Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain1
Laboratory for Microbiology, Department of Biology, Philipps University, MarburgD-35032, Marburg, Germany2
Author for correspondence: Joaquín J. Nieto. Tel: +34 95 4556765. Fax: +34 954 628162. e-mail: jjnieto{at}cica.es
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ABSTRACT |
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Keywords: moderate halophiles, Halomonas elongata, bet genes, osmoregulation, compatible solutes
Abbreviations: BADH, glycine betaine aldehyde dehydrogenase; CDH, choline dehydrogenase
The EMBL accession number for the sequence reported in this paper is AJ238780.
a Present address: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain.
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INTRODUCTION |
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Like most other bacteria, H. elongata maintains its internal osmolarity and generates turgor in media of high salinity by accumulating a limited number of metabolically inert, organic compounds named compatible solutes (Ventosa et al., 1998 ). It is able to synthesize de novo ectoine and hydroxyectoine when grown in media lacking osmoprotectants (Cánovas et al., 1997
). The genes involved in this biosynthetic pathway have recently been isolated and characterized in two H. elongata strains (Cánovas et al., 1998a
; Göller et al., 1998
). This bacterium can also accumulate the compatible solute glycine betaine and structurally related osmoprotectants by transport from the external medium. It was found that glycine betaine suppressed de novo ectoine synthesis partially or completely, depending on the NaCl concentration in the growth medium (Cánovas et al., 1996
). In addition to uptake from the environment, accumulation of glycine betaine can also be achieved by oxidation from its precursor, choline (Cánovas et al., 1996
). We have recently characterized a very efficient system for choline transport and its subsequent oxidation to glycine betaine. The system is mainly regulated by salinity and also by the availability of glycine betaine (Cánovas et al., 1998b
). In this study, we describe the cloning, molecular characterization and expression of the genes responsible for the choline-glycine betaine biosynthetic pathway in H. elongata DSM 3043.
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METHODS |
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Conjugal transfer of plasmids.
Plasmids were conjugated between E. coli strains by triparental matings on LB medium as described by Vargas et al. (1997) by using pRK600 (Kessler et al., 1992
) as helper plasmid.
DNA manipulation and construction of plasmids.
Plasmid DNA manipulations were carried out by standard techniques (Ausubel et al., 1989 ; Sambrook et al., 1989
). The construction of a H. elongata DSM 3043 gene bank has been described previously (Cánovas et al., 1997
). Plasmids pDC4 and pDC7 (Fig. 1
) were constructed by partial digestion with Sau3AI of pDC1, a cosmid clone from the gene bank, and subsequent ligation into the BamHI-digested low-copy-number plasmid pHSG575 (Takeshita et al., 1987
). pDC5 was constructed by inserting a 5·7 kb BamHIEcoRI fragment (carrying the E. coli betIBA genes) from pJB005 (J. Boch & E. Bremer, unpublished data) into BamHI/EcoRI-digested pHSG575. pDC8 and pDC9 were constructed by deleting a 3·6 kb EcoRI and a 2·2 kb HindIII fragment, respectively, from pDC4 and religation of the plasmid backbones. pDC10 and pDC11 are derivatives of the high-copy-number vector pGEM5Zf (Promega) carrying a 4·3 kb and a 1·3 kb PstI fragment from pDC4, respectively. pDC12 was generated by deletion of a 0·4 kb SalI region from pDC10. pDC13 was obtained by subcloning a 4·8 kb BamHISalI fragment, carrying the H. elongata betA gene, from pDC9 into the low-copy-number expression vector pPD100 carrying the phage T7
10 promoter (Dersch et al., 1994
). For the expression of the H. elongata betIB under the control of the same promoter, a 3·4 kb SacIEcoRI fragment from pDC8 was subcloned in the polylinker of pPD101 (Dersch et al., 1994
), resulting in the plasmid pDC14. To obtain pDC26, the 7·4 kb SalI fragment containing the H. elongata betIBA region was transferred from pDC4 into the broad-host-range vector pML123 (Labes et al., 1990
) to give pDC15. pDC26 was subsequently generated by inserting a Tet cassette from pOB26 (O. Schmidt-Kittler & E. Bremer, unpublished data) between the HindIII and EcoRI sites of pDC15.
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Assay for conversion of intracellular accumulated choline into glycine betaine.
E. coli cells carrying the corresponding plasmids were grown to exponential phase at 37 °C in M63 medium with 0·5 M NaCl. Radiolabelled [methyl-14C]choline (10 µM; 0·55 kBq) was added to the medium and the cells were incubated at room temperature for 1 h. Samples (0·5 ml) were taken and the cells were collected after 2 min of centrifugation in a microfuge. The cell pellet of each sample was extracted with 50 µl 80% methanol, and glycine betaine and choline were separated by TLC on Whatman Silica Gel AL-SIL-G plates with 90:10:4 (by vol.) methanol/acetone/hydrochloric acid as the running solvent. The radioactive metabolites were visualized by autoradiography and identified by comparison with [14C]glycine betaine and [14C]choline standards.
Expression of the betIB and betA gene products under the control of the T710 promoter.
E. coli strain PD141(DE3) (Bet-), carrying the gene for the T7 RNA polymerase in the chromosome under lacPO control, was used as the host strain. Transconjugants of this strain containing pJB004 (E. coli betT) plus pDC13 (H. elongata betA), pJB004 plus pDC14 (H. elongata betIB) or pJB004 plasmids were grown in T7 medium supplemented with 0·5 M NaCl and 30 µg chloramphenicol ml1 to an OD600 of 0·7. The bet genes were overexpressed after induction with 1 mM IPTG, following the procedure described by Dersch et al. (1994)
.
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RESULTS |
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To delimit the region in pDC1carrying the genes for the synthesis of glycine betaine, this plasmid was partially digested with Sau3A1 and ligated into the low-copy-number vector pHSG575. The resulting ligation mixture was used to transform E. coli MKH13(pJB004) and strains that were osmotolerant in the presence of choline were selected. Two plasmids, pDC4 and pDC7 (Fig. 1), containing a 7·4 kb and an approximately 17 kb fragment from pDC1, respectively, were found to confer osmoprotection to E. coli MKH13(pJB004).
Conversion of choline into glycine betaine mediated by the H. elongata bet genes
To confirm that pDC1 and its derivative pDC4 conferred osmoprotection to E. coli by mediating the synthesis of glycine betaine, transconjugants of E. coli MKH13(pJB004) harbouring pDC1 or pDC4 were grown in M63 with 0·5 M NaCl to exponential-growth phase and then incubated with 10 µM [methyl-14C]choline (0·55 kBq) for 1 h. Subsequently, the radiolabelled solutes were analysed by TLC and autoradiography. E. coli MKH13(pJB004) carrying pDC5, a derivative of pHSG575 containing the E. coli betIBA genes, and strain MKH13(pJB004) carrying the vector pHSG575, were used as positive and negative controls, respectively. In the same experiment, E. coli MKH13 harbouring only pDC1 or pDC4 was also included to check if these plasmids were also able to mediate the transport of choline by H. elongata. As shown in Fig. 2, both pDC1 and pDC4 mediated the enzymic conversion of choline into glycine betaine when the E. coli choline transporter BetT was present. However, none of them allowed E. coli to take up choline from the medium, suggesting that in H. elongata the choline transport gene(s) is not linked to the glycine betaine biosynthetic genes in the chromosome.
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Analysis of the DNA sequence revealed the existence of four ORFs, three complete ones (betIBA) in the same orientation and a fourth and incomplete one divergent from the others (Fig. 1). The three complete ORFs were identified as the H. elongata betIBA genes. The betI gene starts with a GTG codon at bp 659 and ends with a TAA codon at bp 1280. It encodes a 207 residue protein with a deduced molecular mass of 22·8 kDa. The deduced amino acid sequence showed a high percentage of charged amino acids (26%) and is rather basic, with a net positive charge of 10. The betB gene starts with an ATG codon at bp 1293 and ends with a TAA codon at bp 2760. It encodes a 489 residue protein with a calculated molecular mass of 52·3 kDa. The betA gene starts with an ATG codon at position 2818 and ends with a TAG codon at position 4492. The predicted coding region encodes a 559 residue protein with a calculated molecular mass of 62·0 kDa. Divergently transcribed from the betIBA genes, the incomplete ORF (orf1) starts with an ATG codon at position 465. This ORF did not show any homology with sequences deposited in the public databases. All the ORFs were preceded by putative ribosome-binding sites. Downstream of the betA gene there is an inverted repeat which may function as a Rho-independent transcriptional terminator for the betIBA gene cluster. In addition, between positions 550 and 590 there are two sequences that display a high homology with the consensus sequences of the -10 and -35 regions of the
70-dependent promoters of E. coli.
The betI gene encodes a putative regulatory protein
Computer searches revealed a high homology of the product encoded by the H. elongata betI gene with the BetI protein of E. coli (56% identity) and Si. meliloti (40% identity). Moreover, a significant similarity with the N-terminal regions of other regulatory proteins, such as the TetR repressor of plasmid pSC101 (28% over 61 amino acids) and the MtrR repressor of Neisseria gonorrhoeae (27% over 63 amino acids) was found. All these homologies cover the helixturnhelix motif typically found in DNA-binding proteins (Fig. 3a). This would suggest that the H. elongata betI gene encodes a regulatory protein which might, like its E. coli counterpart, serve as a repressor protein mediating bet expression in response to the availability of choline in the growth medium (Røkenes et al., 1996
).
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BetA is a choline dehydrogenase (CDH) homologue
The deduced amino acid sequence of BetA showed high homology with different CDHs of prokaryotes, such as E. coli (74% identity), St. xylosus (52%) and Si. meliloti (49%), with the eukaryote Rattus rattus (51%) and with the choline oxidase from Arthrobacter globiformis (31%) (Fig. 3c).The N-terminal region of BetA displayed the so-called glycine box, containing a conserved motif (GXGXXG) and a series of amino acids that are characteristic features of flavoproteins (Lamark et al., 1991
; Pocard et al., 1997
; Wierenga et al., 1986
). We concluded from these homologies that the H. elongata BetA protein might be a CDH.
The H. elongata BetA alone mediates the conversion of choline into glycine betaine by E. coli
In E. coli, the membrane-bound CDH encoded by the betA gene mediates not only the oxidation of choline but also the conversion of glycine betaine aldehyde into glycine betaine (Landfald & Strøm, 1986 ). To check if the H. elongata BetA protein can also catalyse the second step of glycine betaine synthesis, the Bet- E. coli strain PD141(
DE3) containing pJB004 (E. coli betT) plus pDC13 (betA), pJB004 plus pDC14 (betIB), or pJB004 was used to express the corresponding proteins under the control of the T7
10 promoter. After induction with 1 mM IPTG for 1 h, cells were incubated with [methyl-14C]choline for 20 min at room temperature, and radiolabelled accumulated solutes were extracted and analysed by TLC. As shown in Fig. 4
, only the strain carrying the betA+ plasmid pDC13 plus the E. coli choline transporter gene betT converted choline into glycine betaine. An additional radioactive species might correspond to the intermediate in glycine betaine synthesis, glycine betaine aldehyde (Fig. 4
). This is probably due to the short incubation time (20 min) with [methyl-14C]choline used in the assay. These data strongly suggest that the H. elongata CDH is able to catalyse both steps of glycine betaine synthesis.
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DISCUSSION |
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Analysis of the H. elongata bet region revealed the presence of three genes (betIBA), which can be functionally expressed in E. coli and seem to be genetically organized in an operon. This organization differs from that found in E. coli (betTIBA; Lamark et al., 1991 ), Si. meliloti (betICBA; Øster
s et al., 1998
), B. subtilis (gbsAB; Boch et al., 1996
) and St. xylosus (cudTCAB; Rosenstein et al., 1999
). Among these gene arrangements, the E. coli and St. xylosus regions carry the choline transport (betT or cudT) genes linked to the synthesis genes. We did not detect, either upstream or downstream of the betIAB cluster of H. elongata, an ORF(s) that could encode a choline transport system. Moreover, the cosmid clone pDC1, which comprises approximately 35 kb of the H. elongata chromosome, including the betIBA region, was unable to mediate the conversion of choline by E. coli strain MKH13 unless the E. coli choline transporter BetT was present. We have previously reported the existence of a high-affinity transport system for choline (Km=10 µM) in H. elongata (Cánovas et al., 1998b
). This transporter must therefore be encoded elsewhere in the H. elongata chromosome. This situation may be similar to that found in B. subtilis, where choline uptake is mediated by two evolutionarily closely related ABC transport systems (OpuB and OpuC), whose genes are separated from the glycine betaine biosynthetic gene locus (gbsAB) (Kappes et al., 1999
).
The betICBA operon in Si. meliloti includes a gene encoding a choline sulfatase (betC), allowing the utilization of this ester as a precursor for glycine betaine production (Østers et al., 1998
). We have previously reported that H. elongata can also use choline-O-sulfate as an osmoprotectant (Cánovas et al., 1996
), but the mechanism by which this osmoprotection is achieved remains unknown. It is possible that in H. elongata choline-O-sulfate does not need to be metabolized to glycine betaine and plays an osmotic role by itself, a situation that has recently been reported for the effective use of choline-O-sulfate as a metabolically inert compatible solute in B. subtilis (Nau-Wagner et al., 1999
). However, the absence of a choline sulfatase gene in the Halomonas betIBA region does not rigorously exclude the possibility that choline-O-sulfate could be hydrolysed to choline and then converted into glycine betaine in this moderate halophile.
By analogy with the corresponding proteins of E. coli (Lamark et al., 1991 ) and Si. meliloti (Øster
s et al., 1998
), we conclude that the betI gene product is most probably a regulatory protein. In E. coli, the divergently overlapping betT and betI promoters are regulated in the same manner by three external stimuli: osmolarity, presence of choline and oxygen. Both promoters remain fully osmotically regulated, but not choline-regulated, in a betI mutant (Lamark et al., 1996
). In vivo (Lamark et al., 1996
) and in vitro (Røkenes et al., 1996
) studies showed that the choline-sensing repressor BetI regulates bet gene expression negatively in response to choline by binding to a 41 bp DNA fragment containing the -10 and -35 regions of both bet promoters. In H. elongata, both the transport and the oxidation of choline to glycine betaine are much faster at high (2 M NaCl) than at reduced (0·5 M NaCl) salinity, indicating that osmolarity is a major factor in the regulation of the choline-glycine betaine pathway (Cánovas et al., 1998b
). Moreover, the end product glycine betaine exerted a slight inhibition of choline uptake and a considerable inhibition of the oxidation of choline to glycine betaine, especially at high salinity (Cánovas et al., 1998b
). Whether or not BetI is involved in these regulatory effects and the role of choline in the regulation of the H. elongata bet genes is presently under investigation. Like Si. meliloti (Smith et al., 1988
), H. elongata can use both choline and glycine betaine as the sole carbon and nitrogen source (Cánovas et al., 1996
), whereas glycine betaine is metabolically inert in E. coli (Landfald & Strøm, 1986
) and B. subtilis (Boch et al., 1994
). This requires additional regulatory circuits in H. elongata to avoid a futile cycle of glycine betaine biosynthesis and degradation under high osmolarity growth conditions.
To cope with osmotic stress caused by the presence of high salt concentration, halophilic aerobic archaea and the anaerobic bacteria of the order Haloanaerobiales maintain high intracellular salt concentrations. In these extremophiles, enzymes and structural cell components have to be adapted to high salinity and show unique molecular adaptations (Oren, 1999 ). Examination of the first crystal structures of proteins from the archaeum Haloarcula marismortui suggests that an abundance of acidic residues distributed over the protein surface is a key determinant of adaptation to high-salt conditions (Elcock & McCammon, 1998
). In contrast, halophilic micro-organisms using the compatible solute strategy maintain low salt concentrations within their cytoplasm. Therefore, it is expected that no special adaptation of their intracellular proteins is required. To test this prediction for the BetIBA proteins of the moderate halophile H. elongata, their charge and amino acid distribution were analysed and compared with the same proteins of the non-halophilic bacteria E. coli and Si. meliloti. Although the enzymes from Halomonas had a slightly higher percentage of charged amino acids compared to those from E. coli and Si. meliloti, there were no substantial differences in amino acid composition of the Bet proteins from the three micro-organisms. BetI proteins showed a relatively high percentage of basic amino acids, with a positive net charge ranging from 9+ (for Si. meliloti BetI) to 12+ (for E. coli BetI). Basic and acidic amino acids were present in approximately equivalent numbers in the BetA enzymes of H. elongata (net charge 3+) and E. coli (net charge 1-). However, Si. meliloti BetA was predominantly basic, with a net charge of 13+. Only BetB exhibited a net negative (13- for the H. elongata enzyme) charge. This holds true for the three BetB enzymes, although BetB from H. elongata had a higher negative charge than the enzymes from E. coli (6-) and Si. meliloti (6-). From all these data we conclude that, as far as the betaine synthesis machinery is concerned, no special adaptations to salt seem to exist in the cytoplasmic proteins of the moderate halophile H. elongata.
The synthesis of glycine betaine, one of the most powerful osmoprotectants found in nature (Le Rudulier et al., 1984 ; Csonka & Hanson, 1991
; Kempf & Bremer, 1998
), also plays an important role in the adaptation process of H. elongata to a high-osmolarity environment. The bet genes characterized in this work will be used for the construction of single and double mutants affected in the synthesis of glycine betaine and/or other compatible solutes, such as ectoine. These mutants will be of invaluable help in elucidating the global regulation of the osmoadaptive mechanisms in this extremophilic micro-organism.
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ACKNOWLEDGEMENTS |
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Received 30 July 1999;
revised 18 October 1999;
accepted 2 November 1999.