Project description:Fitness of bacteria is determined both by how fast cells grow when nutrients are abundant and by how well they survive when conditions worsen. Here, we study how prior growth conditions affect the death rate of Escherichia coli during carbon starvation. We control the growth rate prior to starvation either via the carbon source or via a carbon-limited chemostat. We find a consistent dependence where death rate depends on the prior growth conditions only via the growth rate, with slower growth leading to exponentially slower death. Breaking down the observed death rate into two factors, maintenance rate and recycling yield, reveals that slower growing cells display a decreased maintenance rate per cell volume during starvation, thereby decreasing their death rate. In contrast, the ability to scavenge nutrients from carcasses of dead cells (recycling yield) remains constant. Our results suggest a physiological trade-off between rapid proliferation and long survival. We explore the implications of this trade-off within a mathematical model, which can rationalize the observation that bacteria outside of lab environments are not optimized for fast growth.
Project description:BACKGROUND: Growth substrates, aerobic/anaerobic conditions, specific growth rate (?) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA. RESULTS: We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of ? and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing ? and carbon wasting to non-CO2 by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on ?, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. CONCLUSIONS: It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.
Project description:We studied the proteome changes in Escherichia coli growing in rich media, and compared the changes between exponential phase cells and those underwent sudden carbon starvations.
Project description:The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan. Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression. A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression. It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction. These data are discussed in relation to our present knowledge of stringent and heat-shock responses.
Project description:Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections.
Project description:Effects of stimulus length on reaction times (RTs) in the lexical decision task are the topic of extensive research. While slower RTs are consistently found for longer pseudo-words, a finding coined the word length effect (WLE), some studies found no effects for words, and yet others reported faster RTs for longer words. Moreover, the WLE depends on the orthographic transparency of a language, with larger effects in more transparent orthographies. Here we investigate processes underlying the WLE in lexical decision in German-English bilinguals using a diffusion model (DM) analysis, which we compared to a linear regression approach. In the DM analysis, RT-accuracy distributions are characterized using parameters that reflect latent sub-processes, in particular evidence accumulation and decision-independent perceptual encoding, instead of typical parameters such as mean RT and accuracy. The regression approach showed a decrease in RTs with length for pseudo-words, but no length effect for words. However, DM analysis revealed that the null effect for words resulted from opposing effects of length on perceptual encoding and rate of evidence accumulation. Perceptual encoding times increased with length for words and pseudo-words, whereas the rate of evidence accumulation increased with length for real words but decreased for pseudo-words. A comparison between DM parameters in German and English suggested that orthographic transparency affects perceptual encoding, whereas effects of length on evidence accumulation are likely to reflect contextual information and the increase in available perceptual evidence with length. These opposing effects may account for the inconsistent findings on WLEs.
Project description:The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.
Project description:The project aims at elucidating the effects of carbon starvation on the qualitative and quantitative composition of the Escherichia coli HT115-derived SLE1 strain proteome as determined by the combination of label-free and metabolic labeling-based proteomics.
Project description:To cope with harsh circumstances, bacterial cells must initiate cellular stress response programs, which demands the de novo synthesis of many stress defense proteins. Reactive oxygen species (ROS) is a universal environmental stressor for both prokaryotic cells and eukaryotic cells. However, the physiological burden that limits the survival of bacterial cells during oxidative stress remains elusive. Here we quantitatively characterize the cell growth and translational elongation rate of Escherichia coli cells treated with different doses of hydrogen peroxide. Cell growth is immediately arrested by low to moderate levels of hydrogen peroxide, but completely recovers after a certain lag time. The lag time depends positively on the dose of hydrogen peroxide. During the lag time, translational elongation rate drops by as much as ∼90% at initial stage and recovers to its normal state later, a phenomenon resulting from the dramatic alteration in cellular tRNA pools during oxidative stress. However, translational elongation is completely stalled at a certain threshold-level of hydrogen peroxide, at which cells ultimately fail to resume growth. Although the mRNA transcription of oxidative defense genes in oxyR regulon is dramatically induced upon hydrogen peroxide treatment, the extreme slow-down of translational elongation during high levels of hydrogen peroxide has severely compromised the timely synthesis of those oxidative defense proteins. Our study demonstrates that the tRNA-limited translational elongation is a key physiological bottleneck that the bacteria must overcome to counteract ROS, and the maintenance of translational elongation rate for timely synthesis of stress defense proteins is crucial for cells to smoothly get over the oxidative stress.
Project description:ObjectivesSlowed speaking rate was examined for its effects on speech intelligibility, its interaction with the benefit of contextual cues, and the impact of these factors on listening effort in adults with cochlear implants.DesignParticipants (n = 21 cochlear implant users) heard high- and low-context sentences that were played at the original speaking rate, as well as a slowed (1.4× duration) speaking rate, using uniform pitch-synchronous time warping. In addition to intelligibility measures, changes in pupil dilation were measured as a time-varying index of processing load or listening effort. Slope of pupil size recovery to baseline after the sentence was used as an index of resolution of perceptual ambiguity.ResultsSpeech intelligibility was better for high-context compared to low-context sentences and slightly better for slower compared to original-rate speech. Speech rate did not affect magnitude and latency of peak pupil dilation relative to sentence offset. However, baseline pupil size recovered more substantially for slower-rate sentences, suggesting easier processing in the moment after the sentence was over. The effect of slowing speech rate was comparable to changing a sentence from low context to high context. The effect of context on pupil dilation was not observed until after the sentence was over, and one of two analyses suggested that context had greater beneficial effects on listening effort when the speaking rate was slower. These patterns maintained even at perfect sentence intelligibility, suggesting that correct speech repetition does not guarantee efficient or effortless processing. With slower speaking rates, there was less variability in pupil dilation slopes following the sentence, implying mitigation of some of the difficulties shown by individual listeners who would otherwise demonstrate prolonged effort after a sentence is heard.ConclusionsSlowed speaking rate provides release from listening effort when hearing an utterance, particularly relieving effort that would have lingered after a sentence is over. Context arguably provides even more release from listening effort when speaking rate is slower. The pattern of prolonged pupil dilation for faster speech is consistent with increased need to mentally correct errors, although that exact interpretation cannot be verified with intelligibility data alone or with pupil data alone. A pattern of needing to dwell on a sentence to disambiguate misperceptions likely contributes to difficulty in running conversation where there are few opportunities to pause and resolve recently heard utterances.