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Applied and Environmental Microbiology, June 2005, p. 3014-3024, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3014-3024.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

sacB–5-Fluoroorotic Acid–pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus

Takahiro Yano,1* Carsten Sanders,2 John Catalano,1 and Fevzi Daldal2*

Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania,1 Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 191042

Received 30 September 2004/ Accepted 15 December 2004

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 (5FOAr). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOAr to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOAr 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, 5FOAr 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.

* Corresponding author. Mailing address for Takahiro Yano: Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. Phone: (215) 898-2939. Fax: (215) 573-3748. E-mail: yano{at}mail.med.upenn.edu. Mailing address for Fevzi Daldal: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104. Phone: (215) 898-4394. Fax: (215) 898-8780. E-mail: fdaldal{at}sas.upenn.edu.

Applied and Environmental Microbiology, June 2005, p. 3014-3024, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3014-3024.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sanders, C., Deshmukh, M., Astor, D., Kranz, R. G., Daldal, F. (2005). Overproduction of CcmG and CcmFHRc Fully Suppresses the c-Type Cytochrome Biogenesis Defect of Rhodobacter capsulatus CcmI-Null Mutants. J. Bacteriol. 187: 4245-4256 [Abstract] [Full Text]  


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