Activators of the glutamate-dependent acid resistance system alleviate deleterious effects of YidC depletion in Escherichia coli.

The function of the essential inner membrane protein (IMP) YidC in Escherichia coli has been studied for a limited number of model IMPs and primarily using targeted approaches. These studies suggested that YidC acts at the level of insertion, folding, and quality control of IMPs, both in the context of the Sec translocon and as a separate entity. To further our understanding of YidC's role in IMP biogenesis, we screened a random overexpression library for factors that rescued the growth of cells upon YidC depletion. We found that the overexpression of the GadX and GadY regulators of the glutamate-dependent acid resistance system complemented the growth defect of YidC-depleted cells. Evidence is presented that GadXY overexpression counteracts the deleterious effects of YidC depletion on at least two fronts. First, GadXY prepares the cells for the decrease in respiratory capacity upon the depletion of YidC. Most likely, GadXY-regulated processes reduce the drop in proton-motive force that impairs the fitness of YidC-depleted cells. Second, in GadXY-overproducing cells increased levels of the general chaperone GroEL cofractionate with the inner membranes, which may help to keep newly synthesized inner membrane proteins in an insertion-competent state when YidC levels are limiting.

Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations.

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

Dissipation of proton motive force is not sufficient to induce the phage shock protein response in Escherichia coli.

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H(+)-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response.

Overexpression of HAP4 in glucose-derepressed yeast cells reveals respiratory control of glucose-regulated genes.

A link between control of respiration and glucose repression in yeast is reported. The HAP4 gene was overexpressed in a Delta mig1 deletion background, generating a mutant in which respiratory function is stimulated and glucose repression is diminished. Although this combination does not result in derepression of genes encoding proteins involved in respiratory function, it nevertheless generates resistance against 2-deoxyglucose and hence contributes to more derepressed growth characteristics. Unexpectedly, overexpression of HAP4 in the Delta mig1 deletion strain causes strong repression of several target genes of the Mig1p repressor. Repression is not restricted to glucose growth conditions and does not require the glucose repressors Mig2p or Hxk2p. It was observed that expression of the SUC2 gene is transiently repressed after glucose is added to respiratory-growing Delta mig1 cells. Additional overexpression of HAP4 prevents release from this novel repressed state. The data presented show that respiratory function controls transcription of genes required for the metabolism of alternative sugars. This respiratory feedback control is suggested to regulate the feed into glycolysis in derepressed conditions.

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.