Recombinant antibodies and antibody fragments have become powerful tools for therapy, in vivo and in vitro diagnostics, and laboratory research. However, the production of antibody fragments in high yield for preclinical and clinical trials can be a serious bottleneck in drug discovery. This dissertation describes the development of novel screening systems for isolating antibody fragments and alternatively, E. coli genes, that facilitate expression in E. coli. In the first part of this work, we have developed a screening system for isolating Fab mutants exhibiting 4~5 fold higher expression level at 37oC compared to the parental Fab, by utilizing the APEx 2-hybrid system and multi-color FACS as a screening tool. In the APEx 2-hybrid system, the bacterial periplasm constitutes the milieu for the association of membrane-anchored bait protein and solubly expressed, epitope-tagged prey protein. Upon disruption of the outer membrane, only prey proteins that bind to the bait remain cell-associated and are detected by flow cytometry using fluorescently labeled anti-epitope antibodies. In the second part of this work we developed a new strategy to engineer scFv that can be expressed in soluble and active form in the absence of disulfide bonds. This was achieved using a strain incapable of forming disulfide bonds in proteins expressed in its periplasm in combination with the APEx 2-hybrid system. The selected clones exhibited higher solubility, activity, and stability than that of the wild type scFv in the reducing condition of the cytoplasm. Finally, we sought to isolate E. coli gene fragments that can enhance IgG production in the periplasm of E. coli by a newly developed screening system based on soluble expression of IgG and E. coli genomic fragments. The isolated gene fragments, which are located between moeA and iaaA in the E. coli chromosome, improved the total expression of polypeptides of IgG and also assembly of IgG. / text

Drug efflux represents an important protection mechanism against antibiotics and environmental toxic compounds in bacteria. Efflux genes constitute from 6% to 18% of all transporters in bacterial genomes, yet their regulation, natural substrates, and physiological functions are poorly understood. Among the 20 chromosomally encoded efflux genes in Escherichia coli K-12, only the AcrAB-TolC efflux system is constitutively expressed under the ordinary laboratory growth of E. coli. To explore conditions and circumstances that trigger the expression of additional efflux genes as well as their physiological functions, I examined the expression of all 20 efflux genes under a physiologically relevant circumstance for E. coli, which is anaerobic condition in this study. I found that expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed that the anaerobically induced expression of mdtEF is subject to the regulation of the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance assay showed that anaerobically grown E. coli cells display an increased efflux activity and enhanced drug tolerance in an MdtEF dependent manner, confirming the functional up-regulation of the efflux pump MdtEF in the anaerobic physiology of E. coli. Since the up-regulation of mdtEF by anaerobic growth occurs in the absence of antibiotics and drugs, I speculate that MdtEF has physiological functions under the anaerobic growth of E. coli. To explore this, I first compared the viability of ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell survival during prolonged anaerobic growth of E. coli. Interestingly, this defect became more pronounced when cells grow in the presence of 10 mM nitrate, but no defect was observed in ΔmdtEF strain when cells grow in the presence of 40 mM fumarate under the same anaerobic conditions, suggesting that MdtEF has physiological roles relevant to the anaerobic respiration of nitrate. I further found that E. coli cells harboring the deletion of mdtEF are susceptible to indole nitrosative derivatives, a class of toxic by-products formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions, and deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate. Taken together, I conclude that the multidrug efflux pump MdtEF expels the nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since the production and accumulation of nitrosyl indole derivatives is ascribed to the reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the up-regulated multidrug efflux pump MdtEF functions to protect E. coli from nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Not available / text

Made available in DSpace on 2014-08-13T14:50:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-26Bitstream added on 2014-08-13T18:01:12Z : No. of bitstreams: 1 000735330_20140826.pdf: 26004 bytes, checksum: 80491d16dd561bc066d6dacbdc900451 (MD5) / A piometra canina é uma enfermidade caracterizada pela inflamação do útero com acúmulo de exsudatos, acometendo principalmente fêmeas adultas. Ocorre na fase lútea do ciclo estral em decorrência de alterações hormonais e infecção bacteriana. É reconhecida como uma das principais causas de morte em cadelas e a Escherichia coli é o principal patógeno associado a esta doença. O objetivo deste estudo foi isolar e identificar cepas de E. coli provenientes de conteúdo intra uterino, boca e fezes de cadelas diagnosticadas com piometra, estudar a prevalência de genes codificadores de fatores de virulência uropatogênicos das cepas obtidas testando ainda a semelhança genética entre as cepas isoladas de conteúdo intra uterino e boca e avaliar a susceptibilidade “in vitro” das bactérias isoladas frente a 12 agentes antimicrobianos. Setenta cepas de E. coli, 25 provenientes do conteúdo intra uterino, 26 provenientes da boca e 19 provenientes das fezes, isoladas de 6 cadelas foram examinadas por PCR. Entre as cepas examinadas, 67 (95,7%) foram positivas para o gene fim, 19 (27,1%) foram positivas para iss, 18 (25,7%) foram positivas para hly , 13 (18,5%) foram positivas para iuc e 12 (17,1%) foram positivas para usp. Três animais apresentaram grande similaridade entre as cepas de E. coli isoladas do conteúdo intra uterino e da boca, através da análise da diversidade genética realizada mediante emprego da técnica REP-PCR, ERIC-PCR e BOX-PCR. Resistência antimicrobiana predominante foi detectada para cefalotina (67,1%), ampicilina (65,7%), tetraciclina (61,4%) e nitrofurantoina (58,5%) entre as cepas isoladas. Resistência a múltiplos antimicrobianos foi detectada em 12 (48,0%) dos isolados do conteúdo intra uterino, em 22 (84,6%) dos isolados da boca e em 14 (73,6%) dos isolados das fezes... / Canine pyometra is a disease characterized by the inflammation of the uterus with accumulation of purulent discharge, affecting mainly adult animals. Occurs in the luteus phase of the estrus cycle in result of hormone alterations and generally is associated with bacterial infections. It is recognized as one of the main causes of disease and death in the bitch and Escherichia coli is the major pathogen associated with this disease. The aim of this study was to research, isolation and identification of Escherichia coli strains from intrauterine contents, mouth and feces of bitches diagnosed with pyometra, studying the prevalence of uropathogenic virulence factors genes in strains isolated, still testing the genetic similarity among strains isolated from intrauterine contents and mouth and evaluate the susceptibility in vitro of the isolated bacteria to 12 antimicrobial drugs. Seventy E. coli strains, 25 from intrauterine contents, 26 from mouth and 19 from feces isolated from six bitches were examined by PCR. Among the strains 67 (95.7%) were positive for fim, 19 (27.1%) were positive for iss, 18 (25.7%) were positive for hly, 13 (18.5%) were positive for iuc and 12 (17.1%) were positive for usp. Multiple antimicrobial resistance was detected for cephalothin (68.0%), nalidixic acid (56.0%) and ampicillin (56.0%) among the pyomera E. coli isolates. Multidrug resistance was found in 12 (48.0%) of the isolates from pyometra, in 22 (84.6%) of the isolates from mouth and in 14 (73.6%) of the isolates from feces...

Orientador: José Moacir Marin / Banca: Janete Apparecida Desidério / Banca: Maria de Fátima Martins / Resumo: A piometra canina é uma enfermidade caracterizada pela inflamação do útero com acúmulo de exsudatos, acometendo principalmente fêmeas adultas. Ocorre na fase lútea do ciclo estral em decorrência de alterações hormonais e infecção bacteriana. É reconhecida como uma das principais causas de morte em cadelas e a Escherichia coli é o principal patógeno associado a esta doença. O objetivo deste estudo foi isolar e identificar cepas de E. coli provenientes de conteúdo intra uterino, boca e fezes de cadelas diagnosticadas com piometra, estudar a prevalência de genes codificadores de fatores de virulência uropatogênicos das cepas obtidas testando ainda a semelhança genética entre as cepas isoladas de conteúdo intra uterino e boca e avaliar a susceptibilidade "in vitro" das bactérias isoladas frente a 12 agentes antimicrobianos. Setenta cepas de E. coli, 25 provenientes do conteúdo intra uterino, 26 provenientes da boca e 19 provenientes das fezes, isoladas de 6 cadelas foram examinadas por PCR. Entre as cepas examinadas, 67 (95,7%) foram positivas para o gene fim, 19 (27,1%) foram positivas para iss, 18 (25,7%) foram positivas para hly , 13 (18,5%) foram positivas para iuc e 12 (17,1%) foram positivas para usp. Três animais apresentaram grande similaridade entre as cepas de E. coli isoladas do conteúdo intra uterino e da boca, através da análise da diversidade genética realizada mediante emprego da técnica REP-PCR, ERIC-PCR e BOX-PCR. Resistência antimicrobiana predominante foi detectada para cefalotina (67,1%), ampicilina (65,7%), tetraciclina (61,4%) e nitrofurantoina (58,5%) entre as cepas isoladas. Resistência a múltiplos antimicrobianos foi detectada em 12 (48,0%) dos isolados do conteúdo intra uterino, em 22 (84,6%) dos isolados da boca e em 14 (73,6%) dos isolados das fezes... / Abstract: Canine pyometra is a disease characterized by the inflammation of the uterus with accumulation of purulent discharge, affecting mainly adult animals. Occurs in the luteus phase of the estrus cycle in result of hormone alterations and generally is associated with bacterial infections. It is recognized as one of the main causes of disease and death in the bitch and Escherichia coli is the major pathogen associated with this disease. The aim of this study was to research, isolation and identification of Escherichia coli strains from intrauterine contents, mouth and feces of bitches diagnosed with pyometra, studying the prevalence of uropathogenic virulence factors genes in strains isolated, still testing the genetic similarity among strains isolated from intrauterine contents and mouth and evaluate the susceptibility "in vitro" of the isolated bacteria to 12 antimicrobial drugs. Seventy E. coli strains, 25 from intrauterine contents, 26 from mouth and 19 from feces isolated from six bitches were examined by PCR. Among the strains 67 (95.7%) were positive for fim, 19 (27.1%) were positive for iss, 18 (25.7%) were positive for hly, 13 (18.5%) were positive for iuc and 12 (17.1%) were positive for usp. Multiple antimicrobial resistance was detected for cephalothin (68.0%), nalidixic acid (56.0%) and ampicillin (56.0%) among the pyomera E. coli isolates. Multidrug resistance was found in 12 (48.0%) of the isolates from pyometra, in 22 (84.6%) of the isolates from mouth and in 14 (73.6%) of the isolates from feces... / Mestre

Over the past few years, the use of enzymes as catalysts for the preparation of novel organic molecules has received a steadily increasing amount of attention. Lipolytic enzymes are widely distributed in nature and attract great attention because of their biotechnological potential, as they catalyse the enantio- and regioselective hydrolysis and synthesis of a broad range of natural and non-natural esters. Bacteria produce different lipolytic enzymes, such as esterases (EC 3.1.1.1), which hydrolyse ester-containing molecules at least partly soluble in water, and lipases (EC 3.1.1.3), which hydrolyse water-insoluble long-chain triglycerides. In this study, a bacterial isolate, B. coagulans strain 81-11, isolated from popcorn seeds, was characterized with the specific aim of isolating and characterizing genes encoding novel lipolytic enzymes. A genomic library of B. coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4-kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723-bp open reading frame (ORF), designated estCl, encoding a protein of 240 amino acids with an estimated molecular mass of 27 528 Da and a pI of 9.15. The deduced amino acid sequence of the estCl gene exhibited significant amino acid sequence identity with carboxyl esterases and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrate confirmed the anticipated esterase activity. EstCl exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50°C, although the enzyme was active in the pH range 7-9 and displayed activity at temperatures up to 55°C. Since bacterial esterases are potentially important for a variety of biotechnological applications, there is a considerable industrial interest to produce these enzymes at a larger scale. Among the many systems that are available for heterologous protein production, attempts were made to over express the newly identified B. coagulans estCI esterase¬encoding gene in different Gram-positive bacteria, as they are well known for their important contribution to food biotechnology and as production organisms for industrial enzymes. A recombinant expression vector was thus constructed (pMG36-EstCl) and introduced in Lactococcus lactis, Lactobacillus plantarum and Bacillus subtilis strains 154 and lA297. Of these different bacterial hosts, high levels of intracellular esterase activity were detected in B. subtilis lA297 only. In an attempt to increase extracellular expression of the B. coagulans EstCl esterase, a recombinant secretion plasmid (pNW-EstClaps) was constructed that contained an alkaline protease promoter and signal sequence from a Bacillus species. Following introduction of the construct in B. subtilis lA297, the derived recombinant strain displayed 2.3-fold higher extracellular esterase-activity levels than the parent B. coagulans strain, and the extracellular esterase activity represented 82% of the total esterase activity. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Three overlapping DNA fragments from the lower operon of Pseudomonas putida TOL plasmid pDK1, covering the xy1IH genes and downstream flanking region, were cloned into pUC19. They include a 2.8 kbp XhoI fragment, a 2.7 kbp PstI fragment and a 2.0 kbp EcoRI-HindIII fragment. They were subjected to DNA sequence analysis. The xy1I (4-oxalocrotonate decarboxylase) and xy1H (4-oxalocrotonate tautomerase) genes were found to possess coding regions of 792 and 189 nucleotides, respectively. A possible transcriptional terminator resembling E. coli rho-independent terminators was identified downstream of the translational stop of xy1H. An additional stem and loop structure was found in the intergenic region between xy1I and xy1H. The individual ORF's of the oxalocrotonate branch (xy1G, xy1I and xy1H) have been cloned into pUC18/19. The expression of the xy1G gene in E. coli was successfully assayed spectrophotometrically.

In Cellulomonas fimi the cenB gene encodes a secreted endoglucanase (EngB) involved in the degradation of cellulose. The cenB gene carried on a 5.6 kb C fimi DNA fragment encodes a polypeptide of Mr 110,000 in Escherichia coli. The level of expression of the gene was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. The intact EngB polypeptide is not required for enzymatic activity: active polypeptides of Mr 95,000 and 82,000 also appear in E. coli and a deletion mutant of cenB encodes an active polypeptide of Mr 72,000. The intact and truncated EngB both bind to microcrystalline cellulose. A simple, rapid affinity chromatography procedure on Avicel was developed for the purification of intact EngB and of the 72,000 deletion derivative. Alignment of the amino-terminal amino acid sequence of the purified intact EngB from E. coli with the partial nucleotide sequence of the cloned C. fimi DNA showed that the mature EngB is preceded by a sequence encoding a putative signal polypeptide of 32 amino acids, a translational initiation codon and a sequence resembling an E. coli ribosome binding site 4 nucleotides before the initiation codon. The signal peptide functions and is correctly processed in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of β-galactosidase. The truncation of EngB does not affect its export to the periplasm of E.coli. In the intact EngB, 25% of the residues are hydroxyamino acids. It displays features common to endo-β-1 ,4-glucanases, since it has a high activity on carboxymethylcellulose. The kinetic parameters for carboxymethylcellulose hydrolysis of both intact and truncated EngB are not significantly different. C. fimi protease cleaves intact EngB, in a specific manner, to generate two polypeptides of Mr 65,000 and 43,000; the former has the capacity to bind Avicel. A polyclonal antibody raised against the purified intact EngB recognizes a C. fimi extracellular protein of M 110,000 as well as 5 polypeptides of lower molecular weight. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

All of the glp genes of Escherichia coli are subject to negative regulation by the glpR-encoded repressor (GlpR). Comparison of the repressor binding affinity for consensus and altered consensus operator regions showed that positions 3, 4, 5, 7 and 8 bp removed from the center of operator symmetry are most important for repressor binding. Cooperative binding of repressors to tandem operators was demonstrated. Cooperativity was maximal when two 20-base pair operators were directly repeated and decreased with the deletion of 2 base pairs or the addition of 4 base pairs between the operators. Cooperative binding was eliminated by a 6 base pair insertion between tandem operators. / Master of Science