Browsing by Subject "Plasmids"
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Item Functional interactions of chromosome segregation factors with the 2 micron plasmid : possible evolutionary link between the plasmid portioning locus and the budding yeast centromere(2011-05) Huang, Chu-Chun; Jayaram, Makkuni; Dean, Appling R.; Arlen, Johnson W.; Paull, Tanya T.; Stevens, Scott W.The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence.Item Genetic characterization of an Escherichia coli plasmid associated with hydrogen sulfide production and drug resistance(Texas Tech University, 1977-05) Thai, Le PhamNot availableItem Recognition of oriT at the termination of conjugal transfer by MobA, the R1162 DNA strand transferase(2002-05) Becker, Eric Christian; Meyer, Richard John, Ph. D.R1162 is efficiently mobilized during conjugation by IncP-1 plasmids such as RK2 and R751. Transfer is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized by the plasmid encoded protein MobA. For strand rejoining, MobA covalently linked to the 5' end of the strand rejoins the ends by a reversible transesterification reaction. The minimal oriT fragment of R1162 contains a highly conserved 12 base region (core) including the cleavage site and a ten base imperfect inverted repeat (IR) that is not highly conserved. From those oligonucleotides with a partially degenerate oriT core base sequence, the subpopulations that are bound by MobA, cleaved and rejoined by this protein and support termination of transfer were identified. Both the IR and the adjacent core bases TAA, are needed for tight binding to MobA, whereas the location of the dinucleotide YG determines the site of strand cleavage. At the IR MobA stabilizes duplex DNA during gel electrophoresis and binds weakly to oligonucleotides lacking the outer arm of the inverted repeat, supporting a model where secondary structure at IR provides a duplex region needed for binding during termination (Zhang and Meyer 1995). Significantly altered IR sequences did allow strong binding to MobA yet completely different IR sequences did not, indicating the IR serves a structural role for binding with low base specificity. A 184 residue aminoterminal MobA fragment capable of binding and cleaving oriT was identified by using phage display and partial enzymatic digestion of the protein. No smaller fragments that could bind or cleave oriT were identified. An active nucleoprotein intermediate consisting of MobA covalently linked to the 5' end of the cleaved oriT was used to show that a single molecule of MobA is sufficient to carry out all the DNA processing steps during transfer.Item Relevance of tolC and expression of acrAB and emrAB in Erwinia chrysanthemi(Texas Tech University, 2003-12) Barabote, Ravi DamodarPlants produce a repertoire of antimicrobial chemicals, some of which are produced in response to an infection. Although much is understood about the virulence capabilities of many bacterial plant pathogens, little is known about the mechanisms employed by phytopathogens to survive die onslaught of the plant chemical environment. Here, die first report of the role of TolC in phytopathogenesis is presented. TolC is the outer membrane component of several multi-drug resistance (MDR) efflux pumps, such as AcrAB and EmrAB in E. coli, and plays an important role in the survival and virulence of many bacterial animal pathogens. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective m the efflux of berberine, a model antimicrobial plant chemical. These results suggest that the E. chrysanthemi to/C plays an important role in the survival and colonization of the pathogen in plant tissue by conferring resistance to the antimicrobial compounds produced by plants. Therefore, in order to assess the plausible implication of MDR during plant disease, the expression of the E. chrysanthemi acrAB and emrAB homologs was investigated using their respective promoter-fusions to reporter genes. Both pumps appear to be expressed in planta and in vitro in the presence of several plant-derived molecules. Plant-derived molecules, such as salicylic acid, hydrogen peroxide, paraquat, and genistein have previously been shown to stimulate antibiotic resistance in other bacteria. Interestingly, jasmonate, which is produced in plants as part of the plant defense response to pathogen invasion, was found to stimulate expression of both the pumps in E. chrysanthemi. This is the first report of jasmonate-dependent expression of bacterial efflux pump genes. Avirulence of the E. chrysanthemi tolC mutant and expression of acrAB and emrAB pumps in planta as well as in vitro in the presence of plant-defense related molecules suggest that MDR efflux pumps may play an important role in pathogenesis of this bacterium.Item Selfishness in moderation for self-propagation : the yeast plasmid purloins the host mitotic apparatus for its segregation(2003-12) Mehta, Shwetal Vatsal, 1973-; Jayaram, MakkuniThe 2 micron circle of Saccharomyces cerevisiae is a high-copy, ‘selfish’ extrachromosomal DNA element that resides in the nucleus and propagates itself stably in the cell population. The plasmid segregates as a single cluster with the assistance of an active partitioning machinery, consisting of just two plasmid encoded proteins Rep1p and Rep2p and a cis-acting element STB. The work presented in this thesis reveals the molecular strategies by which the plasmid channels the chromosome segregation machinery towards its own partitioning. The yeast cohesin complex, a multiprotein molecular glue that keeps duplicated sister chromatids together until they are ready to be separated, plays an important role in plasmid segregation. During the cell cycle, the partitioning proteins mediate the recruitment of cohesin to STB. The timely association of cohesin with the plasmid during S phase as well as the timely dissociation of cohesin from it during anaphase are essential for equal partitioning of the plasmid to daughter cells. The plasmid exploits the host mitotic machinery in a second and quite unexpected manner. The mitotic spindle specifies the precise nuclear localization of the plasmid cluster, facilitates its compact organization, and is essential for the enlistment of the cohesin complex. When the spindle is restored from an initially depolymerized state of the microtubules, the cohesin complex can be assembled at STB, but is unable to support equal segregation of the plasmid. This finding underscores the importance of cell cycle timing in the functional association between cohesin and the plasmid. We propose that the cohesin complex plays analogous roles in chromosome and plasmid segregation: to pair and unpair sister chromatids in the former case, and sister clusters in the latter. In one plausible model, cohesin mediated pairing occurs between two clusters containing roughly equal numbers of replicated plasmid molecules. In the second, cohesin pairs each molecule in one cluster with its sister molecule in the second cluster, concomitant with DNA replication. One might look upon the segregation entity consisting of the sixty or so plasmid copies as “the plasmosome”, a non-essential yeast chromosome, in the manner in which binary partitioning units are segregated one to one.Item The stability system of the yeast 2 micron plasmid: analysis of plasmid and host encoded components(2002) Yang, Xianmei; Jayaram, MakkuniThe work presented in this thesis aims to understand the molecular strategies used by an extrachromosomal selfish DNA element for its stable, high copy persistence. The model system studied here is the 2 micron plasmid found nearly ubiquitously in Saccharomyces yeast. By a combination of mutational and functional analysis of a plasmid coded protein Rep1p, an essential component of the stability system, we have provided support for the DNA-protein and proteinprotein interactions predicted to be important in plasmid maintenance. Using cell biological and molecular genetic methods, we have unveiled an apparent coupling of the pathways for plasmid and chromosome segregation. Mutations that affect equal partitioning of the chromosomes also affect the plasmid, and the two tend to missegregate in tandem. We have identified host factors that interact with components of the plasmid stability system, and may thus play a potential role in plasmid partitioning. In particular, we have found that the yeast cohesin complex, that bridges sister chromatids until they are ready to be unpaired and distributed to the daughter cells arising from a division event, may serve an analogous function in plasmid segregation. Our preliminary results suggest that the plasmid stability system follows the ‘recruitment model’, in which a functional complex is assembled by the sum of different sets of DNA-protein and protein-protein interactions. It is possible to reconstitute an active partitioning complex through an alternative set of interactions.Item Strand replacement of plasmid R1162 and transport of MobA during conjugative transfer(2007-05) Parker, Christopher Todd, 1972-; Meyer, RichardR1162 is a broad-host range, mobilizable plasmid conferring resistance to streptomycin and sulfonamides. Efficient conjugative mobilization of R1162 requires three plasmid-encoded proteins: MobA, MobB and MobC. MobA binds plasmid DNA at the origin of transfer (oriT), nicks the subsequently transferred strand and ligates the ends of the strand after transfer into the recipient. The N-terminal region of this protein carries out this DNA processing. The C-terminal half is a primase required to initiate DNA synthesis at two single-stranded priming sites sites, oriL and oriR, during vegetative plasmid replication. The primase region of MobA is not necessary for DNA processing by the N-terminal part of the protein, however its role in strand replacement during conjugation is not clearly defined. This study demonstrates that R1162 can undergo multiple rounds of transfer from a single plasmid molecule. The presence of oriL increases the frequency of second-round transfer, presumably due to initiation of replacement strand synthesis at this site by R1162 primase in the donor. Priming at oriR by the primase region of MobA is required for efficient replacement strand synthesis in the recipient when the plasmid is transferred to Salmonella. When the plasmid is transferred into E. coli, the plasmid-encoded priming system is not required for strand replacement in the recipient, presumably due to a host-encoded mechanism capable of priming the transferred strand. Transport of MobA through the R751 conjugative pore was also investigated. The two domains of MobA can be transported to recipient cells independently of each other. However, MobB is required for the transport of either fragment. Two sites, named the R-site and the P-site, are located in the relaxase and primase domains of MobA, respectively, and make up part of the signals required for MobA transport. Unlike previously described type IV transport signals, domain structure is required for the MobA transport signals to be active.