Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.
In contrast to what happens in Agrobacterium tumefaciens and Rhizobium meliloti, synthesis of periplasmic cyclic 1,2-β-glucan in Brucella spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-β-glucan synthetase of Brucella spp. was not inhibited by 05 M KCl or potassium glutamate ; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The Brucella abortus cyclic 1,2-β-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-β-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (025 M NaCl or 05 M mannitol). On the other hand, when the A. tumefaciens cyclic 1,2-β-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-β-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that Brucella cyclic 1,2-β-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in Agrobacterium or Rhizobium backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-β-glucan synthesis in Brucella is due to two convergent mechanisms : a) the presence of a cyclic 1,2-β-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1,2-β-glucan synthetase.
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