We have developed a new software, MetaboliteDetector, for the efficient and automatic analysis of GC/MS-based metabolomics data. Starting with raw MS data, the program detects and subsequently identifies potential metabolites. Moreover, a comparative analysis of a large number of chromatograms can be performed in either a targeted or nontargeted approach. MetaboliteDetector automatically determines appropriate quantification ions and performs an integration of single ion peaks. The analysis results can directly be visualized with a principal component analysis. Since the manual input is limited to absolutely necessary parameters, the program is also usable for the analysis of high-throughput data. However, the intuitive graphical user interface of MetaboliteDetector additionally allows for a detailed examination of a single GC/MS chromatogram including single ion chromatograms, recorded mass spectra, and identified metabolite spectra in combination with the corresponding reference spectra obtained from a reference library. MetaboliteDetector offers the ability to operate with highly resolved profile mass data. Finally, all analysis results can be exported to tab delimited tables. The features of MetaboliteDetector are demonstrated by the analysis of two experimental metabolomics data sets. MetaboliteDetector is freely available under the GNU public license (GPL) at http://metabolitedetector.tu-bs.de.

Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr-and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

In Pseudomonas aeruginosa, the narK 1 K 2 GHJI operon encodes two nitrate/nitrite transporters and the dissimilatory nitrate reductase. The narK 1 promoter is anaerobically induced in the presence of nitrate by the dual activity of the oxygen regulator Anr and the N-oxide regulator Dnr in cooperation with the nitrate-responsive two-component regulatory system NarXL. The DNA bending protein IHF is essential for this process. Similarly, narXL gene transcription is enhanced under anaerobic conditions by Anr and Dnr. Furthermore, Anr and NarXL induce expression of the N-oxide regulator gene dnr. Finally, NarXL in cooperation with Dnr is required for anaerobic nitrite reductase regulatory gene nirQ transcription. A cascade regulatory model for the finetuned genetic response of P. aeruginosa to anaerobic growth conditions in the presence of nitrate was deduced.The most efficient way for the gram-negative bacterium Pseudomonas aeruginosa to generate energy in the absence of oxygen is through denitrification. During this process, molecular oxygen is replaced by nitrate as the terminal electron acceptor. Nitrate (NO 3 Ϫ ) is reduced in four consecutive steps, via nitrite (NO 2 Ϫ ), nitric oxide (NO), and nitrous oxide (N 2 O) to dinitrogen (N 2 ). This process is vital for growth and survival under microaerobic and anaerobic conditions as found in biofilms and microcolonies of infectious P. aeruginosa (1, 25a). The majority of earlier investigations focused on the enzymology and regulation of nitrite (NO 2 Ϫ )-to-dinitrogen (N 2 ) conversion (27). Here, the regulatory network for the onset of nitrate respiration under oxygen-limiting conditions was elucidated using reporter gene fusions, strains carrying mutated regulatory genes, and site-directed mutagenesis of potential regulator binding sites.Importance of narGHJI, narXL, anr, and dnr for anaerobic growth of P. aeruginosa. In order to confirm the importance of the nitrate reductase genes narGHJI and the regulatory genes anr, dnr, and narXL for the anaerobic growth of P. aeruginosa, knockout mutants were characterized concerning their growth behavior. P. aeruginosa Anr is the oxygen-sensing regulatory protein homologue to Escherichia coli Fnr (19,26). Dnr of P. aeruginosa belongs to the Crp-Fnr superfamily of transcriptional regulators and was reported to activate transcription of the genes nir, nor, and nos (6, 9). In Pseudomonas stutzeri, DnrD was shown to detect NO (13,22). NarXL is a nitrateresponding two-component regulatory system (14). All investigated P. aeruginosa mutant strains failed to grow under anaerobic nitrate respiratory conditions (data not shown). They did not reveal any growth phenotype when tested under aerobic conditions (data not shown). These experiments identify narL, anr, dnr, and narG as key players in the anaerobic growth of P. aeruginosa.Transcriptional control of the nar locus is mediated by the narXL-narK 1 intergenic region. In E. coli and P. stutzeri, Fnrand NarXL-dependent transcription of the narGHJI operon is mediated by the narG ups...

Recently, we identified a pyruvate fermentation pathway in Pseudomonas aeruginosa sustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol. 186:4596-4604, 2004). Anaerobic long-term survival of P. aeruginosa might be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis of P. aeruginosa cells during a 7-day period of pyruvate fermentation revealed the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72-and 22-fold, respectively. Both belong to the group of universal stress proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the P PA3309 -lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter activity was detected in the deeper layers of a P. aeruginosa biofilm using a P PA3309 -gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria.