for DNA binding. Biochemical experiments with the truncated GyrB lacking the extra stretch reveal the importance of this stretch for stable YacG-GyrB interaction. E. coli topoisomerase IV is also resistant to YacG mediated inhibition, probably due to the absence of the extra stretch in ParE subunit, which is otherwise highly similar to GyrB. Further, YacG homologues from other proteobacterial members (Sinorhizobium meliloti and Haemophilus influenzae homologues sharing 35% and 63 % identity with E. coli YacG respectively ) also inhibits E. coli DNA gyrase at comparable levels. YacG thus emerges as a proteobacteria specific inhibitor of DNA gyrase. The occurrence of both YacG and the gyrase extra stretch only in proteobacteria, suggest co-evolution of interacting partners in proteobacteria.
In Chapter 4, the study of endogenous gyrase modulators is extended to Mycobacterium sp. glutamate racemase (MurI) from E. coli has been shown earlier to be an inhibitor of DNA gyrase. However, nothing much was known about its mode of action. MurI is an important enzyme in the cell wall biosynthesis pathway, which catalyses the conversion of L-glutamate to D-glutamate, an integral component of the bacterial cell wall. In this chapter, it is demonstrated that M. tuberculosis MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species specific as E. coli gyrase is also inhibited. However, it is gyrase specific as topoisomerase I activity remains unaltered. The mechanism of inhibition by MurI has been elucidated for the first time and it is shown that MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase.
Chapter 5 is the extension of the studies on glutamate racemase into another species, i.e. Mycobacterium smegmatis. DNA gyrase inhibition seems to be an additional attribute of some of the glutamate racemases, but not all, as Glr isozyme from B. subtilis has no effect on gyrase activity in spite of sharing a high degree of similarity with the gyrase inhibitory glutamate racemases. It is shown that like the M. tuberculosis MurI, M. smegmatis enzyme is also a bifunctional enzyme. It inhibits DNA gyrase in addition to its racemization activity. Further, overexpression of the enzyme in M. smegmatis provides protection to the organism against fluoroquinolones. DNA gyrase inhibitory property thus appears to be a typical characteristic of these MurI and seems to have evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double strand breaks in the genome.
In the above chapters, it is shown that besides its crucial role in cell wall biosynthesis, mycobacterial MurI moon lights as DNA gyrase inhibitor. That the two activities exhibited by M. tuberculosis MurI are unlinked and independent of each other is demonstrated in Chapter 6. Racemization function of MurI is not essential for its gyrase inhibitory property as mutants compromised in racemization activity retain gyrase inhibition property. MurI- DNA gyrase interaction influences gyrase activity but has no effect on racemization activity of MurI. MurI expression in mycobacterial cells provides protection against the action of ciprofloxacin, thereby suggesting a role of MurI in countering external agents targeting DNA gyrase. Further M. tuberculosis MurI overexpressed in near homologous expression system of M. smegmatis yields highly soluble enzyme which can be further used for structural and functional studies.
In conclusion, the studies reveal that the endogenous inhibitors essentially influence the enzyme activity by sequestering the enzyme away from DNA. None of them cause cytotoxicity, which usually arises as a result of DNA damage caused by accumulation of gyrase-DNA covalent intermediate. On the contrary they provide protection against such gyrase poisons. Comparative analysis of these proteinaceous inhibitors, however, does not reveal a common motif or structural fold, required for their ability to inhibit DNA gyrase. Based on these studies, it can be proposed that these endogenous proteins exist to serve as cellular defense strategies against external abuse and also to modulate the intracellular activity of DNA gyrase as and when required, for accurate division, functioning and survival of the cells
Inhibition of DNA gyrase activity by Mycobacterium smegmatis MurI
Get PDFGlutamate racemase (MurI) catalyzes the interconversion of L-glutamate to D-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA gyrase activity by preventing DNA binding of gyrase. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double-strand breaks in the genome
Inhibition of DNA gyrase activity by Mycobacterium smegmatis MurI
No full textGlutamate racemase (MurI) catalyzes the interconversion of L-glutamate to D-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA gyrase activity by preventing DNA binding of gyrase. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double-strand breaks in the genome
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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