Molekulargenetische Untersuchungen zum Ethanol-oxidierenden System in Pseudomonas aeruginosa

Einige Gram-negative Bakterien bilden beim Wachstum auf Alkohol als einziger Kohlenstoff- und Energiequelle interessante Enzymsysteme mit Quinoprotein-Alkoholdehydrogenasen. Diese Quinoproteine besitzen eine ungewöhnliche prosthetische Gruppe, Pyrrolochinolinchinon, sind im Periplasma lokalisiert und übertragen die Reduktionsäquivalente auf die Elektronentransportkette. Pseudomonas aeruginosa ATCC 17933 bildet beim Wachstum auf Ethanol ebenfalls ein Quinoprotein-Enzymsystem. Zwei Komponenten dieses Systems, die Quinoprotein-Ethanoldehydrogenase und das Cytochrom c550 waren zu Beginn der vorliegenden Arbeit bekannt. Das Ziel der Arbeit war es, mit einem molekulargenetischen Ansatz weitere Komponenten des Ethanol-oxidierenden Systems zu identifizieren, die Anzahl und Organisation der beteiligten Gene abzuschätzen und ihre Regulation zu studieren. Zur Identifizierung der beteiligten Gene wurden 21 Pseudomonas aeruginosa Mutanten isoliert, die nicht mehr auf Ethanol wachsen konnten. Diese Mutanten konnten biochemisch charakterisiert und gemäß ihres Phänotyps in vier Gruppen eingeteilt werden. Mit einer Cosmidgenbank wurden alle Mutanten komplementiert und zehn verschiedene Cosmide isoliert. Das Gen des Cytochrom c550 wurde durch Hybridisierung mit einer Gensonde auf einem DNA-Fragment eines komplementierenden Cosmids identifiziert und sequenziert. Die abgeleitete Aminosäuresequenz wies überraschenderweise nur geringe Identitäten zu anderen c-Typ Cytochromen auf. Unerwartet war auch die Umgebung des Cytochrom c550 Gens: Vor dem Cytochrom c550 Gen und zu diesem divergent orientiert liegt das Gen der Quinoprotein Ethanoldehydrogenase. Dem Cytochrom c550 Gen folgt ein Gen für eine cytoplasmatische NAD+-abhängige Aldehyddehydrogenase. Die Regulation der divergent zueinander orientierten Promotoren von Cytochrom c550 und der Quinoprotein Ethanoldehydrogenase wurde durch transkriptionelle Fusionen mit dem lacZ Gen untersucht. Hierbei konnte ein Zwei-Komponenten System identifiziert werden, welches die Expression der Quinoprotein Ethanoldehydrogease unabhängig vom Cytochrom c550 Gen kontrolliert. Insgesamt wurden sieben Regulationsmutanten identifiziert. Die Zusammenfassung aller Ergebnisse dieser Arbeit zeigten, daß P. aeruginosa zum Wachstum auf Ethanol ein unerwartet komplex aufgebautes und reguliertes Ethanol-oxidierendes System benötigt: Mindestens 17 Gene sind am Ethanol oxidierende System in P. aeruginosa beteiligt, davon sind sieben Gene allein für die Regulation notwendig

Sequencing and Characterization of Pseudomonas aeruginosa phage JG004

Phages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection

Genetic tools for the investigation of Roseobacter clade bacteria

<p>Abstract</p> <p>Background</p> <p>The <it>Roseobacter </it>clade represents one of the most abundant, metabolically versatile and ecologically important bacterial groups found in marine habitats. A detailed molecular investigation of the regulatory and metabolic networks of these organisms is currently limited for many strains by missing suitable genetic tools.</p> <p>Results</p> <p>Conjugation and electroporation methods for the efficient and stable genetic transformation of selected <it>Roseobacter </it>clade bacteria including <it>Dinoroseobacter shibae</it>, <it>Oceanibulbus indolifex</it>, <it>Phaeobacter gallaeciensis</it>, <it>Phaeobacter inhibens</it>, <it>Roseobacter denitrificans </it>and <it>Roseobacter litoralis </it>were tested. For this purpose an antibiotic resistance screening was performed and suitable genetic markers were selected. Based on these transformation protocols stably maintained plasmids were identified. A plasmid encoded oxygen-independent fluorescent system was established using the flavin mononucleotide-based fluorescent protein FbFP. Finally, a chromosomal gene knockout strategy was successfully employed for the inactivation of the anaerobic metabolism regulatory gene <it>dnr </it>from <it>D. shibae </it>DFL12^T.</p> <p>Conclusion</p> <p>A genetic toolbox for members of the <it>Roseobacter </it>clade was established. This provides a solid methodical basis for the detailed elucidation of gene regulatory and metabolic networks underlying the ecological success of this group of marine bacteria.</p

Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas aeruginosa </it>causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, <it>P. aeruginosa </it>cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic <it>P. aeruginosa </it>infections. However, little is known about the factors which influence phage infection of <it>P. aeruginosa </it>under infection conditions and suitable broad host range phages.</p> <p>Results</p> <p>We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental <it>P. aeruginosa </it>strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity.</p> <p>Conclusions</p> <p>Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.</p

JProGO: a novel tool for the functional interpretation of prokaryotic microarray data using Gene Ontology information

A novel program suite was implemented for the functional interpretation of high-throughput gene expression data based on the identification of Gene Ontology (GO) nodes. The focus of the analysis lies on the interpretation of microarray data from prokaryotes. The three well established statistical methods of the threshold value-based Fisher's exact test, as well as the threshold value-independent Kolmogorov–Smirnov and Student's t-test were employed in order to identify the groups of genes with a significantly altered expression profile. Furthermore, we provide the application of the rank-based unpaired Wilcoxon's test for a GO-based microarray data interpretation. Further features of the program include recognition of the alternative gene names and the correction for multiple testing. Obtained results are visualized interactively both as a table and as a GO subgraph including all significant nodes. Currently, JProGO enables the analysis of microarray data from more than 20 different prokaryotic species, including all important model organisms, and thus constitutes a useful web service for the microbial research community. JProGO is freely accessible via the web at the following address

SYSTOMONAS — an integrated database for systems biology analysis of Pseudomonas

To provide an integrated bioinformatics platform for a systems biology approach to the biology of pseudomonads in infection and biotechnology the database SYSTOMONAS (SYSTems biology of pseudOMONAS) was established. Besides our own experimental metabolome, proteome and transcriptome data, various additional predictions of cellular processes, such as gene-regulatory networks were stored. Reconstruction of metabolic networks in SYSTOMONAS was achieved via comparative genomics. Broad data integration is realized using SOAP interfaces for the well established databases BRENDA, KEGG and PRODORIC. Several tools for the analysis of stored data and for the visualization of the corresponding results are provided, enabling a quick understanding of metabolic pathways, genomic arrangements or promoter structures of interest. The focus of SYSTOMONAS is on pseudomonads and in particular Pseudomonas aeruginosa, an opportunistic human pathogen. With this database we would like to encourage the Pseudomonas community to elucidate cellular processes of interest using an integrated systems biology strategy. The database is accessible at

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O-2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2

The Pseudomonas aeruginosa Universal Stress Protein PA4352 Is Essential for Surviving Anaerobic Energy Stress

During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection