Funktionale Genomanalyse von Pseudomonas putida KT2440

Pseudomonas putida KT2440 ist ein metabolisch vielseitiges, Gram-negatives Bodenbakterium, das als effizienter Besiedler von Pflanzenwurzeln gilt. Gene und Genprodukte, die für das Überleben des Organismus in der Rhizosphäre von Weizenkeimlingen bedeutsam sind, wurden identifiziert. Das methodische Vorgehen gliederte sich in folgende Teilbereiche: Zunächst erfolgte der Aufbau einer P. putida KT2440-Mutantenbibliothek unter Verwendung eines neuartigen Transposonsystems. Dadurch war die schnelle Bestimmung der Transposonintegrationsstelle im Genom durch direkte DNA-Sequenzierung möglich. Durch die weitergehende geno- und phänotypische Analyse (Adhäsion, Siderophorbildung, oxidative Stressantwort, Beweglichkeit) der Mutanten konnten die für die Besiedlung relevanten Gene und Genprodukte identifiziert werden. So hatte z.B. die Transposonintegration in dem flagellar motor switch protein fliG für die Mutante neben der Unbeweglichkeit auch eine signifikant herabgesetzte Fähigkeit zur Besiedlung der Weizenkeimlingswurzeln in Konkurrenz zum Wildtyp zur Folge. Proteomanalytische Untersuchungen des Wachstums von KT2440 in der Rhizosphäre im Vergleich zu Kontrollen (in Pflanzennährlösung) sowie mit Weizenwurzelexudaten und im Vollmedium zeigten unterschiedliche Proteinexpressionsmuster. Hierbei wurden vor allem Proteine des Energie- und Aminosäurestoffwechsels sowie Transport- und Bindeproteine differentiell exprimiert.Pseudomonas putida strain KT2440 is a metabolically versatile plant root-colonising Gram-negative soil bacterium. Gene and gene products which are important for the organism´s lifestyle in the rhizosphere of wheat seedlings were identified. The experimental design was devided into different approaches. First a P. putida KT2440 mutant library was constructed using a new transposon mutagenesis technique that allows the direct sequencing of DNA sequence flanking the transposon in the genome. The identification of genes and gene products that are relevant for the colonisation was done by phenotypical (adhesion, siderophore production, oxidative stress response, flagellar mediated movement) and genotypical analysis of the transposon mutants. A transposon integration in the flagellar motor switch protein fliG resulted e.g. in a mutant impaired in the flagellar mediated movement, that additionally showed a significantly decreased ability to colonise the roots of wheat seedlings in competition to the wild type. Different protein patterns were obtained when the strain was grown in the rhizosphere in comparison to controls (in plant nutrient solution) as well as incubated with root exudates and in complex medium. Here proteins belonging to the energy and amino acid metabolism as well as transport and binding proteins were differently expressed

Genome-Scale Reconstruction and Analysis of the Pseudomonas putida KT2440 Metabolic Network Facilitates Applications in Biotechnology

A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, 13C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype–phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential

Proteomic Insights into Metabolic Adaptations in Alcanivorax borkumensis Induced by Alkane Utilization

Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This “hydrocarbonoclastic” bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating β-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats

Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis

Schneiker-Bekel S, dos Santos VAPM, Bartels D, et al. Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium Alcanivorax borkumensis. Nature Biotechnology. 2006;24(8):997-1004