No description available.

Genetic exclusion in phage T4 is the prime responsibility of the imm and sp genes. The map region containing imm does not allow sufficient bps to encode for proteins the size reported for the imm gp. After assaying 30 mutants of the genes adjacent to imm, I found 7 in gene 42 that were defective in the imm phenotype. Upon reverting amNG411(42), the mutant most defective exclusion, for its gene 42 phenotype the exclusion phenotype also changed. When assayed in UGA suppressor hosts, imm+ phage showed a decreased exclusion ability indicating that an opal codon was involved in production of the functional imm gp. I concluded that imm and gene 42 overlap in an out-of-phase orientation with the involvement of an opal readthrough. This overlap has implications in the genetic regulation of this region. This region of T4 also encodes several other genes important in phage intra- and interspecific competition. They are B-gt, 42 and sp. Using recombinant DNA techniques, I precisely located the sp gene to a region between 21.647 and 22.014 kbp on the T4 restriction map and determined its molecular weight as approximately 15 kDa. This same region of T4 was purported to contain gene 40. Complementation and marker rescue experiments with sp+ plasmids indicated that genes sp and 40 are the same. Gene 40 mutants also were found to be defective in sp function. Genes sp and 40 were redesignated gene sp/40 thus linking an early expressing gene with the morphogenic pathway of prohead assembly. Functionally, host enzymes exo III and exo V were found as participants in gp imm mediated exclusion. Presumably gp imm alters the pilot protein of the superinfecting DNA thus exposing it. Gp sp functions by an anti-lysozyme action. But the pleiotrophic effects of sp/40 are best explained by a temperature induced conformational rearrangement hypothesis. This work links molecular genetics to the ecological concept of competition and provides insights into the function and the evolutionary significance of the competition cluster genes. The competition cluster encodes fundamental adaptive strategies found universally in nature.

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Heartwater is a major threat to livestock in Africa due to its high mortality rate. The intracellular nature of the causative organism, Cowdria ruminantium, makes it difficult to study, hence an effective and user-friendly vaccine has been extremely difficult to obtain. Two C. ruminantium DNA libraries have recently been constructed, the lambda GEM11 bacteriophage DNA library and the lambda ZAPII bacteriophage DNA library, and this has lead to a renewed search for protective genes that could be used as a vaccine against heartwater. In this study, several molecular techniques including PCR, cloning and sequencing were used to identify genes in the lambda GEM11 bacteriophage DNA library that code for proteins, which could be used as vaccines to protect susceptible animals against heartwater. The lambda GEM11 library was screened with a rickettsial secretory protein gene sequence, known as seeD. One positive colony was selected from which the bacteriophage DNA was isolated. The C. ruminantium DNA was amplified from the bacteriophage DNA by using PCR and C. ruminantium-specific primers. The C. ruminantium DNA was screened with Mycoplasma, bovine and Cowdria DNA probes. The amplified DNA was subeloned into two vectors and the clones were screened by restriction analysis to identify clones containing inserts. The appropriate clones were sequenced and overlapping sequences matched, ordered and aligned. Two sequences were continuous with a short sequence of unidentified bases in between. Oligonucleotide primers were designed to amplify the DNA sequence between the two contiguous sequences. This led to the identification of the entire sequence of the C. ruminantium genome contained within the bacteriophage plaque. The single contiguous sequence was analysed and the putative protein-coding sequences were obtained and compared to DNA sequences of known organisms using the BLAST program. Five open reading frames were identified with homology to genes encoding specific proteins in bacteria. Two open reading frames showed homology to the genes encoding the transporter proteins, FtsY and the ABC transporter, and three open reading frames were found to be homologous to genes encoding the essential enzymes dethiobiotin synthetase, pro lipoprotein diacylglycerol transferase and the putative NADH-ubiquinone oxidoreductase subunit. The five open reading frames encode for genes, which are essential for the normal functioning of the C. ruminantium organism. However, these open reading frames might not be effective for use in a DNA vaccine since none of the open reading frames showed homology to obvious genes that could play a role in immunity and therefore confer protection. The open reading frames can be used in mutagenesis studies to produce attenuated strains of the organism that possess mutated versions of these proteins. These attentuated strains could be used for the vaccination of cattle, and thereby confer protection against viable pathogenic C. ruminantium isolates. / AFRIKAANSE OPSOMMING: Hartwater is 'n bedreiging vir vee in Afrika weens die hoë mortaliteitssyfer verbonde aan die siekte. Die intrasellulêre aard van die organisme wat hartwater veroorsaak, Cowdria ruminantium, bemoeilik navorsing aangaande die organisme. Dit het tot gevolg dat 'n effektiewe en gebruikersvriendelike entstof moeilik bekombaar is. Daar is onlangs sukses behaal met die konstruksie van twee C. ruminantium DNA genoteke, die lambda GEM11 bakteriofaag genoteek en die lambda ZAPII bakteriofaag genoteek. Dit het gelei tot 'n herlewing in die soektog na beskermende gene, wat in 'n entstof teen hartwater gebruik kan word. In hierdie studie is verskeie molekulêre tegnieke insluitende PKR, klonering en geenopeenvolging bepaling, gebruik om gene te identifiseer in die lambda GEM11 bakteriofaag genoteek wat kodeer vir proteïene wat in entstowwe gebruik kan word as beskerming teen hartwater. Die secD geen is gebruik om die lambda GEM11 bakteriofaag genoteek te sif. Een positiewe plaak is gevind waarna die DNA uit die bakteriofaag plaak geïsoleer en die C. ruminantium DNA vanuit die bakteriofaag plaak geamplifiseer is deur gebruik te maak van PKR en spesifieke C. ruminantium inleiers. Die C. ruminantium DNA is gesif met Mycoplasma, bees en Cowdria radioaktief gemerkte DNA peilers. Die C. ruminantium DNA is vervolgens in twee vektore gekloneer. Die klone is gesif deur middel van restriksie analise. Die DNA volgorde van die klone is bepaal en twee ononderbroke sekwense is geïdentifiseer met 'n gaping in die middel tussen die twee sekwense. Oligonukleotied inleiers is daarna ontwerp om die geenopeenvolging van die gaping tussen die twee sekwense te vul. Hierdeur kon die volledige geenopeenvolging van die genoom van C. ruminantium wat in die lambda GEM 11 bakteriofaag plaak voorkom, bepaal word. Hierdie volledige geenopeenvolging is vervolgens geanaliseer en die oop leesrame wat daarin voorkom geïdentifiseer. Vyf leesrame is gevind om homologie met gene wat kodeer vir proteïene wat in bakterieë voorkom, te toon. Twee leesrame het homologie met die gene wat kodeer vir transport proteïene, FtsYen die ABC transporter getoon, en drie leesrame het homologie met gene wat kodeer vir die essensiële ensieme detiobiotin sintetase, prolipoproteïen diasielgliserol transferase en die NADHubikinoon oksidoreduktase subeenheid getoon. Dié vyf leesrame het die potensiaal om as entstowwe gebruik te word aangesien al vyf leesrame kodeer vir gene wat 'n belangrike rol speel in die oorlewing van die C. ruminantium organisme. Alhoewel die leesrame moontlik nie so effektief sal wees in 'n DNA entstof nie, toon dit potensiaal om in mutasieeksperimente gebruik te word. Organismes wat die gemuteerde weergawe van die geen besit sal nie-funksionele proteïene produseer, wat 'n invloed kan hê op die normale fisiologiese funksies van die organisme en dus sal lei tot 'n minder virulente organisme. Die geattenueerde organisme kan moontlik gebruik word om diere te immuniseer en daardeur immuniteit aan diere lewer wat beskerming sal bied teen patogeniese C. ruminantium isolate.

Bacteriophage genomes show pervasive mosaicism, indicating that horizontal gene exchange plays a crucial role in their evolution. Phage genomes represent unique combinations of modules, each of them with a different phylogenetic history. Thus, a web-like, rather than a hierarchical scheme is needed for an appropriate representation of phage evolutionary relationships. Part of the virology community has long recognized this fact and calls for changing the traditional taxonomy that classifies tailed phages according to the type of genetic materials and phage tail and head/capsid morphologies. Moreover, based on morphological features, the current system depends on inspection of phage virions under the electron microscope and cannot directly classify prophages. With the genomic era, many phages have been sequenced that are not classified, calling for development of an automatic classification procedure that can cope with the sequencing pace. The ACLAME database provides a classification of phage proteins into families and assigns the families with at least 3 members to one or several functions. In the first contribution of this work, the relative contribution of those different protein families to the similarities between the phages is assessed using pair-wise similarity matrices. The modular character of phage genomes is readily visualized using heatmaps, which differ depending on the function of the proteins used to measure the similarity. Next, I propose a framework that allows for a reticulate classification of phages based on gene content (with statistical assessment of the significance of number of shared genes). Starting from gene/protein families, we built a weighted graph, where nodes represent phages and edges represent phage-phage similarities in terms of shared families. The topology of the network shows that most dsDNA phages form an interconnected group, confirming that dsDNA phages share a common gene pool, as proposed earlier. Differences are observed between temperate and virulent phages in the values of several centrality measures, which may correlate with different constraints to rampant recombination dictated by the phage lifestyle, and thus with a distinct evolutionary role in the phage population. To this graph I applied a two-step clustering method to generate a fuzzy classification of phages. Using this methodology, each phage is associated with a membership vector, which quantitatively characterizes the membership of the phage to the clusters. Alternatively, genes were clustered based on their ‘phylogenetic profiles’ to define ‘evolutionary cohesive modules’. Phages can then be described as composite of a set of modules from the collection of modules of the whole phage population. The relationships between phages define a network based on module sharing. Unlike the first network built from statistical significant number of shared genes, this second network allows for a direct exploration of the nature of the functions shared between the connected phages. This functionality of the module-based network runs at the expense of missing links due to genes that are not part of modules, but which are encoded in the first network. These approaches can easily focus on pre-defined modules for tracing one or several traits across the population. They provide an automatic and dynamic way to study relationships within the phage population. Moreover, they can be extended to the representation of populations of other mobile genetic elements or even to the entire mobilome. Finally, to enrich the phage sequence space, which in turn allows for a better assessment of phage diversity and evolution, I devise a prophage prediction tool. With this methodology, approximately 800 prophages are predicted in 266 among 800 replicons screened. The comparison of a subset of these predictions with a manually annotated set shows a sensitivity of 79% and a positive predictive value of 91%, this later value suggesting that the procedure makes few false predictions. The preliminary analysis of the predicted prophages indicates that many may constitute novel phage types. This work allows tracing guidelines for the classification and analysis of other mobile genetic elements. One can foresee that a pool of putative mobile genetic elements sequences can be extracted from the prokaryotic genomes and be further broken down in groups of related elements and evolutionary conserved modules. This would allow widening the picture of the evolutionary and functional relationships between these elements.

Recombinational repair is a widespread mechanism for dealing with DNA damage. It is found in both prokaryotes and eukaryotes which implies that it is an ancient process which arose early in the evolutionary history of life on Earth. In addition, it has been implicated as a driving force in the evolution of sexual reproduction. In this dissertation I report experimental results which clarify the role of recombinational repair in bacteriophage (phage) T4. The Luria-Latarjet effect is an increase in resistance to DNA damage by phage T4 during infection. It has often been assumed to involve recombinational repair, but this has never been actually demonstrated. Using eleven phage T4 mutants, I have obtained evidence that the Luria-Latarjet effect is due to three repair pathways--excision repair, post-replication-recombinational-repair (PRRR) and multiplicity reactivation (MR), a form of recombinational repair. My results show that the Luria-Latarjet effect develops in two stages. The first stage starts soon after infection. Damage which occurs during the first stage can be repaired by excision repair or PRRR. The second stage appears to start after the first round of DNA replication is complete. Damage which occurs during this stage can apparently be repaired by MR as well as the other two repair pathways. I have also transferred the yeast RAD50 gene, which is required for recombinational repair, into an E. coli expression vector. After demonstrating expression of the protein, I used this construct to test for complementation by the RAD50 gene of E. coli and phage T4 mutants defective in recombinational repair. I was unable to demonstrate complementation in five different assays. Based on the results discussed above and what is known about the phage T4 life cycle, I propose a model for the Luria-Latarjet effect in phage T4. Further, I propose that recombinational repair has been selected to ensure progeny phage genomes are packaged with minimum damage. Since numerous other viruses also show a Luria-Latarjet effect type resistance to DNA damage, I suggest that the conclusions from this phage T4 study may have wide applicability to other viruses.

There has been an increase in number of human infections by mycobacteria and opportunistic pathogens of the closely related nocardioform bacteria. Frequent multiple drug resistance in these organisms makes it desirable to identify novel targets for antimicrobial agents. Bacteriophages offer one way to do this as analysis of their DNA reveals great diversity in their genetic makeup, suggesting variety in the way they interfere with host cells. Four novel nocardiophages were therefore isolated from soil and characterized. Libraries of their nucleic acid were constructed and screened for clones inhibitory to a nocardioform of the genus Rhodococcus. Nine clones were characterized, and minimum necessary DNA for inhibitory activity sequenced. Of 18 ORFs predicted on these DNAs, 13 could not be assigned a function. Genes similar to ones in databases apparently interfered with DNA metabolism, protein synthesis, or integrity of plasma membrane. This genetic approach may be an efficient and effective way to discover novel targets for antibiotics.

Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

Graduation date: 1999

A crucial factor in determining the accuracy of DNA replication is maintenance of a balanced supply of deoxyribonucleoside triphosphates (dNTPs) at replication forks. Perturbation of dNTP biosynthesis can induce dNTP pool imbalance with deleterious genetic consequences, including increased mutagenesis, recombination, chromosomal abnormalities and cell death. Using the T4 bacteriophage system, I investigated the molecular basis of mutations induced by imbalanced dNTP pools in vivo. Two approaches were adopted to disturb dNTP biosynthesis: 1) using mutations which affect the deoxyribonucleotide biosynthesis pathway; 2) exogenously supplying mutagenic deoxyribonucleoside analogs which are then taken up by cells and are metabolized to dNTPs. The levels of dNTPs under different conditions were measured in crude extracts of phage-infected cells, while mutagenic effects were quantitated by analysis of certain rII mutations, thought to revert to wild type along either GC-to-AT or AT-to-GC transition pathways. The mutation pathways stimulated by dNTP pool perturbations were confirmed by direct DNA sequencing after amplification of template by the polymerase chain reaction (PCR). By replacing phage ribonucleotide (rNDP) reductase with the host, Escherichia coli, rNDP reductase, in phage-infected cells, I examined the mechanism of mutation induced by the thymidine analog 5- bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to- AT transition mutations were stimulated many hundred-fold when cells were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions were stimulated predominantly when T4 reductase was active, while ATto- GC transitions were stimulated more when E. coli reductase was active. By examining the control by dNTPs on CDP reduction, I found that the T4 rNDP reductase is substantially inhibited by either BrdUTP or dTTP in crude enzyme extracts. These experimental results are consistent with the hypothesis that mutagenic effects of BrdUrd are based on dNTP perturbations, supporting the model that rNDP reductase is a major determinant of BrdUrd mutagenesis. I also studied the mutator phenotype of one temperature-sensitive conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4 deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions induced by ts LB3 at a semipermissive temperature (34° C). Sequence analysis of revertants from the most sensitive gene marker, rII SN103, showed that either cytosine within the mutated triplet can undergo change to either thymidine or adenine, supporting a model in which mutagenesis induced by ts LB3 at a semipermissive temperature is based on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine triphosphate (hm-dCTP) caused by the temperature-labile dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations. However, no significant dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected cells even when cells were grown at the semi-permissive temperature, suggesting that imbalanced dNTP pools occurred only locally, close to replication forks. These results support a model of dNTP "functional compartmentation", in which DNA replication is fed by a small and rapidly depleted pool, with the bulk of measurable dNTP in a cell representing a replication-inactive pool. To further characterize the mutagenic specificity and DNA site specificity induced by T4 ts LB3, I developed a fast forward mutation approach using thymidine kinase as a marker gene. The studies confirmed that the principal mutagenic effect induced by ts LB3 is C-to- T transition, while C-to-A transversion mutagenesis also occurs. Analysis of DNA sequences around each mutation also suggests that local DNA context influences mutation frequency. / Graduation date: 1991

Most bacteriophage escape the confines of the host bacterium by compromising the integrity of its cell wall, an event that results in rupture (lysis) of the cell. The lysis strategy of bacteriophage Qβ is inhibition of cell wall biosynthesis while the cell is growing. To elicit lysis, the maturation protein (A2) of Qβ inhibits the catalytic activity of MurA, an essential, induced fit enzyme in the cell wall biosynthetic pathway. Consequent lysis releases progeny phage into the environment. The research in this dissertation addresses how lysis timing is integrated into Qβ’s life cycle and discerns the molecular basis of the lytic event. Working off the notion that, as displayed by the mature virion, A2 inhibits MurA, we developed an in vivo bioassay to resolve the amount of inhibitory A2 during infection. We found that the amount of free A2 is vastly greater than the amount of virion-associated A2 and that both forms inhibit MurA. Additionally, the amount of A2 correlates to lysis time and the burst size, as mutant Qβ with upregulated expression of A2 (Qβpor) elicit host cell lysis faster and release fewer mature virions than with the wildtype level of A2. This further suggests that protection from Qβ lysis afforded by MurAL138Q is due to perturbed affinity between A2 and MurA. Yeast two-hybrid analysis supports that A2 and MurAL138Q interact with weaker affinity by rendering small colonies compared to yeast containing interacting A2-MurAwt. Scanning mutagenesis of MurA’s surface near L138 identified residues that may be important for A2 contact in the inhibitory complex. Potentially important residues map to a contiguous area on the surface of MurA that spans both lobes on the flexible loop face of the enzyme, suggesting that A2 prevents the induced fit mechanism of MurA in an uncompetitive manner. Subsequent truncation analysis reveals that the aminoterminal half of A2 is sufficient to mediate host cell lysis. Together, these findings insinuate a model in which the amino-terminus of free A2 interacts with, and inhibits MurA. Then, when the infected cell initiates division, septal catastrophe ensues causing the cell to lyse and liberate progeny bacteriophage Qβ.