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J. Biol. Chem., Vol. 279, Issue 11, 9892-9898, March 12, 2004
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From the
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal, the ¶Departamento de Bioquímica and Centro de Neurociências e Biologia Molecular, Universidade de Coimbra, 3000 Coimbra, Portugal, and the ||Nestlé Research Center, CH-1000 Lausanne 26, Switzerland
Rhodothermus marinus responds to fluctuations in the growth temperature and/or salinity by accumulating mannosylglycerate (MG). Two alternative pathways for the synthesis of MG have been identified in this bacterium: a single-step pathway and a two-step pathway. In this work, the genetic and biochemical characterization of the two-step pathway was carried out with the goal of understanding the function of the two pathways and their regulatory mechanisms. Mannosyl-3-phosphoglycerate synthase (MPGS) of the two-step pathway was purified from R. marinus. Sequence information led to the isolation of two contiguous genes, mpgs (encoding MPGS) and mpgp (encoding mannosyl-3-phosphoglycerate phosphatase). The recombinant MPGS had a low specific activity compared with other homologous MPGSs and contained
30 additional residues at the C terminus. Truncation of this extension produced a protein with a 10-fold higher specific activity. Moreover, the activity of the complete MPGS was enhanced upon incubation with R. marinus cell extracts, and protease inhibitors abolished activation. Therefore, the C-terminal peptide of MPGS was identified as a regulatory site for short term control of MG synthesis in R. marinus. The control of gene expression by heat and osmotic stress was also studied; the level of mannosylglycerate synthase involved in the single-step pathway was selectively enhanced by heat stress, whereas MPGS was overproduced in response to osmotic stress. The concomitant changes in the level of MG were assessed as well. We conclude that the two alternative pathways for the synthesis of MG are differently regulated at the level of expression to play specific roles in the adaptation of R. marinus to two different types of aggression. This is the only example of pathway multiplicity being rationalized in terms of the need to respond efficiently to distinct environmental stresses.
Received for publication, November 6, 2003 , and in revised form, December 15, 2003.
* This work was funded by European Commission 5th Framework Programme Project QLK3-CT-2000-00640 and Fundação para a Ciência e a Tecnologia and FEDER, Portugal PRAXIS/P/BIO/12082/1998, and POCTI/35715/BIO/2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY271294
Recipient of Ph.D. Grant 19868/99 from PRAXIS XXI.
** Recipient of Ph.D. Grant 21665/99 from PRAXIS XXI.

To whom correspondence should be addressed. Tel.: 351-214469828; Fax: 351-214428766; E-mail: santos{at}itqb.unl.pt.
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