Chez les organismes procaryotes, l’adaptation rapide aux changements de l’environnement est synonyme de survie. Un des moyens utilisés pour s’adapter rapidement est un contrôle strict et efficace de l’expression génétique. Par exemple, un changement rapide de pH entraînera l’arrêt de synthèse de certains canaux dans le but de conserver un pH cellulaire interne stable se situant entre 7,2 et 7,8 chez la bactérie Escherichia coli.(Slonczewski et al. 1981) Alors, quand ces changements de l’environnement surviennent, la bactérie se doit de posséder des détecteurs efficaces qui peuvent réguler l’expression des gènes rapidement. Un des nombreux moyens utilisés chez les procaryotes pour détecter et contrôler l’expression génétique est l’utilisation des riborégulateurs. Ces ARN structurés sont localisés dans le 5’ non codant de certains gènes bactériens et permettent une régulation efficace de l’opéron avec lequel ils sont associés. Les riborégulateurs lient directement un métabolite, appelé ligand, ce qui entraîne un changement dans la structure d’ARN et permet une régulation du (des) gène (s) en aval du riborégulateur. Pour accomplir ces deux étapes, soit la détection et la régulation, les riborégulateurs se doivent d’être très structurés pour effectuer une reconnaissance spécifique du ligand et pour réguler l’expression génétique. Un des riborégulateurs les plus structurés est le riborégulateur btuB chez la bactérie E. coli. En effet, ce riborégulateur lie l’adénosylcobalamine (AdoCbl) et est constitué de quatorze tiges boucles ainsi que d’une structure particulière nommée pseudonoeud. Cette structure a été montrée par le passé comme étant importante pour la réponse au ligand dans un contexte in vivo.(Nahvi et al. 2002) Par contre, son rôle dans le changement de structure lors de la liaison du ligand est encore méconnu. Le but ce projet de maîtrise était de déterminer le rôle de ce pseudonoeud dans le changement de structure du riborégulateur lors de la liaison de l’AdoCbl à ce dernier. Les résultats de cette étude ont non seulement permis de déterminer que le pseudonoeud est important pour la communication de la liaison du ligand, mais qu’il est aussi important pour la liaison. De plus, nous avons aussi confirmé que le contexte transcriptionnel est essentiel pour que le pseudonoeud puisse être formé, comme suggéré par les précédents travaux de Perdrizet et al, (2012).

Early events occurring during induction of the SOS response in Escherichia coli are poorly understood. In order to understand the early steps in SOS induction more fully, we have isolated several mutations which constitutively express the SOS regulon. Using a Mud(Apᴿ,lac) fusion to the SOS regulated gene sulA, we isolated Lac⁺ colonies as mutants in which RecA protein is constitutively activated for repressor cleavage. The mutations map to four loci: dam, lig, uvrD and recA. The extent of constitutive SOS induction in these mutants varied greatly, indicating different levels or types of signal in the cell. The mutations isolated demonstrate two early steps in SOS induction. The first step in SOS induction is signal generation and includes mutations found in dam, lig and uvrD genes. The mutant gene products presumably alter DNA metabolism to produce an inducing signal. These non-lethal mutations lead to sub-induction and probably generate very specific signals, such as abnormally unwound DNA in the case of DNA helicase II mutants or unsealed DNA nicks that result from deficient ligation in lig mutants. Greater induction may require quantitatively more signal or different types of signal generated by severe defects leading to cell death. These mutations also show that signal is a variable quantity, allowing the cell to fine tune the levels of SOS repair activity according to the amount or type of signal (damage) perceived. In some cases (such as dam mutations), blocking the SOS response by lexA(Ind⁻) alleles leads to cell death. In this type of constitutively activated strain, the increased level of repair from SOS induction is required to allow the cell to tolerate potentially lethal DNA structures generated by the mutant gene product. The second step in induction is the interaction of signal with RecA protein and is shown by isolating 8 recA mutants. Mutant recA alleles caused the strongest SOS induction in any mutants obtained, similar to the level found in strains lacking repressor (lexA(Def) mutants). This full induction in the absence of lethal DNA damage underscores the pivotal role of RecA protein in regulating the SOS response.

The penicillin tolerance of ammo acid-deprived re/A+ Escherichia coli is attributed to the stringent response. The β-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic snglycerol 3-phosphate dehydrogenase). Therefore, β-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. Both the priming and the lysis induction stages of β-lactam-induced lysis were shown to require phospholipid synthesis. It has been known for some time that phospholipid synthesis is inhibited by the stringent reponse. These results indicate that the inhibition of peptidoglycan synthesis and the induction of penicillin tolerance during the stringent response are both secondary consequences of the inhibition of phospholipid synthesis. Direct experimental evidence is presented for the first time indicating that the penicillin tolerance of amino acid-deprived E coli was directly attributable to action of guanosine 3',5'-bispyrophosphate (ppGpp) and not to some other effect of amino acid deprivation. The overproduction of ppGpp resulted in the inhibition of peptidoglycan and phospholipid synthesis and in penicillin tolerance. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment of the bacteria with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB gene, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis. The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This also indicates that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly , treatment with β-lactam antibiotics by itself induced re/A-dependent ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance. It was al so demonstrated that penicillin tolerance was induced when phospholipid synthesis was inhibited in normal growing E. coli. This penicillin tolerance was not the result a simple inhibition of growth or a decrease in the membrane levels of individual phospholipids (e.g., acidic phospholipids), but rather the direct result of the inhibition of net phospholipid synthesis. A number of factors that interfere with β-lactam-induced lysis were investigated. (i) It was demonstrated that de-energization of the E. coli cytoplasmic membrane resulted in penicillin tolerance due to the inhibition of both the priming and the lysis induction stages. (ii) Inhibition of protein synthesis in the absence of the stringent response promoted both the priming and the lysis induction stages resulting in a faster onset of β-lactam-induced lysis. (iii) The temperature sensitivity of β-lactam-induced lysis in amino acid-deprived E. coli was re-investigated. Penicillin tolerance resulting from a temperature up-shift was not due to the induction of the heat-shock response, as previously reported, but from a reversible inhibition of unidentified thermosensitive enzyme(s) involved in the lysis induction stage. / Graduate

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.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / David G. Renter / Shiga toxin-producing Escherichia coli serogroups (O26, O45, O103, O111, O121, O145, and O157; STEC-7) are recognized as major food-borne pathogens with outbreaks, human infections, and occasional deaths associated with the consumption of contaminated foods. Cattle are recognized as the primary reservoir for STEC-7 and shed these bacteria in their feces, which are considered a principal source of contamination of cattle hides and carcasses at harvest. Pre-harvest interventions that effectively reduce fecal shedding of STEC-7 have the potential to reduce the public health concerns and economic impact of these bacteria and enhance food safety. In the research presented in this dissertation, distinct study designs were used to evaluate the impact of commercially available pre-harvest interventions and develop a better understanding of the epidemiology of STEC-7 in commercial feedlot cattle. A randomized pen-level trial indicated that a commercially available vaccine significantly reduced the fecal prevalence of STEC O157 and prevalence of high shedders compared to unvaccinated pens. However, there was no evidence of a direct-fed microbial (DFM) effect on either measure of STEC O157 shedding. In a continuum of the efficacy study, the performance and carcass characteristics associated with these pre-harvest interventions were quantified. Results indicated that feeding the DFM to cattle improved performance, whereas the vaccine negatively impacted performance during the intervention period, though most of these attributes were not reflected at the time the animals were harvested. Later, a cross-sectional observational study was used to determine the regional-, feedlot- and pen-level fecal prevalence of enterohemorrhagic Escherichia coli (EHEC), a subset of STEC, in commercial feedlot cattle. Results indicated that EHEC serogroup O157 was detected more frequently than non-O157 serogroups of EHEC; however, all feedlots had at least one sample positive for both O157 and non-O157 EHEC. Further, risk factors associated with non-O157 serogroups of EHEC were identified; further evaluation of these factors as potential control points may enable the ability to positively impact public health concerns and food safety by reducing the pathogen load prior to harvest. Overall, the research described in this dissertation provides an assessment of pre-harvest interventions and multi-level prevalence estimates of STEC-7 in commercial feedlot operations.

Escherichia coli has evolved in environments which may commonly be acidic and thus developed adaptive mechanisms to minimise acid-induced damage. It has previously been observed that adapted bacteria to moderately acidic conditions can grow in media considerably below their optimum growth pH. To explain this phenomenon, a hypothesis which suggested that diffusible molecules (alarmones) may serve as early warning systems of acidic conditions was proposed. Alarmones are thought to be produced upon exposure to mildly-acidic conditions. They then diffuse in the environment and elicit a protective response against acid in recipient cells. The protective activity of those putative alarmones against lethal acid was investigated. The main aim of this project is to determine the mode of action of those alarmones at the molecular level. Preliminary experiments confirmed acid resistance conferred by alarmones to populations of E. coli C600. The stability of those alarmones at different temperatures and following proteinase K treatment was investigated. Moreover, investigations into whether alarmones are autoinducer-2 (AI-2) molecules and whether alarmones increase the percentage of persisters in an E. coli population were undertaken. Subsequently, microarray analyses of both alarmone-induced and non-induced cultures were performed to reveal E. coli genes induced by alarmones. Moreover, proteomic studies using two-dimensional gel electrophoresis were conducted to reveal proteins induced by alarmones. Supernatants from alarmone-induced cultures conferred statistically significant protection (p<0.01) on recipient cultures against lethal acid (pH3). Alarmones were inactivated by heat (60oC) and by proteinase K. The autoinducer-2 (AI-2) assay revealed that alarmones are not AI-2 molecules. In addition, alarmones did not increase the percentage of persisters. In order to elucidate potential mechanisms for alarmone-mediated protection, the genomic expression and protein induction of alarmone-induced cells using microarray analysis and two-dimensional gel electrophoresis, respectively, were performed. Two-dimensional gel electrophoresis of transduced cultures indicated that around 13 proteins were induced in the alarmone-protected populations of E. coli C600. Mass-spectrometric analysis revealed that these alarmone-inducible proteins include the acid stress chaperone HdeB and the DNA-binding transcriptional dual regulator, H-NS which plays an important role in stress adaptation. Microarray analyses of transduced cultures indicated that 671 open reading frames (ORFs) were significantly differentially expressed between alarmone-protected and control populations (p<0.05). 508 ORFs were upregulated in the induced cells including 10 genes related to acid-resistance and 36 different genes related to multidrug efflux system proteins whereas 163 ORFs including the autoinducer-2 system were downregulated. E. coli releases diffusible signalling compounds which mediate adaptation to acid stress of recipient cells. Microarray and proteomic data show that two acid fitness island genes (hdeB and hdeD) and three genes that encode the antiporters of the three amino-acid-dependent acid resistance mechanisms (gadC, adiC and cadB) were among the upregulated genes. This work confirms that there is communication between bacterial cells in general and warning messages amongst E. coli C600 cells in particular in the presence of stress.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Escherichia coli phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxylase (transphorsphorylating) EC 4.1.1.49) catalyzes the decarboxylation and subsequent phosphorylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of Mg2+ATP and synergistic catalysis has been observed in the presence of Ca2+ or Mn2+. Structural analyses have shown that active site residues Arg333, Ser250 and Tyr207 are coordinated differently in E. coli PCK structures complexed with Mg2+ATP-oxalate, Mg2+ATP-Mn2+-pyruvate and Mg2+ATP-Ca2+-pyruvate; hence, we hypothesize that the function of Arg333, Ser250 and Tyr207, depends on the absence or presence of Ca2+ or Mn2+ during catalysis by E. coli phosphoenolpyruvate carboxykinase (PCK). In order to verify this hypothesis, site directed mutagenesis of the pckA gene was used to convert Arg333 to Gln, Ser250 to Ala and Tyr207 to Phe, while 14CO2 exchange assay and x-ray crystallography were used to determine the effects of these mutations on catalysis by E. coli PCK in the presence of OAA and Mg2+ATP with Ca2+ or Mn2+ metal ions. Kinetic analysis showed that the Tyr207Phe mutation decrease kcat by 1.7 fold, while Ser250Ala and Arg333Gln reduced kcat by 10.8 and 4,555 fold respectively in the presence of Mg2+ATP and OAA. In the presence of Mg2+ATP, OAA and Ca2+, Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 11,688, 44 and 2 fold respectively. In the presence of Mg2+ATP, OAA and Mn2+ Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 2,880, 4 and 5.5 fold respectively. The crystal structure of Ser250Ala complexed with Mg2+ATP-Mn2+-pyruvate, showed that in the presence of Mn2+, Ser250Ala mutation reduced the angle between the γ-phosphate of ATP and residue 250 by 6.2 Å and increased the distance between the hydroxyl group of Tyr207 and the CH2 group of pyruvate by 0.5 Å. As a result we conclude that Arg333 is important for oxaloacetate decarboxylation and phosphorylation. During catalysis in the presence of Mg2+ATP with or without Ca2+ or Mn2+, Ser250 functions to maintain one γ-phosphate oxygen of ATP in an eclipsed conformation, while Tyr207 functions to drive oxaloacetate decarboxylation during catalysis in the presence of Mn2+ ion. Kinetic and structural studies of E. coli PCK have previously been used to show that Asp269 is involved in metal coordination, while Lys254 and Arg65 are important for Mg2+ATP and OAA binding to E. coli PCK respectively. In this study the E. coli PCK Asp269Asn-Mg2+ATP-Ca2+-pyruvate crystal structure showed that the Asp269Asn mutation reduced the number of ligands coordinating Ca2+ from seven to three, while no electron density was observed for Mg2+ATP and OAA in Lys254Ser and Arg65Gln crystal structures respectively.

The lexA41 (formerly tsl-l) mutant was previously isolated as a UV-resistant, temperature-sensitive derivative of its UV-sensitive lexA3(Ind⁻) parent. Cells exhibit a so-called "split-phenotype", a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments. In this work, lexA41 has been cloned and sequenced; the mutant gene retains the lexA3 mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 132). LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32° and 42°C. β-galactosidase activities of lac fusions to thirteen different SOS promoters were measured at 30° and 42° to determine levels of expression and were found to vary considerably. LexA41 protein is deficient in repressor function. The temperature sensitive phenotype is due to increased expression of sulA, which encodes a division inhibitor, at 42°. Excision repair genes, including uvrA, uvrB and uvrD, are constitutively expressed at 30° accounting for the UV resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuDC, is not adequately derepressed explaining the failure to induce mutagenesis in this background. This differential expression of SOS genes gives a plausible explanation of the "split-phenotype" associated with lexA41. In another set of experiments, I have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam⁻). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD, and dinF) were found to express higher levels of (beta)-galactosidase in dam⁻ strains than in isogenic dam⁺ strains. The attempted construction of dam⁻ strains that were also mutant in one of several SOS genes indicated that viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for viability of dam⁻ strains.

The LexA repressor of Escherichia coli represses a set of genes that are expressed in response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires RecA protein. Under physiological conditions in vitro, RecA-dependent cleavage also occurs. At alkaline pH, however, the specific cleavage reaction occurs spontaneously without RecA, a reaction which is termed autodigestion. The LexA repressor is, therefore, thought to cleave itself with RecA acting to stimulate autodigestion. A set of lexA (Ind⁻) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function were isolated. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction. Many of the mutant proteins were purified in order to further characterize their properties in both autodigestion and RecA-mediated cleavage. All of these mutant proteins are found to be deficient in both cleavage reactions. A mutant protein, replacing Lys-156 to Arg, requires a higher pH condition than the wild-type protein does for both cleavage reactions. The results suggest that deprotonation of Arg-156, and by inference Lys-156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and that in the latter reaction RecA acts to reduce the pKa of Lys-156, allowing efficient cleavage of wild-type repressor under physiological conditions. Finally, several mutant proteins affecting amino acids around the cleavage site and the proposed nucleophile in the cleavage reaction (Ser-119) could not efficiently act as a competitive inhibitor in the RecA-mediated cleavage of wild-type repressor, presumably because they affect RecA binding.