Itaconic acid degradation in Aspergillus niger: the role of unexpected bioconversion pathways.

BACKGROUND: Itaconic acid (IA), a C5-dicarboxylic acid, has previously been identified as one of the top twelve biochemicals that can be produced by biotechnological means. IA is naturally produced by Aspergillus terreus, however, heterologous production in the related species Aspergillus niger has been proposed earlier. Remarkably, we observed that during high producing conditions and elevated titers A. niger detoxifies the extracellular medium of IA. In order to determine the genes responsible for this decline in IA titers a transcriptome analysis was performed. RESULTS: Transcriptome analysis has led to the identification of two novel and previously unknown IA bioconversion pathways in A. niger. One pathway is proposed to convert IA into pyruvate and acetyl-CoA through the action of itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA) and citramalyl-CoA lyase, similar to the pathway identified in A. terreus. Another pathway putatively converts IA into 1-methyl itaconate through the action of trans-aconitate methyltransferase (TmtA). Upon deleting the key genes ictA and ichA we have observed increased IA production and titers and cessation of IA bioconversion. Surprisingly, deletion of tmtA lead to strong reduction of heterologous IA production. CONCLUSION: Heterologous IA production in A. niger induces the expression of IA bioconversion pathways. These pathways can be inhibited by deleting the key genes ictA, ichA and tmtA. Deletion of ictA and ichA resulted in increased IA production. Deletion of tmtA, however, resulted in almost complete cessation of IA production.

Evaluating novel synthetic compounds active against Bacillus subtilis and Bacillus cereus spores using Live imaging with SporeTrackerX.

An empirical approach was taken to screen a novel synthetic compound library designed to be active against Gram-positive bacteria. We obtained five compounds that were active against spores from the model organism Bacillus subtilis and the food-borne pathogen Bacillus cereus during our population based experiments. Using single cell live imaging we were able to observe effects of the compounds on spore germination and outgrowth. Difference in sensitivity to the compounds could be observed between B. subtilis and B. cereus using live imaging, with minor difference in the minimal inhibitory and bactericidal concentrations of the compounds against the spores. The compounds all delayed the bursting time of germinated spores and affected the generation time of vegetative cells at sub-inhibitory concentrations. At inhibitory concentrations spore outgrowth was prevented. One compound showed an unexpected potential for preventing spore germination at inhibitory concentrations, which merits further investigation. Our study shows the valuable role single cell live imaging can play in the final selection process of antimicrobial compounds.

RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress.

Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene were found to be hypersensitive to the lipophilic weak organic acid. RodZ is involved in determining the cell's rod-shape and believed to interact with the bacterial actin-like MreB cytoskeleton. Since rodZ lies upstream in the genome of the essential gene pgsA (phosphatidylglycerol phosphate synthase) we hypothesized that expression of the latter might also be affected in rodZ mutants and hence contribute to the phenotype observed. We show that both genes are co-transcribed and that both the rodZ::mini-Tn10 mutant and a conditional pgsA mutant, under conditions of minimal pgsA expression, were sensitive to sorbic and acetic acid. Both strains displayed a severely altered membrane composition. Compared to the wild-type strain, phosphatidylglycerol and cardiolipin levels were lowered and the average acyl chain length was elongated. Induction of rodZ expression from a plasmid in our transposon mutant led to no recovery of weak acid susceptibility comparable to wild-type levels. However, pgsA overexpression in the same mutant partly restored sorbic acid susceptibility and fully restored acetic acid sensitivity. A construct containing both rodZ and pgsA as on the genome led to some restored growth as well. We propose that RodZ and PgsA play intertwined roles in membrane homeostasis and tolerance to weak organic acid stress.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.

Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pHi at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pHi in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pHi and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pHi regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. IMPORTANCE: This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth behavior upon the addition of weak organic acid food preservatives. Generally, these data are gathered in the form of plate counting of samples incubated with the acids. Here, we visualize the underlying heterogeneity in cellular pH homeostasis, opening up avenues for mechanistic analyses of the heterogeneity in the weak acid stress response. Thus, microbial risk assessment can become more robust, widening the scope of use of these well-known weak organic acid food preservatives.

Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry. Its effect on spore germination and outgrowth in a combined, 'hurdle', preservation setting has gained limited attention. Therefore, the effects of mild sorbic acid (3 mM), heat-treatment (85 °C for 10 min) and a combination of both mild stresses on germination and outgrowth of B. subtilis 1A700 spores were analysed at single spore level. The heat-treatment of the spore population resulted in a germination efficiency of 46.8% and an outgrowth efficiency of 32.9%. In the presence of sorbic acid (3 mM), the germination and outgrowth efficiency was 93.3% and 80.4% respectively whereas the combined heat and sorbic acid stress led to germination and outgrowth efficiencies of 52.7% and 27.0% respectively. The heat treatment clearly primarily affected the germination process, while sorbic acid affected the outgrowth and generation time. In addition a new 'burst' time-point was defined as the time-point at which the spore coat visibly breaks and/or is shed. The combined stresses had a synergistic effect on the time of the end of germination to the burst time-point, increasing both the mean and its variation more than either of the single stresses did.

Reinforcement of Bacillus subtilis spores by cross-linking of outer coat proteins during maturation.

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically (15)N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination.

Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3'-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis. To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool "SporeTracker". In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3'-digallate, clearly affected the growth of emerging vegetative cells.

Comparative physiological and transcriptional analysis of weak organic acid stress in Bacillus subtilis.

The advent of 'omics' techniques bears significant potential for the assessment of the microbiological stability of foods. This requires the integration of molecular data with their implication for cellular physiology. Here we performed a comparative physiological and transcriptional analysis of Bacillus subtilis stressed with three different weak organic acids: the commonly used food preservatives sorbic- and acetic-acid, plus the well-known uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). The concentration of each compound needed to cause a similar reduction of the growth rate negatively correlated with their membrane solubility, and positively with the concentration of undissociated acid. Intracellular acidification was demonstrated by expressing a pH-sensitive GFP derivative. The largest drop in intracellular pH was observed in CCCP-stressed cells and was accompanied by the transcriptional induction of the general stress response (GSR) and SigM regulon, responses known to be induced by acidification. The GSR was induced by acetate, but not by sorbate in mildly-stressed cells. Microarray analysis further revealed that all three acids activate transcriptional programs normally seen upon nutrient limitation and cause diverse responses indicative of an adaptation of the cell envelope. Based on the responses observed and the utilized pH measurements, the inhibitory effect of sorbic acid seems to be more focused on the cell membrane than that of acetic acid or CCCP.

Basic regulatory principles of Escherichia coli's electron transport chain for varying oxygen conditions.

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen.

The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations, and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy, and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes, and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.

In pursuit of protein targets: proteomic characterization of bacterial spore outer layers.

Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two Bacilli and one Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.