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W2029942689 abstract "The moderate halophile Halomonas elongata Deustche Sammlung für Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 m NaCl). Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated. Mutant CHR62 could not grow above 0.75 m NaCl, and CHR63 did not grow above 1.5m NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13C NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-γ-acetyldiaminobutyric acid. These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC). Salt sensitivity of the mutants at 1.5–2.5 m NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-γ-acetyldiaminobutyric acid. This is the first evidence for the role of N-γ-acetyldiaminobutyric acid as osmoprotectant. Finally, a cosmid from the H. elongata genomic library was isolated which complemented the Ect− phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine. The moderate halophile Halomonas elongata Deustche Sammlung für Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 m NaCl). Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated. Mutant CHR62 could not grow above 0.75 m NaCl, and CHR63 did not grow above 1.5m NaCl. These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13C NMR and mass spectroscopy. Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-γ-acetyldiaminobutyric acid. These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC). Salt sensitivity of the mutants at 1.5–2.5 m NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-γ-acetyldiaminobutyric acid. This is the first evidence for the role of N-γ-acetyldiaminobutyric acid as osmoprotectant. Finally, a cosmid from the H. elongata genomic library was isolated which complemented the Ect− phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine. Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes.Journal of Biological ChemistryVol. 273Issue 19PreviewIn the period from the submission to the publication of this work, a study on the characterization of genes from the biosynthetic pathway of ectoine in Marinococcus halophilus was reported by Louis and Galinski (Louis, P., and Galinski, E. A. (1997) Microbiology 143, 1141–1149). To follow the same nomenclature, the provisional designations that have been used in our paper for the loci encoding the three enzymes of ectoine synthesis should be changed as follows: ectA(instead of ectB) encoding the diaminobutyric acid acetyltransferase, ectB (instead of ectA) encoding l-diaminobutyric acid transaminase, andectC for the gene encoding the ectoine synthase. Full-Text PDF Open Access Halomonas elongata is a moderately halophilic bacterium that can grow over a wide range of salinity, from ∼0.1 to ∼4m NaCl (1Vreeland R.H. Litchfield C.D. Martin E.L. Elliot E. Int. J. Syst. Bacteriol. 1980; 30: 485-495Crossref Scopus (332) Google Scholar, 2Ventosa A. Priest F.G. Ramos-Cormenzana A. Tindall B.J. Bacterial Diversity and Systematics. Plenum Publishing Corp., New York1994: 231-242Crossref Google Scholar). This property makes this halophile an excellent model to study the osmoregulatory mechanisms in this group of extremophilic organisms. Moreover, H. elongata has recently received considerable interest because of its potential for use in biotechnology. Thus, it is a good source for halophilic enzymes as well as the compatible solutes ectoine and hydroxyectoine that can be used as protecting agents for enzymes and whole cells (3Ventosa A. Nieto J.J. World J. Microbiol. & Biotechnol. 1995; 11: 85-94Crossref PubMed Scopus (222) Google Scholar). Although genetic tools for moderate halophiles have been developed recently (4Fernández-Castillo R. Vargas C. Nieto J.J. Ventosa A. RuizBerraquero F. J. Gen. Microbiol. 1992; 138: 1133-1137Crossref PubMed Scopus (26) Google Scholar, 5Kunte H.J. Galinski E.A. FEMS Microbiol. Lett. 1995; 128: 293-299Crossref PubMed Google Scholar, 6Mellado E. Asturias J.A. Nieto J.J. Timmis K.N. Ventosa A. J. Bacteriol. 1995; 177: 3443-3450Crossref PubMed Google Scholar, 7Vargas C. Fernández-Castillo R. Cánovas D. Ventosa A. Nieto J.J. Mol. Gen. Genet. 1995; 246: 411-418Crossref PubMed Scopus (44) Google Scholar), the genetic basis of the osmoregulatory mechanisms in these bacteria remains unclear. As most other bacteria, moderate halophiles maintain their internal osmolality and generates turgor in media of high salinity by accumulating organic compatible solutes (8Kushner D.J. Kamekura M. Rodrı́guez-Valera F. Halophilic Bacteria. I. CRC Press, Inc., Boca Raton, FL1988: 109-140Google Scholar). When grown in media lacking osmoprotectants, H. elongata synthesizes ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylate) as its major compatible solute (9Galinski E.A. Adv. Microb. Physiol. 1995; 37: 273-328Crossref Scopus (399) Google Scholar). It can also accumulate glycine betaine and related osmoprotectants by transport from the medium (9Galinski E.A. Adv. Microb. Physiol. 1995; 37: 273-328Crossref Scopus (399) Google Scholar, 10Cánovas D. Vargas C. Csonka L.N. Ventosa A. Nieto J.J. J. Bacteriol. 1996; 178: 7221-7226Crossref PubMed Google Scholar). Glycine betaine has been shown to suppress the accumulation of ectoine partially or completely in H. elongata, depending on the NaCl concentration and the strain (9Galinski E.A. Adv. Microb. Physiol. 1995; 37: 273-328Crossref Scopus (399) Google Scholar, 10Cánovas D. Vargas C. Csonka L.N. Ventosa A. Nieto J.J. J. Bacteriol. 1996; 178: 7221-7226Crossref PubMed Google Scholar). Although H. elongata is typical among bacteria in that it accumulates glycine betaine in response to high salinity stress, the biochemical basis for its unusual NaCl tolerance is not clear.Escherichia coli and Salmonella typhimurium, which served as model organisms for the elucidation of basic principles of osmoregulation, also use glycine betaine as the preferred osmoprotectant (11Csonka L.N. Hanson A.D. Annu. Rev. Microbiol. 1991; 45: 569-606Crossref PubMed Scopus (647) Google Scholar). However, although glycine betaine can elicit dramatic stimulation of growth in media of inhibitory osmolality in these organisms, it can support growth of the latter organisms only to about 1.2 m NaCl, considerably less that the maximum salinity that can be tolerated by H. elongata. Ectoine has been discovered as a compatible solute in the extremely halophilic bacteria Ectothiorhodospira halochloris (12Galinski E.A. Pfeiffer H.-P. Trüper H.G. Eur. J. Biochem. 1985; 149: 135-139Crossref PubMed Scopus (303) Google Scholar) and subsequently shown in H. elongata (13Peters P. Galinski E.A. Truper H.G. FEMS Microbiol. Lett. 1990; 71: 157-162Crossref Google Scholar). The biosynthetic pathway of this compound is shown in Fig.1 (13Peters P. Galinski E.A. Truper H.G. FEMS Microbiol. Lett. 1990; 71: 157-162Crossref Google Scholar). Because the organisms that can synthesize ectoine are generally halophilic or marine bacteria (14Severin J. Wohlfarth A. Galinski E.A. J. Gen. Microbiol. 1992; 138: 1629-1638Crossref Scopus (162) Google Scholar), it has been suggested that high salinity tolerance could be connected with the ability to synthesize this compatible solute. To test whether there is such a causal connection between halotolerance and the synthesis of ectoine and to identify the ectoine biosynthetic genes, we isolated mutants of H. elongata DSM 3043 that are blocked in the synthesis of this compound. This work describes the isolation and characterization of these mutants as well as the isolation of the genes involved in the biosynthesis of ectoine in H. elongata. Bacterial strains and plasmids used in this study are listed in Table I. H. elongata strains were routinely grown in SW-10 medium, which contained 10% (w/v) total salts (15Nieto J.J. Fernández-Castillo R. Márquez M.C. Ventosa A. Ruiz-Berraquero F. Appl. Environ. Microbiol. 1989; 55: 2385-2390Crossref PubMed Google Scholar) and 0.5% (w/v) yeast extract (Difco). Salt-sensitive mutants were isolated on a modified version of this medium, which contained 2% total salts (designated SW-2 medium). The complex LB medium was used for the growth of E. coli (16Davis R.W. Botstein D. Roth J.R. Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1980Google Scholar). M63 (17Cohen G.N. Rickenberg R.H. Ann. Inst. Pasteur (Paris). 1956; 91: 693-720PubMed Google Scholar), containing 20 mm glucose as the sole carbon source, was used as the minimal medium. The osmotic strength of M63 was increased by the addition of 0.5 to 4 m final concentrations of NaCl. When used, glycine betaine (Aldrich) was added to a final concentration of 1 mm. The pH of all media was adjusted to 7.2 with KOH. Solid medium contained 20 g/liter Bacto-agar (Difco). When used, filter-sterilized antibiotics were at the following final concentrations (μg/ml): Ap, 1The abbreviations used are: Ap, ampicillin; Km, kanamycin; NADA, N-γ-acetyldiaminobutyric acid; DA, diaminobutyric acid; bp, base pair(s); kb, kilobase pair(s); GC-MS, gas chromatography-mass spectrometry. 100; chloramphenicol, 25; Km, 75; rifampicin, 25; tetracycline, 15. Liquid cultures were incubated at 37 °C in an orbital shaker at 200 rpm. Growth was monitored as the optical density of the culture at 600 nm with a Perkin-Elmer 551S UV/VIS spectrophotometer.Table IBacterial strains and plasmids used in this studyStrain/plasmidsRelevant featuresRef. or sourceH. elongata DSM 3043Wild-type1Vreeland R.H. Litchfield C.D. Martin E.L. Elliot E. Int. J. Syst. Bacteriol. 1980; 30: 485-495Crossref Scopus (332) Google Scholar CHR61Spontaneous Rfr mutant of DSM 3043This study CHR62Mutant of CHR61 defective in the synthesis of ectoineThis study CHR63Mutant of CHR61 defective in the synthesis of ectoineThis study CHR64Salt-sensitive mutant of CHR61This study CHR65Salt-sensitive mutant of CHR61This studyE. coli HB101Δ(gpt-proA)62 leuB6 thi-1 lacY1hsdS B 20 recA rpsL20 (Strr) ara-14 galKZ dxyl-5 mtl-1 supE44 mcrB B31Boyer H.W. Roulland-Dussoix D. J. Mol. Biol. 1969; 41: 459-472Crossref PubMed Scopus (2574) Google Scholar SM10thi thr leu tonA lacY sup E recA Muc+18Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar DH5αsupE44ΔlacU169 (φ80lacZΔM15)hsdR17 recA1endA1 gyrA96 thi-1 relA121Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarPlasmids pSUP102−Gmr, Kmr, vector for transposon mutagenesis18Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar, 19Ubben D. Schmitt R. Gene (Amst.). 1986; 41: 145-152Crossref PubMed Scopus (90) Google ScholarGm::Tn1732 pVK102Tcr, Kmr, cosmid vector, Inc P22Knauf V.C. Nester E.W. Plasmid. 1982; 8: 45-54Crossref PubMed Scopus (331) Google Scholar pKS(−)AprStratagene pRK600Cmr, helper plasmid, ColE132Kessler B. de Lorenzo V. Timmis K.N. Mol. Gen. Genet. 1992; 233: 293-301Crossref PubMed Scopus (240) Google Scholar pDE123-kb SalI fragment from CHR63 DNA carried in pKS(−)This study pDE38.6-kb SalI fragment from pDE1 cloned in pKS(−)This study pDE9pVK102-derivative carrying the H. elongata genes encoding the ectoine synthase and diaminobutyric acid acetyltransferaseThis study pDE10pVK102 derivative carrying the H. elongatagene for the ectoine synthaseThis study pDE11Same as pDE10This study Open table in a new tab Transposon mutagenesis was performed by conjugal transfer of pSUP102-Gm::Tn1732 from E. coli SM10 (18Simon R. Priefer U. Pühler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5658) Google Scholar, 19Ubben D. Schmitt R. Gene (Amst.). 1986; 41: 145-152Crossref PubMed Scopus (90) Google Scholar) to H. elongata strain CHR61. Matings were carried out by mixing the donor and recipient cultures at a ratio of 1:4 (100 μl of donor, 400 μl of recipient). The mixed cultures were washed with sterile SW-2 medium to eliminate the antibiotics. The pellet was resuspended in 100 μl of SW-2 and placed on a 0.45-μm pore filter on SW-2 solid media (which allows the growth of E. coli and the putative salt-sensitive mutants of H. elongata). After overnight incubation at 30 °C, cells were resuspended in 20% (v/v) sterile glycerol and, after appropriate dilutions, inoculated on SW-2 + rifampicin + Km plates at a density resulting in about 100–200 colonies per plate. Colonies from these master plates were transferred with sterile toothpicks to duplicate M63 plates, one contained 2.7m NaCl and the other contained 0.5 m NaCl. Plates were incubated at 37 °C and inspected for colonies that had grown at 0.5 m but not at 2.7 m NaCl. Chromosomal DNA from H. elongata was isolated as described by Ausubel et al.(20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.R. Struhl K. Current Protocols in Molecular Biology. Greene Associates/Wiley Interscience, New York1989Google Scholar). Plasmid DNA was isolated from E. coli with the alkaline lysis method (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Restriction enzyme digestion and ligations were performed as recommended by the manufacturers. Probes used for plasmid, genomic DNA, and colony hybridization were generated by using the non-radioactive digoxigenin DNA labeling and detection kit from Boehringer Mannheim. For genomic DNA hybridization, genomic DNA was isolated from the wild-type strain and salt-sensitive mutants of H. elongata, digested with restriction enzymes, separated by agarose gel electrophoresis and transferred to nylon filters (Amersham Corp.) as described by Sambrook et al. (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). An internal 1-kb HindIII fragment of the transposon Tn1732was used as a probe. For colony hybridization, 3,000 single colonies of the H. elongata genomic library were allowed to grow 12 h at 37 °C on LB + Km plates. After growth, plates were chilled for 1 h at 4 °C and transferred to nylon filters, as described by Sambrook et al. (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For colony hybridization, cellular debris was removed before hybridization to avoid background. Hybridization was in 3 × SSC, 0.1% SDS with shaking for 2 h at 68 °C. Hybridization, washes, and detection were done according to the instructions of the kit provided by Boehringer Mannheim. To clone the DNA region flanking the Tn1732 insertion in mutant CHR63, genomic DNA of CHR63 was digested with BglII, ligated toBamHI-digested pKS−, and transformed into DH5α. Transformants were selected on LB + Km + Ap. From one KmrApr colony, the plasmid pDE1, containing a 23-kbBglII fragment from CHR63 DNA, was isolated. An 8.6-kbSalI fragment of pDE1 was subcloned in pKS−, giving pDE3. AH. elongata gene bank was constructed in the broad host range cosmid pVK102 (22Knauf V.C. Nester E.W. Plasmid. 1982; 8: 45-54Crossref PubMed Scopus (331) Google Scholar), a low copy number cosmid that can replicate in both E. coli and Halomonas (23Vargas C. Coronado M.J. Ventosa A. Nieto J.J. Syst. Appl. Microbiol. 1997; 20: 173-181Crossref Scopus (27) Google Scholar). H. elongata DNA was partially digested with SalI, and DNA fragments in the size range of 23–30 kb were separated in sucrose gradients and cloned into the pVK102 vector, which had been linearized with SalI and treated with alkaline phosphatase. The ligation mix was packed in vitro into bacteriophage lambda heads by using a kit from Amersham Corp. and transduced into E. coli HB101. Out of 3,000 Kmr transductants, 30 colonies were analyzed, and all proved to have inserts of an average size of 27 kb, which guarantees a 99.8% probability of finding a given sequence in the bank (24Clark L. Carbon J. Methods Enzymol. 1979; 68: 396-408Crossref PubMed Scopus (31) Google Scholar). To isolate the genes responsible for the synthesis of ectoine, a total of 3,000 colonies of the H. elongata genomic library were screened by using as a probe a 370-bp EcoRI fragment from the 1.9-kb region carried in pDE3. This fragment, gel-isolated after EcoRI digestion of pDE3, was selected because it is adjacent to the Tn1732insertion in mutant CHR63. In fact, one of the EcoRI sites used to generate the probe lies in the right inverted repeat of Tn1732 (see Fig. 7 B). Therefore, the 370-bpEcoRI region should contain part of the H. elongata ectoine synthase gene (ectC). Plasmids isolated after library screening were conjugated from E. coli to H. elongata by triparental matings on SW-2 by using pRK600 as helper plasmid. Wild-type and salt-sensitive mutants of H. elongata were grown in M63 containing the maximal NaCl concentration that they could tolerate. At mid-exponential phase, cells were harvested and washed twice with the growth medium without any carbon source. To extract the cytoplasmic solutes, cells were resuspended in 10 ml of double distilled water and incubated for 5 h at room temperature. Cell debris was removed by centrifugation, and the supernatant was filtered through a 0.65-μm pore membrane filter. Cell extracts were lyophilized and resuspended in 0.5 ml of D2O. Natural abundance 13C NMR spectra were recorded on a Brucker ac200 spectrometer at 50 MHz with probe temperature of 20–22 °C. Signals due to glutamate, glutamine, and diaminobutyrate were identified by comparison with the spectra of each of these pure compounds. Signals generated by N-acetyldiaminobutyrate were deduced from the spectrum of DA signals. Ectoine was identified by comparison of chemical shifts with published values (25Fernández-Linares L. Faure R. Bertrand J.C. Gauthier M. Lett. Appl. Microbiol. 1996; 22: 169-172Crossref Scopus (7) Google Scholar, 26Wohlfarth A. Severin J. Galinski E.A. J. Gen. Microbiol. 1990; 136: 705-712Crossref Scopus (121) Google Scholar). Wild-type and mutants CHR63 and CHR62 were grown in 1-liter cultures of M63 plus the maximal NaCl that they can tolerate toA 600 of 1.8, 0.6, and 0.8, respectively, and extracted in 20 ml of distilled H2O, as described above. To 3-ml samples, 250 nmol of internal standards, α-amino-n-butyrate and α-aminoadipic acid, were added. Samples were applied to 3.5 × 1-cm columns of Dowex-1-acetate (100–200 mesh), equilibrated with H2O, and washed with 5 ml of H2O. The acidic amino acids, glutamate and aspartate, retained on this column were eluted with 6 ml of 2 m acetic acid. The water wash that contained the neutral and basic amino acids was applied to 3.5 × 1-cm columns of Dowex-50W-H+(100–200 mesh) equilibrated with H2O. The latter columns were washed with 8 ml of H2O, and neutral and basic amino acids were eluted with 8 ml of 6 m NH4OH. Fractions were evaporated to dryness under a stream of air and redissolved in 0.8 ml of 30% methanol, and aliquots were analyzed by TLC on Whatman Silica Gel AL-SIL-G (aluminum-backed) plates (20 × 20-cm; 250-μm layer), developed in n-butyl alcohol/acetic acid/water (60:20:20, v/v/v). After development and drying, the plates were sprayed with ninhydrin (0.15% w/v in ethanol) and amino acids visualized by heating in a drying oven (140 °C for 1–2 min). The above protocol was first tested with an aqueous extract from mutant CHR63, without internal standards; analyses of the acidic and neutral + basic amino acid fractions by GC-MS (below) revealed that these extracts were devoid of α-amino-n-butyrate and α-aminoadipic acid, hence the choice of these compounds as internal standards for the neutral + basic and acidic amino acid fractions, respectively. NADA was purified free of DA from the neutral amino acid fraction obtained from extracts of the mutant CHR63 by the following procedure. Aqueous extract was applied to a 4 × 1.5-cm column of Dowex-1-acetate, equilibrated with H2O, to first remove acidic amino acids. Columns were washed with 10 ml of H2O, and the water wash was applied to 4 × 1.5-cm columns of Dowex-50W-NH4+ (100–200 mesh) equilibrated with H2O. This effectively removed basic amino acids, including DA. The water wash from the latter column, containing neutral amino acids, was then applied to 4 × 1.5-cm columns of Dowex-50-W-H+ (100–200 mesh), washed with 24 ml of H2O, and the neutral amino acids eluted with 18 ml of 6m NH4OH. The basic amino acids were eluted from the Dowex-50W-NH4+ with 18 ml of 6m NH4OH. Fractions containing the desired amino acids were evaporated to dryness under a stream of air. The neutral amino acid fraction was dissolved in a small volume of 60% methanol and applied to the origin of a preparative 20 × 20-cm Whatman Silica Gel 150A glass-backed TLC plate (100-μm layer). The plate was developed in n-butyl alcohol/acetic acid/water (60:20:20, v/v/v). After development and drying, the central portion of the TLC plate was covered with a glass plate, and the plate edges were sprayed with ninhydrin (0.15% w/v in ethanol) and amino acids visualized by heating the edges of the plate with a hot air gun. The zone corresponding to NADA (the most abundant amino acid in the neutral fraction of mutant CHR63) was scraped from the central portion of the plate, and the NADA was then eluted from the silica gel with water. A small aliquot (2 μl) of this material was then re-analyzed by analytical TLC (above) before and after acid hydrolysis (1.25n HCl, 110 °C, 2 h). Amino acids purified by ion exchange chromatography and preparative TLC were derivatized toN(O,S)-heptafluorobutyryl (N-HFBI) amino acid derivatives, essentially as described by Rhodes et al. (27Rhodes D. Hogan A.L. Deal L. Jamieson G.C. Haworth P. Plant Physiol. (Bethesda). 1987; 84: 775-780Crossref PubMed Google Scholar). Briefly, this procedure entails reaction of the dried amino acid sample with 200 μl of freshly prepared isobutyl alcohol/acetyl chloride (5:1, v/v) at 120 °C for 20 min, evaporation to dryness, followed by reaction with 100 μl of heptafluorobutyric anhydride at 120 °C for 10 min, and evaporation to incipient dryness. The samples are finally redissolved in 100 μl of ethyl acetate/acetic anhydride (1:1, v/v) for GC-MS analysis (see below). Authentic ectoine did not produce a volatile derivative in this procedure, as determined by GC-MS. An authentic standard of NADA was not available. However, tests with authenticN-acetylornithine indicated that substantial hydrolysis to ornithine occurred during derivatization, suggesting that hydrolysis of NADA to DA is likely to occur in this protocol. Electron ionization and chemical ionization GC-MS of the amino acid derivatives were performed as described previously (27Rhodes D. Hogan A.L. Deal L. Jamieson G.C. Haworth P. Plant Physiol. (Bethesda). 1987; 84: 775-780Crossref PubMed Google Scholar), except that the column used was a DB-1 fused silica capillary column (30 m × 0. 25 mm inner diameter) and the oven temperature program was 100 °C for 4 min to 280 °C at 12 °C/min. H. elongata DSM 3043, formerly named strain 1H11 (1Vreeland R.H. Litchfield C.D. Martin E.L. Elliot E. Int. J. Syst. Bacteriol. 1980; 30: 485-495Crossref Scopus (332) Google Scholar), has a broad salinity range in M63 minimal medium, being able to grow from 0.5 to 3 m NaCl (10Cánovas D. Vargas C. Csonka L.N. Ventosa A. Nieto J.J. J. Bacteriol. 1996; 178: 7221-7226Crossref PubMed Google Scholar). The type strain of H. elongata ATCC 33173 has been shown to synthesize both ectoine and hydroxyectoine as compatible solutes in response to osmotic stress (26Wohlfarth A. Severin J. Galinski E.A. J. Gen. Microbiol. 1990; 136: 705-712Crossref Scopus (121) Google Scholar). To test whether this is also true forH. elongata DSM 3043, the latter organism was grown in M63 plus 3 m NaCl, and the composition of its internal solutes was analyzed by 13C NMR. Major signals corresponded to ectoine and hydroxyectoine, and glutamate and glutamine were also detectable at lower levels (Fig. 2). Salt-sensitive mutants of H. elongata were isolated by transposon mutagenesis, as described under “Experimental Procedures.” Putative salt-sensitive mutants were identified as those that were unable to grow on M63 plus 2.7 m NaCl plates but still able to grow on M63 containing 0.5 m NaCl. Out of ∼4,000 Kmr colonies screened, four showed this phenotype (Table II). Each was able to grow on M63 + 2.7 m NaCl + 1 mm betaine. This result indicated that the mutations did not cause a general NaCl sensitivity and suggested that the mutants might be defective in the synthesis of a compatible solute. Strain CHR63 could not grow above 1.5 mNaCl in the absence of betaine. Strain CHR62 was affected more severely, being unable to grow above 0.75 m NaCl without betaine. Strains CHR64 and CHR65 could not grow above 2.0 mNaCl. We showed (see below) that strains CHR62 and CHR63 were blocked in the synthesis of ectoine; these two strains have been designated as Ect−. The target site of the mutations in the other two strains, CHR64 and CHR65, has not yet been identified.Table IIGrowth of H. elongata salt-sensitive mutants in M63 with different NaCl concentrationsMutant strainNaCl0.50.7511.522.72.7 + betaine2-aGrowth in M63 + 2.7 m NaCl + 1 mm betaine.mCHR62++−−−−+CHR63++++−−+CHR64+++++−+CHR65+++++−+2-a Growth in M63 + 2.7 m NaCl + 1 mm betaine. Open table in a new tab The phenotypes of the highly NaCl-sensitive mutants CHR62 and CHR63 were characterized more extensively in liquid cultures. Fig.3 shows the growth rates of these mutants in M63 with different salinities, in the presence or absence of exogenous betaine. Both grew more slowly than the wild-type at any salinity. Partial growth at high salinity was restored by betaine for both mutants, although this osmoprotectant could not restore wild-type growth rate at ≥2.5 m for CHR63 or at ≥1.5 mfor CHR62. To check that the mutant phenotype was due to a single transposition event in each of the mutants, hybridization analysis was performed with an internal fragment of the transposon Tn1732 as a probe against genomic DNA of the mutants digested with the restriction enzymes SalI or BglII, which do not have any recognition site in Tn1732. As shown in Fig.4, unique hybridization signals were detected in the mutant DNAs, confirming that the salt-sensitive phenotype was due to single insertions of the transposon. Mutants CHR62 and CHR63 were grown in M63 glucose minimal medium containing the maximal NaCl concentration that they could tolerate (0.75 mfor CHR62 and 1.5 m for CHR63), and their major cytoplasmic solutes were analyzed by 13C NMR (Fig.5). Signals corresponding to ectoine were absent from the extracts of both mutants, indicating that they were defective in the synthesis of this compatible solute. Hydroxyectoine could not be detected either, suggesting a common biosynthetic pathway for both solutes. Signals corresponding to DA at 29, 37, 51, and 174 ppm were found in the CHR62 extract (Fig. 5 A), suggesting that this mutant accumulated this compound. The spectrum of the CHR63 extract showed major signals around 25, 32, 37, 53, 174, and 175 ppm (Fig. 5 B). A comparison with the chemical shifts of DA data suggested that those signals could correspond to a derivative of DA. Signals at 174 and 175 ppm could be attributed to the carbonylic moiety of the carboxylic and acetyl groups, and a signal at 25 ppm was typical of the methyl moiety of an acetyl group. These data were consistent with those expected for NADA and suggested the accumulation of this compound by CHR63. Electron ionization and chemical ionization GC-MS analyses of N-HFBI amino acid derivatives confirmed that the major amino acid constituents of mutant CHR62 was DA (HFBI derivative molecular weight = 566 (Fig.6); CI protonated molecular ion =m/z 567; major EI fragment ions =m/z 252 and 240), whereas that of mutant CHR63 was NADA (HFBI derivative molecular weight = 412; CI protonated molecular ion = m/z 413; major EI fragment ions = m/z 269 and 311) (Fig. 6). Initial analyses of the neutral + basic amino acid fraction of mutant CHR63 revealed high levels of DA in addition to NADA. However, the vast majority of this DA probably originated from hydrolysis of NADA during derivatization. This was confirmed by purifying NADA free of DA from extracts of CHR63 by ion exchange chromatography and preparative TLC. Approximately 80% of the purified NADA was converted" @default.
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W2029942689 title "Isolation and Characterization of Salt-sensitive Mutants of the Moderate Halophile Halomonas elongata and Cloning of the Ectoine Synthesis Genes" @default.
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