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In-Depth Information
Results
In this study, the expression of λ Red genes in P. ananatis was found to be
highly toxic. A screening was performed to select mutants of P. ananatis that
were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was
identified that grew well under conditions of simultaneous expression of λ
gam, bet, and exo genes. Using this strain, procedures for fast introduction of
multiple rearrangements to the Pantoea ananatis genome based on the λ Red-
dependent integration of the PCR-generated DNA fragments with as short as
40 bp flanking homologies have been demonstrated.
Conclusion
he λ Red-recombineering technology was successfully used for rapid genera-
tion of chromosomal modifications in the specially selected P. ananatis recipi-
ent strain. The procedure of electro-transformation with chromosomal DNA
has been developed for transfer of the marked mutation between different P.
ananatis strains. Combination of these techniques with λ Int/Xis-dependent
excision of selective markers significantly accelerates basic research and con-
struction of producing strains.
background
Pantoea ananatis belongs to the Enterobacteriacea family. The P. ananatis strain
AJ13355 (SC17) was isolated from soil in Iwata-shi (Shizuoka, Japan) as a bacte-
rium able to grow at acidic pH and showing resistance to high concentrations of
glutamic acid [1]. These physiological features made this organism an interesting
object for biotechnological studies, and for this reason its genome has been se-
quenced by Ajinomoto Co. (unpublished results). Nevertheless, up to the recent
past, the absence of efficient genetic tools has hampered manipulations of this
bacterium and retarded both basic research and applied investigations.
Over the last decade [2-7], the most powerful method for generating a wide
variety of DNA rearrangements in E. coli has been termed “recombineering” (re-
combination-mediated genetic engineering) [8]. The term generally refers to in
vivo genetic engineering with DNA fragments carrying short homologies with a
bacterial chromosome, using the proteins of a homologous recombination system
of the bacteriophage λ ( λ Red system). Lambda red operon includes only three
genes encoding Exo, Beta and Gam proteins. Gam inhibits the host nucleases,
RecBCD and SbcCD, thereby protecting the dsDNA substrate for recombination
[9,10]; Exo degrades linear dsDNA from each end in a 5' 3' direction, creat-
ing dsDNA with 3' single-stranded DNA tails [11-14]; and Beta stably binds a
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