| Summary: | 碩士 === 國立中興大學 === 植物學系 === 86 === Abstract: The gram-negative bacterium Xanthomonas campestris pv.
campestris is an important plant pathogen, causing black rot in
crucifers worldwide resulting in tremendous loss. It is also a
producer of xanthan gum, a substance which has a variety of
applications in petroleum production, cosmetics and food
industries. We were interested in expression and regulation of
the genes involved in xanthan gum synthesis. We have
previoumutagenesis. Using pulsed-field gel electrophoresis
(PFGE) and Southern hybridization, the mutations leading to the
non-mucoid phenotypes have been located at eight loci in the
Xc17 chromosome, which were named eps1, eps2, eps3, eps4, eps5,
eps6, eps7 and eps8, respectively.
This study focused on the non-mucoid mutant AY61E. One
recombinant cosmid, pEXO40, was cloned from the genomic bank of
Xc17. After deletion mapping, a smallest clone, pR2-2, was
obtained which contained a 2.2-kb insert. Mucoid phenotype was
restored to AY61E harboring pR2-2, indicating that the pR2-2
insert contained the gene responsible for the mutation in AY61E.
Nucleotide sequence analysis of the pR2-2 insert revealed an
open reading frame (orf482) able to encode a polypeptide with a
calculated molecaa weight of 54,013 dal. The deduced amino acid
sequence of the C-terminal 153 residues of ORF482 exhibited 99%
identity to the gene 1 of X. campestris pv. campestris strain.
In Northern hybridization of the total RNA extracted from Xc17,
only one transcript with the size similar to that of the orf482
coding region (1.5 kb) was detected. Primer extension indicated
the transcription start site to be the base T at 232 nt upstream
from the orf482 codon region. A 458-bp PstI fragment before the
orf482 start cod was found to efficiently express the lacZ
reporter when cloned as a transcriptional fusion unit in the
promoter-proving vector pFY13-9. Expression of the pR2-2 insert
in vitro using E. coli S30 transcription/translation system
produced a protein of 54 kDa which is similar to the MW deduced
from nucleotide sequence. The promoter of gum operon, a cluster
of 12 genes required for xanthan synthesis, cloned as a
transcriptional fusion expressed normally in AY61E indicating
that its expression is not regulated bORF482. AY61E exhibited
higher resistance to chloramphenicol and salt. The amounts of
the xanthan gum produced by AY61E and AY61E(pR2-2) were about
29.6% and 54.6%, respectively, of that produced by Xc17. AY61E
showed attenuated virulence in causing black rot in chinese
cabbage and pakchoi. The transcription direction of orf482 was
determined to be counterclockwise on the Xc17 chromosome map.
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