Validation of a High-Throughput Sausage Casing Model for the Assessment of Bacterial Inactivation Affected by Salt Concentration, pH, and Temperature.

Previous studies have shown the efficacy of high concentrations of salt as the main preservative against vegetative bacteria present on natural sausage casings. These studies were limited in the number of variables and the interactions between these variables that were assessed. To remedy this situation, a MicroCasing high-throughput model was developed and validated to study the inactivation kinetics of various combinations of parameters (salt concentration, pH, and temperature) on eight bacterial isolates of Salmonella enterica, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes over a prolonged period. A Weibullian power model was the best fit to show the trends in sensitivity of each bacterial isolate to salt, pH, and temperature over time. The inactivation kinetics generated with this novel approach could serve as a predictive model for the required salting period for casings. The actual bacterial contamination of the product can vary with the respective production step during processing from animal intestine into sausage casings (initial level, ∼105 CFU/g; level after salting, <102 CFU/g). Subsequent selection and grading of these casings will require complete removal of all salt, and upon completion of this production step, the casings will be resalted. By determining the actual contamination level before the salting process, the minimum storage period in salt can be calculated and potentially optimized by adjusting the pH and temperature. As a result, a standard holding period of at least 30 days may no longer be necessary to produce salted natural casings in accordance with validated quality and food safety criteria.

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression.

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

Monte-Carlo modeling of the central carbon metabolism of Lactococcus lactis: insights into metabolic regulation.

Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.

The redox potential of the plastoquinone pool of the cyanobacterium Synechocystis species strain PCC 6803 is under strict homeostatic control.

A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH2) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH2 content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH2 content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH2-PQ ratio then followed from comparison of the PQH2 signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH2 extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal.

Molecular physiology of the dynamic regulation of carbon catabolite repression in Escherichia coli.

We report on the use of the chemostat as an optimal device to create time-invariant conditions that allow accurate sampling for various omics assays in Escherichia coli, in combination with recording of the dynamics of the physiological transition in the organism under study that accompany the initiation of glucose repression. E. coli cells respond to the addition of glucose not only with the well-known transcriptional response, as was revealed through quantitative PCR analysis of the transcript levels of key genes from the CRP (cAMP receptor protein) regulon, but also with an increased growth rate and a transient decrease in the efficiency of its aerobic catabolism. Less than half of a doubling time is required for the organism to recover to maximal values of growth rate and efficiency. Furthermore, calculations based on our results show that the specific glucose uptake rate (qs) and the H(+)/e(-) ratio increase proportionally, up to a growth rate of 0.4 h(-1), whilst biomass yield on glucose (Yx / s) drops during the first 15 min, followed by a gradual recovery. Surprisingly, the growth yields after the recovery phase show values even higher than the maximum theoretical yield. Possible explanations for these high yields are discussed.

Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone.

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Uncoupling of substrate-level phosphorylation in Escherichia coli during glucose-limited growth.

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

On the function of the various quinone species in Escherichia coli.

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Role of phosphate in the central metabolism of two lactic acid bacteria--a comparative systems biology approach.

Lactic acid-producing bacteria survive in distinct environments, but show common metabolic characteristics. Here we studied the dynamic interactions of the central metabolism in Lactococcus lactis, extensively used as a starter culture in the dairy industry, and Streptococcus pyogenes, a human pathogen. Glucose-pulse experiments and enzymatic measurements were performed to parameterize kinetic models of glycolysis. Significant improvements were made to existing kinetic models for L. lactis, which subsequently accelerated the development of the first kinetic model of S. pyogenes glycolysis. The models revealed an important role for extracellular phosphate in the regulation of central metabolism and the efficient use of glucose. Thus, phosphate, which is rarely taken into account as an independent species in models of central metabolism, should be considered more thoroughly in the analysis of metabolic systems in the future. Insufficient phosphate supply can lead to a strong inhibition of glycolysis at high glucose concentrations in both species, but this was more severe in S. pyogenes. S. pyogenes is more efficient at converting glucose to ATP, showing a higher tendency towards heterofermentative energy metabolism than L. lactis. Our comparative systems biology approach revealed that the glycolysis of L. lactis and S. pyogenes have similar characteristics, but are adapted to their individual natural habitats with respect to phosphate regulation.

Growth rate-dependent control in Enterococcus faecalis: effects on the transcriptome and proteome, and strong regulation of lactate dehydrogenase.

Enterococcus faecalis V583 was grown in a glucose-limited chemostat at three different growth rates (0.05, 0.15, and 0.4 h⁻¹). The fermentation pattern changed with growth rate, from a mostly homolactic profile at a high growth rate to a fermentation dominated by formate, acetate, and ethanol production at a low growth rate. A number of amino acids were consumed at the lower growth rates but not by fast-growing cells. The change in metabolic profile was caused mainly by decreased flux through lactate dehydrogenase. The transcription of ldh-1, encoding the principal lactate dehydrogenase, showed very strong growth rate dependence and differed by three orders of magnitude between the highest and the lowest growth rates. Despite the increase in ldh-1 transcript, the content of the Ldh-1 protein was the same under all conditions. Using microarrays and quantitative PCR, the levels of 227 gene transcripts were found to be affected by the growth rate, and 56 differentially expressed proteins were found by proteomic analyses. Few genes or proteins showed a growth rate-dependent increase or decrease in expression across the whole range of conditions, and many showed a maximum or minimum at the middle growth rate (i.e., 0.15 h⁻¹). For many gene products, a discrepancy between transcriptomic and proteomic data were seen, indicating posttranscriptional regulation of expression.

Dynamic regulation of mitochondrial respiratory chain efficiency in Saccharomyces cerevisiae.

To adapt to changes in the environment, cells have to dynamically alter their phenotype in response to, for instance, temperature and oxygen availability. Interestingly, mitochondrial function in Saccharomyces cerevisiae is inherently temperature sensitive; above 37 °C, yeast cells cannot grow on respiratory carbon sources. To investigate this phenomenon, we studied the effect of cultivation temperature on the efficiency (production of ATP per atom of oxygen consumed, or P/O) of the yeast respiratory chain in glucose-limited chemostats. We determined that even though the specific oxygen consumption rate did not change with temperature, oxygen consumption no longer contributed to mitochondrial ATP generation at temperatures higher than 37 °C. Remarkably, between 30 and 37 °C, we observed a linear increase in respiratory efficiency with growth temperature, up to a P/O of 1.4, close to the theoretical maximum that can be reached in vivo. The temperature-dependent increase in efficiency required the presence of the mitochondrial glycerol-3-phosphate dehydrogenase GUT2. Respiratory chain efficiency was also altered in response to changes in oxygen availibility. Our data show that, even in the absence of alternative oxidases or uncoupling proteins, yeast has retained the ability to dynamically regulate the efficiency of coupling of oxygen consumption to proton translocation in the respiratory chain in response to changes in the environment.

Effects of fluconazole on the secretome, the wall proteome, and wall integrity of the clinical fungus Candida albicans.

Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of (14)N-labeled query walls relative to a reference standard mixture of (15)N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.

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.

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.

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs.

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.

Algal photosynthesis as the primary driver for a sustainable development in energy, feed, and food production.

High oil prices and global warming that accompany the use of fossil fuels are an incentive to find alternative forms of energy supply. Photosynthetic biofuel production represents one of these since for this, one uses renewable resources. Sunlight is used for the conversion of water and CO2 into biomass. Two strategies are used in parallel: plant-based production via sugar fermentation into ethanol and biodiesel production through transesterification. Both, however, exacerbate other problems, including regional nutrient balancing and the world's food supply, and suffer from the modest efficiency of photosynthesis. Maximizing the efficiency of natural and engineered photosynthesis is therefore of utmost importance. Algal photosynthesis is the system of choice for this particularly for energy applications. Complete conversion of CO2 into biomass is not necessary for this. Innovative methods of synthetic biology allow one to combine photosynthetic and fermentative metabolism via the so-called Photanol approach to form biofuel directly from Calvin cycle intermediates through use of the naturally transformable cyanobacterium Synechocystis sp. PCC 6803. Beyond providing transport energy and chemical feedstocks, photosynthesis will continue to be used for food and feed applications. Also for this application, arguments of efficiency will become more and more important as the size of the world population continues to increase. Photosynthetic cells can be used for food applications in various innovative forms, e.g., as a substitute for the fish proteins in the diet supplied to carnivorous fish or perhaps--after acid hydrolysis--as a complex, animal-free serum for growth of mammalian cells in vitro.

The ArcBA two-component system of Escherichia coli is regulated by the redox state of both the ubiquinone and the menaquinone pool.

ArcBA is a two-component regulatory system of Escherichia coli involved in sensing oxygen availability and the concomitant transcriptional regulation of oxidative and fermentative catabolism. Based on in vitro data, it has been postulated that the redox state of the ubiquinone pool is the determinant for ArcB kinase activity. Here we report on the in vivo regulation of ArcB activation, as determined using a lacZ reporter specifically responsive to phosphorylated ArcA. Our results indicate that upon deletion of a ubiquinone biosynthetic enzyme, regulation of ArcB in the anaerobic-aerobic transition is not affected. In contrast, interference with menaquinone biosynthesis leads to inactivation of ArcB during anaerobic growth; this phenotype is fully rescued by addition of a menaquinone precursor. This clearly demonstrates that the menaquinones play a major role in ArcB activation. ArcB shows a complex pattern of regulation when E. coli is titrated through the entire aerobiosis range; ArcB is activated under anaerobic and subaerobic conditions and is much less active under fully aerobic and microaerobic conditions. Furthermore, there is no correlation between ArcB activation and the redox state of the ubiquinone pool, but there is a restricted correlation between the total cellular ubiquinone content and ArcB activity due to the considerable increase in the size of the ubiquinone pool with increasing degrees of aerobiosis. These results lead to the working hypothesis that the in vivo activity of ArcB in E. coli is modulated by the redox state of the menaquinone pool and that the ubiquinone/ubiquinol ratio in vivo surely is not the only determinant of ArcB activity.

ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli.

Understanding the biogenesis of iron-sulfur (Fe-S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe-S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli an A-type Fe-S protein that we named ErpA. Remarkably, erpA was found essential for growth of E. coli in the presence of oxygen or alternative electron acceptors. It was concluded that isoprenoid biosynthesis was impaired by the erpA mutation. First, the eukaryotic mevalonate-dependent pathway for biosynthesis of isopentenyl diphosphate restored the respiratory defects of an erpA mutant. Second, the erpA mutant contained a greatly reduced amount of ubiquinone and menaquinone. Third, ErpA bound Fe-S clusters and transferred them to apo-IspG, a protein catalyzing isopentenyl diphosphate biosynthesis in E. coli. Surprisingly, the erpA gene maps at a distance from any other Fe-S biogenesis-related gene. ErpA is an A-type Fe-S protein that is characterized by an essential role in cellular metabolism.

The role of two-component regulation systems in the physiology of the bacterial cell.

Two-component regulation systems (TCRSs) are the dominant type of signal transduction system in prokaryotes that are used to inform the cellular trancriptional machinery (and additional targets for regulation, like the motility apparatus) about actual changes in the extracellular physicochemical conditions. We now review their molecular structure and enzymatic characteristics, their mutual interactions and its implications, and their role in cellular physiology. Specific emphasis is placed on the ArcB/A system, a representative of the phosphorelay type of TCRS, and a key player in the adjustment of the cellular make-up of enterobacteria in response to alterations in the oxygen availability. Also some applied aspects of the TCRSs are discussed, i.e. their role as a target to develop new anti-bacterials and their application in biotechnology (or: 'synthetic biology').