<p>These studies are based on the isolation of ë replication defective mutants that had acquired multiple point mutations within ë replication initiation genes O and P in a cryptic prophage (Hayes et al., 1998). Each mutant cell arose after shifting wild type cells with a cI[Ts] cryptic ë prophage deleted for int-kil, and from ren into E. coli, from 30oC to 42oC. Derepression of the trapped cryptic prophage kills the host cells (designated as RK+). Rare colony forming units survive and were designated as RK- mutants. This led to a hypothesis that ë replication-triggered cell stress provokes mutator activity, i.e., increases the frequency of replication errors within the simultaneously replicating chromosome of the host E. coli cells. We tested this hypothesis by asking three questions: (1) Do unselected, untargeted (with no link to ë fragment) auxotrophic mutations appear within the RK- mutant population selected from RK+ culture cells? (2) Is replication initiation from the cryptic ë fragment, or, alternatively, just expression of one or more ë genes required for the appearance of the unselected auxotrophic mutations? (3) Do E. coli functions participate in the appearance of unselected auxotrophic mutations within the RK- mutant population? Our results indicate that auxotrophic mutations unlinked to the ë fragment appeared at high frequency within RK- mutants. RK- auxotrophs arising on rich medium were identified by screening the survivor clones for growth on minimal medium. The appearance of RK- auxotrophic colonies at high frequency (>1 per 100 RK- mutants) leads us to conclude that auxotrophic mutations arise during the independent selection for RK- mutants. Conditions that inhibited ë fragment induction fully suppressed the mutator phenotype. Mutation of host dnaB such that the helicase does not support replication initiation from the induced ë fragment completely suppressed host cell killing, but not the appearance of auxotrophic mutations. We asked if E. coli error-prone polymerases IV and V, or gene functions regulated as part of the host SOS response contributed to the provoked mutator phenotype and observed no close correlation. We demonstrated that the RK+ starting cells did not have a distinct intrinsic mutator activity in several ways, including moving the cryptic ë fragment to different E. coli host cells, blocking ë fragment induction by the addition of a cI+ plasmid to eliminate ë gene expression at high temperatures, and independent assays for spontaneous rifampicin resistance. We found that the induced mutator phenotype associated with the appearance of untargeted auxotrophs was linked to the expression of lambda gene P, and did not require replication initiation from the cryptic ë prophage. We also found that the mutator phenotype of the induced cryptic ë fragment increased the frequency of rifampicin resistant colonies among the RK- mutant population. </p>

The bacteriophage lambda tail protein gpK is required for tail assembly. The activity of the protein can be found at the assembling tail tip and is believed to be localized to this structure. GpK is a 27 kDa protein that has sequence identity to two families of proteins: the Mov34 family of peptidases and the NlpC/P60 family of peptidoglycan endopeptidases. Point substitutions and complementation data confirm that gpK possesses each of these domains and that they can function in trans. When the Mov34 domain is inactivated tail assembly is disrupted whereas when the NlpC/P60 domain is inactivated tails assemble but are inactive. Evidence is presented here that the C-terminal domain possesses lytic activity in isolation but not when part of the full-length protein.

Since the World Wide Web Consortium (W3C) has recommended XQuery as the standard XML query language, the interest in using existing relational technology to query the XML data has dramatically increased. The most significant challenge of the relational approach is how to fully support XQuery semantics in XQuery-to-SQL translation. To eliminate the implicit semantics of XQuery, an XQuery fragment must be defined with simple syntax and explicit semantics. XQ is proposed as an XQuery fragment to express XML queries. <br /><br /> In this thesis, XQ is intensively investigated. It is encoded by <em>System F</em>, a second-order lambda calculus with a considerable expressive power and a strong normalization property. Since XML data is defined as inductive data types, XML tree and XML forest, in <em>System F</em>, all basic XML operators in XQ have been successfully encoded. Also, the semantics of XQ are represented in <em>System F</em> where XQ's semantics environment is encoded by an <em>Environment</em> data type with the corresponding operators. The successful encoding of XQ by <em>System F</em> ensures the termination of XQ query evaluation. <br /><br /> Moreover, an extension of XQ by a new tree operator Xtree and a vertical Vfor clause is proposed in this thesis to express some <em>undefinable</em> XQ queries. It is demonstrated that this extension still allows XQ to retain its XQ-to-SQL translation property that ensures the polynomial evaluation time complexity, and its <em>System F</em> encodable property that ensures the termination of query evaluation.

Closed loop fuel injection have been in use for two decades but it's not until the recent five years that the wide band lambda sensor have been utilized. The goal is to explain wide band and discrete lambda sensors in a simple but powerful way. Both sensors are modeled by simple mathematics and accounts for Oxygen, Hydrogen and Carbon monoxide influences. The focus is not just on the output from the sensors, but also on the underlying function. This means that all explanations are thorough and methodical. The function of a wide band lambda sensor is more complicated than a discrete type lambda sensor, therefore it's harder to get correct readings. The model of the wide band lambda sensor is used to evaluate different problems in preparation for the development of an observer. Several potential problem sources are tested and investigated, these include calibration error, pressure error, air leak error, gas sensitivity and fuel errors. To evaluate the potential problems and their ability to explain differences between actual lambda and sensor output, two sensors with differing outputs have been used. The final result is implemented in an ECU. The models indicate that the difference between the two sensors is most likely explained by different sensitivity for CO, O2 and H2. This can in turn have one or several explanations. It is suggested that different ability to pump oxygen, different nernst cells or even different controllers can cause this. The reason is not investigated further as this would require a very deep research on the two sensors. Because no usable explanation is found an observer that estimates the offset at stoichiometric conditions, where lambda equals one, is constructed. The observer uses the fact that the switch point of a discrete lambda sensor is insensitive to disturbances. The offset calculation is performed in real time on an ECU. Tools for calibration of the observer are also developed. With the observer the error for the two sensors is roughly halved over the whole spectrum and at stoichiometric conditions, which is the normal operation for an engine, the error was too small to measure. Although the wide band lambda sensor is a very complex sensor it is shown that it can be understood with simple mathematics and basic knowledge in chemistry. The developed model agrees well with the real sensor for steady state conditions. For transient conditions, however, the model needs to be refined further. The question why the two sensors differ is discussed but the true origin of the cause remains unsolved. The conclusion is that the error can be drastically reduced with just an offset. It is also shown that when building a lambda sensing device the controller is of equal importance as the sensor element itself. This is due to the sensitivity of surrounding factors that the controller must be able to handle. These effects are specially important for engines running at lambda not equal to 1, for example diesel engines.

Since the World Wide Web Consortium (W3C) has recommended XQuery as the standard XML query language, the interest in using existing relational technology to query the XML data has dramatically increased. The most significant challenge of the relational approach is how to fully support XQuery semantics in XQuery-to-SQL translation. To eliminate the implicit semantics of XQuery, an XQuery fragment must be defined with simple syntax and explicit semantics. XQ is proposed as an XQuery fragment to express XML queries. <br /><br /> In this thesis, XQ is intensively investigated. It is encoded by <em>System F</em>, a second-order lambda calculus with a considerable expressive power and a strong normalization property. Since XML data is defined as inductive data types, XML tree and XML forest, in <em>System F</em>, all basic XML operators in XQ have been successfully encoded. Also, the semantics of XQ are represented in <em>System F</em> where XQ's semantics environment is encoded by an <em>Environment</em> data type with the corresponding operators. The successful encoding of XQ by <em>System F</em> ensures the termination of XQ query evaluation. <br /><br /> Moreover, an extension of XQ by a new tree operator Xtree and a vertical Vfor clause is proposed in this thesis to express some <em>undefinable</em> XQ queries. It is demonstrated that this extension still allows XQ to retain its XQ-to-SQL translation property that ensures the polynomial evaluation time complexity, and its <em>System F</em> encodable property that ensures the termination of query evaluation.

<p>These studies are based on the isolation of ë replication defective mutants that had acquired multiple point mutations within ë replication initiation genes O and P in a cryptic prophage (Hayes et al., 1998). Each mutant cell arose after shifting wild type cells with a cI[Ts] cryptic ë prophage deleted for int-kil, and from ren into E. coli, from 30oC to 42oC. Derepression of the trapped cryptic prophage kills the host cells (designated as RK+). Rare colony forming units survive and were designated as RK- mutants. This led to a hypothesis that ë replication-triggered cell stress provokes mutator activity, i.e., increases the frequency of replication errors within the simultaneously replicating chromosome of the host E. coli cells. We tested this hypothesis by asking three questions: (1) Do unselected, untargeted (with no link to ë fragment) auxotrophic mutations appear within the RK- mutant population selected from RK+ culture cells? (2) Is replication initiation from the cryptic ë fragment, or, alternatively, just expression of one or more ë genes required for the appearance of the unselected auxotrophic mutations? (3) Do E. coli functions participate in the appearance of unselected auxotrophic mutations within the RK- mutant population? Our results indicate that auxotrophic mutations unlinked to the ë fragment appeared at high frequency within RK- mutants. RK- auxotrophs arising on rich medium were identified by screening the survivor clones for growth on minimal medium. The appearance of RK- auxotrophic colonies at high frequency (>1 per 100 RK- mutants) leads us to conclude that auxotrophic mutations arise during the independent selection for RK- mutants. Conditions that inhibited ë fragment induction fully suppressed the mutator phenotype. Mutation of host dnaB such that the helicase does not support replication initiation from the induced ë fragment completely suppressed host cell killing, but not the appearance of auxotrophic mutations. We asked if E. coli error-prone polymerases IV and V, or gene functions regulated as part of the host SOS response contributed to the provoked mutator phenotype and observed no close correlation. We demonstrated that the RK+ starting cells did not have a distinct intrinsic mutator activity in several ways, including moving the cryptic ë fragment to different E. coli host cells, blocking ë fragment induction by the addition of a cI+ plasmid to eliminate ë gene expression at high temperatures, and independent assays for spontaneous rifampicin resistance. We found that the induced mutator phenotype associated with the appearance of untargeted auxotrophs was linked to the expression of lambda gene P, and did not require replication initiation from the cryptic ë prophage. We also found that the mutator phenotype of the induced cryptic ë fragment increased the frequency of rifampicin resistant colonies among the RK- mutant population. </p>

The purpose of the work described in this dissertation is to better understand the role of Rz and Rz1 function with respect to phage lysis. We determined using both a genetic and biochemical approach that the Rz protein is an inner membrane protein containing a single N-terminal transmembrane domain (TMD) with an Nin/Cout topology. Consistent with previous work on Rz1, the Rz1 lipoprotein was found to be localized to the outer membrane (OM). Following localization, both Rz and Rz1 form homodimers in vivo due to intermolecular disulfide formation. Despite being localized to apposing membranes, the two proteins form a complex. A small number of phages encode a potential single protein equivalent of Rz-Rz1. This protein, termed a spanin, is predicted to tether the inner and outer membranes by a single polypeptide chain. Based on complementation, it was concluded that gp11 from the phage T1 is a functional equivalent of Rz-Rz1. Gp11, and by analogy the Rz-Rz1 two-component spanin complex, threads the meshwork of the PG layer. The presence of an Rz-Rz1 complex, which forms in the presence of peptidoglycan (PG), is supported by in vivo results. The soluble periplasmic domains of Rz and Rz1, which are dimeric and monomeric respectively, were purified. Circular dichroism analysis indicates that Rz is structured, with significant α-helical content, whereas Rz1, in which 10 out 39 residues are proline, is unstructured. Mixing the proteins results in the formation of a complex with significant new α-helical content. Negative-stain images reveal ~ 25 nm x ~ 4 nm rod-shaped structures. Holin independent activity of Rz and Rz1 is found to disrupt whole cells. Furthermore, time lapse microscopy of λ and λRzam lysis allows us to conclude that Rz and Rz1 are essential for lysis. These results suggest a model for Rz-Rz1 function which begins with Rz and Rz1 forming a complex through direct interaction prior to holin and endolysin function. Holin-mediated hole formation allows the endolysin to degrade PG which sterically hinders Rz-Rz1 activity. Removal of PG by endolysin degradation thus triggers Rz-Rz1 OM disruption via fusion of the inner and outer membranes.

The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.

The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.

The assembly of λ tail requires the action of 11 gene products which must interact in an organized fashion to assemble infectious tail particles. GpL is an essential protein for the formation of the tail tip complex and necessary for the assembly of λ tail. The work described here has shown that gpL and its homologues contain two domains where the C-terminal domain coordinates an oxygen-sensitive [4Fe-4S] 2+ cluster using 4 highly conserved cysteines. This is the first report of a bacteriophage morphogenetic protein to coordinate a [4Fe-4S]2+ cluster. Through two individual cysteine mutants, C184A and C228A, it was determined that these mutant proteins coordinate a [2Fe-2S]2+ cluster also using 4 cysteines; the fourth cysteine being non-conserved. λ tails assembled with cysteine mutant gpL resulted in a 1000-fold decrease in the titer of active tails and tail particles could not be detected by TEM indicating that λ tails cannot be assembled with cysteine mutant gpL. I propose that the coordination of a [4Fe-4S] cluster with the four conserved cysteines maintains a conformation in gpL that can optimally interact with other tail proteins for efficient tail assembly.