No description available.

No description available.

No description available.

In this study, we report that expression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon is induced in stationary phase cultures and that this increase is largely dependent on RpoS, the alternative stress sigma factor. Using combinatorial 𝘢𝘳𝘨𝘙 and 𝘳𝘱𝘰𝘚 mutants, we evaluated the relative contributions of these two regulators to the expression of 𝘢𝘳𝘨𝘏 using operon 𝘭𝘢𝘤𝘡 fusions. While ArgR was found to be the main factor responsible for de-repression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon, RpoS was required for full expression of this biosynthetic operon at concentrations below 10 μg arginine ml⁻¹, a level at which growth of an arginine auxotroph was arginine limited. At high arginine concentrations (>10 μg ml⁻¹) 𝘢𝘳𝘨𝘊𝘉𝘏 expression was strongly repressed as expected by ArgR. 𝘢𝘳𝘨𝘊𝘉𝘏 expression was 30 fold higher in Δ𝘢𝘳𝘨𝘙 mutants relative to a wild type fully repressed strain and this expression was independent of RpoS. These results indicate that RpoS plays an important role in the regulation of arginine biosynthesis, particularly when the operon is partially de-repressed as would be the case in starvation conditions. / Thesis / Master of Science (MS)

The Ad5 E1B antigens of MW 58000 and 19000 are known to be involved in oncogenic transformation of mammalian cells. To obtain sufficient quantities of these proteins for biochemical studies on their mechanism of action, I have attempted to express the Ad5 E1B genes in Escherichia coli. Using the strategy developed by Guarente et al. (1980), I have constructed plasmids which have the trancriptional and translational controls of the E. coli lac operon linked 5' of the 19k and 58k coding sequences. One plasmid was shown to synthesize high levels of a stable, immunoreactive 19k analogue consisting of 19k with 29 adenovirus-coded ribosome-binding sequence is functional in directing translation of this protein. Synthesis of 58k was not demonstrated, perhaps the result of [protein instability in E. coli. However, immunoreactive proteins which may correspond to the amino terminal region of 58k were demonstrated. / Thesis / Master of Science (MS)

No description available.

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes. / The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer.

O pEO5, que codifica hemolisina, foi isolado de uma amostra de EPEC do sorotipo O26. Este plasmídio mostrou ser conjugativo e compatível com o pO157, e pelos testes de hibridização observou-se que estes plasmídios não são geneticamente relacionados. Para o estudo comparativo de similaridade foi seqüenciada uma região de 9227 pb de DNA do pEO5 que compreende todo o operon hlyCABD e suas regiões a montante e a jusante. A região do operon hemolitico (7225 pb) e a região promotora do operon foram similares às mesmas regiões do pHly152, que em uma amostra de E. coli isolada de roedor, codifica uma a hemolisina. No entanto, verificou-se a presença de elementos de inserção na região a montante do gene hlyC no pHly152. O pEO5 mostrou ser semelhante a outros plasmídios que também codificam a hemolisina em cepas de EPEC O26 de origem humana e de bovinos. A presença de estruturas semelhantes a transposons em ambas as extremidades do operon a hemolítico do pEO5 indica que esse fator de virulência provavelmente foi adquirido por transferência horizontal de genes. / The conjugative pEO5 encoding haemolysin in strains of EPEC O26 was investigated for its relationship with EHEC haemolysin-encoding of EHEC O26 and O157 strains. pEO5 was found to be compatible with EHEC virulence plasmids and did not hybridize in Southern blots with pO157, indicating that both plasmids were unrelated. A 9227 bp stretch pEO5 DNA encompassing the entire operon hlyCABD was sequenced and compared for similarity to plasmid and chromosomally inherited hly determinants. The a hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of pHly152, in particular, the structural a-hlyCABD and hlyR regions. pEO5 and hly of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural a-hlyCABD. The major difference found between the hly regions of pHly152 and pEO5 is caused by the IS2 upstream of the hlyC in pHly152. The presence of transposonlike structures at both ends of hly sequence indicates that pEO5 was probably acquired by horizontal gene transfer.

Escherichia coli (E. coli) are a diverse species of bacteria that reside, often harmoniously and beneficially, in the gastrointestinal tracts of humans and other mammals. However, some strains are associated with serious intestinal and extra-intestinal disease and are considered pathogens. The main differences between strains of these different E. coli pathotypes can be explained by the acquisition of genetic information introduced by mobile genetic elements, in particular bacteriophage. In enterohaemorrhagic E. coli (EHEC) O157:H7 strain EDL933, a pathotype of E. coli containing prophage-encoded Shiga toxins associated with severe gastrointestinal and systemic disease in humans, these horizontally acquired elements have been termed O-islands (OIs) and include both fully functional and cryptic prophages. The overall aim of this research was to try and determine what these OIs are actually doing for the bacteria. Systems pertinent in the life cycle and virulence of this pathogen were therefore investigated by phenotypically screening a large library of OI deletions in EHEC strain TUV93-0, a Shiga toxin-negative derivative strain of EDL933, and then comparing these with the parent strain. These analyses highlighted a subset of OIs with the potential to regulate motility and type III secretion (T3S), the latter being an essential colonisation factor for EHEC that is encoded by the locus of enterocyte effacement (LEE). Deletion of OI-51, a 14.93 Kb cryptic prophage designated as CP-933C, significantly reduced persistence of faecal shedding in sheep and levels of T3S expression in vitro. Cloning and complementation together with targeted allelic replacements in OI-51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the sequenced E. coli O157:H7 strain Sakai that is present but not annotated in the EDL933 sequence. Functionally important residues of ECs1581 were identified by site-directed mutagenesis based on phenotypic variants present in strains from different E. coli pathotypes, including strains not harbouring a LEE-encoded T3S system. This regulator was subsequently termed RgdR based on a motif demonstrated to be important for stimulation of gene expression from LEE1. Purified RgdR protein was able to form multiple complexes on a PCR generated LEE1 promoter fragment, and activation of this operon appeared to require this DNA binding capacity as a non-T3S inducing variant was unable to bind this same LEE1 promoter fragment. RgdR did not directly activate LEE1 transcription in vitro, nor did it activate transcription by relieving H-NS repression as proposed for the global regulator Ler (LEE-encoded regulator). However, RgdR activation did require a wild type LEE1 promoter and the Ler auto-induction cycle to induce LEE2-5 expression and T3S. RgdR was able to increase binding to Congo red and was capable of repressing bacterial motility. Further analyses demonstrated that RgdR did not regulate T3S and cell motility via GrlA (global regulator of LEE activator) and QseC (quorum sensing E. coli regulator C), two established regulators in E. coli that control LEE gene expression and motility in conjunction with their partners, GrlR (global regulator of LEE repressor) and QseB (quorum sensing E. coli regulator B) respectively. RgdR is therefore identified as a novel regulator able to co-ordinate T3S and motility expression. This research has identified OI-51 as being important for EHEC O157:H7 colonisation in sheep and has identified a completely new family of small bacterial regulators that control surface factor expression in E. coli.

Publicación a texto completo no autorizada por el autor / El documento digital no refiere un asesor / Identifica y genotipifica cepas de STEC aisladas en puestos de venta de carne de pollo en un distrito de Lima. Para ello, se tomó hisopados de la superficie de manos, tablas de picar y mesa de expendio de 50 puestos de venta de carne de pollo en el distrito de San Juan de Miraflores, se realizó el aislamiento microbiológico estándar, identificación molecular de los genes stx1, stx2 y eaeA mediante PCR y la subtipificación genética mediante electroforesis en campo pulsatil (PFGE). El 84% (42/50) y 66% (33/50) de los puestos de venta poseían al menos una de las superficies contaminadas con E. coli y STEC, respectivamente. El 42%(63/150) y 25.3%(38/150) de las muestras fueron positivas a E. coli y STEC, respectivamente. 43 de las 63 cepas de E. coli aisladas fueron patógenas por presentar al menos un gen evaluado. 38 cepas fueron STEC y presentaron los genes stx1 (19.1%;12/63), stx2 (14.3%;9/63) y las asociaciones: stx1 y stx2 (12.7%;8/63); stx1, stx2 y eaeA (6.3%;4/63); stx2 y eaeA (4.8%;3/63); y, stx1 y eaeA (3.2%;2/63). También se identificó E. colieaeA (7.9%;5/63). Veintitrés cepas de STEC fueron evaluadas mediante PFGE las cuales no mostraron ser cepas genéticamente idénticas, perose llegaron a establecer cinco clusters y nueve cepas poco relacionadas epidemiológicamente. Se observaron prácticas de higiene deficientes en el puesto de venta y durante el expendio. Se confirma que los puestos de venta de carne de pollo evaluados son una fuente de contaminación de STEC cuyas cepas difícilmente proceden de un origen común. Es presumible que la manipulación en el puesto de venta y el manejo en expendio favorezcan la contaminación de carne de pollo con STEC en nuestro medio, debiendo fortalecerse las medidas de control a este nivel para salvaguardar la salud pública del consumidor. / Tesis