Abstract
The major uptake carrier for the compatible solute glycine betaine in Corynebacterium glutamicum is the secondary transport system BetP. It is effectively regulated by the external osmolality both on the level of expression and of activity. BetP carries highly charged domains both at the N and at the C terminus. We investigated the role of these extensions in the regulatory response to hyperosmotic stress. Mutants of the betP gene coding for proteins with truncated N- and C-terminal extensions were expressed in the C. glutamicum betP deletion strain DHP1 and were functionally characterized with respect to regulation of activity. The optimum of activation at 1.3 osmol/kg in wild type was shifted in the recombinant strains to about 2.6 osmol/kg in mutants with deletions in the N-terminal part. Deletions in the C-terminal domain resulted in a complete loss of regulation. The altered response to changes in osmolality led to severe consequences in the cellular adaption to hyperosmotic stress. Whereas in the wild type, the steady state level of glycine betaine accumulation is maintained by activity regulation of the BetP system itself, in the mutant with BetP proteins carrying truncations in the C-terminal domain, the observed steady state betaine accumulation was found to be due to a kinetic balance of unregulated glycine betaine uptake by the modifed BetP and efflux via the mechanosensitive efflux channel for compatible solutes at the same time.
Highlights
IntroductionTwo uptake systems for compatible solutes have been identified in these organisms, namely the binding protein-dependent transporter ProU and the secondary carrier ProP
Sively studied in the Gram-negative Escherichia coli and Salmonella typhimurium
We have previously described that the glycine betaine carrier BetP, the major osmo-reactive uptake system in C. glutamicum, effectively responds to osmotic changes within a time range of seconds or less (14)
Summary
Two uptake systems for compatible solutes have been identified in these organisms, namely the binding protein-dependent transporter ProU and the secondary carrier ProP. Whereas ProU is regulated both on the level of transcription and activity (4, 5), ProP is mainly regulated on the activity level (6 – 8) Another well studied organism in this respect is the Gram-positive Bacillus subtilis. In this organism, glycine betaine is taken up via three osmo-regulated transport systems, belonging either to the ABC-type or to the class of secondary carriers (9 –11). The secondary structure of BetP was predicted to carry two extensions at the cytoplasmic face, a negatively charged N-terminal and a positively charged Cterminal extension (13) The function of these domains in sensing osmotic stress was studied by construction of deletion mutants and functional characterization of the gene products
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