Identification and Characterization of EctR1, a New Transcriptional Regulator of the Ectoine Biosynthesis Genes in the Halotolerant Methanotroph Methylomicrobium alcaliphilum 20Z

Abstract

Mineralization of small saline and soda lakes can vary significantly depending on the season and weather conditions. The ability to rapidly adjust intracellular concentrations of key osmolytes (also known as compatible solutes) to changes in external salinity is an important property of microorganisms inhabiting these biotopes (8,18,34). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was found to be a major compatible solute in many halophilic or halotolerant bacteria isolated from alkaline, moderately hypersaline environments (14). This organic solute can be synthesized de novo or taken up from the environment when available (15, 18). The biochemistry and genetics of ectoine synthesis have been described for several bacteria (15,28,29,32). However, little is known about the transcriptional regulation of the ectoine biosynthetic pathway. Comprehensive analysis of the ectoine gene cluster ectABC in Chromohalobacter salexigens showed four putative transcription initiation sites upstream of the ectA start codon. Two 70 -dependent, one S -dependent, and one 32 -dependent promoter were identified and shown to be involved in ectABC transcription in this bacterium (6). Transcription of the ectA, ectB, and ectC genes from Marinococcus halophilus was initiated from three individual 70 / A -dependent promoter sequences located upstream of each gene (3). In Bacillus pasteurii, the ectABC genes are organized in a single operon preceded by a typical A -dependent promoter region (21). The halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z is capable of growth at a salinity as high as 2 M NaCl (19). It was demonstrated that in response to the elevated salinity of the growth medium, M. alcaliphilum cells accumulate ectoine as a major osmoprotective compound (20). The ectoine biosynthesis pathway in M. alcaliphilum 20Z is similar to the pathway employed by halophilic/halotolerant heterotrophs and involves three specific enzymes: diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC) (7,21,24,30,32,49). In M. alcaliphilum 20Z, the ectoine biosynthetic genes were shown to be organized in the ectABC-ask operon containing the additional ask gene, encoding aspartokinase (32). Here we describe the transcriptional organization of the ectoine biosynthetic genes in M. alcaliphilum 20Z. We identify a new MarR-like transcriptional regulator (EctR1) and show that EctR1 represses the expression of the ectABC-ask operon from the ectAp 1 promoter by binding at the putative Ϫ10 sequence. These results demonstrate the presence of a new, previously uncharacterized regulatory system for ectoine biosynthesis in the salt-tolerant methanotroph. MATERIALS AND METHODSBacterial strains and culture conditions. The M. alcaliphilum and Escherichia coli strains, plasmids, and primers used in this study are listed in Tables S1 and S2 in the supplemental material. M. alcaliphilum strains were grown at 30°C under a methane-air atmosphere (1:1) or in the presence of 0....

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

“…A subset of the ectoine producers hydroxylate ectoine to 5-hydroxyectoine (9,17,48) in a stereospecific fashion using the ectoine hydroxylase EctD (8,9,50). The structural genes (ectABC) for the ectoine biosynthetic enzymes are typically organized as a gene cluster whose transcription can be induced by increased osmolarity of the growth medium (9,11,32,33,40,43). In some microorganisms (e.g., in several bacilli) the ectABC genes are transcribed as an operon (8,32,33), but more complex patterns of ect transcription have been reported for Chromohalobacter salexigens (17) and Halomonas elongata (57).…”

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

“…3). The predicted -10 (TACTAT) and -35 (TGGACA) regions of ectAр1 showed rather high level of identity with the consensus sequence of the E. coli σ 70 -recognized promoter (Reshetnikov et al, 2006;Mustakhimov et al, 2010). The putative -10 and -35 sequences of the ectAр2 promoter differ from the respective regions of the E. coli σ 70 -promoter thus suggesting that expression from ectAр2 may be less effective, than from ectAр1.…”

“…In Bacillus pasteurii (Kuhlmann and Bremer, 2002), Marinococcus halophilus (Bestvater and Galinski, 2002), Halobacillus halophilus (Saum and Muller, 2008b) and S. salexigens (Bursy et al, 2007), transcription of the ectABC-ask or ectABC genes proceeded as polycistronic mRNAs and subjected to osmotic conditions. The osmoregulated expression of ectABC was also revealed in gramnegative Brevibacterium epidermis (Onraedt et al, 2004), Mm alcaliphilum (Reshetnikov et al, 2006;Mustakhimov et al, 2010) and Chromohalobacter salexigens (Calderon et al, 2004).…”

“…The autoregulation was described for some other MarR proteins. The purified EctR1 specifically binds to the promoter region of ectABC-ask operon (Mustakhimov et al, 2010). The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp suggesting dimeric association of the EctR1 with the DNA where each protein subunit binds with an inverted repeat (Mustakhimov et al, 2010).…”

“…Despite low identity, the protein has a structure analogous to the MarR-family regulators: it has the HelixTurn-Helix (HTH) DNA-binding motif flanking the "wing 1" region (Mustakhimov et al, 2010;Hong et al, 2005;Wilkinson and Grove, 2006). Regulatory function of EctR1 in ectoine biosynthesis was elucidated by characterization of the ectR1 knockout mutant.…”