Biomedical subjects

Thomas Nyström

Publications and source records attributed to Thomas Nyström.

At least 37 records · Page 2Linked to original sources

PubMed2004

Growth versus maintenance: a trade-off dictated by RNA polymerase availability and sigma factor competition?

The regulatory design of higher organisms is proposed to comprise a trade-off between activities devoted to reproduction and those devoted to cellular maintenance and repair. Excessive reproduction will inevitably limit the organism's ability to resist stress whereas excessively devoted stress defence systems may increase lifespan but reduce Darwinian fitness. The trade-off is arguably a consequence of limited resources in any one organism but the nature and identity of such limiting resources are ambiguous. Analysis of global control of gene expression in Escherichia coli suggests that reproduction and maintenance activities are also at odds in bacteria and that this antagonism may be a consequence of a battle between transcription factors for limiting RNA polymerase. The outcome of this battle is regulated and depends on the nutritional status of the environment, the levels of the alarmone ppGpp, and RNA polymerase availability. This paper reviews how the concentration of RNA polymerase available for transcription initiation may vary upon shifts between growth and growth-arrest conditions and how this adjustment may differentially affect genes whose functions relate to reproduction and maintenance.

Animals↗

PubMed2004

Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli.

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i). the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii). heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii). basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

Culture Media↗

PubMed2004

Stationary-phase physiology.

Bacteria enjoy an infinite capacity for reproduction as long as they reside in an environment supporting growth. However, their rapid growth and efficient metabolism ultimately results in depletion of growth-supporting substrates and the population of cells enters a phase defined as the stationary phase of growth. In this phase, their reproductive ability is gradually lost. The molecular mechanism underlying this cellular degeneration has not been fully deciphered. Still, recent analysis of the physiology and molecular biology of stationary-phase E. coli cells has revealed interesting similarities to the aging process of higher organisms. The similarities include increased oxidation of cellular constituents and its target specificity, the role of antioxidants and oxygen tension in determining life span, and an apparent trade-off between activities related to reproduction and survival.

Bacterial Physiological Phenomena↗

PubMed2003

Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations.

A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fed-batch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.

Bacterial Proteins↗

PubMed2003

Analysis of molecular diffusion in ftsK cell-division mutants using laser surgery.

Escherichia coli cells that lack the carboxy-terminal part of FtsK fail to segregate their chromosomes properly during cytokinesis and tend to form chains. These chains are possibly formed as a result of DNA being trapped in the division planes or a failure to fuse the membrane during septum formation. If so, small molecules might diffuse between the apparent cell compartments. To investigate this theory, we developed an optical workstation that allows simultaneous imaging of and surgical operations on cellular objects in the sub-micrometre range. By surgical incisions of E. coli cell poles, diffusion of propidium iodide (PI) can be followed in real time. This analysis showed that PI was unable to diffuse from one cell equivalent to another in chain-forming ftsK mutants. Thus, the cytoplasm of the cell compartments in the chains seems to be fully separated.

Cell Division↗

PubMed2003

The oncogenic RAS2(val19) mutation locks respiration, independently of PKA, in a mode prone to generate ROS.

The RAS2(val19) allele, which renders the cAMP-PKA pathway constitutively active and decreases the replicative life-span of yeast cells, is demonstrated to increase production of reactive oxygen species (ROS) and to elevate oxidative protein damage. Mitochondrial respiration in the mutant is locked in a non-phosphorylating mode prone to generate ROS but this phenotype is not linked to a constitutively active PKA pathway. In contrast, providing RAS2(val19) cells with the mammalian uncoupling protein UCP1 restores phosphorylating respiration and reduces ROS levels, but does not correct for PKA-dependent defects. Thus, the RAS2(val19) allele acts like a double-edged sword with respect to oxidation management: (i). it diminishes expression of STRE element genes required for oxidative stress defenses in a PKA-dependent fashion, and (ii). it affects endogenous ROS production and the respiratory state in a PKA-independent way. The effect of the oncogenic RAS allele on the replicative life-span is primarily asserted via the PKA-dependent pathway since Pde2p, but not UCP1, overproduction suppressed premature aging of the RAS2(val19) mutant.

Alleles↗

PubMed2003

Asymmetric inheritance of oxidatively damaged proteins during cytokinesis.

Carbonylated proteins were visualized in single cells of the budding yeast Saccharomyces cerevisiae, revealing that they accumulate with replicative age. Furthermore, carbonylated proteins were not inherited by daughter cells during cytokinesis. Mother cells of a yeast strain lacking the sir2 gene, a life-span determinant, failed to retain oxidatively damaged proteins during cytokinesis. These findings suggest that a genetically determined, Sir2p-dependent asymmetric inheritance of oxidatively damaged proteins may contribute to free-radical defense and the fitness of newborn cells.

Actins↗

PubMed2003

Limiting factors in Escherichia coli fed-batch production of recombinant proteins.

Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

Acetic Acid↗

PubMed2003

Nonculturable bacteria: programmed survival forms or cells at death's door?

Upon starvation and growth arrest, Escherichia coli cells gradually lose their ability to reproduce. These apparently sterile/nonculturable cells initially remain intact and metabolically active and the underlying molecular mechanism behind this sterility is something of an enigma in bacteriology. Three different models have been proposed to explain this phenomenon. The first theory suggests that starving cells become nonculturable due to cellular deterioration, are moribund, and show some of the same signs of senescence as aging organisms. The two other theories suggest that genetically programmed pathways, rather than stochastic deterioration, trigger nonculturability. One "program" theory suggests that nonculturability is the culmination of an adaptive pathway generating dormant survival forms, similar to spore formation in differentiating bacteria. The other "program" theory states that starved cells lose viability due to activation of genetic modules mediating programmed cell death. The different models will be reviewed and evaluated in light of recent data on the physiology and molecular biology of growth-arrested E. coli cells.

Apoptosis↗

PubMed2003

The bacterial universal stress protein: function and regulation.

The universal stress protein A (UspA) superfamily encompasses an ancient and conserved group of proteins that are found in bacteria, Archea, fungi, flies and plants. The Escherichia coli UspA is produced in response to a large number of different environmental onslaughts and UspA is one of the most abundant proteins in growth-arrested cells. Although insights into the regulation of the E. coli uspA gene have been gained, the exact roles of the Usp proteins and Usp domains remain enigmatic; they appear, in some cases, to be linked to resistance to DNA-damaging agents and to respiratory uncouplers.

Bacterial Proteins↗

PubMed2003

Differential oxidative damage and expression of stress defence regulons in culturable and non-culturable Escherichia coli cells.

Potentially pathogenic bacteria, such as Escherichia coli and Vibrio cholerae, become non-culturable during stasis. The analysis of such cells has been hampered by difficulties in studying bacterial population heterogeneity. Using in situ detection of protein oxidation in single E. coli cells, and using a density-gradient centrifugation technique to separate culturable and non-culturable cells, we show that the proteins in non-culturable cells show increased and irreversible oxidative damage, which affects various bacterial compartments and proteins. The levels of expression of specific stress regulons are higher in non-culturable cells, confirming increased defects relating to oxidative damage and the occurrence of aberrant, such as by amino-acid misincorporation, proteins. Our data suggest that non-culturable cells are produced due to stochastic deterioration, rather than an adaptive programme, and pinpoint oxidation management as the 'Achilles heel' of these cells.

Animals↗

PubMed2003

Evaluation of inducible promoters on the secretion of a ZZ-proinsulin fusion protein in Escherichia coli.

Four inducible promoters, uspA, uspB, lacUV5 and malK, were evaluated in the expression of the fusion protein ZZ-proinsulin by Escherichia coli. The aim was to select for their effects on the most appropriate expression system (promoter and culture medium) for secretion of ZZ-proinsulin to the periplasmic space and culture medium. All the expression vectors contained the RNase III cleavage site to ensure that the mRNA translation rate remained independent of 5'-untranslated regions thus making promoter strength comparisons more accurate. The highest ZZ-proinsulin secretion yields were 6.2 mg/g of dry cell weight in the periplasmic space and 2.6 mg/g of dry cell weight in the culture medium using the malK promoter. It was also demonstrated that the use of M9 minimal medium favours secretion.

ATP-Binding Cassette Transporters↗

PubMed2003

Conditional senescence in bacteria: death of the immortals.

Like ageing insects, worms and mammals, growth-arrested Escherichia coli cells accumulate oxidatively damaged proteins. In the early stages of the E. coli stationary phase, this oxidation is caused by an increased production of aberrant proteins, which are especially susceptible to oxidative attack. This route of oxidation appears to elude the classical oxidative defence proteins. The failure of growth-arrested cells fully to combat oxidative damage may also be linked to a trade-off between proliferation activities (primarily directed by the housekeeping sigma factor, sigma70) and maintenance (primarily directed by sigmaS). This trade-off is regulated by the alarmone ppGpp such that elevated ppGpp levels allow sigmaS, and other alternative sigma factors, to work in concert with sigma70 by shifting their relative competitiveness for RNA polymerase binding. However, even during elevated ppGpp levels and stasis, E. coli cells maintain a basal transcription of housekeeping sigma70-dependent genes, and resources are thus partly diverted from maintenance and stress defences to activities relating to proliferation. An alternative view argues for ppGpp being involved in programmed cell death upon growth arrest by regulating chromosomally located toxin-antitoxin loci. Thus, models of bacterial senescence, like those dealing with ageing in higher organisms, encompass both stochastic deterioration theories and programming theories. This review summarizes and evaluates these models.

Bacteria↗

PubMed2002

The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.

Some promoters, including the DmpR-controlled sigma(N)-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping sigma(D)-factor can restore sigma(N)-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivo transcription competition assays, we show that all the four sigma(D) mutant proteins are defective in their ability to compete with sigma(N) for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of sigma(D) or overproduction of the anti-sigma(D) protein Rsd were also found to allow (p)ppGpp-independent transcription from the sigma(N)-Po promoter. Together with data from the direct effects of (p)ppGpp on sigma(N)-dependent Po transcription and sigma-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative sigma-factor competition to adapt to changing cellular nutritional demands.

Base Sequence↗

PubMed2002

Regulation of sigma factor competition by the alarmone ppGpp.

Many regulons controlled by alternative sigma factors, including sigma(S) and sigma(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of sigma(S)-dependent promoters because underproduction of sigma(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-sigma(70)) restored expression from sigma(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type sigma(70) to compete with sigma(32) for core binding and the mutant sigma(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type sigma(70). Similarly, an in vivo competition assay showed that the ability of both sigma(32) and sigma(S) to compete with sigma(70) is diminished in cells lacking ppGpp. Consistently, the fraction of sigma(S) and sigma(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of sigma factors in accordance with cellular demands during physiological stress.

Bacterial Proteins↗

PubMed2002

The Escherichia coli ftsK1 mutation attenuates the induction of sigma(S)-dependent genes upon transition to stationary phase.

A mutation in the cell division gene ftsK causes super-induction of sigma(70)-dependent stress defense genes, such as uspA, during entry of cells into stationary phase. In contrast, we report here that stationary phase induction of sigma(S)-dependent genes, uspB and cfa, is attenuated and that sigma(S) accumulates at a lower rate in ftsK1 cells. Ectopic overexpression of rpoS restored induction of the rpoS regulon in the ftsK mutant, as did a deletion in the recA gene. Thus, a mutation in the cell division gene, ftsK, uncouples the otherwise coordinated induction of sigma(S)-dependent genes and the universal stress response gene, uspA, during entry into stationary phase.

Bacterial Proteins↗

PubMed2002

Aging in bacteria.

Analysis of senescent Escherichia coli cells reveals a link between protein oxidation and the fidelity of the translational apparatus. This model system has also provided a mechanistic molecular explanation for a trade-off between reproduction and survival activities, which may inspire proponents of the disposable soma theory and antagonistic pleiotropy hypothesis of aging.

Bacterial Physiological Phenomena↗

PubMed2002

Translational fidelity, protein oxidation, and senescence: lessons from bacteria.

Senescence of growth-arrested Escherichia coli cells is like mandatory aging in higher eukaryotes, accompanied by increased oxidative modifications of macromolecules. Similar to aged flies, this senescence-related oxidation targets enzymes of the Krebs cycle. Additional targets include the universal stress protein A, the Hsp70 chaperone DnaK, translation elongation factors, and histone-like proteins. However, during the early stages of stasis, protein oxidation is more general and affects a large number of proteins. Attempts to correlate this protein oxidation with a reduced activity of the antioxidant defense systems or an increased tendency of the respiratory apparatus to produce reactive oxygen species (ROS) have generated conflicting results. Instead, recent data indicate that oxidation of proteins may occur in the absence of an increased oxidative stress and is rather caused by an increased production of substrates available for oxidative attack. Misfolded and aberrant proteins appear to be such substrates and the production of aberrant polypeptides surge during the early stages of stasis due to a reduced translational fidelity. The data point to a novel mechanism involved in protein oxidation that has not been considered previously in aging research.

Aging↗