Enterococcus faecalis is a Gram-positive bacterial species that is typically a member of the human gastrointestinal tract microbiota. However, E. faecalis is also a nosocomial pathogen, which is involved in urinary tract infections, soft tissue infections and endocarditis. In recent times, the occurrence of antibiotic resistance has complicated the treatment of these infections. One of the major differences between commensal and pathogenic strains of E. faecalis is that pathogens contain multiple mobile elements such as plasmids, transposons and integrative conjugative elements (ICE). These elements allow for the acquisition and transfer of virulence factors and resistance genes. Conjugative plasmids are a class of plasmids present in E. faecalis whose transfer to host cells is induced by a small pheromone peptide, cCF10 (LVTLVFV). This peptide is initially encoded as a 22-amino acid precursor (pre-cCF10) from the signal sequence of the chromosomal ccfA gene and is then proteolytically cleaved by signal peptidase II and Eep. Once pCF10 has been transferred a host E. faecalis cell, it is exceptionally stable. A replicon clone is maintained in greater than 85% of host cells over 100 generations in the absence of selection, suggesting the stability of pCF10 is intrinsic to the replicon. Three unique features of the replication initiation protein PrgW may contribute to this stability: (a) the interaction of PrgW with pre-cCF10, (b) disulfide bond formation at three conserved cysteines (C78, C275, and C307) in PrgW, and (c) processing of the nascent PrgW protein. Replication initiation proteins associated with theta replicons, such as pCF10, are often self-contained units. To initiate plasmid replication, the replication initiation protein (PrgW) binds to direct repeats (oriV) in its own coding sequence (prgW). In silico analysis of PrgW suggests the existence of three distinct domains within the protein. The first 122 amino acids are homologous to a conserved domain present in related replication initiation proteins, which includes a Helix-Turn-Helix (HTH) DNA binding domain. This suggests that this domain of PrgW has a DNA-binding function and binds to the oriV site in prgW. The following 61 amino acids are not similar to any known sequence, and are encoded by the DNA sequence containing the direct repeats in the oriV site. This domain may or may not have a distinct function. The remaining sequence forms a domain that contains cysteines C275 and C307, and is also similar to no known structure. It is hypothesized that this domain is related to the stability of pCF10. C307 appears to be critical, as previous experiments indicate that its mutation alone affects plasmid stability. Secondary structure analysis of this domain revealed the presence of multiple alpha-helices that contain distinct hydrophobic domains that possibly contribute to pre-cCF10 binding and PrgW tertiary structure. The positions of the conserved cysteines within these alpha-helices may stabilize a hydrophobic binding pocket that could potentially facilitate interaction with pre-cCF10. PrgW has a predicted molecular weight of 38.6 KDa and can be detected in Western blots as a band with an apparent approximate molecular weight (mw) of 36,000. Previous data from our lab indicates that, when overexpressed in E. faecalis, four bands of PrgW are present with observed molecular weights of 40,000, 36,000, 24,000 and 18,000. Time course experiments revealed that the 40,000 mw form is converted to a 36,000 mw form independent. The 40,000 mw form is unstable (with a complete turnover in 30 minutes) while the 36,000 mw form has a half-life of greater than 4 hours. The 24,000 mw band does not have a DNA binding motif and is likely a turnover product. When the three conserved cysteines (and only cysteines) in PrgW are replaced with alanine, the 40,000 mw form is still processed to the 36,000 mw form. However, the cysteine to alanine mutants accumulate the 36,000 mw form. / Microbiology and Immunology

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

No description available.

The retinoblastoma tumor suppressor protein (RB) is an important regulator of the cell cycle and development. Significantly, RB is inactivated in a majority of human cancers. Thus, elucidating the function of RB will give us a better understanding of how it prevents cancer. Many decades of research have yielded a detailed understanding of the role of RB in cell proliferation through transcriptional repression of target genes. However, the precise mechanisms of its action in many cellular pathways are poorly understood, including the control of DNA replication and post-transcriptional control of gene expression. Drosophila melanogaster presents a simplified genetic system to study cancer genes. Several published observations have suggested a role for RB in regulating DNA replication. Interestingly, other data indicate that RB associates with RNA processing factors. I have characterized novel protein-protein interactions with the Drosophila retinoblastoma tumor suppressor homologue Rbf, with an emphasis on its poorly characterized N-terminal domain. I describe the interaction of Rbf with the origin recognition complex, indicating a unique connection to DNA replication control. I also show that Rbf interacts with the RNA binding protein Squid, and review the literature that suggests potential role of RB/E2F in the control of RNA processing. The ability to control RNA processing may be an additional, unappreciated mode of gene regulation by RB. A focused study of the uncharacterized amino-terminal domain of Rbf has revealed new details about the retinoblastoma tumor suppressor in cell regulation, including DNA replication and RNA processing.

Bacterial cells reproduce by doubling in size and dividing. The molecular control systems which regulate the cell cycle must do so in a manner which maintains a similar cell size over many generations. A cell can under conditions of fast growth conclude cell cycles in shorter time than the time required to replicate its chromosome. Under such conditions several rounds of replication are maintained in parallel and a cell will inherit replication processes which were initiated by an ancestor. To accomplish this the cell has to initiate and terminate one round of replication during each cell cycle. To investigate the effects of the cell cycle on gene-regulation in the gut bacterium Escherichia coli, an experimental method combining microfluidics, single molecule fluorescence microscopy and automated analysis capable of acquiring an arbitrary number of complete cell cycles per experiment was developed. The method allowed for the rapid exchange of the chemical environment surrounding the cells. Using this method it was possible to measure the dissociation time of the transcription factor molecule, LacI-Venus, from the native lactose operator sequence, lacO1, and an artificially strong operator, lacOsym, in vivo. The results indicated that regulation of gene-expression from the lactose operon does not occur at equilibrium in living cells. Furthermore, by studying the intracellular location of non-specifically interacting transcription factor molecules it was possible to determine that these do not form long-lived gradients inside the cell as was previously proposed. By studying the replication machinery and the origin of replication it was found that replication is initiated according to a cell volume per origin which did not vary over different growth conditions. Further, division timing was found to be determined by the initiation event to occur after a fixed time-delay. A consequence of this mode of regulation is an uncertainty relation between the size at birth and the cell cycle time, in which cells will vary more in in the cycle time during conditions of slow growth as compared to fast growth and vary more in birth length during conditions of fast growth as compared to slow growth.

The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.

Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions. A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated. The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos. The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV. In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.