title: Analysis of lipid export in hydrocarbonoclastic bacteria of the genus Alcanivorax: identification of lipid export-negative mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9.
abstract: Triacylglycerols (TAGs), wax esters (WEs), and polyhydroxyalkanoates (PHAs) are the major hydrophobic compounds synthesized in bacteria and deposited as cytoplasmic inclusion bodies when cells are cultivated under imbalanced growth conditions. The intracellular occurrence of these compounds causes high costs for downstream processing. Alcanivorax species are able to produce extracellular lipids when the cells are cultivated on hexadecane or pyruvate as the sole carbon source. In this study, we developed a screening procedure to isolate lipid export-negative transposon-induced mutants of bacteria of the genus Alcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants of A. jadensis and seven of A. borkumensis impaired in lipid secretion were isolated. All isolated mutants were still capable of synthesizing and accumulating these lipids intracellularly and exhibited no growth defect. In the A. jadensis mutants, the transposon insertions were mapped in genes annotated as encoding a putative DNA repair system specific for alkylated DNA (Aj17), a magnesium transporter (Aj7), and a transposase (Aj5). In the A. borkumensis mutants, the insertions were mapped in genes encoding different proteins involved in various transport processes, like genes encoding (i) a heavy metal resistance (CZCA2) in mutant ABO_6/39, (ii) a multidrug efflux (MATE efflux) protein in mutant ABO_25/21, (iii) an alginate lyase (AlgL) in mutants ABO_10/30 and ABO_19/48, (iv) a sodium-dicarboxylate symporter family protein (GltP) in mutant ABO_27/29, (v) an alginate transporter (AlgE) in mutant ABO_26/1, or (vi) a two-component system protein in mutant ABO_27/56. Site-directed MATE, algE, and algL gene disruption mutants, which were constructed in addition, were also unable to export neutral lipids and confirmed the phenotype of the transposon-induced mutants. The putative localization of the different gene products and their possible roles in lipid excretion are discussed. Beside this, the composition of the intra- and extracellular lipids in the wild types and mutants were analyzed in detail.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany.
authors.forename: Efraín
authors.initials: E
authors.lastname: Manilla-Pérez
authors.forename: Christina
authors.initials: C
authors.lastname: Reers
authors.forename: Meike
authors.initials: M
authors.lastname: Baumgart
authors.forename: Stephan
authors.initials: S
authors.lastname: Hetzler
authors.forename: Rudolf
authors.initials: R
authors.lastname: Reichelt
authors.forename: Ursula
authors.initials: U
authors.lastname: Malkus
authors.forename: Rainer
authors.initials: R
authors.lastname: Kalscheuer
authors.forename: Marc
authors.initials: M
authors.lastname: Wältermann
authors.forename: Alexander
authors.initials: A
authors.lastname: Steinbüchel
mesh_terms: D053521:Alcanivoraceae / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics / Q000502:physiology*; D002855:Chromatography, Thin Layer; D050356:Lipid Metabolism; D046529:Microscopy, Electron, Transmission; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins