Thesis (Ph. D.)--University of Wisconsin--Madison, 1951. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-53).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1964. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

L'objectif de cette thèse consistait à analyser la biodiversité des bactériophages infectant Lactococcus lactis. Les bactériophages sont l'une des principales causes d'inhibition de plusieurs procédés de fermentation. L'entrée continuelle de virions via le lait cru et l'absence d'une méthode efficace d'inactivation complète des phages dans l'industrie laitière empêche leur élimination. L'étude de la biodiversité des phages et de leur évolution permettra une action ciblée et une gestion plus intelligente des stratégies anti-phages actuellement disponibles. Depuis quelques années, la classification courante des phages de lactocoques fait l'objet de critiques. Ainsi, les bactériophages de référence représentant les espèces reconnues dans la littérature ainsi que des phages dont l'espèce n'avait pu être identifiée par diverses méthodes ont été étudiés. Une nouvelle proposition de classification contenant dix espèces de phages de L. lactis dont deux nouvelles jamais décrites auparavant a été soumise. De plus, des modifications ont été apportées à la méthode de détection par PCR multiplex des trois espèces prédominantes de phages de L. lactis. La seconde partie du projet porte sur la biodiversité des phages de L. lactis dans une usine québécoise fabriquant du fromage Cheddar. Ainsi, 25 bactériophages de L lactis ont été isolés, classés dans l'une des espèces connues de phages de L. lactis et comparés par leur profil RFLP et leur spectre lytique. Dans la troisième section, l'isolement de huit phages de l'espèce 936 infectant des souches de lactocoques productrices d'exopolysaccharides (EPS) a démontré que les EPS ne représentent pas une barrière efficace contre les phages de cette espèce. Des modifications ont également été proposées pour la méthode de classification des souches EPS+ de L. lactis par analyse du polymorphisme de la taille des fragments de restriction (RFLP). Finalement, bien que l'espèce 936 soit la plus fréquemment rencontrée, uniquement deux séquences génomiques complètes étaient disponibles au début de ce projet. L'analyse de la séquence nucléotidique de trois génomes additionnels apporte de l'information supplémentaire sur la biodiversité des phages à l'intérieur d'une même espèce.

The nature of the cell surface components involved in donor cell clumping (Clu+) and the relationship of Clu+ to high frequency conjugal transfer of lactose utilization (Lac) in Lactococcus lactis subsp. lactis ML3 was examined. Lactose positive (Lac+), Clu+ transconjugants, containing a novel 104 kilobase Lac plasmid, were obtained by mating ML3 with LM2301. When used as Lac+ donors in second round matings, these transconjugants transferred Lac at high frequencies ranging from 10-2 to 10-4 transconjugants per donor CFU. Treatment of donor cells with EDTA and EGTA containing solutions or proteolytic enzymes (proteinase K and chymotrypsin A) resulted in a loss of Clu+. By using a direct plate conjugation technique, these treatments also decreased the capacity for transferring Lac at high frequency. Analysis of cell-surface proteins by SOS-PAGE identified a novel protein of approximately 125 kDa which was present in Clu+ transconjugants, but not in non-clumping transconjugants. These results suggest that Clu+ is required for high frequency Lac transfer in ML3 transconjugants, and at least one large protein is involved in Clu+. De novo synthesis requirements of donor cells for conjugal transfer of Lac were tested on direct plate conjugation technique. Results indicate that de novo protein synthesis and RNA synthesis are not required for conjugal transfer of Lac.

Lactococcus lactis subsp. cremoris Ropy 352 and L. lactis subsp. cremoris Hollandicus produce an exopolysaccharide (EPS) that imparts commercially desirable textural and rheological properties to fermented milk products. This ropy phenotype is expressed under specific environmental conditions. A mucoid EPS phenotype, also expressed under specific environmental conditions, but not involved in the fermentation of ropy milk was identified. The two EPS phenotypes can be expressed individually or concurrently. Genetic regulators involved in expression of the EPS phenotypes were sought. DNA probes and polyclonal antiserum specific to two regulators of EPS in Escherichia coli, Lon protease and RcsA protein, were used to probe ropy and non-ropy strains of L. lactis. The two ropy strains of L. lactis subsp. cremoris, Ropy 352 and Hollandicus, expressed significantly less of the Lon protein than non-ropy strains. Southern and Western blot analysis was extended to a number of Gram negative and Gram positive bacteria. All of the Gram negative bacteria probed contained DNA sequences that hybridized to the Ion and rcsA gene probes, and all of these bacteria has at least one protein that reacted with antiserum to E. coli Lon and RcsA proteins. Two of the Gram positive bacteria contained DNA sequences that hybridized to the E. coli rcsA probe. None of the other Gram positive organisms contained DNA sequences that hybridized to the rcsA or the Ion probes. However, all the Gram positive bacteria contained one high molecular weight protein that reacted with Lon antiserum. In addition, Streptococcus salivarius expressed a protein that reacted with RcsA antiserum. In the course of this study, a second RcsA protein was identified in E. coll. The two RcsA proteins are expressed from one rcsA gene. One RcsA protein is not the proteolytic product of the other RcsA protein. Limited peptide digest profiles of each RcsA protein reveals almost identical peptides indicating the two proteins share a high degree of homology but are not identical. Ferguson plot analysis strongly suggests that the two RcsA proteins differ by size not by charge. Neither RcsA protein can be detected in cells mutant for Ion and rcsB. / Graduation date: 1997

Thèse (M.Sc.)--Université Laval, 2009. / Bibliogr.: f. 71-86. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA. / text

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Proefschrift Rijksuniversiteit Groningen. / Datum laatste controle: 16-12-1997. Lit.opg. - Met samenvatting in het Nederlands.