66 research outputs found
Reversal of the glucose inhibition of histidase biosynthesis in Aerobacter aerogenes
No full textGlucose inhibits the biosynthesis of several inducible enzymes in Aerobacter aerogenes. Previ-ous studies (Neidhardt and MIagasanik, 1956a) have indicated that this inhibitory effect is not due to (a) failure of the inducing agents to pene-trate the cell, (b) the high growth rate of the cells in a glucose-containing medium, or (c) a deficit of the substances known to be essential for protein formation. Attempts to reverse this inhibition of enzyme biosynthesis by enrichment of the mini-mal medium with amino acid, purine-pyrimidine, or vitamin supplements have been unsuccessful (Neidhardt and M\Iagasanik, 1956a). The studies reported in this paper describe a different ap-proach to this problem: rather than enriching the glucose medium with organic supplements, the organism was placed in a situation in which the formation of an inducible enzyme in the presence of glucose was a prerequisite for growth. MATERIALS AND METHODS Organism and media. Two strains of A. aeroge-nes were employed in these experiments. One was strain XXXV, the wild type previous4y de-scribed (Neidhardt and M1agasanik, 1956a) and the other was strain A215B-19, an L-glutamic acid-requiring mutant kindly supplied by Dr
The effect of temperature on the protein synthetic capacity of Escherichia coli.
No full textThe growth rate of Escherichia coli depends on growth medium and temperature. At constant temperature, only enough of the protein synthetic machinery is made to meet cellular needs. The primary goal of this thesis is to determine how E. coli regulates its protein synthetic capacity with respect to changes in temperature. Within the normal range (23 to 37\sp\circC), where growth rate increases with increasing temperature as though it were a simple chemical reaction, cells were found to contain a constant number of ribosomes, and average peptide chain elongation rate was found to increase with increasing temperature parallel to growth rate. At high temperature (42\sp\circC), cells were found to contain the same number of working ribosomes as within the normal range, but peptide chain elongation rate was found to increase with temperature even though growth rate does not. These findings indicate that peptide chain elongation is not rate limiting for growth at 42\sp\circC, and that these cells contain an apparent excess of protein synthetic capacity. One possible explanation is that protein degradation increases at high temperature. Temperature shift experiments show that peptide chain elongation rate increases immediately after an increase in temperature. These findings support a mechanism of heat shock response induction in which an increase in newly synthesized, unfolded protein induces this response. Cells growing at low temperature ({<}20\sp\circC) contain a larger pool of ribosomal subunits than cells growing in the normal range. Peptide chain elongation rate is not rate limiting for growth at low temperature and varies more than 50% among individual proteins. This is not the result of variation in the usage of rare codons, since all the proteins tested were highly expressed and contain few rare codons. At all temperatures the elongation rates measured by the time it takes to make the first molecule of -galactosidase were consistently slower than the rates obtained from pulse-labeling experiments. A model involving transcriptional or translational pausing is proposed to explain these results.PhDCellular and Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103577/1/9332054.pdfDescription of 9332054.pdf : Restricted to UM users only
Analysis of htpI, a conditionally essential heat shock gene of Escherichia coli.
No full textThe heat shock response of E. coli involves the induction of at least 17 genes under the positive transcriptional control of an alternative sigma factor, -32. Eight of the genes have been at least partially characterized and have been shown to be involved in most of the major cellular functions: protein turnover, transcription-translation, cell division, and macromolecule assembly. This study explores one of the heat shock genes and its protein product. A set of random, temperature-inducible Mud-Lac fusion strains were examined by two-dimensional gel electrophoresis of cell extracts. This analysis revealed a previously unknown heat shock protein, D48.5. Heat shock induction of both protein D48.5 and -galactosidase expressed from the lacZ fusion in its gene, htpI, was found to be dependent on -32. Like that of other heat shock proteins, synthesis of protein D48.5 after a heat shock was transient. Using Hfr strains and P1 transduction, the htpI::lacZ fusion was mapped to near minute 98, within an 18-minute region rich in heat shock genes. The fusion was cloned and its expression was found to be dependent on -32. Analysis of the nucleotide sequence of the promoter region of the cloned fusion revealed two areas similar to the consensus sequence for -32 promoters. Both protein D48.5 and fusion-expressed -galactosidase were induced roughly 3-fold on a shift from 28 to 42\sp\circC and 4-fold on a shift to 46\sp\circC. The relative levels of this polypeptide and fusion-expressed -galactosidase increased across the temperature range from 28 to 46\sp\circC. When the growth rate of the cell increased as a function of media composition, the relative level of protein D48.5 also increased, making it a member of the Ic regulatory class. Proteins of this class are involved in replication, transcription, and translation. Interestingly, the fusion strain was temperature sensitive for growth after a shift from 28 to 46\sp\circC. Both the parental strain and an Hfr recombinant strain that had a restored protein D48.5 grew after such a shift, suggesting an essential function for this protein at high temperatures.PhDBiological SciencesBiologyMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128235/2/8821635.pd
Modulation of the heat shock and stringent responses of Escherichia coli by a mutation in theglyA gene.
No full textA genetic screen, designed to isolate mutants of Escherichia coli altered in the ability to induce the heat shock response, identified a strain unable to induce the heat shock proteins in a defined medium lacking methionine after exposure to 2,4-dinitrophenol (DNP). The strain also exhibited a relaxed-like phenotype, i.e. it continued to synthesize translational machinery rapidly during the period of inhibited growth following exposure to DNP, whereas wild-type cells decreased the rate at which the translational apparatus was made. Also, this strain also grew slowly at 28\sp\circC, and linearly at 42\sp\circC. The mutation responsible for this phenotype mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylene-tetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. The abnormal phenotypes associated with the glyA mutation were reversed when methionine was included in the growth medium. Because fixed one-carbon units, as methionine, allowed mutant cells to heat shock after exposure to DNP, a one-carbon restriction was considered possibly responsible for the phenotypes described above. It was postulated that the relaxed-like phenotype arose from a partial block in translational initiation, resulting from low levels of formylated initiator tRNA, and that the abnormal heat shock was a result of changes in physiology caused by the relaxed-like state. Measurements of f-met-tRNA levels in the glyA mutant were consistent with this hypothesis. However, an fmt mutant, which is unable to formylate initiator tRNA, failed to mimic the glyA mutant in these respects, suggesting that the phenotype associated with the glyA mutant is not the result of a low level of f-met-tRNA. In addition, a mutant failed to exhibit either the relaxed-like or abnormal heat shock phenotypes. This suggests that the stringent response seen following DNP addition may arise via a ppGpp independent mechanism. The results with this glyA mutant establish a nutritional modulation of the heat shock response that is connected with a novel form of the stringent response network.PhDCellular and Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103586/1/9332063.pdfDescription of 9332063.pdf : Restricted to UM users only
The involvement of the stringent response in temperature physiology of Escherichia coli.
No full textShifting an exponentially growing culture of E. coli from 37 to 10\sp\circC, a temperature just above the minimum temperature for growth, results in inhibition of protein, DNA and RNA synthesis for a 4-hour growth lag period. This present study was initiated to investigate changes in gene expression following downshifts in temperature to determine the possible involvement of an adaptive response for growth. The primary technique employed was to label cells with a radioactive precusor to protein (usually (\sp{35}S) methionine) and then to resolve the complex mixture of total cell protein by the technique of two-dimensional polyacrylamide gel electrophoresis. Changes in gene expression following different downshifts in temperature were monitored and compared. Because the nucleotides guanosine 5\sp\primetriphosphate-3\sp\primediphosphate (pppGpp) and guanosine 5\sp\primediphosphate-3\sp\primediphosphate (ppGpp), collectively abbreviated (p)ppGpp, have been reported to decrease following a downshift in temperature, the effects of overproduction and underproduction of (p)ppGpp on gene expression and growth following a downshift in temperature were investigated, along with effects on steady state growth at low temperature. The study revealed that any major downshift in temperature results in the induction of several proteins termed the cold shock response, continued synthesis of transcriptional and translational proteins, and repression of heat shock proteins. This study also showed that (p)ppGpp, a pleiotropic effector of gene expression, is a negative effector of the synthesis of cold shock proteins and transcriptional and translational proteins, and a positive effector of heat shock proteins following a downshift in temperature. The combined results indicate that the decrease in the (p)ppGpp level through the resultant changes in gene expression contributes to cold adaptation. In contrast to growth at high temperatures, adaptation to growth at low temperature, as well as the final steady state growth rate at low temperature, is negatively affected by (p)ppGpp-mediated regulation in E. coli.PhDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103694/1/9034449.pdfDescription of 9034449.pdf : Restricted to UM users only
Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage.
No full textFor many years bacterial physiologists have studied how bacteria adapt and grow in various environments, while other microbiologists have studied pathogenic bacteria to try to determine what special characteristics these bacteria possess that make them virulent. The current work combined elements of these two fields of study. The project had two goals: to characterize the bacterial response to the physiological conditions found within the mammalian host cell, and in so doing, to shed light on the nature of the intracellular environment. The primary tool used in these experiments was two-dimensional polyacrylamide gel electrophoresis. First, the pattern of proteins made by Salmonella typhimurium situated within the intracellular environment of macrophage-like U937 cells was determined. The levels of approximately 40 proteins increased in the intracellular condition, while approximately 100 proteins exhibited repressed levels relative to those of an extracellular control culture. Second, the patterns of proteins made by S. typhimurium while the bacteria were exposed to various environmental conditions in the laboratory were determined. Third, the intracellular protein pattern was carefully compared to the various protein patterns that resulted from the exposure of the bacteria to several environmental conditions. This analysis revealed that, as expected, the intracellular environment imposes numerous stresses on the bacteria, but an unexpected finding was that the macrophage induced response was not a simple sum of individual stress responses displayed during extracellular growth. The use of several methods to determine the growth rate of S. typhimurium within the host macrophages revealed that these bacteria exist in the intracellular environment in at least two populations. One population consists of bacteria that are growing and dividing rapidly. A second population consists of viable cells that are in some growth arrested state. Experiments in which bacterial protein synthesis was inhibited immediately after invasion of the macrophages revealed that the ability to synthesize the macrophage induced proteins was important for intracellular survival of the bacteria.PhDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105647/1/9208481.pdfDescription of 9208481.pdf : Restricted to UM users only
Stress proteins in biology and medicine : Edited by R. Morimoto, A. Tissieres, and C. Georgopoulos. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. (1990). 450 pp. $97.00
No full textPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28305/1/0000059.pd
Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest
No full textThe synthesis of the small, cytoplasmic protein UspA universal stress protein A) of Escherichia coli is induced as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth. The increase in UspA synthesis appears to be the result of Induction of the monocistronic uspA gene. Induction of this gene during a heat-shock treatment is demonstrated to be the result of transcriptional activation of Σ 70 -dependent promoter which has previously been shown to be activated also during carbon starvation-induced growth arrest. Mutant cells lacking UspA grow at rates indistinguisible from the isogenic parent at different temperatures and in the presence of different growth inhibitors but are impaired in their ability to survive prolonged periods of complete growth inhibition caused by a variety of diverse stresses, including CdCl 2 , H 2 O 2 , DNP, CCCP exposure, and osmotic shock. Moreover, the uspA mutation results in an increased sensitivity of cells to carbon-source starvation (i.e. glucose, glycerol or succinate depletion). Also, the mutation causes a marked alteration in the timing of starvation protein expression but protein expression during steady-state growth appears to be normal. The results presented have prompted us to postulate that UspA may have a general protective function related to the growth arrest state.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73757/1/j.1365-2958.1994.tb00334.x.pd
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