Open AccessAnalysis of Lipid Export in Hydrocarbonoclastic Bacteria of the Genus Alcanivorax : Identification of Lipid Export-Negative Mutants of Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9
Author(s) -
Efraín ManillaPérez,
Christina Reers,
Meike Baumgart,
Stephan Hetzler,
Rudolf Reichelt,
Ursula Malkus,
Rainer Kalscheuer,
Marc Wältermann,
Alexander Steinbüchel
Publication year - 2010
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.00700-09
Subject(s) - biology , mutant , transposable element , biochemistry , atp binding cassette transporter , symporter , operon , gene , microbiology and biotechnology , transporter
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 genusAlcanivorax for identification of genes required for lipid export by employing the dyes Nile red and Solvent Blue 38. Three transposon-induced mutants ofA. jadensis and seven ofA. 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 theA. 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 theA. 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-directedMATE ,algE , andalgL 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.