Molecular characterization of catabolite repression by succinate in the nodulating bacterium Sinorhizobium meliloti Page: 2 of 7
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2. We cloned, and over-expressed, the AgpT gene in E. coi, and showed that crude extracts made
from the over-expressing strain were able to bind to the promoter region of the melA-agp operon.
3. We made transcriptional fusions between the melA-agp promoter region and gfp. These fusions
are convenient tools that allowed us to easily monitor expression of the melA-agp promoter region.
4. Because of its useful characteristics (high levels of expression induced by a-galactosides, and
low levels of expression when succinate is present and a-galactosides are absent) the melA-agp
promoter is being used by the rhizobium research community as a promoter for heterologous
expression is S. me/iloti.
5. S. me/iloti carrying melA-gfp reporter plasmids were used as whole-cell biosensors to study the
release of galactosides from plant roots that were grown in non-sterilized soil. This was the first
time that anyone was able to non-destructively, and at micrometer spatial scales, monitor the
release of a specific class of sugars from roots growing in non-sterilized soil. The fluorescent
reporters also allowed visualization of trophic interactions because protists that consumed
fluorescent bacteria, became fluorescent themselves.
6. Studies of melA-agp promoter control revealed that a gene down stream of agpT, that encoded a
LacI-type repressor, affected expression of the melA-agp operon. This gene was subsequently
shown by us to be LacR, which controls the expression of the lac genes which are next to the
melA-agp operon. These genes are required for the transport and utilization of f3-galactoside, and
they are also downregulated by SMCR.
7. Biochemical and genetic studies utilizing the lac and melA-agp genes showed that SMCR
occurs, at least in part through inducer exclusion. That is succinate blocks the transport of
secondary carbon sources, such as raffinose, when both are present together. This was the first time
that anyone had elucidated any of the molecular mechanisms by which SMCR works in rhizobia or
8. Epistatic analysis of HprK, ManX(EIIA) and Hpr. Our current model postulates that the
enhanced SMCR phenotypes of the AhprK and AmanX(EIIA) mutants are brought about because
they cause Hpr to exist mainly as Hpr-His22-P. If this is true, then AhprK Ahpr and AmanX(EIIA)
Ahpr double mutants should exhibit the same phenotype as a Ahpr mutant -- they should show
weakened SMCR rather than enhanced SMCR (AhprK and AmanX(EIIA) phenotypes). This set of
experiments was thought to be particularly important because it should reveal whether ManX
(EIIA) and HprK affect SMCR only through their effects on Hpr, or whether they can affect
SMCR independently of Hpr. The double, unmarked, AmanX(EIIA) Ahpr mutant has been
constructed using sacB-gene replacement methods similar to those used to make the in-frame
mutans described in the main proposal. This strain, CAP32(AmanX(EIIA)Ahpr), showed weak
SMCR, similar to the Ahpr mutant upon growth on XGal-succinate + lactose + Xgal plates. Our
hypothesis that SMCR phenotype of the AmanX(EIIA) mutation was caused by its effect on Hpr is
consistent with the observation described above that the Ahpr mutation is epistatic to the AmanX
(EIIA) mutation. Construction of the AhprK Ahpr double mutant is in progress.
9. We have not identified the protein responsible for phosphorylating Hpr. In other
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Gage, Daniel. Molecular characterization of catabolite repression by succinate in the nodulating bacterium Sinorhizobium meliloti, report, September 27, 2006; United States. (digital.library.unt.edu/ark:/67531/metadc881860/m1/2/: accessed April 19, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.