Osmotically Regulated Synthesis of the Compatible Solute Ectoine in Bacillus pasteurii and Related Bacillus spp

ABSTRACT By using natural-abundance 13C-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography (HPLC) analysis we have investigated the types of compatible solutes that are synthesized de novo in a variety of Bacillus species under high-osmolality growth conditions. Five different patterns of compatible solute production were found among the 13 Bacillus species we studied. Bacillus subtilis, B. licheniformis, and B. megaterium produced proline; B. cereus, B. circulans, B. thuringiensis, Paenibacillus polymyxa, and Aneurinibacillus aneurinilyticus synthesized glutamate; B. alcalophilus, B. psychrophilus, and B. pasteurii synthesized ectoine; and Salibacillus (formerly Bacillus) salexigens produced both ectoine and hydroxyectoine, whereas Virgibacillus (formerly Bacillus) pantothenticus synthesized both ectoine and proline. Hence, the ability to produce the tetrahydropyrimidine ectoine under hyperosmotic growth conditions is widespread within the genus Bacillus and closely related taxa. To study ectoine biosynthesis within the group of Bacillus species in greater detail, we focused on B. pasteurii. We cloned and sequenced its ectoine biosynthetic genes (ectABC). The ectABC genes encode the diaminobutyric acid acetyltransferase (EctA), the diaminobutyric acid aminotransferase (EctB), and the ectoine synthase (EctC). Together these proteins constitute the ectoine biosynthetic pathway, and their heterologous expression in B. subtilis led to the production of ectoine. Northern blot analysis demonstrated that the ectABC genes are genetically organized as an operon whose expression is strongly enhanced when the osmolality of the growth medium is raised. Primer extension analysis allowed us to pinpoint the osmoregulated promoter of the B. pasteurii ectABC gene cluster. HPLC analysis of osmotically challenged B. pasteurii cells revealed that ectoine production within this bacterium is finely tuned and closely correlated with the osmolality of the growth medium. These observations together with the osmotic control of ectABC transcription suggest that the de novo synthesis of ectoine is an important facet in the cellular adaptation of B. pasteurii to high-osmolarity surroundings.

[1]  J A Chudek,et al.  The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis. , 1990, Journal of general microbiology.

[2]  M. Jebbar,et al.  Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics , 1992, Journal of bacteriology.

[3]  E. Bremer.  Coping with osmotic challenges : osmoregulation through accumulation and release of compatible solutes in bacteria , 2000 .

[4]  M. Jebbar,et al.  Osmoadaptation in rhizobia: ectoine-induced salt tolerance , 1994, Journal of bacteriology.

[5]  Y. Murooka,et al.  Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata , 1999, Journal of bacteriology.

[6]  L N Csonka,et al.  Physiological and genetic responses of bacteria to osmotic stress. , 1989, Microbiological reviews.

[7]  S. Takeshita,et al.  High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. , 1987, Gene.

[8]  H. Gainer,et al.  Osmoregulation. , 1972, Folia medica Neerlandica.

[9]  T. Arakawa,et al.  The stabilization of proteins by osmolytes. , 1985, Biophysical journal.

[10]  E. Bremer,et al.  Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B , 1997, Molecular microbiology.

[11]  H. G. Trüper,et al.  The biosynthesis of ectoine , 1990 .

[12]  H. G. Trüper,et al.  Microbial behaviour in salt‐stressed ecosystems , 1994 .

[13]  J. Hansen,et al.  Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168 , 1995, Journal of bacteriology.

[14]  L. Wu,et al.  Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA , 1996, Journal of bacteriology.

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  D. W. Bolen,et al.  Osmolyte-driven contraction of a random coil protein. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Casadaban,et al.  Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. , 1976, Journal of molecular biology.

[18]  A. Burkovski,et al.  Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP , 1998, Journal of bacteriology.

[19]  E. Bremer,et al.  Response of Bacillus subtilis to high osmolarity: uptake of carnitine, crotonobetaine and γ-butyrobetaine via the ABC transport system OpuC. , 1998, Microbiology.

[20]  S. Salzberg,et al.  DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae , 2000, Nature.

[21]  E. Bremer,et al.  Ectoine functions as an osmoprotectant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC , 1997 .

[22]  J. M. Wood,et al.  Osmoadaptation by rhizosphere bacteria. , 1996, Annual review of microbiology.

[23]  R. Bernlohr,et al.  Changes in Free Amino Acid Production and Intracellular Amino Acid Pools of Bacillus licheniformis as a Function of Culture Age and Growth Media , 1972, Journal of bacteriology.

[24]  Antonio Ventosa,et al.  Biology of Moderately Halophilic Aerobic Bacteria , 1998, Microbiology and Molecular Biology Reviews.

[25]  E. Galinski,et al.  Enzyme stabilization be ectoine-type compatible solutes: protection against heating, freezing and drying , 1992, Applied Microbiology and Biotechnology.

[26]  Y. Kho,et al.  Sporosarcina aquimarina sp. nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosarcina globispora comb. nov., Sporosarcina psy , 2001, International journal of systematic and evolutionary microbiology.

[27]  C. Harwood,et al.  Molecular biological methods for Bacillus , 1990 .

[28]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[29]  J. J. Nieto,et al.  Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043. , 1998, Systematic and applied microbiology.

[30]  H. G. Trüper,et al.  1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. , 1985, European journal of biochemistry.

[31]  J. Boch,et al.  High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute inBacillus subtilis , 1999, Applied and Environmental Microbiology.

[32]  J. J. Nieto,et al.  Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine , 1999, Applied and Environmental Microbiology.

[33]  Bacillus salexigens sp. nov., a new moderately halophilic Bacillus species. , 1997, International journal of systematic bacteriology.

[34]  E. Bremer,et al.  Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A‐ and sigma B‐dependent stress‐responsive promoters , 1998, Molecular microbiology.

[35]  E. Galinski,et al.  Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. , 1997, Microbiology.

[36]  E. Bremer,et al.  Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments , 1998, Archives of Microbiology.

[37]  M. Roberts,et al.  Osmoadaptation and osmoregulation in archaea. , 2000, Frontiers in bioscience : a journal and virtual library.

[38]  M. Marahiel,et al.  Engineered Biosynthesis of the Peptide Antibiotic Bacitracin in the Surrogate Host Bacillus subtilis * , 2001, The Journal of Biological Chemistry.

[39]  E. Galinski,et al.  Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis. , 1998, FEMS microbiology letters.

[40]  H. G. Trüper,et al.  A modified FMOC-method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines) , 1993 .

[41]  E. Bremer,et al.  OpuA, an Osmotically Regulated Binding Protein-dependent Transport System for the Osmoprotectant Glycine Betaine in Bacillus subtilis(*) , 1995, The Journal of Biological Chemistry.

[42]  J. Boch,et al.  Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis , 1999, Molecular microbiology.

[43]  M. Besnard,et al.  Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells* , 1998, The Journal of Biological Chemistry.

[44]  Eugene W. Myers,et al.  Basic local alignment search tool. Journal of Molecular Biology , 1990 .

[45]  J. J. Nieto,et al.  Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes* , 1997, The Journal of Biological Chemistry.

[46]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[47]  Long-Fei Wu,et al.  Glycine Betaine-assisted Protein Folding in a lysAMutant of Escherichia coli * , 2000, The Journal of Biological Chemistry.

[48]  E. Bremer.  Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes in B. subtilis , 2000 .

[49]  R. Reed,et al.  Determination of turgor pressure in Bacillus subtilis: a possible role for K+ in turgor regulation. , 1990, Journal of general microbiology.

[50]  J. Boch,et al.  Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes , 1996, Journal of bacteriology.

[51]  J. J. Nieto,et al.  Chromohalobacter salexigens sp. nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174. , 2001, International journal of systematic and evolutionary microbiology.

[52]  J. D. Helmann,et al.  Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA , 1995, Nucleic Acids Res..

[53]  Y. Nakamura,et al.  Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. , 2000, Nucleic acids research.

[54]  I. Booth,et al.  Managing hypoosmotic stress: aquaporins and mechanosensitive channels in Escherichia coli. , 1999, Current opinion in microbiology.

[55]  I. Booth,et al.  Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity , 1999, The EMBO journal.

[56]  A. D. Brown,et al.  Microbial water stress. , 1976, Bacteriological reviews.