L'enzyme glutamyl-queuosine-ARNt synthétase, présente chez plusieurs bactéries et codée par le gène yadB chez Escherichia coli, catalyse la formation de glutamylqueuosine- ARNtAsp. Le rôle de cet aminoacyl-ARNt est inconnu, mais la cotranscription de yadB et de dksA suggère une implication dans la réponse générale au stress. yadB a été remplacé dans une souche de Escherichia coli [delta]lac par lacZ, sans modification des éventuels éléments transcriptionnels de yadB. L'activité [béta]-galactosidase a été utilisée comme rapporteur pour déterminer le niveau d'expression de yadB dans diverses conditions de croissance et de stress. Les différences phénotypiques entre les souches sauvage et [delta]yadB ont été investiguées. Nous avons découvert que yadB est un gène non-essentiel chez Escherichia coli. Sa faible expression suggère un rôle dans le métabolisme basal de la cellule. Cette expression est sensiblement plus élevée pendant la phase stationnaire de croissance. Aucune différence phénotypique n'a été découverte entre les souches sauvage et [delta]yadB.

L'enzyme étudiée est la glutamyl-ARNt synthetase (GluRS) de la bactérie Escherichia coli. Son activité consiste principalement à charger l'acide glutamique sur l'ARNtGlu. Cette GluRS se compose de 5 domaines structuraux, dont les deux derniers (#4 et 5) situés dans la partie C-terminale ont été acquis durant l'évolution et sont responsables de la reconnaissance de la boucle de l'anticodon de l'ARNtGlu. Le domaine 4 est lié au domaine 5 par une charnière mobile (4-5) permettant à ce dernier de s'incliner et de s'adapter à l'anticodon de l'ARNtGlu (Nureki et al., 1995). Cette GluRS bactérienne, qui joue un rôle essentiel dans la biosynthèse des protéines, est considérée comme une nouvelle cible de l'antibiothérapie grâce à l'existence d'analogues du glutamyl-AMP qui inhibent plus des GluRS bactériennes que la GluRS cytopiasmique des mammifères (Balg et al., 2007). La première partie de ce projet consiste à étudier le rôle de la charnière 4-5 sur l'activité de la GluRS en produisant par mutagenèse dirigée du gène gltX de E. coli des variants E366A, E455R et D333A, altérés dans les résidus qui pourraient influencer les orientations relatives des domaines 4 et 5. Les propriétés cinétiques de ces variants dans la réaction de formation du glutamyl-ARNt montrent qu'il n'y a pas d'effet majeur de ces substitutions sur les Km sauf pour D333A. La constante de spécificité de l'enzyme sauvage reste plus grande que celle des variants ce qui suggère que la flexibilité de la charnière n'est pas grandement affectée par la substitution d'un seul de ces résidus. Accidentellement, un variant double charnière (#dc) a été obtenu dont le Km pour l'ARNtGlu est 7,7 fois plus grand que celui de la GluRS sauvage, et dont la constante de spécificité est 15 fois plus faible. Ce résultat suggère que la longueur de la charnière a peut-être autant d'influence que la nature de ces résidus pour le bon fonctionnement de la GluRS. La deuxième étape du projet est de tester l'hypothèse selon laquelle la flexibilité de la charnière 4-5 de la GluRS est importante pour l'activité catalytique de cette enzyme. Des formes tronquées de ces variants ne contenant que les 2 derniers domaines 4 et 5 ont été surproduites et purifiées pour la mesure de la flexibilité de la charnière 4-5 par résonance magnétique nucléaire (RMN). Une analyse préliminaire de la GluRS tronquée sauvage par RMN a montré qu'elle est bien repliée. Notre étude structure-fonction de la charnière 4-5 facilitera le design rationnel de nouveaux inhibiteurs plus efficaces de la GluRS. Ceux-ci pourraient être des composés de départ (« lead compounds ») pour la conception d'un nouvel antibiotique.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 152-162).

The prevalence and antibiotic resistant profiles of shiga-toxin producing Escherichia coli 0157 strains isolated from faeces samples of cattle, pigs and human stool samples were determined. The strains were further characterised by molecular methods for the presence of shiga-toxin virulence genes and antibiotic resistant genes. Seventy-six Escherichia coli 0157 strains were isolated and the prevalence was higher among E. coli isolated from faeces from pigs (44.2% to 50%) than those from cattle faeces (5.4% to 20.0%) or human stool samples (7 .5%). On testing E. coli 0157 isolates for their resistance to 9 antimicrobial agents, multiple antibiotic resistance (MAR) was observed in all of the isolates arising from resistance to three or more antibiotics. Seventy (92.1 %) of the E. coli 0157 isolated from humans, cattle and pigs were resistant to tetracycline. 73 (96.1 %) were resistant to sulphamethoxazole, 63 (82.9%) were resistant to erythromycin. 40 (52.6%) were resistant to streptomycin and 26 (34.2%) were resistant to ampicillin. The highest frequency of resistance was observed among the human isolates (n=3 ), where 3 (I 00%) of the isolates were resistant to tetracycline, sulphamethoxazole, erythromycin and ampicillin. Furthermore, among the pig isolates (n=60), 58 (96. 7%) were resistant to tetracycline, 57 (95%) were resistant to sulphamethoxazole, 47 (78.3%) were resistant to erythromycin. 38 (63.3%) were resistant to streptomycin and 22 (36. 7%) were resistant to ampicillin. The MAR phenotypes S-Smx-T-E, Smx-T-Ap and Smx-T-E were the dorminant phenotypes among the E. coli 0157 isolated from the faeces samples of communal pigs in 30.4%, 21 .7% and 17.4% of these isolates, respectively. However, phenotypes Smx-T -E and S-Smx-T-E-Ne were identified at I6.2% and 10.8%, respectively within the isolates obtained from commercial pig faeces. The phenotype Smx-T-E was the only MAR phenotype identified among the E. coli 0157 isolated from the faecal samples of commercial cattle at Lichtenburg. Furthermore, MAR phenotypes Smx-T-E-C, K-S-Smx-T-E, S-Smx-T-E and Smx-T-E-Ap were obtained at 25%, respectively for the isolates obtained from communal cattle at Mogosane while Smx-T-E-Ap was the dorminant (66.7%) phenotype among the isolates of human origin. The phenotype Smx-T fom1ed the basis of all the MAR phenotypes obtained and this was similar to the percentage antibiotic resistance data. The distribution of the resistant determinants for tetracycline was determined by PCR analysis in resistant isolates. A tetB gene was detected in E. coli 0157 of pig origin. Based on the characterisation of 30 isolates for the presence of STEC virulence genes by PCR, 18 (60%) possessed the hlyA gene, 7 (23.7%) possessed the eae gene and 5 ( 16. 7%,) harboured both genes. The average MAR indices for pig, cattle and human E. coli 0157 isolates were 0.4n2, 0.3419 and 0.4814, respectively. Among the cattle isolates, the group MAR index was highest for the communal (Mogosane) population while the values for the commercial populations at Lichtenburg and Rustenburg were 0.33 and 0.22, respectively. £. coli 0157 isolated from pigs revealed MAR index results that were 0.508 and 0.415 for the commercial and communal populations respectively and 0.1851 for the E. coli control strains. Characterisation by cluster analysis to determine the commonness and resolve differences between the E. coli 0157 isolated from the Various sources revealed a close association between pig (Tlapeng and Mareetsane), cattle (Mogosane) and human isolates. Interestingly, E. coli 0157 isolated from pigs occurred at the highest frequency in all the clusters. which suggested their role in the dissemination of resistant determinants. / MSc. (Agric.) North-West University, Mafikeng Campus, 2006

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 111-127).

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 81-88.

No description available.

yes / Background The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. Results We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. Conclusions A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology. / Food Standards Agency

The genome of Escherichia coli harbors several catabolic operons involved in the utilization of a wide variety of natural compounds as carbon sources. The chitobiose (chu) operons of E.coli Is involved in the utilization of chitobiose(disaccharide of N-acety1-D-glucosamine) and cellbiose (disaccharide of glucose) derived from the two most abundant naturally occurring carbon sources on earth, chitin and cellulose respectively. The operon consists of the chbBCARFG genes coding for transport, regulation and hydrolysis functions required to utilize these compounds; the chuyBCA genes code for a multi-subuni PTS transporter ; the chuR codes for a dual function repressor/activator of the operon; the chbF codes for a phospho-glucosidase and the chbG codes for a protein of unknown function. The chu operon Is regulated by three transcription factors; NagC, a key regulator of the nag genes involved in amino sugar metabolism; ChbR, a dual function operon-specific regulator; and CRP_cAMP. The operon is repressed by NagC and ChbR in the absence of catabolic substrate. In the presence of chitobiose, expression is induced by the abrogation of NagC-mediated repression by GlcNAc-6-P generated by the hydrolysis of chitobiose-6-P and subsequent activation of transcription by ChbR and CPR-cAMP. Wild type E.coli connot utilize cellbiose due to the inability of cellbiose to induce expression from the operon. The simultaneous presence of a loss of function mutation in nagC and a gain –of-function mutation in chbR is necessary and sufficient to allow cellbiose to induce expression and confer on E.coli the ability to utilize cellbiose. The activation step by ChbR and CPR-cAMP requires an inducer that is recognized by ChbR. The chemical identity of the inducer and the mechanism of transcriptional activation by ChbR and CPR-cAMP are not understood. The studies described in the chapter 2 shows that chbG is essential for the utilization of the acetylated sugars chitobiose and chitotriose while it is dispensable for the sugars lacking the acety1group such as cellobiose and chitosan dimer, a disaccharide of N-glucosamine. ChbG is produced as a cytosolic protein and removes one acety1 group from chitobiose and chitotriose thus shows a mono-decetylase activity. Taken together, the observing suggest that ChbG deacetylates chitobiose-6-P and chitotriose-6-P producing the mono-decetylated from of the sugars. The deacetylateion is necessary for their recognition both as inducers by ChbR to activate transcription along with CRP-cAMP and as substractes by phosop-glucosidase ChbF. Cellobiose positive(Cel+) mutants carrying nagC delection and different gain-of-function mutations in chbR are independent of chbG for induction by chitobiose suggesting that the mutations in ChbR can allow it to recognize the acetylated form of chitobiose-6-P. Despite normal induction, the mutants to grow on chitobiose without chbG are consistant with the requirement of deacetylation for hydrolysis by ChbF. The prediction active site of chbG was validated by demonstrating the loss of chbG function upon alanine substitution of the putative metal binding residues. Vibro cholerace ChbG can complement the function of E.coli ChbG indicating that ChbG is conserved in both the organisms. The studies presented in chapter 3 address the mechanism of transcriptional activation of the chb operon by ChbR and CPR-cAMP. ChbR and CPR-cAMP function in a synergistic manner in response to the induction signal. The synergy is not because of their cooperative binding to the DNA. The role of CRP as a class I activator via the known mechanism involving interaction between the Activation region1 (AR1) and the C-terminal domain of the alpha subunit of RNA polymerase (CTD) was not crucial for the chb operon. A direct interaction between the two activators in virto was observed. Based on these results and the close spacing of the synergy is due to interaction between the two regulators bound to DNA that is enhanced in the presence of the inducer, binding about an optimal confirmation in ChbR required to interact with RNA polymerase. ChbR contacts different residues in the subunit in response to cellbiose and chitobiose; whereas it utilizes the known residues in the presence cellbiose, it appears to require different and unknown residues for induction in the presence of chitobiose. In conclusion, the studies reported in chapter 2 and 3 provide an understanding of the regulation of the chitin oligosaccharides utilization in E.coli at different levels. The broad implications of these studies and possible future directions are discussed in chapter 4. ChbG is an evolutionary conserved protein found in both prokaryotes and enkayotes including humans. ChbG homologs have been implicated in inflammatory bowel disorders in humans and development in metazoans. Therefore, the studies on chbG described in this thesis have been broader significance.

Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.