title: Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains.
abstract: For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as Gluconobacter in biotechnological processes, a simple markerless deletion system is essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an upp mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the codA gene from Escherichia coli, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The codA-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal upp gene of Gluconobacter. We found that the presence of E. coli codB, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a codA gene is absent and an upp gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the Acetobacteraceae.
authors: Lehrstuhl für Mikrobiologie, Technische Universität München, Emil-Ramann-Strasse 4, D-85354, Freising, Germany.
authors: David
authors: D
authors: Kostner
authors: Björn
authors: B
authors: Peters
authors: Markus
authors: M
authors: Mientus
authors: Wolfgang
authors: W
authors: Liebl
authors: Armin
authors: A
authors: Ehrenreich
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005819:Genetic Markers; D005821:Genetic Techniques; D020576:Gluconobacter / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; D026901:Membrane Transport Proteins; C075818:codB protein, E coli; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D043525:Cytosine Deaminase; C468827:codA protein, E coli