The role of the cell envelope in cell division has been examined using mutants of Escherichia coli which are temperature-sensitive in the process of cell division. These mutants grow normally at 30 C but exhibit morphological changes at 42 C. Two assays for peptidoglycan-specific autolytic enzymes have been developed. One depends on the prevention of synthesis of autolytic enzymes using chloramphenicol, while the autolytic capacity is determined by observing the rate of cell lysis in the presence of low levels of ampicillin. The second assay is based on the in vitro release of specific radioactive fragments from peptidoglycan previously labelled with radioactive diaminopimelic acid. The majority of cells in an exponential population of E. coli B/r/l are not lysed by penicillin if chloramphenicol is added. However, larger cells are particularly susceptible to lysis. Three types of lysis have been defined by observing rates of lysis of synchronous cultures at different ages and at different growth rates: (A) lysis associated with cross-wall formation during cell division; (B) lysis associated with initiation and segregation of the replication of DNA; and (C) lysis associated with general expansion of the cell wall. Filamentous mutants of E. coli which segregate DNA exhibit lysis in excess of type C while filaments formed by inhibition of DNA synthesis exhibit lysis only of type C. The autolytic enzymes of E. coli appear to be tightly bound to localized areas of the cell envelope as determined by the in vitro assay. Qualitative differences between the autolytic activities of normal cells and of filaments formed by inhibition of DNA synthesis are described. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A temperature-sensitive division mutant, Escherichia coli BUG-6, has been investigated. This organism divides normally when grown at 30 C but fails to divide at 42 C. Growth continues at 42 C to produce very long, multinucleate filamentous cells. When returned to 30 C, or a high osmotic environment is added at 42 C, the filamentous cells divide rapidly to produce cells of normal size. The kinetics of cell division of the filaments at 30 C depends on the period at 42 C. During filament formation, DNA, RNA and protein synthesis continue as measured by radio-isotopic incorporation and chemical cell fractionation. DNA segregation occurs as shown by autoradiography. The rapid division of filaments replaced at 30 C cannot be prevented by novobiocin or cycloserine but is prevented by vancomycin and penicillin suggesting that de novo synthesis of cell wall precursors is not required for division but that positioning and cross-linking of the precursors is required. Filaments growing at 42 C were treated with nalidixic acid for different lengths of time. On returning these filaments to 30 C in nalidixic acid the number of divisions was proportional to the length of time at 42 C in the absence of nalidixic acid, ie. proportional to the amount of DNA synthesized at 42 C. Inhibition of protein synthesis, by chloramphenicol, does not prevent the division of filaments on replacing at 30 C provided that the period of filamentation at 42 C was greater than 6 minutes and less than 110 minutes. The maximum amount of division in the absence of protein synthesis occurred after a longer lag and slower than in non-inhibited control cultures. If protein synthesis was inhibited in filaments at 42 C the ability of such treated cells to divide at 30 C was rapidly lost. This loss of 'division potential’ has a half-life of about 0.5 minutes, ie. 0.5 minutes of protein inhibition at 42 C reduces the subsequent division at 30 C by 50%. The normal presence of 'division potential', therefore requires the synthetic doubling rate to be in excess of 0.5 minutes. Very short periods at 42 C indicate that 10 minutes incubation at 42 C is required to produce this extremely fast synthetic rate. A model for the production and expression of 'division potential' is presented. A biochemical analysis of the cell envelope of the filamentous cells and of normally dividing cells is presented. The major phospholipid compositions are the same. However, the fatty acid contents differ especially with regard to the cyclic fatty acids. When the filaments are allowed to divide by replacing at 30 C their fatty acid composition very rapidly reverts to that of normally dividing cells. The rates of individual phospholipid syntheses appear to change during the rapid cell division phase, however this may be an artifact resulting from an overall increase in the rate of phospholipid synthesis during this period. An analysis of the proteins within the cell envelope by radio-active double labelling techniques and followed by gel electrophoresis indicates that a protein(s) of molecular weight 80,000 - 90,000 exists in the envelope of filamentous cells which is not in the envelope of normal cells or is made at a much slower rate in normal cells. The protein is not incorporated into septa during the division of filaments at 30 C and little turnover occurs in the major proteins synthesized at 42 C when these cells are placed at 30 C. The possibility exists, however, that this protein is a product of filamentation and not the temperature sensitive gene product. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The regulation of RNA polymerase subunit synthesis and its relationship to the expression of ribosome component genes have been investigated in strains of Escherichia coli having a mutation in one of the genes specifying either the β or β' subunit of the core enzyme.' Particular attention has been focused on the L10 transcriptional unit (organization: P[sub= L10] – rp1J(LlO) - rplL(L7/L12) - attenuator - rpoB(β) -rpoC(β ') ). The mutant strain XH56 produces a defective β ' subunit which renders the RNA polymerase inactive in transcription initiation at 42°C; at somewhat lower temperatures, the RNA polymerase activity is only partially restricted. A temperature shift of this strain from 30°C to 39°C resulted in a rapid 5-fold increase in the transcription of the rpo β and CI genes and in the synthesis rates of the β and β' subunits, indicating that β and β' synthesis is regulated primarily at the transcriptional level. Transcription of the α subunit gene, located in the spc-str region of the chromosome, was also enhanced. Transcription of the lacZ gene (coding for β -galactosidase) was decreased to undetectable levels, indicating that the dramatic increase of rpo β and C. transcription occurred at the expense of transcription of other operons. The mutant strains Ts4 and A2R7 produce defective β' and β subunits respectively which are unable to assemble into core RNA polymerase at the nonpermissive temperature. In these strains RNA polymerase assembled prior to a temperature shift from 30°C to 42°C retains its activity but little or no enzyme is assembled after the shift. Prolonged incubation of these strains after such a shift produced a gradual 1.5- to 2-fold increase in the transcription of the rpoβ and C genes and in the synthesis rates of the β and β' subunits. During the restrictions, transcription of ribosome component genes was essentially unchanged. RNA polymerase assembly was also inhibited in strains carrying both a temperature-sensitive amber suppressor mutation and an amber mutation in the rpoβ gene. Under permissive conditions these amber mutations are suppressed by insertion of serine into the β protein at the UAG codon. After a temperature shift to 42°C, core RNA polymerase synthesis is restricted due to the failure to produce 3 in the non-polar amber strain MX515 and both β and β' in the polar amber strain MX515. Core enzyme synthesized prior to the shift retains its activity. Inhibition of core enzyme synthesis in this manner resulted in a gradual stimulation of rpoβ and C transcription; in the polar strain this was accompanied by a concomitant increase in the synthesis rate of the β' subunit protein. The increase of rpoβ and C transcription involved both increased initiation at P[sub L10] and relaxed termination in the rp1L-rpoβ intergenic space. It was also observed that transcription of the a subunit gene was specifically stimulated during the restriction, suggesting that the regulatory mechanisms are specific for genetic units containing core RNA polymerase genes. These results therefore indicate that the mechanisms which govern the transcriptional frequency of operons containing RNA polymerase genes are coupled to the demand for active RNA polymerase; a sudden restriction of enzyme activity produces a rapid and dramatic increase of rpoβ and C transcription whereas a slow restriction results in only a gradual and less extensive induction. The regulatory mechanisms operating within the L10 transcription unit were accentuated by introducing the composite colEl plasmid pJC701 into the RNA polymerase activity mutant strain XH56.. All of the genes in the L10 transcription unit except the distally located rpoC are present on this plasmid and therefore were amplified in the transformed bacteria. The partial temperature inactivation of RNA polymerase activity in this strain allowed us to modulate the transcription of the proximal rp1J-rp1L genes and the distal rpoβ gene over a 10-fold and 30-fold range respectively. The observed imbalance in transcription between the proximal and distal portions of the L10 transcription unit strongly. suggest that the restriction has :two distinct effects: (i) it stimulates initiation at the major L10 promotor and (ii) it reduces termination at the attenuator located within or near the rp1L-rpoβ intergenic space. The synthesis rates of L7/L12 and β subunit proteins were also measured and compared to their respective mRNA levels under these conditions. The synthesis rate of L7/L12 protein and β protein varied by less than 2-fold and by 15-fold respectively. These measurements clearly indicate that translation of excess L7/L12 ribosomal protein mRNA is severely restricted and contributes to maintaining the balanced synthesis of ribosome components. The translational efficiency of 3 mRNA was also reduced by about 50%. Under the above conditions, β protein is produced in large excess relative to β' subunit protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Escherichia coli can produce several respiratory chains which transfer electrons to oxygen, nitrate or other acceptors. Cytochromes are amongst the electron carriers of these chains, and can be studied by a number of spectroscopic techniques. Of particular interest is the formate-nitrate reductase respiratory pathway which is composed of two complexes, formate dehydrogenase and nitrate reductase each with a cytochrome associated. The cytochromes of E. coli after growth under a variety of conditions have been studied by spectrophotometry redox titration and other spectroscopic methods. These have shown that, contrary to some previous reports, there are at least six cytochromes produced irrespective of the culture conditions used. This emphasizes the need for simplified systems and two of these have been investigated. By fractionation of cytochromes prior to spectroscopic analysis a cytochrome b of redox potential OmV was identified in cells grown 556 aerobically. In addition the association of two cytochromes of potential +20mV and +120mV with nitrate reductase was demonstrated. Secondly the formate-nitrate reductase pathway has been investigated by spectroscopic studies of chi mutants which are defective in this activity. Three phenotypes of cytochrome production were observed. Mutants at loci associated with the production of the cofactor for both nitrate reductase and formate dehydrogenase were shown to produce the same cytochromes as the wild-type. Mutants mapping at the chlC locus and defective in nitrate reductase but not formate dehydrogenase were found to lack only the two cytochromes associated with nitrate reductase. They had high leyels of a cytochrome of redox potential -100mV which was shown to be associated with formate dehydrogenase. A third class of pleiotropic regulatory mutants was identified which was not related with a specific genotype. These produced none of the anaerobic respiratory pathways but overproduced the aerobic respiratory pathway leading to cytochrome d. The second aerobic respiratory pathway leading to cytochrome o was most evident in double mutants lacking both of the cytochromes associated with nitrate reductase and that associated with formate dehydrogenase. On the basis of these results a model for the arrangement of the cytochromes in the respiratory chains of E. coli is proposed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Two major proteins of the outer membrane of Escherichia coli, the matrix protein, A (M.W. 36,500) and the heat-modifiable protein, B were purified and partially characterized. Both have a low content of cysteine, . an excess of acidic amino acids over basic and a moderate content of hydrophobic amino acids. Protein B (M.W. 28,500) was converted to form B* (M.W. 33j^00) upon heating in the presence of sodium dodecyl sulfate at temperatures higher than 50°C. Physical studies showed that protein B unfolds upon heating without a large increase in binding of sodium dodecyl sulfate. It is proposed that protein B as extracted from the membrane contains some native structure which is lost upon heating. The level of protein A1, a major outer membrane protein in glucose-grown cells, was decreased in cells grown on other carbon sources with a concomitant increase in the amount of protein A2. Both proteins were tightly associated with the peptidoglycan and had similar amino acid composition, suggesting that they play the same role in the outer membrane. The organization of proteins in the outer membrane of E. coli was studied by proteolytic digestion, covalent labelling and crosslinking. The proteins of the outer membrane were inaccessible to pronase in intact cells and the cells altered in their lipopolysaccharide component. The protein components of isolated outer membrane preparations varied in their rates of digestion and labelling with fluoresca- ' mine, suggesting that they are asymmetrically arranged in the membrane. The proteins most rapidly degraded (proteins B,C-,D1 and E) were judged to be exposed at the surface of the membrane, while those resistant to digestion (proteins A1,A2 and D2) must be protected by their arrangement in the membrane. Digestion of outer membrane preparations with pronase left a fragment derived from protein B (protein Bp) embedded in the membrane. This fragment was not enriched in hydrophobic amino acids relative to protein B. Protein B could be reassociated with itself j without phospholipid or lipopolysacchari.de such that pronase digestion of the reassociated material gave protein Bp. These results suggest that protein B may not be held in the membrane primarily by hydrophobic interactions. The resistance of proteins A1 and A2 to protease digestion is likely due to protein-protein interactions since oligomers of protein A could be isolated. Treatment of protein A1- or A2-peptidogly-can complexes with dithiobis (succinimidyl propionate) or glutaraldehyde produced dimer, trimer and higher oligomers of protein A. No crosslinking of protein A to the peptidoglycan was detected. The proteins of the isolated outer membrane varied in their ease of crosslinking. Protein B, but not the pronase-resistant fragment, protein Bp, was readily crosslinked to give high molecular weight oligomers, while protein A formed dimers and trimers under the same conditions. No crosslinking of protein A to B was detected. Crosslinking of cell wall preparations showed that protein B and the free form of the lipoprotein, F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. These results suggest that specific protein-protein interactions occur in the outer membrane. A model for the arrangement of the proteins in the outer membrane of E. coli, summarizing the results of proteolytic digestion, covalent labelling and crosslinking, is presented. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

During amino acid deprivation, the amount of meso-diaminopimelic acid (Dap) incorporated into peptidoglycan by dap lys amino acid auxotrophs of Escherichia coli was found to be dependent on the activity of the relA gene product. In relA⁺ bacteria, the- incorporation was substantially reduced, whereas the incorporation in relA⁻ bacteria was essentially equal to that in the unstarved control. The inhibition of Dap incorporation in relA⁺ bacteria was readily overcome by restoration of the required amino acid or by addition of chloramphenicol (CAM). Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is the product of the reaction between the relA gene product and idling ribosomes in stringent cells. In vitro experiments indicated that physiological levels of ppGpp inhibited at least two steps in peptidoglycan biosynthesis. One was the phospho-N-acetylmuramoyl-pentapeptide transferase (EC2.7.1.13) reaction and the other inhibition was probably at the transfer of peptidoglycan precursors from the glycosyl carrier lipid (GCL) to the nascent peptidoglycan. Quantitation of the peptidoglycan precursors and the net peptidoglycan in relA⁺ control and amino acid-deprived bacteria indicated that peptidoglycan accumulation was inhibited. There was as much UDP-MurNAc-pentapeptide and GCL-linked intermediates in the amino acid-deprived bacteria as in the control bacteria. This suggests that the transfer of lipid-linked precursors to nascent acceptor is the site of inhibition of Dap incorporation. In addition, the pool of soluble nucleotide-linked precursors was found to accumulate when relA⁻ bacteria were deprived of required amino acids. This suggests that the size of the precursor pool is also regulated by the activity of the relA gene product. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Le cancer colorectal est un des cancers les plus fréquents en France. Les patients présentant une inflammation chronique de l'intestin sont à haut risque de développer un cancer colorectal. Il a récemment été démontré que certaines bactéries de l'espèce Escherichia coli pouvaient être impliquées dans la genèse de maladies inflammatoires intestinales, par des mécanismes d'adhésion et d'invasion aux cellules épithéliales intestinales faisant intervenir le récepteur CEACAM6, marqueur tumoral reconnu dans le cancer colorectal. Nous avons montré que certaines bactéries de l'espèce E. coli colonisaient la muqueuse colique de patients atteints de cancer colorectal et possédaient des propriétésd'adhésion et d'invasion dans les cellules épithéliales intestinales. Ces souches synthétisent des cyclomodulines variées susceptibles de jouer un rôle dans la carcinogenèse colique, et colonisent davantage les tumeurs les plus évoluées. Une corrélation a été observée entre la colonisation de la muqueuse colique par un clone unique de ces E. coli et l'expression du récepteur CEACAM6. Nous avons montré in vitro que certains clones de pouvaient induire l'expression de CEACAM6 par des cellules épithéliales intestinales en culture. Enfin, sur modèle animal transgénique exprimant le récepteur humain CEACAM6, ces mêmes clones ont montré des capacités de colonisation très supérieures à desE. coli non pathogènes, et l'induction d'une surexpression de marqueurs de prolifération au niveau de la muqueuse colique. Les E. coli associés à la muqueuse colique pourraient ainsi participer à la promotion du cancer colorectal via l'induction d'une surexpression du récepteur CEACAM6 et d'une hyperprolifération des cellules épithéliales. / No abstract available

Orientador : Domingos da Silva Leite / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-31T17:42:47Z (GMT). No. of bitstreams: 1 Silva_AlexSouzada_M.pdf: 2809054 bytes, checksum: a7208782bc39f37dd5ae5103b7c97476 (MD5) Previous issue date: 2002 / Mestrado

Orientadora : Marlene Braide Serafim / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-16T07:14:56Z (GMT). No. of bitstreams: 1 Said_AparecidaCellideAlmeida_M.pdf: 2680850 bytes, checksum: 6c29a5202e3546b45fb97f05fb19511b (MD5) Previous issue date: 1985 / Resumo: Trabalhamos com 64 amostras Escherichia coli, já sabidamente produtoras de enterotoxina termolábil (LT), provenientes de diversas fontes, (humanas, suínas, alimentos e água de rio). Essas amostras foram examinadas nos testes de imunohemólise passiva (IHP) e ELEK modificado (Biken), procurando-se comparar o desempenho desses dois testes na detecção de LT, utilizando-se para isso os antissoros: anti LT humano (anti-LTh); anti LT suíno (anti-LTs); antitoxina colérica (anti TC) e anti coleragenóide (anti-Cg). As toxinas LT de origem humana (LTh) e de origem suína (LTs), necessárias para obtenção dos respectivos antissoros, foram purificadas segundo o método de Clementes & Finkelstein (6), e posteriormente inoculadas em coelhos seguindo-se o esquema de imunização proposto por Honda et alii (33). Já o soro ant-Cg, e a toxina colérica purificada nos foram doados, sendo o soro ant-TC obtido através de inoculação da mesma em cavalo, seguindo-se o mesmo esquema de imunização utilizado para a obtenção dos antissoros anti-LTh e anti-LTs (33). Os antissoros foram então titulados nos testes de IHP e Biken, frente as amostras padrão de Escherichia coli produtora de LT, de origem suína. Uma vez conhecidos os títulos dos mesmos, passamos ao exame das 64 amostras com cada um dos antissoros, em ambos os testes, IHP e Biken ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Not informed. / Mestrado / Mestre em Ciências Biológicas

Orientador: Antonio Fernando Pestana de Castro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-16T22:05:00Z (GMT). No. of bitstreams: 1 Ricci_LucilaCostallat_D.pdf: 10174303 bytes, checksum: ecb6e9b94df041a1820a718f3691aec2 (MD5) Previous issue date: 1987 / Resumo: Nas reações sorológicas de imuno-hemólise passiva (IHP), imuno-hemólise radial (IHR) e hemaglutinação indireta (IH), usadas na detecção das enterotoxinas LTs de Escherichia coli, de origem humana e suína e toxina colérica, estas se absorvem naturalmente as hemácias de carneiro, sem a necessidade de qualquer agente ligante. Esta ligação só poderia ser explicada, pela presença de receptores específicos para estas essas enterotoxinas nessas células, que até o momento, não haviam sido identificados e caracterizados. Como o propósito de esclarecermos a natureza desses receptores, nos propusemos, no presente trabalho, a estudar os seguintes itens: a) Verificar a sensibilização de eritrócitos de galinha cobaia, boi e do homem, pelas enterotoxinas LTs, de origem humana e suína e TC, a fim de obtermos subsídios para podermos comparar eventuais diferenças nos receptores para estas toxinas em hemácias, além de uma provável adaptação das técnicas sorológicas de IHP, IHR e HI, à essas células. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Not informed. / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular