Food web and metabolic interactions of the lung inhabitants Streptococcus pneumoniae and Pseudomonas aeruginosa.

Bacteria that successfully adapt to different substrates and environmental niches within the lung and overcome the immune defence can cause serious lung infections. Such infections are generally complex, and recognized as polymicrobial in nature. Both Pseudomonas aeruginosa and Streptococcus pneumoniae can cause chronic lung infections and were both detected in cystic fibrosis (CF) lung at different stages. In this study, single and dual species cultures of Pseudomonas aeruginosa and Streptococcus pneumoniae were studied under well-controlled planktonic growth conditions. Under pH-controlled conditions, both species apparently benefited from the presence of the other. In co-culture with P. aeruginosa, S. pneumoniae grew efficiently under aerobic conditions, whereas in pure S. pneumoniae culture, growth inhibition occurred in bioreactors with dissolved oxygen concentrations above the microaerobic range. Lactic acid and acetoin that are produced by S. pneumoniae were efficiently utilized by P. aeruginosa. In pH-uncontrolled co-cultures, the low pH triggered by S. pneumoniae assimilation of glucose and lactic acid production negatively affected the growth of both strains. Nevertheless, ammonia production improved significantly, and P. aeruginosa growth dominated at later growth stages. This study revealed unreported metabolic interactions of two important pathogenic microorganisms and shed new lights into pathophysiology of bacterial lung infection.

Co-cultures with integrated in situ product removal for lactate-based propionic acid production.

Propionic acid (PA) is a valuable organic acid for the food and feed industry, but no bioproduction at industrial scale exists so far. As product inhibition is a major burden for bioprocesses producing organic acids, in situ product removal (ISPR) is desirable. Here, we demonstrate a new strategy to produce PA with a co-culture coupled with ISPR using electrodialysis. Specifically, Bacillus coagulans first produces lactic acid (LA) from sugar(s) and LA is converted to PA using Veillonella criceti. Applying ISPR to the mentioned co-culture, the specific PA yield was increased from 0.35 to 0.39 g g-1 compared to no ISPR usage. Furthermore, the productivity was increased from 0.63 to 0.7 g L-1 h-1 by applying ISPR. Additionally, it was shown that co-consumption of xylose and glucose led to a higher PA productivity of 0.73 g L-1 h-1, although PA yield was only increased slightly up to 0.36 g g-1.

Enhanced electron transfer of different mediators for strictly opposite shifting of metabolism in Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor.

Microbial electrosynthesis or electro-fermentation in bioelectrochemical systems (BES) have recently received much attention. Here, we demonstrate with the glycerol metabolism by Clostridium pasteurianum that H 2 from in situ water electrolysis, especially in combination with a redox mediator, provides a simple and flexible way for shifting product selectivity and enhancing product yield in the fermentation process. In particular, we report and quantify for the first time strictly different effects of Neutral Red (NR) and the barely studied redox mediator Brilliant Blue (BB) on the growth and product formation of C. pasteurianum grown on glycerol in a newly developed BES. We were able to switch the product formation pattern of C. pasteurianum with a concentration-dependent addition of NR and BB under varied iron availability. Interestingly, NR and BB influenced the glycerol metabolism in a strictly opposite manner concerning the formation of the major products 1,3-propanediol (1,3-PDO) and n-butanol (BuOH). Whereas, NR and iron generally enhance the formation of BuOH, BB favors the formation of 1,3-PDO. In BES the metabolic shifts were enhanced, leading to a further increased yield by as high as 33% for BuOH in NR fermentations and 21% for 1,3-PDO in BB fermentations compared with the respective controls. For the first time, the electron transfer mediated by these mediators and their recycle (recharge) were unambiguously quantified by excluding the overlapping effect of iron. BB has a higher capacity than NR and iron. The extra electron transfer by BB can account for as high as 30-75% of the total NAD + regeneration under certain conditions, contributing significantly to the product formation.

Metabolic and proteomic analyses of product selectivity and redox regulation in Clostridium pasteurianum grown on glycerol under varied iron availability.

BACKGROUND: Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes significantly with the composition of the culture medium. Among others iron content in the medium was shown to strongly affect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses. RESULTS: 1,3-PDO is most effectively produced under iron limited condition (Fe-), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe+). With increased iron availability the BuOH/1,3-PDO ratio increased significantly from 0.27 mol/mol (at Fe-) to 1.4 mol/mol (at Fe+). Additionally, hydrogen production was enhanced significantly under Fe+ condition. Proteomic analysis revealed differentiated expression of many proteins including several ones of the central carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydrogenases, and several electron transfer flavoproteins was found to be strongly up-regulated under Fe+ condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently their influences on the product selectivity in C. pasteurianum. Of particular significance is the finding that H2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which significantly affects the redox balance and thus the product selectivity. CONCLUSIONS: The metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under different iron conditions.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

BACKGROUND: Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. RESULTS: Controlling the dissolved oxygen concentration (pO2) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO2-limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13C-based fluxomics levels. CONCLUSION: Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.

Fermentation of mixed substrates by Clostridium pasteurianum and its physiological, metabolic and proteomic characterizations.

BACKGROUND: Clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as n-butanol and 1,3-propanediol. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. Although C. pasteurianum can grow efficiently with either glucose or glycerol alone, under certain conditions, glucose limitation in the mixed substrate fermentation leads to growth cessation. To understand this phenomenon and for process optimization, fermentation experiments were performed in the presence of excess glycerol but with varied initial concentrations of glucose which were followed by physiological, metabolic and proteomic analyses. RESULTS: Physiological characterization showed that the observed cease of growth is not due to the toxicity of n-butanol. Furthermore, the growth can be resumed by addition of glucose or the intermediate oxaloacetate. Proteomic analysis shed more light on the system-level regulation of many proteins directly or indirectly associated with this phenomenon. Surprisingly, it is found that the specific growth rate of C. pasteurianum in the different growth phases (e.g. before and after glucose limitation) correlated well with the expression level of the ATP dependent pyruvate carboxylase and with the expression level of biotin synthase which provides the cofactor biotin for the formation of oxaloacetate from pyruvate. Bioenergetic analysis based on the formation rates of metabolites further show that ATP supply is not a limiting factor for the pyruvate carboxylation to oxaloacetate. CONCLUSIONS: The results of physiological and proteomic analyses clearly show that the anaplerotic synthesis of oxaloacetate plays a key role in determining the growth behaviour of C. pasteurianum in fermentations with mixed substrates of glucose and glycerol. This study provides interesting targets for metabolic engineering of this emerging industrial microorganism.

Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates.

BACKGROUND: Microbial lipids (triacylglycerols, TAG) have received large attention for a sustainable production of oleochemicals and biofuels. Rhodosporidium toruloides can accumulate lipids up to 70% of its cell mass under certain conditions. However, our understanding of lipid production in this yeast is still much limited, especially for growth with mixed substrates at the level of metabolic network. In this work, the potentials of several important carbon sources for TAG production in R.toruloides are first comparatively studied in silico by means of elementary mode analysis followed by experimental validation. RESULTS: A simplified metabolic network of R.toruloides was reconstructed based on a combination of genome and proteome annotations. Optimal metabolic space was studied using elementary mode analysis for growth on glycerol, glucose, xylose and arabinose or in mixtures. The in silico model predictions of growth and lipid production are in agreement with experimental results. Both the in silico and experimental studies revealed that glycerol is an attractive substrate for lipid synthesis in R. toruloides either alone or in blend with sugars. A lipid yield as high as 0.53 (C-mol TAG/C-mol) has been experimentally obtained for growth on glycerol, compared to a theoretical maximum of 0.63 (C-mol TAG/C-mol). The lipid yield on glucose is much lower (0.29 (experimental) vs. 0.58 (predicted) C-mol TAG/C-mol). The blend of glucose with glycerol decreased the lipid yield on substrate but can significantly increase the overall volumetric productivity. Experimental studies revealed catabolite repression of glycerol by the presence of glucose for the first time. Significant influence of oxygen concentration on the yield and composition of lipids were observed which have not been quantitatively studied before. CONCLUSIONS: This study provides for the first time a simplified metabolic model of R.toruloides and its detailed in silico analysis for growth on different carbon sources for their potential of TAG synthesis. Experimental studies revealed the phenomenon of catabolite repression of glycerol by glucose and the importance of oxygen supply on the yield and composition of lipids. More systematic studies are needed to understand the mechanisms which should help to further optimize the lipid production in this strain of industrial interest.

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation.

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH = 6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L × h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L × h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.

Microbial community composition and dynamics in high-temperature biogas reactors using industrial bioethanol waste as substrate.

Stillage, which is generated during bioethanol production, constitutes a promising substrate for biogas production within the scope of an integrated biorefinery concept. In this study, a microbial community was grown on thin stillage as mono-substrate in a continuous stirred tank reactor (CSTR) at a constant temperature of 55 °C, at an organic loading rate of 1.5 goTS/L*d and a retention time of 25 days. Using an amplicon-based dataset of 17,400 high-quality sequences of 16S rRNA gene fragments (V2-V3 regions), predominance of Bacteria assigned to the families Thermotogaceae and Elusimicrobiaceae was detected. Dominant members of methane-producing Euryarchaeota within the CSTR belonged to obligate acetoclastic Methanosaetaceae and hydrogenotrophic Methanobacteriaceae. In order to investigate population dynamics during reactor acidification, the organic loading rate was increased abruptly, which resulted in an elevated concentration of volatile fatty acids. Acidification led to a decrease in relative abundance of Bacteria accompanied with stable numbers of Archaea. Nevertheless, the abundance of Methanosaetaceae increased while that of Methanobacteriales decreased successively. These findings demonstrate that a profound intervention to the biogas process may result in persistent community changes and reveals uncommon bacterial families as process-relevant microorganisms.

Metabolomic and kinetic investigations on the electricity-aided production of butanol by Clostridium pasteurianum strains.

In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms.

Kinetic studies on the expression of alginate and extracellular proteins by Pseudomonas aeruginosa FRD1 and PAO1.

Pseudomonas aeruginosa is characterized by its capability to produce extracellular virulence proteins and to establish biofilm-based infections that do not respond easily to conventional treatments. However, the physiological conditions that decrease the fitness of such a persistent pathogen would assist the host to defend itself and reduce the infection prevalence. Therefore, developing treatments against P. aeruginosa requires a quantitative understanding of the relationship between bacterial growth kinetics and secretion of alginate and proteins, in addition to the ecological factors that control their synthesis. For this purpose, we examined various environmental factors that affect the specific product yield coefficients (expressed as g product/OD600) of alginate and extracellular proteins using a mucoid (FRD1) and a non-mucoid (PAO1) clinical isolate of P. aeruginosa, respectively. The results suggested magnesium sulfate, trace elements and hydrogen peroxide as significant variables that positively affect alginate synthesis by the FRD1 cells. However, the production of extracellular proteins by PAO1 was negatively affected by the concentration of ferrous sulfate. For understanding the kinetics of expressing alginate and extracellular proteins by the cells, a well-controlled 5 L tank bioreactor was used. The results suggested that under the bioreactor controlled conditions, both alginate and extracellular proteins are expressed parallel to biomass increase in the cells of P. aeruginosa.

Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation.

Clostridium pasteurianum produces industrially valuable chemicals such as n-butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum.

Expression of extracellular polysaccharides and proteins by clinical isolates of Pseudomonas aeruginosa in response to environmental conditions.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.

Glucose-mediated regulation of glycerol uptake in Rhodosporidium toruloides: Insights through transcriptomic analysis on dual substrate fermentation.

Rhodosporidium toruloides is a robust oleaginous yeast that can accumulate lipids to more than 70% of its dry cell mass. Even though it is extensively studied for its fermentation of substrates like glucose and glycerol, limited information is available about its metabolism of mixture of glucose and glycerol. During growth on mixture of glucose and glycerol a typical diauxic growth and higher lipid yields were observed. To understand this phenomenon, RNA-seq analysis was implemented to study the gene expression profiles during growth on mixtures mainly to elucidate regulation of glycerol metabolism. Insights into lipid biosynthesis on mixed substrates are provided at a systems level. Among others, transcriptional profiles showed that glycerol might be produced intracellularly and glycerol kinase (GUT1) and glycerol 3-phosphate dehydrogenase (GUT2) enzymes were not downregulated in the presence of glucose. Transcriptional analysis also showed that the regulation of glycerol uptake in the presence of glucose at transcriptional level is different from that observed in Saccharomyces cerevisiae.

Co-cultivation of Lactobacillus zeae and Veillonella cricetifor the production of propionic acid.

: In this work a defined co-culture of the lactic acid bacterium Lactobacillus zeae and the propionate producer Veillonella criceti has been studied in continuous stirred tank reactor (CSTR) and in a dialysis membrane reactor. It is the first time that this reactor type is used for a defined co-culture fermentation. This reactor allows high mixing rates and working with high cell densities, making it ideal for co-culture investigations. In CSTR experiments the co-culture showed over a broad concentration range an almost linear correlation in consumption and production rates to the supply with complex nutrients. In CSTR and dialysis cultures a strong growth stimulation of L. zeae by V. criceti was shown. In dialysis cultures very high propionate production rates (0.61 g L-1h-1) with final titers up to 28 g L-1 have been realized. This reactor allows an individual, intracellular investigation of the co-culture partners by omic-technologies to provide a better understanding of microbial communities.

Draft Genome Sequence of Type Strain Clostridium pasteurianum DSM 525 (ATCC 6013), a Promising Producer of Chemicals and Fuels.

Clostridium pasteurianum, an anaerobic bacterium able to utilize atmospheric free nitrogen for biosynthesis, has recently been proven to be a promising producer of chemicals and fuels, such as 1,3-propanediol and n-butanol. Here, we report the high-quality draft genome sequence of DSM 525, a type strain of C. pasteurianum.

Microbial production of diols as platform chemicals: recent progresses.

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.

Use of 3D Potential Field and an Enhanced Breadth-first Search Algorithms for the Path Planning of Microdevices Propelled in the Cardiovascular System.

Potential field algorithms often used in path finding applications on a 2D plane are expanded onto a 3D map trajectories for navigation planning of a microdevice designed to be propelled through the cardiovascular system using magnetic gradients generated by a clinical MRI system. This system assembles a 3D reconstruction of a cardiovascular system through magnetic resonance angiography images. The method also allows the extraction of the physiological properties of the given network.

Physiological responses of Pseudomonas aeruginosa PAO1 to oxidative stress in controlled microaerobic and aerobic cultures.

Pseudomonas aeruginosa PAO1 was found to exhibit several remarkable physiological responses to oxidative stress upon its growth in a computer-controlled suspension culture. First, it strongly reduced the transfer rate of oxygen from the gas into the liquid phase, causing oxygen-limited or microaerophilic conditions in the culture after a short period of cultivation, even at high aeration rates with pure oxygen. Second, PAO1 that was previously classified as 'non-mucoid' formed a clear polysaccharide capsule on the cell surface (mucoid phenotype) under oxidative-stress conditions. Third, the strain showed a reduced growth rate and a longer lag phase under high oxygen tension. Finally, P. aeruginosa PAO1 released a high amount of proteins into the culture broth. The release of some virulence factors by PAO1, such as elastase, was significantly enhanced or only occurred under microaerobic conditions (i.e. dissolved oxygen tension value around 1% of air saturation). Hence, it is concluded that P. aeruginosa PAO1 prefers microaerobic conditions for growth and for the formation of some of its virulence factors. PAO1 can create such growth conditions by at least two mechanisms: (i) blockage of the transfer of oxygen and (ii) formation of a polysaccharide capsule on the cell surface. It is postulated that the blockage of oxygen transfer may play an important role in the defence of this pathogen against reactive oxygen intermediates.

Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface. In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena. The physiological responses of two P. aeruginosa PAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations. In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed. The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient, kLa, significantly decreased under iron-limited conditions. The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration. The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way for P. aeruginosa PAO1 to adapt to growth conditions of iron limitation. Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation. The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions. These results have implications for lung infection of cystic fibrosis patients by P. aeruginosa in view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death.