Orientadores: Antonio Fernando Pestana de Castro, Jorge Blanco Alvarez / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:25:24Z (GMT). No. of bitstreams: 1 Aidar_Leila_M.pdf: 4916322 bytes, checksum: 5ce886241ad2f530966d97c38fe5219c (MD5) Previous issue date: 1999 / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

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Escherichia coli (E. coli) frequently experiences changes in environmental osmolarity thus requiring homeostatic adjustments for proper cell function. In a hypoosmotic shock the central players are the mechanosensitive channels (MS channels), considered the major routes for the release of cytoplasmic solutes to achieve rapid reduction of turgor pressure. In the present study a combination of in vivo and in vitro techniques were used to investigate three of the seven MS channels present in the genome of E. coli, these channels being MscL, MscS and MscK. The first chapter focused on three residues (R46, R59 and K60) in the transmembrane helices TM1 and TM2 of MscS. It has been hypothesized that these residues act as part of the tension sensor mechanism, interacting either with membrane phospholipids or ions within the pore of the channel. In addition to MscS, the second chapter included MscL and MscK and analyzed the precise localization and diffusion rate of these proteins in the cytoplasmic membrane by the use of a super resolution fluorescence microscopy technique. The same technique was used in the third chapter to quantify the two main MS channels, MscL and MscS. As well as providing a single cell census these analyses provided a correlation between abundance of channels and cells survival during a downshock, the situation in which these channels gate and protect cells. Quantification revealed a high abundance of these proteins, thus MS channels were also investigated for an alternative role, namely the release of solutes during hyperosmotic shocks. This last chapter focused specifically on glutamate excretion, which is part of the hyperosmotic response. In summary, the results presented in this thesis substantially increased our knowledge of MS channels, both from a functional and a quantitative perspective.

The cytochrome o and cytochrome d oxidase complexes provide twin termini for the branched respiratory chain of aerobically grown Escherichia coli. Combined use of mutant strains, modulated growth conditions and high resolution analytical techniques enabled cytochromes to be resolved, identified and partially characterized. The cytochrome complement of everted membrane vesicles and detergent extracts fractionated by liquid chromatography is more complex than previously recognised. Multiple type-b cytochromes were resolved by potentiometry and by high resolution spectrophotometry in membrane vesicles from mutant strains lacking the cytochrome d oxidase complex and grown under conditions minimising respiratory chain diversity. Cytochrome o was identified with Em = +235 mV (vs. NHE) as were low potential cytochromes associated with dehydrogenases. Spectrally distinct components of the cytochrome d complex yielded Em values of +125 mV (cytochrome 6595) and +187 mV (cytochrome d). The latter displayed atypical redox behaviour with extreme hysteresis during potentiometric titrations. Several cytochromes b₅₅₆ displaying single, symmetrical redox α-bands at 77 K were resolved from detergent extracts of vesicles. Mutant strains identified one with Mr = 52500 (gel filtration) and Em = +20 mV as the sdhC gene product, a component of succinate dehydrogenase. DL-lactate induced another while a hydroperoxidase, Mr = 386000 (gel filtration) with twin Em values of -2mV and -121 mV and a split Soret absorption band at 77 K (λ[symbol omitted]max= 426.0 nm + 434.0 nm) was produced under limited oxygen tension. The Triton-solubilized and purified cytochrome 0 complex exhibited Mr = 516000 (gel filtration) with five component peptides of Mr= 55000, 32000, 31000, 21000 and 16000 (SDS-PAGE). It displayed mid-point potentials of -58 mV, +127 mV and +260 mV and three a-absorption maxima at 77 K : 554.5 nm, 557.0 nm and 563.5 nm. These components were reduced equivalently during poised-potential low temperature spectrophotometric analyses. Carbon monoxide binding changed the complex's redox α-absorption spectrum minimally but shifted the high potential Em to approximately +420 mV. Quinone analogues inhibited both reduction and reoxidation of the complex. Cytochrome o complex prepared from cloned sources contained a significantly greater proportion of the component with mid range electrochemical potential absorbing at 554.0 nm. These results are discussed in relation to possible structures of the complex, its respiratory interactions and the identity of cytochrome o itself. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The acetohydroxy acid synthetase levels in streptomycin-sensitive, -dependent and -resistant mutants have been studied in four different strains of Escherichia coli. The activity of the ∝-acetolactate-forming system was found to be greater both at pH 6.0 and at pH 8.0 in streptomycin-dependent mutants than in the corresponding streptomycin-sensitive cultures. In general, streptomycin-resistant mutants demonstrated enzyme activities within the range found for streptomycin-sensitive organisms regardless of whether they were grown in the presence or absence of antibiotic. The acetohydroxy acid synthetase activity of streptomycin-sensitive and -resistant revertants was observed to be lower than that of the dependent Escherichia coli culture from which they were derived by back-mutation. Mutation to streptomycin-resistance or -dependence had no effect on glucokinase and glutamic dehydrogenase activities. The addition of the coenzyme flavin adenine dinucleotide to the incubation mixtures markedly stimulated the activities of all the extracts. This enhancement of acetohydroxy acid synthetase activity had little or no effect on the ratio of activities of this enzyme in the dependent and sensitive Escherichia coli strains investigated. ∝-Acetohydroxybutyrate formation was found to be greater in extracts from the streptomycin-dependent organism than in extracts prepared from the same strain of sensitive and resistant Escherichia coli. The degree of elevation of ∝-acetohydroxybutyrate paralleled that of ∝-acetolactate formation in the dependent mutant. It was concluded from these observations that excretion of L-valine by streptomycin-dependent Escherichia coli was a consequence of the elevated acetohydroxy acid synthetase activity of these mutants. In the dependent organism, it was postulated that streptomycin functioned as a wde-repressorw of acetohydroxy acid synthetase thus permitting the biosynthetic pathway leading to L-valine to serve as an important route of pyruvate dissimilation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The compound α-acetolactate has been prepared by the chemical method of Krampltz (1948) and by an enzymatic procedure. The products obtained by each method were characterized chromatographically and spectrophotometrically by conversion to their 2,4—dinitrophenylhydrazone derivatives as well as by conversion to acetoin. The Identity of the hydrazone of enzymatically prepared α-acetolactate with hydrazones of the chemical product was established by comparison of R[subscript: F] values, the identical absorption maxima, and by the infrared spectra of these compounds. The α-acetolactate so obtained was used as substrate in a comparison of the activity in streptomycin mutants of the reductoisomerase enzyme which catalyzes the rearrangement and reduction of α-acetolactate to α,β-dihydroxyisovaleric acid. This reaction is the second step in valine biosynthesis. Streptomycin-dependent mutants of Escherichia coli previously has been shown to be derepressed in acetohydroxy acid synthetase, the enzyme which initiates valine biosynthesis. In addition, it had been reported previously that in streptomycin-dependent E. coli K-12, threonine dehydratase, the enzyme which initiates biosynthesis of isoleucine, also is derepressed. In contrast, reductoisomerase, which is common to both the valine and isoleucine pathway, has been found in this work to be normal (i.e., not derepressed) in streptomycin mutants. An additional enzyme of the common pathway, transaminase B, was found to be about 2 to 3 times higher in streptomycin-dependent mutants than in sensitive or resistant strains. The structural genes for both transaminase B and threonine dehydratase of E. coli K-12 have been shown by genetic studies (Ramakrishnan and Adelberg, 1965b) to be coordinately regulated (i.e., on the same operon). The observations made in this study with streptomycin-dependent E. coli K-12 support the observations of these workers. That is, transaminase B of streptomycin-dependent E. coli K-12 is derepressed coordinately with threonine dehydratase. However, the degree of derepression of transaminase B (about 2 to 3 fold) was much less than that of threonine dehydratase (about 9 fold, according to Desai and Polglase, 1966). It may be concluded from these studies that the type of derepression of certain enzymes which has been observed in streptomycin-dependent E. coli has contrasting features to the type of derepression which would be expected on the basis of the Jacob and Monod model (1961) from a nonfunctional regulatory gene or product thereof. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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