The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities. / Department of Biology

Thesis (M.S.)--State University of New York at Binghamton, Biological Sciences Department, 2007. / Includes bibliographical references.

Orientador: Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T12:42:44Z (GMT). No. of bitstreams: 1 Amstalden_MariaCeciliaKrahenbuhl_M.pdf: 4328649 bytes, checksum: efc5c151f8382e97f2def6576eb633b6 (MD5) Previous issue date: 2013 / Resumo: Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Note: The complete abstract is available with the full electronic document / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestra em Biociências e Tecnologia de Produtos Bioativos

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This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer. / Department of Biology

Vibrio parahaemolyticus is an opportunistic human pathogen that occurs naturally in a non-pathogenic form in coastal estuarine and marine environments worldwide. Following the acquisition of virulence-associated genes, V. parahaemolyticus has emerged as a significant pathogen causing seafood-borne illnesses. The mechanisms and conditions that promote the emergence of disease causing V. parahaemolyticus strains are not well understood. In addition, V. parahaemolyticus clinical strains isolated from disease-associated samples and environmental strains from sediment, water, and marine organisms have been identified with considerable diversity; however, the evolutionary relationships of disease-causing strains and environmental strains are not known. In the following research, the evolutionary relationships of V. parahaemolyticus clinical and environmental strains are examined. In addition, the contribution of genetic elements and molecular mechanisms such as deficiency of DNA repair to the evolution of V. parahaemolyticus clinical and environmental strains is shown. Molecular analysis of the evolutionary relationships of V. parahaemolyticus clinical and environmental strains demonstrated separate lineages of pathogenic and non-pathogenic strains with the exception of several environmental strains that may represent a reservoir of disease-causing strains in the environment. Sequence characterization of plasmids isolated from diverse environmental Vibrios indicated a role of plasmids in strain evolution by horizontal transfer of housekeeping genes. In addition, analysis of plasmids from V. parahaemolyticus clinical and environmental strains indicated the existence of a plasmid family distributed among V. parahaemolyticus, V. campbellii, and V. harveyi environmental strains. Sequence characterization of a plasmid of this family from a V. parahaemolyticus environmental strain indicated the contribution of these plasmids to the emergence of the clonal pandemic strains. Investigation of the role of molecular mechanisms to the evolution of V. parahaemolyticus strains showed that inactivation of the DNA repair pathway methyl-directed mismatch repair (MMR) increased the accumulation of spontaneous mutations leading to increased nucleotide diversity in select genes. The research findings in the following chapters demonstrate a considerable contribution of genetic elements and molecular mechanisms to the evolution of genetic and phenotypic diversity.

La transformation naturelle bactérienne permet aux micro-organismes d'échanger des informations génétiques pour promouvoir leurs réponses adaptatives pour faire face aux changements environnementaux. De l'ADN extracellulaire est incorporé et recombiné au génome de l'hôte. Ce processus augmente la plasticité des bactéries. Chez S. pneumoniae, un pathogène majeur chez l'Homme engendrant des infections pouvant être mortelles, la transformation bactérienne accentue la transmission de gènes de résistance aux antibiotiques. Chez les bactéries à Gram positif, l'opéron comF encode l'expression de deux protéines. L'une est démontrée comme étant essentielle à la transformation, est décrite pour être membranaire. La seconde n'a pas été étudiée. Cependant ces protéines n'ont pas été étudiées d'un point de vue structural ou fonctionnel. Des mutagenèse et le double hybride bactérien ont permis de mettre en évidence que ses protéines sont indispensables pour l'expression de la compétence et interagissent avec de nombreuses protéines du transformasome. De plus, l'expression des deux protéines de manière hétérologue prouve qu'elles sont solubles et forment des oligomères. L'analyse structurale de ComFA, atteste de la conformation atypique de cette helicase trimerique et hexamerique. En outre, l'activité ATPasique simple brin DNA-dépendant de cette protéine est démontrée. Finalement un complexe protéique a été révélé entre ComFA et ComFC dont l'étude microscopique à hautes résolutions prouve l'apparition d'un anneau via l'assemblage de deux hexamères. Ces résultats suggèrent que ComFA est le moteur tirant l'ADN dans la cellule. Quant à ComFC, elle semble aider à la stabilisation de ComFA. / Bacterial natural transformation allows microorganisms to exchange genetic information to promote their adaptive responses to cope with environmental changes. The extracellular DNA is incorporated and recombined with the genome of the host. This phenomenon increases the plasticity of Gram positive and negative bacteria. S. pneumoniae is a major pathogen for humans, which is causing infections that can be deadly. In this specie, bacterial transformation increases the transmission of antibiotic resistance.In Gram-positive bacteria, comF operon encodes the expression of two proteins. One of them, shown to be essential for natural transformation, is expected to be a membrane protein. The second is not described. However, up to now neither protein has been studied from a structural or functional point of view. Mutagenesis technique and double hybrid bacterial assay allowed to show that both proteins are essential for the expression of the competence and interact with many proteins of the transformasome. In addition, heterologous expresion of both proteins have shown their solubility and the formation of oligomers. Structural analysis of ComFA demonstrates the unique conformation of this hexameric and trimeric helicase. Furthermore, the ATPase single stranded DNA-dependent activity of this protein could be detected. Finally, a protein complex is formed between ComFA and ComF, and high-resolution microscopic study proves the occurrence of a ring via a two-hexamers. These results suggest that ComFA is the engine pulling the DNA in the cell. As for ComFC, this protein seems to help stabilizing of ComFA.

Chapter–I Introduction Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are essentially non-linear waves that propagate at supersonic speeds. Such disturbances occur in steady transonic or supersonic flows, during explosions, earthquakes, tsunamis, lightening strokes and contact surfaces in laboratory devices. Any sudden release of energy (within few μs) will invariably result in the formation of shock wave since it is one of the efficient mechanisms of energy dissipation observed in nature. The dissipation of mechanical, nuclear, chemical, and electrical energy in a limited space will result in the formation of a shock wave. However, it is possible to generate micro-shock waves in laboratory using different methods including controlled explosions. One of the unique features of shock wave propagation in any medium (solid, liquid or gases) is their ability to instantaneously enhance pressure and temperature of the medium. Shock waves have been successfully used for disintegrating kidney stones, non-invasive angiogenic therapy and osteoporosis treatment. In this study, we have generated a novel method to produce micro-shock waves using micro-explosions. Different biological applications were developed by further exploring the physical properties of shock waves. Chapter – II Bacterial transformation using micro-shock waves In bacteria, uptake of DNA occurs naturally by transformation, transduction and conjugation. The most widely used methods for artificial bacterial transformation are procedures based on CaCl2 treatment and electroporation. In this chapter, controlled micro-shock waves were harnessed to develop a unique bacterial transformation method. The conditions have been optimized for the maximum transformation efficiency in E. coli. The highest transformation efficiency achieved (1 × 10-5 transformants per cell) was at least 10 times greater than the previously reported ultrasound mediated transformation (1 × 10-6 transformants per cell). This method has also been successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella Typhimurium. This novel method of transformation has been shown to be as efficient as electroporation with the added advantage of better recovery of cells, economical (40 times cheaper than commercial electroporator) and growth-phase independent transformation. Chapter – III Needle-less vaccine delivery using micro-shock waves Utilizing the instantaneous mechanical impulse generated behind the micro-shock wave during controlled explosion, a novel non-intrusive needleless vaccine delivery system has been developed. It is well established, that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle free devices for epidermal delivery have inherent problems from the perspective of patient safety and comfort. The penetration depth of less than 100 µm in the skin can elicit higher immune response without any pain. Here the efficient utilization of the device for micro-shock wave mediated vaccination was demonstrated. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. The device mediated vaccination elicits better protection as well as IgG response even in lower vaccine dose (ten-fold lesser), compare to other routes of vaccination. Chapter – IV In vitro and in vivo biofilm disruption using shock waves Many of the bacteria secrete highly hydrated framework of extracellular polymer matrix on encountering suitable substrates and get embedded within the matrix to form biofilm. Bacterial colonization in biofilm form is observed in most of the medical devices as well as during infections. Since these bacteria are protected by the polymeric matrix, antibiotic concentration of more than 1000 times of the MIC is required to treat these infections. Active research is being undertaken to develop antibacterial coated medical implants to prevent the formation of biofilm. Here, a novel strategy to treat biofilm colonization in medical devices and infectious conditions by employing shock waves was developed. Micro-shock waves assisted disintegration of Salmonella, Pseudomonas and Staphylococcus biofilm in urinary catheters was demonstrated. The biofilm treated with micro-shock waves became susceptible to antibiotics, whereas the untreated was resistant. Apart from medical devices, the study was extended to Pseudomonas lung infection model in mice. Mice exposed to shock waves responded well to ciprofloxacin while ciprofloxacin alone could not rescue the mice from infection. All the mice survived when antibiotic treatment was provided along with shock wave exposure. These results clearly demonstrate that shock waves can be used along with antibiotic treatment to tackle chronic conditions resulting from biofilm formation in medical devices as well as biological infections. Chapter – V Shock wave responsive drug delivery system for therapeutic application Different systems have been used for more efficient drug delivery as well as targeted delivery. Responsive drug delivery systems have also been developed where different stimuli (pH, temperature, ultrasound etc.) are used to trigger the drug release. In this study, a novel drug delivery system which responds to shock waves was developed. Spermidine and dextran sulfate was used to develop the microcapsules using layer by layer method. Ciprofloxacin was loaded in the capsules and we have used shock waves to release the drug. Only 10% of the drug was released in 24 h at pH 7.4, whereas 20% of the drug was released immediately after the particles were exposed to shock waves. Almost 90% of the drug release was observed when the particles were exposed to shock waves 5 times. Since shock waves can be used to induce angiogenesis and wound healing, Staphylococcus aureus skin infection model was used to show the effectiveness of the delivery system. The results show that shock wave can be used to trigger the drug release and can be used to treat the wound effectively. A brief summary of the studies that does not directly deal with the biological applications of shock waves are included in the Appendix. Different drug delivery systems were developed to check their effect in Salmonella infection as well as cancer. It was shown for the first time that silver nanoparticles interact with serum proteins and hence the antimicrobial properties are affected. In a nutshell, the potential of shock waves was harnessed to develop novel experimental tools/technologies that transcend the traditional boundaries of basic science and engineering.

H. pylori has a panmictic population structure due to high genetic diversity. The homoplasy index for H. pylori is 0.85 (where 0 represents a completely clonal organism and 1.0 indicates a freely recombining organism) which is much higher than homoplasy index for E. coli (0.26) or naturally competent Neisseria meningitides (0.34). It undergoes both inter as well as intra strain transformation. Intergenomic recombination is subject to strain specific restriction in H. pylori. Hence, a high homoplasy index means that competence predominates over restriction in H. pylori. Annotation of the genomes of H. pylori strains 26695 and J99 show the presence of nearly two dozen R-M systems out of which 16 were postulated to be Type II for J99. H. pylori has been described to be an ideal model system for understanding the equilibrium between competing tension of genomic integrity and diversity (42). R-M systems allow some degree of sexual isolation in a population of competent cells by acting as a barrier to transformation. The mixed colonizing population of H. pylori has a polyploidy nature where each H. pylori strain adds to ‘ploidy’ of the colonizing population. Maintenance of polyploidy nature of mixed colonizing population in a selective niche of stomach needs a barrier to free gene flow. Restriction barrier maintains a polyploidy nature of H. pylori population which is considered as yet another form of genetic diversity helping in persistence of infection. Thus, according to the model proposed by Kang and Blaser, where H. pylori are considered as perfect gases like bacterial population, transformation and restriction both add to genetic diversity of the organism. Again, restriction barriers are not completely effective, which could be due to cellular regulation of restriction system. Thus, a perfect balance between restriction and transformation in turn regulates the gene flow to equilibrate competition and cooperation between various H. pylori strains in a mixed population. RecA, DprA and DprB have been shown to be involved in the presynaptic pathway for recombination substrates brought in through the Com system. Biochemical characterization of HpDprA, during this study revealed its ability to bind to ssDNA and dsDNA. Binding of HpDprA to both ssDNA as well as dsDNA results in large nucleoprotein complex that does not enter the native PAGE. However, DNA trapped in the wells could be released by the addition of excess of competitor DNA, illustrating that the complex are formed reversibly and do not represent dead-end reaction products. Transmission electron microscopy for SpDprA interaction with ssDNA established that a large nucleoprotein complex consisting of a network of several DNA molecules bridged by DprA is formed which is retained in the well. A large DNA-protein complex that sits in the well has also been observed with other DNA binding proteins like RecA. It has been observed for ssDNA binding protein (SSB) that they bind non-specifically to dsDNA under low salt condition (20 mM NaCl) in the absence of Mg2+. The non specific binding of SSB to dsDNA was prevented under high salt conditions (200 mM NaCl) or in the presence of Mg2+. HpDprA interaction with both ssDNA and dsDNA was stable under high salt condition (200 mM NaCl) and in the presence of Mg2+ indicating that these interactions are specific. The interaction of HpDprA with dsDNA is significant since dsDNA plays an important role in natural transformation of H. pylori. The pathway of transformation by dsDNA is highly facilitated (nearly 1000 fold) as compared to ssDNA. However, dsDNA is a preferred substrate for REases which are a barrier to horizontal gene transfer. This implies that the decision of ‘restriction’ or ‘facilitation for recombination’ of incoming DNA might be taken before the conversion of dsDNA into ssDNA. The incoming DNA has been shown to be in the double-stranded form in periplasm and in single-stranded form in cytoplasm. Hence, the temporal and spatial events surrounding endonuclease cleavage remain to be understood. Taken together, these results suggest a very important role of dsDNA in natural transformation in H. pylori. Hence, binding and protection of dsDNA by HpDprA is possibly of crucial importance in the success of natural transformation process of the organism. DprA is characterized by presence of a conserved DNA binding domain. The DNA binding domain adopts a Rossman fold like topology spanning most region of the protein. Rossman fold consists of alternating alpha helix and beta strands in the topological order of β-α-β-α-β. It generally binds to a dinucleotide in a pair as a single Rossman fold can bind to a mononucleotide only. All homologous DprA proteins characterized till date show that in addition of the prominent Rossman fold domain they consist one or more smaller domains. RpDprA consists two more domains other than the Rossman fold domain i.e., N- terminal SAM (sterile alpha motif) domain and a C-terminal DML-1 like domain. SpDprA consist of an N-terminal SAM domain other than Rossman fold domain. While the main function of Rossman fold is to bind DNA, the supplementary domains are highly variable in sequences and functions. For example, the SAM domain in S. pneumoniae plays a key role in shut-off of competence by directly interacting with ComE~P. HpDprA consist of an N-terminal Rossman fold domain and a C-terminal DML-1 like domain. Both these domains are found to be prominently α-helical in nature. Amino acid sequence analysis of the protein suggests that NTD is basic and CTD is acidic in nature. NTD is sufficient for binding with ssDNA and dsDNA, while CTD plays an important role in formation of higher order polymeric complex with DNA. For HpDprA and SpDprA, dimerization site was mapped in Rossman fold domain. Gel filtration data revealed an important observation that HpDprA can exist as a monomer (dominant species at lower concentration) as well as a dimer (dominant species at higher concentration) in solution. However, the exchange between these two forms is very fast resulting in a single peak of elution. Since, HpDprA binds to DNA in dimeric form, the dimer species will be favoured in presence of DNA. Hence, even at lower concentrations HpDprA will be mainly a dimer in presence of DNA. Interestingly, both domains of HpDprA i.e., NTD and CTD were able to form dimers but no higher oligomeric form. On the other hand, HpDprA was seen to form oligomeric forms higher than dimer in gluteraldehyde cross linking assay. The strength of CTD dimer was much lower that NTD dimer, therefore it could be proposed that there are two sites of interaction present in HpDprA - a primary interaction site (N-N interaction) and a secondary interaction site (C-C interaction). The N-N interaction is responsible for dimer formation but further oligomerization of HpDprA necessitates the interaction of two dimers using C-C interaction site. It was shown that NTD binds to ssDNA but forms lower molecular weight complex. SPR analysis of DprA and NTD – DNA interaction pointed out that deletion of CTD leads to faster dissociation of the protein from DNA. Concomitantly, reduction in binding affinity was observed for both ss and ds DNA upon deletion of CTD from full length protein. These results suggest that CTD does play an important role in interaction of full length HpDprA with DNA. Two possible roles of CTD were proposed by Wang et al (2014) group to explain their observation of formation of lower molecular weight complex in absence of CTD. (i) CTD possesses a second DNA binding site but much weaker than site present in NTD. (ii) CTD is not involved in DNA binding but mediates nucleoprotein complex formation through protein – protein interaction. EMSA and SPR analysis with purified CTD protein confirmed that there is no secondary DNA binding site present in CTD. As discussed above, it was observed that CTD can mediate interaction between two HpDprA through C-C interaction. Since the interaction is weaker it is lesser likely to be responsible for dimer formation but in trimer or higher oligomeric form of HpDprA, the presence of N-N interaction will facilitate and stabilize C-C interaction. These observations together bring forward an interesting model for HpDprA – DNA interaction. HpDprA forms dimer through N-N interaction (favourably in presence of DNA) and many HpDprA dimers bind to DNA owing to their high affinity and sequence independent nature of binding. These dimers interact with each other through C-C interaction resulting in higher molecular weight nucleoprotein complex. HpDprA - DNA complex formation is slower than NTD – DNA complex but the former one is more stable (Fig. 2). According to the above proposed model there are two binding events (DNA – protein and protein – protein) in case of HpDprA – DNA complex formation and hence it would take longer time than NTD-DNA complex formation which involves only one binding event. But the resulting higher order complex with HpDprA – DNA would be much more stable. NTD is able to offer equally efficient protection from nuclease to ssDNA and dsDNA (Fig. 7). This shows that NTD alone is sufficient to completely coat single molecule DNA. AFM images confirm the difference in binding pattern of HpDprA full length protein and NTD. As can be seen in Fig. 8F, NTD binds a DNA molecule by entirely occupying all the available space but forms nucleoprotein filaments isolated from each other. In contrast to full length HpDprA, which forms tightly packed, condensed, extensively cross linked polynucleoprotein complexes, NTD forms much thinner complexes with DNA. In the electron micrographs of SpDprA – DNA complex, extensive cross filament interaction was observed resulting in a dense molecular aggregate. Similar kinds of complexes with DNA were also observed for Bacillus subtilis DprA in atomic force microscope images. Thus, it could be proposed that HpDprA binds to a single DNA molecule (single strand or double strand) mainly as a dimer formed through N-N interaction. Such multiple individual nucleoprotein filaments come together and interact with each other through C- C interaction resulting in dense and intricate poly – nucleoprotein complex. HpDprA is proposed to undergo conformational changes from closed state to open state in presence of ssDNA. In agreement with this, structural transition (resulting in reduction of α-helicity of the protein) was observed in presence of ssDNA. Similar structural transitions were observed for dsDNA indicating possibly a common mode of interaction for both forms of DNA. Further, mutation of the residues shown to be involved in binding ssDNA from crystallographic data, resulted in decrease of binding affinity with dsDNA as well. The fold reduction in binding affinity of dsDNA was lower than that for ssDNA despite that it is obvious that the same positively charged pocket which is primarily involved in ssDNA interaction is also responsible (atleast partially) for binding with dsDNA. However, the residues crucial for interaction with these two forms of DNA may be different. Both DprA and R-M systems have been shown to have presynaptic role in natural transformation process. While DprA has a protective role, R-M systems have an inhibitory role for incoming DNA suggesting a functional interaction between them. Results of this study show that HpDprA interacts with dsDNA, inhibits Type II restriction enzymes from acting on it and at the same time stimulates the activity of MTases resulting in increased methylation of bound DNA. This observation is of significance from the view of genetic diversity as the only way a bacterial cell discriminates between self and nonself DNA is through the pattern of methylation. Binding of HpDprA to incoming DNA inhibits its access to restriction endonucleases but not to methyltransferases. As a result DNA will be methylated with the same pattern as that of the host cell. Hence, it no longer remains a substrate for restriction enzymes. HpDprA thus, effectively alleviates the restriction barrier. However, it remains to be understood as to how DNA in complex with HpDprA, while not accessible to REases or other cellular nucleases, is accessible to a MTase? A possible explanation could be that HpDprA interacts with MTase and recruits it on DNA. It has been shown that there is a overlap between DprA dimerization and RecA interaction interfaces and in presence of RecA, DprA-DprA homodimer is replaced with DprA-RecA heterodimer allowing RecA nucleation and polymerization on DNA followed by homology search and synapsis with the chromosome. A similar scenario can be thought for interaction of HpDprA with the MTase. R-M systems play an important role in protection of genomic DNA from bacteriophage DNA. Hence, downregulation of restriction barrier by HpDprA may not be desirable by host during the entire life cycle. Therefore, the expression of HpDprA, which is ComK dependent and that which takes place only when competence is achieved is noteworthy. In H. pylori, DNA damage induces genetic exchange via natural competence. Direct DNA damage leads to significant increase in intergenomic recombination. Taken together it can be proposed that when genetic competence is induced, R-M systems are down regulated to allow increased genetic exchange and thus, increasing adaptive capacity in a selective environment of stomach. There is an evolutionary arms race between bacterial genomes and invading DNA molecules. R-M systems and anti-restriction systems have co-evolved to maintain an evolutionary balance between prey and predator. Phages and plasmids employ anti-restriction strategies to avoid restriction barrier by a) DNA sequence alteration, b) transient occlusion of restriction sites and c) subversion of restriction-modification activities. DNA binding proteins have been shown to bind and occlude restriction sites. On the other hand, λ Ral protein alleviates restriction by stimulating the activity of Type IA MTases. The observations of MTase stimulation and site occlusion of restriction sites by HpDprA appears to be analogous to anti restriction strategies, otherwise employed by bacteriophages. Thus, DprA could be a unique bacterial anti-restriction protein used by H. pylori for downregulating its own R-M systems to maintain the balance between fidelity and diversity. In conclusion, HpDprA has unique ability to bind to dsDNA in addition ssDNA but displays higher affinity towards ssDNA. Binding of HpDprA to DNA results in a compact complex that is inert to the activity of nucleases. A novel site of oligomerization for HpDprA was observed which suggests the role of C-C interaction in inter-nucleoprotein filament interaction. It would be interesting to further study the effects of CTD deletion on the transformation efficiency of H. pylori, to understand these mechanisms better. It has been well demonstrated that R-M systems offer a barrier to incoming DNA, but our understanding of the regulation of R-M systems has been poor. While other factors like regulation of cellular concentration of restriction enzymes and conversion of dsDNA into ssDNA might play crucial roles in striking the perfect balance between genome diversity and integrity, one of the factors that regulate R-M systems could be DprA.

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