Downloaded from www.microbiologyresearch.org by IP: 23.22.6.154 On: Fri, 05 Feb 2016 09:22:27 Microbiology (1997), 143, 1141-1 149 Printed in Great Britain Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoreg u lated expression in Escherichia coli Petra Louis and Erwin A. Galinski Author for correspondence: Erwin A. Galinski. Tel: +49 228 733799. Fax: +49 228 737576. lnstitut fur Mikrobiologie & Biotechnologie, R hein isc he Fried r ic h- Wilhelms-Universitat Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl- 4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4 4 kb fragment revealed four major ORFs, which were designated ectA, ectf?, ectC and odA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for ~-2,4-diaminobutyric acid acetyltransferase, ectf? for ~-2,4- diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium. INTROD JCTION Keywords : Marinococcus halophilus, compatible solutes, ectoine genes, osmoregulation, salt stress Saline environments are characterized by high osmotic strength (low water potential). Most halophilic eubac- teria cope with these conditions by accumulating small, highly water-soluble organic compounds, the so-called compatible solutes (Brown, 1976). These osmolytes enable organisms to adapt to a wide range of salt concentrations by adjusting the cytoplasmic solute pool to the osmolarity of the surrounding environment. Ectoines represent the predominant class of osmolytes in aerobic chemoheterotrophic eubacteria (Severin et al., 1992; Frings et al., 1993; Galinski, 1995). The bio- synthetic pathway for ectoine has been elucidated at the enzymological level in Gram-negative eubacterial halo- philes (Peters et al., 1990; Tao et al., 1992; Galinski & Truper, 1994). It comprises three steps, the first being the conversion of aspartate semialdehyde, an inter- mediate in amino acid metabolism, to ~-2,4-diamino- butyric acid. This is followed by acetylation to NY- The GenBank accession number for the sequence reported in this paper is U66614 acetyldiaminobutyric acid. The last step consists of a cyclic condensation reaction to form the tetrahydro- pyrimidine ectoine (Fig. 1). Expression and regulation of genes involved in osmo- adaptation has thus far been investigated almost ex- clusively in non-halophilic bacteria, especially Escher- ichia coli (for recent reviews see Csonka & Hanson, 1991; Lucht & Bremer, 1994; Galinski, 1995). This organism responds to increased salinity with the rapid accumulation of potassium and concomitant synthesis of glutamate as a counter anion. Subsequently, these charged solutes are partially replaced by endogenous trehalose or compatible solutes accumulated from the medium, if present (Dinnbier et al., 1988). Most studies at the molecular level have so far focused on the various solute uptake systems of E. coli (Altendorf & Epstein, 1993; Mellies et al., 1995; Gowrishankar & Manna, 1996). The only investigations concerned with the biosynthesis of compatible solutes covered choline oxidation and trehalose synthesis (Lamark et al., 1996; Strarm & Kaasen, 1993). It has been proposed that relatively non-specific signals, such as intracellular 0002-1424 0 1997 SGM 1141