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Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis
FEMS Microbiology Letters 161 (1998) 293^300
Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis Karin Goëller a , Alexandra Ofer a , Erwin A. Galinski b; * a
Institut fuër Mikrobiologie and Biotechnologie, Rheinische Friedrich-Wilhelms-Universitaët, Meckenheimer Allee 168, 53115 Bonn, Germany b Westfaëlische Wilhelms-Universitaët, Institut fuër Biochemie, Wilhelm-Klemm-StraMe 2, 48149 Muënster, Germany Received 23 November 1997; revised 18 February 1998; accepted 18 February 1998
Abstract Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, L-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Halomonas elongata; Compatible solute; Ectoine biosynthesis; Transposon mutagenesis ; Salt-sensitive mutant
1. Introduction To cope with the osmotic stress imposed by a saline environment, most halophilic and halotolerant eubacteria control cytoplasmic osmolality by the accumulation of organic osmolytes, generally designated compatible solutes [1]. The tetrahydropyrimidines L-ectoine and s,s-L-hydroxyectoine are the most common osmolytes in aerobic chemoheterotrophic eubacteria [2^5] and are known to serve as protective agents for enzymes and whole cells [6,7]. The * Corresponding author. Tel.: +49 (251) 8333042; Fax: +49 (251) 8332122; E-mail: [email protected]
genes for ectoine biosynthesis of the Gram-positive halophilic eubacterium Marinococcus halophilus have recently been identi¢ed and characterized in our laboratory [8]. The enzymatic reactions involved in ectoine biosynthesis have been elucidated in Gram-negative eubacterial halophiles [9,10]. In the ¢rst step, aspartate semialdehyde, an intermediate in amino acid metabolism, is converted into L-2,4-diaminobutyric acid. This reaction is followed by acetylation to NQ -acetyl L-2,4-diaminobutyric acid. A cyclic condensation reaction ¢nally leads to the tetrahydropyrimidine L-ectoine (Fig. 1). We applied transposon mutagenesis to create saltsensitive mutants of Halomonas elongata, a moder-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 8 6 - X
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ately halophilic proteobacterium which is able to synthesize ectoine and hydroxyectoine as compatible solutes. The aim of this study was to characterize a mutant (H. elongata SAA4) which had a defect in the ectoine biosynthetic pathway and to determine the transposon insertion site.
Kunte and Galinski [14]. Transconjugants were considered salt-sensitive when they showed no growth on medium MM63 containing 16% NaCl (w/v) after 5 days incubation at 30³C, but grew well on plates supplemented with ectoine or betaine (1 mM).
2. Materials and methods
Genomic DNA of H. elongata was prepared using Qiagen genomic tips as directed by the manufacturer (Qiagen, Hilden, Germany). Qiaprep spin columns or the Plasmid Midi Kit from Qiagen were used for plasmid preparations of E. coli XL1-Blue. For Southern hybridization analysis, genomic DNA of H. elongata SAA4 was completely digested with SalI and BglII. DNA fragments were separated on an agarose gel and transferred onto a nylon ¢lter by standard techniques [15]. Tn1732-containing fragments were localized with a digoxigenin-labeled Tn1732 probe using the Digoxigenin DNA Labeling and Detection Kit (Boehringer Mannheim, Germany) according to the manufacturer's directions. For cloning experiments, genomic DNA of H. elongata SAA4 was completely digested with SalI and BglII and separated on an agarose gel. Tn1732-containing fragments were recovered by electroelution and ligated into the BamHI and SalI site of pCK01. E. coli XL1-Blue was transformed with recombinant plasmids by the method of Inoue et al. [16]. Colonies were screened on agar plates containing isopropyl L-D-thiogalactopyranoside (IPTG, 0.5 mM), 5-bromo-4-chloro-3-indolyl L-D-galactopyranoside (X-Gal, 40 Wg ml31 ), chloramphenicol (30 Wg ml31 ) to select for the plasmid, and kanamycin (50 Wg ml31 ) to select for the transposon Tn1732. PCR ampli¢cation was performed in a ¢nal volume of 50 Wl containing 150^200 ng of genomic DNA of wild-type H. elongata (DSM 2581T ) as template, 0.5^1 WM of each primer, 200 WM dNTP, and 5 U Taq polymerase (Promega, Madison, WI, USA). The following oligonucleotides (Roth, Karlsruhe, Germany) were used as primers: 5P-ATGACCATAAGCGGCTG-3P (forward), 5P-CTCGCCTTCGATGCAATA-3P (reverse). Forward and reverse primers were derived from the sequence of the mutated gene and the sequence of the ectoine synthase from Halomonas sp. [17], respectively. Double strand sequencing was carried out by
2.1. Bacterial strains, transposon and plasmid Escherichia coli SM10 containing the suicide vector pSUP102-Gem: :Tn1732 was the conjugal transposon donor [11,12]. H. elongata R11, a spontaneous streptomycin-resistant mutant of wild-type H. elongata (DSM 2581T ) was used for transposon mutagenesis experiments. E. coli XL1-Blue (Stratagene, Heidelberg, Germany) was employed for cloning purposes. Plasmid pCK01, a lacZ K-complementing low-copy-number cloning vector (replicon pSC101) which contains the multiple cloning site of pUC18, was kindly provided by V. de Lorenzo (CNB, Madrid, Spain). 2.2. Growth conditions H. elongata was grown aerobically at 30³C or 37³C either in antibiotic broth (AB medium No. 3, purchased from Oxoid, Wesel, Germany) containing 3% (w/v) NaCl, or in medium MM63 [13] with various carbon sources and NaCl concentrations. For supplementation studies, all supplements (betaine, ectoine, NQ -acetyl L-2,4-diaminobutyric acid) were used at a ¢nal concentration of 1 mM. H. elongata SAA4 was grown on antibiotic broth containing 50 Wg ml31 kanamycin and medium MM63 with 3200 Wg ml31 kanamycin to select for the transposon Tn1732. E. coli XL1-Blue was grown aerobically at 37³C in antibiotic broth containing 1% (w/v) NaCl. To select for cells bearing the plasmid pCK01, 30 Wg ml31 chloramphenicol was added. 2.3. Conjugation procedure, transposon mutagenesis and screening Conjugation and transposon mutagenesis were carried out according to the method described by
2.4. Molecular biology methods
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Sequiserve (Munich, Germany). DNA sequences were analyzed with the programs GENEPRO and MACAW. Databank searches were performed through the National Center for Biotechnology Information (NCBI) using the BLAST program [18]. 2.5. Analytical methods For identi¢cation and quanti¢cation of intracellular solutes, cells were harvested (10 000Ug, 20³C) in the late exponential growth phase, freeze-dried and extracted with methanol/chloroform/water (10:5:4) by a modi¢ed technique of Bligh and Dyer [19] as described by Galinski and Herzog [20]. Extracts were analyzed by isocratic and gradient HPLC methods as described previously [20,21]. For 13 C-NMR spectroscopy analysis, cell extracts were dissolved in D2 O supplemented with acetonitrile as internal standard. Samples were measured on a Varian XL 300 spectrometer.
3. Results and discussion 3.1. Salt susceptibility and internal solute content of H. elongata SAA4 We created the salt-sensitive mutant H. elongata SAA4 according to the method of Kunte and Galinski [14]. Single insertion of transposon Tn1732 without cointegration of the vector was proved by southern hybridization analysis using digoxigenin-labeled pSUP102-Gem: :Tn1732 as probe. To assess the ability of H. elongata SAA4 to compensate salt stress, this mutant was grown at 30³C in medium MM63 containing various concentrations of NaCl. H. elongata SAA4 failed to grow at NaCl concentrations higher than 4% (w/v). HPLC analysis of the internal solute content at salinities between 2 and 4% NaCl revealed an elevated level of glutamic acid in comparison to H. elongata R11, as well as the occurrence of glutamine and small amounts of L-2,4-diaminobutyric acid (Fig. 2A). Neither ectoine, usually the major osmolyte under these growth conditions in Halomonas sp., nor NQ -acetyl L-2,4-diaminobutyric acid, the direct precursor of ectoine, could be detected with HPLC or 13 C-NMR measurements (data not shown). In the
Fig. 1. Biosynthetic pathway of ectoine based on enzymological studies [8,9]. 1, L-2,4-diaminobutyric acid transaminase ; 2, L-2,4diaminobutyric acid NQ -acetyltransferase; 3, ectoine synthase.
parent strain H. elongata R11 ectoine was the predominant osmolyte, whereas glutamic acid and NQ acetyl L-2,4-diaminobutyric acid represented minor components (Fig. 2B). Only at elevated temperature (37³C) was hydroxyectoine detectable by HPLC in the parent strain but not in H. elongata SAA4. The total solute content of H. elongata SAA4 increased with increasing salinity to almost the same extent as in the parent strain R11. These results indicate that H. elongata SAA4 is impaired in ectoine biosynthesis, but is able to balance this defect up to 4% NaCl by the increased accumulation of glutamic acid and glutamine. However, whereas both wild-type and ect3 mutant displayed approximately the same growth rate at 2% NaCl (0.23 h31 ), growth was severely impaired at 4% NaCl (less than half the growth rate of the parent strain). Glutamic acid, although frequently employed as an osmolyte in slightly halotolerant species
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Fig. 2. Internal solute content of H. elongata SAA4 (A) and R11 (B) grown in medium MM63 (30³C) at various NaCl concentrations as measured by HPLC. DABA : L-2,4-diaminobutyric acid; N-Ac-DABA : NQ -acetyl L-2,4-diaminobutyric acid.
[22], is not a typical compatible solute. Due to its negative net charge at physiological pH, a concomitant accumulation of a neutralizing cation (K ) is necessary, which may interfere with the cell's physiological functions. Therefore, by using glutamic acid as an osmolyte, only limited osmoadaptation is possible [23]. Glutamine, although uncharged at physiological pH, is poorly soluble in its zwitterionic form. This compound is accumulated to saturation in the moderately halotolerant Corynebacterium glutamicum and C. ammoniagenes [24], but seems to play a secondary role as an osmolyte in truly halophilic bacteria. 3.2. Supplementation experiments Osmoprotectants such as ectoine and glycine be-
taine are known to stimulate the growth of non-halophilic bacteria, e.g. E. coli [25] in media of elevated osmotic strength. It was, therefore, of interest to study the e¡ect of these compounds on the growth of the NaCl-sensitive mutant H. elongata SAA4. Supplementation experiments were performed at 30³C in medium MM63 with 10% NaCl (w/v). The NaCl-tolerant phenotype was restored by the addition of betaine, ectoine and the direct precursor of ectoine, NQ -acetyl L-2,4-diaminobutyric acid, although in this case with a signi¢cantly diminished growth rate (data not shown). Analysis of the internal solute content of H. elongata SAA4 supplemented with NQ -acetyl L-2,4-diaminobutyric acid proved the occurrence of ectoine, and, under appropriate growth conditions (37³C or NaCl concentrations higher than 10%), hydroxyectoine
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Fig. 3. A: Location of insertion site in target DNA and sequences £anking Tn1732 insertion prior to sequence analysis. Inverted repeats of Tn1732 are boxed, 5-bp direct repeats of the target DNA are shown against a dark background. The orientation of the transposon was shown to be in the same direction as the mutated gene. B: Alignment of the sequence of ectA (GenBank AF031489) with the sequence of the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway of M. halophilus (GenBank U66614). Identical positions are shown against a dark background and conservative replacements are boxed. The following were considered conservative replacements: R-K-H, D-E, Q-N, S-T, G-A, F-Y-W, I-L-V-M. The arrow indicates the transposon insertion site.
(data not shown). These results suggest that the enzyme responsible for the acetylation of L-2,4-diaminobutyric acid to NQ -acetyl L-2,4-diaminobutyric acid in the ectoine biosynthetic pathway has been mutated by transposon insertion. The accumulation of diaminobutyric acid in cells grown in unsupplemented medium (see Fig. 2) supports this proposition. H. elongata SAA4 was also grown in medium MM63 (37³C, 8% NaCl w/v) with ectoine or hydroxyectoine provided as sole carbon source (10 mM). Growth occurred with both substances, indicating that biosynthesis and degradation pathways are not identical. When cells were supplemented with one of the two ectoines, the other was (given the appropriate conditions) also detectable in the cells. This demonstrates the presence of a conversion pathway which does not involve the mutated gene.
3.3. Genetic identi¢cation of the mutated gene To determine the molecular nature of the observed NaCl sensitivity, we cloned and sequenced the regions £anking the Tn1732 insertion in the H. elongata SAA4 chromosome. For subsequent sequence analysis it was taken into account that Tn1732 generates 5-bp direct repeats of the target DNA (Fig. 3A). Databank searches using the BLAST algorithm revealed 38% homology between the mutated gene (designated ectA) and the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway of M. halophilus [10] (Fig. 3B). Sequence analysis also revealed some similarity to the ARD1 subunit of the N-terminal acetyltransferase complex of Dictyostelium discoideum. From these sequence comparisons we conclude that the mutated gene encodes the L-2,4-diaminobutyric acid acetyltransferase.
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Fig. 4. A: Sequence alignment of ectB (GenBank AF031489) with the sequence of the transaminase from M. halophilus (GenBank U66614). B : Sequence alignment of ectC (GenBank AF031489) with the sequence of the ectoine synthase from M. halophilus (GenBank U66614). Identical positions are shown against a dark background and conservative replacements are boxed. The following were considered conservative replacements: R-K-H, D-E, Q-N, S-T, G-A, F-Y-W, I-L-V-M.
3.4. Localization and sequencing of the other genes involved in ectoine biosynthesis In M. halophilus, the genes encoding the diaminobutyric acid transaminase and the ectoine synthase are located downstream of the gene for the diaminobutyric acid acetyltransferase (ectA). As we were unable to detect open reading frames with a high degree of identity to ectoine biosynthetic genes
upstream of the mutated gene, we assumed a similar organization of the genes responsible for ectoine biosynthesis in H. elongata. Using PCR we ampli¢ed and subsequently sequenced a 2.2-kb fragment which, in addition to ectA, contained one complete open reading frame (ectB) with 48% similarity to the transaminase and one incomplete open reading frame (ectC) with 46% similarity to the ectoine synthase from the ectoine biosynthetic pathway of M.
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halophilus [8] (Fig. 4). Furthermore, sequence comparison of the ectoine synthase of Halomonas sp. [17] with ectC displayed 100% similarity. We were, therefore, able to demonstrate that the genes involved in ectoine biosynthesis in H. elongata (ectABC) follow the same order as in M. halophilus [8]. As insertion of the transposon into ectA did not a¡ect the expression of the downstream genes, it remains to be shown whether the ectoine genes are organized in an operon.
[4] [5] [6]
[7]
[8]
4. Note added during revision After submission of the manuscript it has come to our attention that Canovas et al. (J. Biol. Chem. 272 (1997) 25794^25801) reported on transposon mutagenesis experiments with H. elongata DSM 3043, using the methods previously applied by our group [14]. Although the authors did not determine the sequence of the ectoine genes, their observations suggested that they were closely linked. The present paper on H. elongata DSM 2581T (type strain) shows that the genes in H. elongata DSM 3043 are organized in the same way (namely ectA, ectB, ectC) and probably share a high degree of identity with those of the type strain. To avoid confusion it is to be noted that Canovas et al. (1997) chose a preliminary gene designation di¡erent from the ¢rst description [8].
[9] [10]
[11]
[12]
[13]
[14]
[15]
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ga 393/3) and by the European Union (Bio4-CT96-0488).
[16]
[17]
[18]
References [1] Brown, A.D. (1976) Microbial water stress. Bacteriol. Rev. 40, 803^846. [2] Severin, J., Wohlfarth, A. and Galinski, E.A. (1992) The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J. Gen. Microbiol. 138, 1629^1638. [3] Wohlfarth, A., Severin, J. and Galinski, E.A. (1990) The spectrum of compatible solutes in heterotrophic halophilic eubac-
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teria of the family Halomonadaceae. J. Gen. Microbiol. 136, 705^712. Galinski, E.A. and Truëper, H.G. (1994) Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev. 15, 95^108. Galinski, E.A. (1995) Osmoadaptation in bacteria. Adv. Microb. Physiol. 37, 273^328. Lippert, K. and Galinski, E.A. (1992) Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl. Microbiol. Biotechnol. 37, 61^65. Louis, P., Truëper, H.G. and Galinski, E.A. (1994) Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Appl. Microbiol. Biotechnol. 41, 684^688. Louis, P. and Galinski, E.A. (1997) Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143, 1141^1149. Peters, P., Galinski, E.A. and Truëper, H.G. (1990) The biosynthesis of ectoine. FEMS Microbiol. Lett. 71, 157^162. Tao, T., Yasuda, N., Ono, H., Shinmyo, A. and Takano, M. (1992) Puri¢cation and characterization of 2,4-diaminobutyric acid transaminase from Halomonas sp. Annu. Rep. Int. Center Cooperative Res. Biotechnol. Jpn. 15, 187^199. Simon, R., Priefer, U. and Puëhler, A. (1983) A broad host range system for in vivo genetic engineering : transposon mutagenesis in Gram negative bacteria. BioTechnology 1, 784^ 791. Ubben, D. and Schmitt, R. (1986) Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. Gene 41, 145^152. Larsen, P.I., Sydnes, L.K., Landfald, B. and StrÖm, A.R. (1987) Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch. Microbiol. 147, 1^7. Kunte, H.J. and Galinski, E.A. (1995) Transposon mutagenesis in halophilic eubacteria : conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata. FEMS Microbiol. Lett. 128, 293^299. Sambrook, J., Fritsch, E.F. and Maniatis, T.E. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Inoue, H., Nojima, H. and Okayama, H. (1990) High e¤cient transformation of Escherichia coli with plasmids. Gene 96, 23^ 28. Min-Yu, L., Ono, H. and Takano, M. (1993) Gene cloning of ectoine synthase from Halomonas sp. Annu. Rep. Int. Center Cooperative Res. Biotechnol. Jpn. 16, 193^200. Altschul, S.F., Gish, W., Miller, M., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. Bligh, E.G. and Dyer, W.J. (1959) A rapid method of lipid extraction and puri¢cation. Can. J. Biochem. Physiol. 37, 911^ 917. Galinski, E.A. and Herzog, R.M. (1990) The role of trehalose as a substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris). Arch. Microbiol. 153, 607^613. Kunte, H.J., Galinski, E.A. and Truëper, H.G. (1993) A modi¢ed FMOC-method for the detection of amino acid-type os-
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molytes and tetrahydropyrimidines (ectoines). J. Microbiol. Methods 17, 129^136. [22] Csonka, L.N. (1989) Physiological and genetic response of bacteria to osmotic stress. Microbiol. Rev. 53, 121^147. [23] Galinski, E.A. and Oren, A. (1991) Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui. Eur. J. Biochem. 198, 593^598.
[24] Frings, E., Kunte, H.J. and Galinski, E.A. (1993) Compatible solutes in representatives of the genera Brevibacterium and Corynebacterium : occurrence of tetrahydropyrimidines and glutamine. FEMS Microbiol. Lett. 109, 25^32. [25] Malin, G. and Lapidot, A. (1996) Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their e¡ect on Escherichia coli in response to osmotic and heat stress. J. Bacteriol. 178, 385^395.
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Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis Karin Goëller a , Alexandra Ofer a , Erwin A. Galinski b; * a
Institut fuër Mikrobiologie and Biotechnologie, Rheinische Friedrich-Wilhelms-Universitaët, Meckenheimer Allee 168, 53115 Bonn, Germany b Westfaëlische Wilhelms-Universitaët, Institut fuër Biochemie, Wilhelm-Klemm-StraMe 2, 48149 Muënster, Germany Received 23 November 1997; revised 18 February 1998; accepted 18 February 1998
Abstract Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine. HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, L-2,4-diaminobutyric acid. Ectoine and hydroxyectoine were not detectable. This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4%. However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained. We cloned and sequenced the regions flanking the transposon insertion in the H. elongata chromosome. Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus. Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M. halophilus. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Halomonas elongata; Compatible solute; Ectoine biosynthesis; Transposon mutagenesis ; Salt-sensitive mutant
1. Introduction To cope with the osmotic stress imposed by a saline environment, most halophilic and halotolerant eubacteria control cytoplasmic osmolality by the accumulation of organic osmolytes, generally designated compatible solutes [1]. The tetrahydropyrimidines L-ectoine and s,s-L-hydroxyectoine are the most common osmolytes in aerobic chemoheterotrophic eubacteria [2^5] and are known to serve as protective agents for enzymes and whole cells [6,7]. The * Corresponding author. Tel.: +49 (251) 8333042; Fax: +49 (251) 8332122; E-mail: [email protected]
genes for ectoine biosynthesis of the Gram-positive halophilic eubacterium Marinococcus halophilus have recently been identi¢ed and characterized in our laboratory [8]. The enzymatic reactions involved in ectoine biosynthesis have been elucidated in Gram-negative eubacterial halophiles [9,10]. In the ¢rst step, aspartate semialdehyde, an intermediate in amino acid metabolism, is converted into L-2,4-diaminobutyric acid. This reaction is followed by acetylation to NQ -acetyl L-2,4-diaminobutyric acid. A cyclic condensation reaction ¢nally leads to the tetrahydropyrimidine L-ectoine (Fig. 1). We applied transposon mutagenesis to create saltsensitive mutants of Halomonas elongata, a moder-
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 8 6 - X
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ately halophilic proteobacterium which is able to synthesize ectoine and hydroxyectoine as compatible solutes. The aim of this study was to characterize a mutant (H. elongata SAA4) which had a defect in the ectoine biosynthetic pathway and to determine the transposon insertion site.
Kunte and Galinski [14]. Transconjugants were considered salt-sensitive when they showed no growth on medium MM63 containing 16% NaCl (w/v) after 5 days incubation at 30³C, but grew well on plates supplemented with ectoine or betaine (1 mM).
2. Materials and methods
Genomic DNA of H. elongata was prepared using Qiagen genomic tips as directed by the manufacturer (Qiagen, Hilden, Germany). Qiaprep spin columns or the Plasmid Midi Kit from Qiagen were used for plasmid preparations of E. coli XL1-Blue. For Southern hybridization analysis, genomic DNA of H. elongata SAA4 was completely digested with SalI and BglII. DNA fragments were separated on an agarose gel and transferred onto a nylon ¢lter by standard techniques [15]. Tn1732-containing fragments were localized with a digoxigenin-labeled Tn1732 probe using the Digoxigenin DNA Labeling and Detection Kit (Boehringer Mannheim, Germany) according to the manufacturer's directions. For cloning experiments, genomic DNA of H. elongata SAA4 was completely digested with SalI and BglII and separated on an agarose gel. Tn1732-containing fragments were recovered by electroelution and ligated into the BamHI and SalI site of pCK01. E. coli XL1-Blue was transformed with recombinant plasmids by the method of Inoue et al. [16]. Colonies were screened on agar plates containing isopropyl L-D-thiogalactopyranoside (IPTG, 0.5 mM), 5-bromo-4-chloro-3-indolyl L-D-galactopyranoside (X-Gal, 40 Wg ml31 ), chloramphenicol (30 Wg ml31 ) to select for the plasmid, and kanamycin (50 Wg ml31 ) to select for the transposon Tn1732. PCR ampli¢cation was performed in a ¢nal volume of 50 Wl containing 150^200 ng of genomic DNA of wild-type H. elongata (DSM 2581T ) as template, 0.5^1 WM of each primer, 200 WM dNTP, and 5 U Taq polymerase (Promega, Madison, WI, USA). The following oligonucleotides (Roth, Karlsruhe, Germany) were used as primers: 5P-ATGACCATAAGCGGCTG-3P (forward), 5P-CTCGCCTTCGATGCAATA-3P (reverse). Forward and reverse primers were derived from the sequence of the mutated gene and the sequence of the ectoine synthase from Halomonas sp. [17], respectively. Double strand sequencing was carried out by
2.1. Bacterial strains, transposon and plasmid Escherichia coli SM10 containing the suicide vector pSUP102-Gem: :Tn1732 was the conjugal transposon donor [11,12]. H. elongata R11, a spontaneous streptomycin-resistant mutant of wild-type H. elongata (DSM 2581T ) was used for transposon mutagenesis experiments. E. coli XL1-Blue (Stratagene, Heidelberg, Germany) was employed for cloning purposes. Plasmid pCK01, a lacZ K-complementing low-copy-number cloning vector (replicon pSC101) which contains the multiple cloning site of pUC18, was kindly provided by V. de Lorenzo (CNB, Madrid, Spain). 2.2. Growth conditions H. elongata was grown aerobically at 30³C or 37³C either in antibiotic broth (AB medium No. 3, purchased from Oxoid, Wesel, Germany) containing 3% (w/v) NaCl, or in medium MM63 [13] with various carbon sources and NaCl concentrations. For supplementation studies, all supplements (betaine, ectoine, NQ -acetyl L-2,4-diaminobutyric acid) were used at a ¢nal concentration of 1 mM. H. elongata SAA4 was grown on antibiotic broth containing 50 Wg ml31 kanamycin and medium MM63 with 3200 Wg ml31 kanamycin to select for the transposon Tn1732. E. coli XL1-Blue was grown aerobically at 37³C in antibiotic broth containing 1% (w/v) NaCl. To select for cells bearing the plasmid pCK01, 30 Wg ml31 chloramphenicol was added. 2.3. Conjugation procedure, transposon mutagenesis and screening Conjugation and transposon mutagenesis were carried out according to the method described by
2.4. Molecular biology methods
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Sequiserve (Munich, Germany). DNA sequences were analyzed with the programs GENEPRO and MACAW. Databank searches were performed through the National Center for Biotechnology Information (NCBI) using the BLAST program [18]. 2.5. Analytical methods For identi¢cation and quanti¢cation of intracellular solutes, cells were harvested (10 000Ug, 20³C) in the late exponential growth phase, freeze-dried and extracted with methanol/chloroform/water (10:5:4) by a modi¢ed technique of Bligh and Dyer [19] as described by Galinski and Herzog [20]. Extracts were analyzed by isocratic and gradient HPLC methods as described previously [20,21]. For 13 C-NMR spectroscopy analysis, cell extracts were dissolved in D2 O supplemented with acetonitrile as internal standard. Samples were measured on a Varian XL 300 spectrometer.
3. Results and discussion 3.1. Salt susceptibility and internal solute content of H. elongata SAA4 We created the salt-sensitive mutant H. elongata SAA4 according to the method of Kunte and Galinski [14]. Single insertion of transposon Tn1732 without cointegration of the vector was proved by southern hybridization analysis using digoxigenin-labeled pSUP102-Gem: :Tn1732 as probe. To assess the ability of H. elongata SAA4 to compensate salt stress, this mutant was grown at 30³C in medium MM63 containing various concentrations of NaCl. H. elongata SAA4 failed to grow at NaCl concentrations higher than 4% (w/v). HPLC analysis of the internal solute content at salinities between 2 and 4% NaCl revealed an elevated level of glutamic acid in comparison to H. elongata R11, as well as the occurrence of glutamine and small amounts of L-2,4-diaminobutyric acid (Fig. 2A). Neither ectoine, usually the major osmolyte under these growth conditions in Halomonas sp., nor NQ -acetyl L-2,4-diaminobutyric acid, the direct precursor of ectoine, could be detected with HPLC or 13 C-NMR measurements (data not shown). In the
Fig. 1. Biosynthetic pathway of ectoine based on enzymological studies [8,9]. 1, L-2,4-diaminobutyric acid transaminase ; 2, L-2,4diaminobutyric acid NQ -acetyltransferase; 3, ectoine synthase.
parent strain H. elongata R11 ectoine was the predominant osmolyte, whereas glutamic acid and NQ acetyl L-2,4-diaminobutyric acid represented minor components (Fig. 2B). Only at elevated temperature (37³C) was hydroxyectoine detectable by HPLC in the parent strain but not in H. elongata SAA4. The total solute content of H. elongata SAA4 increased with increasing salinity to almost the same extent as in the parent strain R11. These results indicate that H. elongata SAA4 is impaired in ectoine biosynthesis, but is able to balance this defect up to 4% NaCl by the increased accumulation of glutamic acid and glutamine. However, whereas both wild-type and ect3 mutant displayed approximately the same growth rate at 2% NaCl (0.23 h31 ), growth was severely impaired at 4% NaCl (less than half the growth rate of the parent strain). Glutamic acid, although frequently employed as an osmolyte in slightly halotolerant species
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Fig. 2. Internal solute content of H. elongata SAA4 (A) and R11 (B) grown in medium MM63 (30³C) at various NaCl concentrations as measured by HPLC. DABA : L-2,4-diaminobutyric acid; N-Ac-DABA : NQ -acetyl L-2,4-diaminobutyric acid.
[22], is not a typical compatible solute. Due to its negative net charge at physiological pH, a concomitant accumulation of a neutralizing cation (K ) is necessary, which may interfere with the cell's physiological functions. Therefore, by using glutamic acid as an osmolyte, only limited osmoadaptation is possible [23]. Glutamine, although uncharged at physiological pH, is poorly soluble in its zwitterionic form. This compound is accumulated to saturation in the moderately halotolerant Corynebacterium glutamicum and C. ammoniagenes [24], but seems to play a secondary role as an osmolyte in truly halophilic bacteria. 3.2. Supplementation experiments Osmoprotectants such as ectoine and glycine be-
taine are known to stimulate the growth of non-halophilic bacteria, e.g. E. coli [25] in media of elevated osmotic strength. It was, therefore, of interest to study the e¡ect of these compounds on the growth of the NaCl-sensitive mutant H. elongata SAA4. Supplementation experiments were performed at 30³C in medium MM63 with 10% NaCl (w/v). The NaCl-tolerant phenotype was restored by the addition of betaine, ectoine and the direct precursor of ectoine, NQ -acetyl L-2,4-diaminobutyric acid, although in this case with a signi¢cantly diminished growth rate (data not shown). Analysis of the internal solute content of H. elongata SAA4 supplemented with NQ -acetyl L-2,4-diaminobutyric acid proved the occurrence of ectoine, and, under appropriate growth conditions (37³C or NaCl concentrations higher than 10%), hydroxyectoine
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Fig. 3. A: Location of insertion site in target DNA and sequences £anking Tn1732 insertion prior to sequence analysis. Inverted repeats of Tn1732 are boxed, 5-bp direct repeats of the target DNA are shown against a dark background. The orientation of the transposon was shown to be in the same direction as the mutated gene. B: Alignment of the sequence of ectA (GenBank AF031489) with the sequence of the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway of M. halophilus (GenBank U66614). Identical positions are shown against a dark background and conservative replacements are boxed. The following were considered conservative replacements: R-K-H, D-E, Q-N, S-T, G-A, F-Y-W, I-L-V-M. The arrow indicates the transposon insertion site.
(data not shown). These results suggest that the enzyme responsible for the acetylation of L-2,4-diaminobutyric acid to NQ -acetyl L-2,4-diaminobutyric acid in the ectoine biosynthetic pathway has been mutated by transposon insertion. The accumulation of diaminobutyric acid in cells grown in unsupplemented medium (see Fig. 2) supports this proposition. H. elongata SAA4 was also grown in medium MM63 (37³C, 8% NaCl w/v) with ectoine or hydroxyectoine provided as sole carbon source (10 mM). Growth occurred with both substances, indicating that biosynthesis and degradation pathways are not identical. When cells were supplemented with one of the two ectoines, the other was (given the appropriate conditions) also detectable in the cells. This demonstrates the presence of a conversion pathway which does not involve the mutated gene.
3.3. Genetic identi¢cation of the mutated gene To determine the molecular nature of the observed NaCl sensitivity, we cloned and sequenced the regions £anking the Tn1732 insertion in the H. elongata SAA4 chromosome. For subsequent sequence analysis it was taken into account that Tn1732 generates 5-bp direct repeats of the target DNA (Fig. 3A). Databank searches using the BLAST algorithm revealed 38% homology between the mutated gene (designated ectA) and the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway of M. halophilus [10] (Fig. 3B). Sequence analysis also revealed some similarity to the ARD1 subunit of the N-terminal acetyltransferase complex of Dictyostelium discoideum. From these sequence comparisons we conclude that the mutated gene encodes the L-2,4-diaminobutyric acid acetyltransferase.
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Fig. 4. A: Sequence alignment of ectB (GenBank AF031489) with the sequence of the transaminase from M. halophilus (GenBank U66614). B : Sequence alignment of ectC (GenBank AF031489) with the sequence of the ectoine synthase from M. halophilus (GenBank U66614). Identical positions are shown against a dark background and conservative replacements are boxed. The following were considered conservative replacements: R-K-H, D-E, Q-N, S-T, G-A, F-Y-W, I-L-V-M.
3.4. Localization and sequencing of the other genes involved in ectoine biosynthesis In M. halophilus, the genes encoding the diaminobutyric acid transaminase and the ectoine synthase are located downstream of the gene for the diaminobutyric acid acetyltransferase (ectA). As we were unable to detect open reading frames with a high degree of identity to ectoine biosynthetic genes
upstream of the mutated gene, we assumed a similar organization of the genes responsible for ectoine biosynthesis in H. elongata. Using PCR we ampli¢ed and subsequently sequenced a 2.2-kb fragment which, in addition to ectA, contained one complete open reading frame (ectB) with 48% similarity to the transaminase and one incomplete open reading frame (ectC) with 46% similarity to the ectoine synthase from the ectoine biosynthetic pathway of M.
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halophilus [8] (Fig. 4). Furthermore, sequence comparison of the ectoine synthase of Halomonas sp. [17] with ectC displayed 100% similarity. We were, therefore, able to demonstrate that the genes involved in ectoine biosynthesis in H. elongata (ectABC) follow the same order as in M. halophilus [8]. As insertion of the transposon into ectA did not a¡ect the expression of the downstream genes, it remains to be shown whether the ectoine genes are organized in an operon.
[4] [5] [6]
[7]
[8]
4. Note added during revision After submission of the manuscript it has come to our attention that Canovas et al. (J. Biol. Chem. 272 (1997) 25794^25801) reported on transposon mutagenesis experiments with H. elongata DSM 3043, using the methods previously applied by our group [14]. Although the authors did not determine the sequence of the ectoine genes, their observations suggested that they were closely linked. The present paper on H. elongata DSM 2581T (type strain) shows that the genes in H. elongata DSM 3043 are organized in the same way (namely ectA, ectB, ectC) and probably share a high degree of identity with those of the type strain. To avoid confusion it is to be noted that Canovas et al. (1997) chose a preliminary gene designation di¡erent from the ¢rst description [8].
[9] [10]
[11]
[12]
[13]
[14]
[15]
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ga 393/3) and by the European Union (Bio4-CT96-0488).
[16]
[17]
[18]
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[24] Frings, E., Kunte, H.J. and Galinski, E.A. (1993) Compatible solutes in representatives of the genera Brevibacterium and Corynebacterium : occurrence of tetrahydropyrimidines and glutamine. FEMS Microbiol. Lett. 109, 25^32. [25] Malin, G. and Lapidot, A. (1996) Induction of synthesis of tetrahydropyrimidine derivatives in Streptomyces strains and their e¡ect on Escherichia coli in response to osmotic and heat stress. J. Bacteriol. 178, 385^395.
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