Characterization of a cbbR allele that suppresses phenotypes associated with a tktA/B double mutant strain in Sinorhizobium meliloti

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Patuwatha Withanage, Ishani
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The genome of Sinorhizobium meliloti contains three genes that are annotated as transketolases. Transketolase activity is necessary for the pentose phosphate pathway, and under normal conditions the protein responsible for transketolase activity is encoded for by the gene tktA. A strain carrying a mutation in this gene is auxotrophic for aromatic amino acids and is also severely debilitated in its ability to establish an effective symbiosis. It has been shown that the phenotypes associated with a mutation in tktA can be partially suppressed by point mutations in a negative regulator that results in the expression of tktB. In the present study, mutants were isolated that suppressed carbon phenotypes associated with a tktA/tktB double mutant. The objective of the study was to identify and characterize this suppressor mutation. The mutation was in cbbR which is annotated as a Lys-R type transcriptional regulator which is recognized as the key regulator of the Calvin Benson Bassham (cbb) operon in CO2 fixing organisms. In S. meliloti, the cbb operon encodes a third putative transketolase-cbbT. The results of qRT-PCR show that the cbbR* mutation is correlated with the upregulation of the 10 genes in the cbb gene cluster which includes cbbR. Introduction of cbbT on a plasmid into a strain carrying mutations in tktA/tktB results in suppression of tktA/tktB associated phenotypes, suggesting that the enzyme encoded by cbbT plays a significant role in suppression of tktA/tktB. When inoculated onto alfalfa plants, strains carrying the cbbR* mutation were able to partially suppress the fix- phenotypes associated with tkt mutations. This suppression resulted in ~50% nitrogen fixing efficiency compared to wild-type. These strains were also highly uncompetitive for nodule occupancy, yet they exhibited nodulation kinetics that were like that of the wildtype. Overall, these observations suggest that the aberrant regulation of cbb genes can affect symbiotic development and nitrogen fixation in S. meliloti.
S. meliloti, Carbon metabolism, Transketolase, Rhizobium, Nitrogen fixation, Sinorhizobium meliloti