title: sacB-5-Fluoroorotic acid-pyrE-based bidirectional selection for integration of unmarked alleles into the chromosome of Rhodobacter capsulatus.
abstract: The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB-5-fluoroorotic acid (5FOA)--pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura-) and hence resistant to 5FOA (5FOA(r)). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA(r) to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA(r) selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA(r) colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. yano@mail.med.upenn.edu
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yano
authors.forename: Carsten
authors.initials: C
authors.lastname: Sanders
authors.forename: John
authors.initials: J
authors.lastname: Catalano
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D009963:Orotic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D011995:Recombination, Genetic; D016354:Rhodobacter capsulatus / Q000235:genetics* / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C112242:HelC protein, bacteria; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D006602:Hexosyltransferases; C021513:levansucrase; D009962:Orotate Phosphoribosyltransferase