Results and Discussion

A PCR strategy was used to isolate BetP, a secondary transporter which belongs to the BCCT (Betaine Choline Carnitine Transporter) subfamily in S. meliloti. The betP gene was localized on the pSymb and the gene product displayed significant identities with the choline transporter BetT of Escherichia coli and with the GB transporters OpuD of Bacillus subtilis and BetP of Corynebacterium glutamicum. Sequence analysis suggested that the BetP protein of S. meliloti contained 12 transmembrane spanning segments and two hydrophile N and C terminal cytoplasmic extensions.

The transport characteristics of S. meliloti BetP were determined in an E. coli mutant strain devoided of choline, GB and proline uptake. The BetP protein was only active under high osmotic conditions and was specifically involved in GB and PB uptake at high affinity, exhibiting a Km of 16 |iM towards GB and 56 fiM towards PB. Using a transcriptional betP-lacZ fusion recombined into the genome of S. meliloti, the betP gene was shown to be constitutively expressed. Consequently, the activation of GB mediated BetP transport by high osmolarity may involve a post-transductional mechanism. Indeed, in the wild-type strain of S. meliloti, betaines transport activity was enhanced by 3.5-fold within five minutes following an osmotic upshock, a feature also observed in presence of chloramphenicol, while no activation could be detected in a betP-Km derivative mutant strain. Thus, the activity of BetP is stimulated by elevation of the osmolarity and may allow S. meliloti to respond rapidly to sudden changes in the osmotic pressure of the environment. The betP mutant showed a clear delay in its growth when transferred from low to high osmolarity medium containing betaines, presenting a much longer lag phase than the wild-type (5 h versus 12 h in media containing GB and 7 h versus 20 h using PB as osmoprotectant). For a long term adaptation in environment of elevated osmolarity, S. meliloti uses at least another betaines uptake system, most probably transcriptionally induced, since 16 h after transfer from low to high osmolarity medium, the betP mutant retains 40% of the betaine transport capacity of the wild-type strain.

The BetP protein appeared to be a major betaines transporter involved in osmoregulation. BetP may play an important role in legume-rhizobia interaction subjected to osmotic stress, as betaines are available within the rhizosphere, particulary proline betaine which is excreted by alfafa roots.

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