1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain
2 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
3 Department of Organic Chemistry and Pharmaceutical Chemistry, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain
4 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA
Correspondence
Joaquín J. Nieto
jjnieto{at}us.es
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ABSTRACT |
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INTRODUCTION |
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Usually, halotolerant (i.e. tolerating but not requiring salt) or halophilic (i.e. requiring salt and also growing at higher salinities) micro-organisms are able to synthesize de novo one or a few compatible solutes. One of the most widespread compatible solutes is ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid). First discovered in the phototrophic sulfur bacterium Ectothiorhodospira halochloris (Galinski et al., 1985), ectoine has been found to be synthesized and accumulated by halophilic/halotolerant representatives of bacteria belonging to three major phylogenetic branches: the actinobacteria (i.e. genera Actinopolyspora, Brevibacterium, Nocardiopsis and Streptomyces) the firmicutes (i.e. genera Bacillus, Salibacillus, Virgibacillus, Halobacillus, Marinococcus and Sporosarcina), and the proteobacteria (i.e. genera Chromohalobacter, Ectothiorhodospira, Halomonas, Methylobacter, Pseudomonas, Rhodovulum and Salinivibrio) (Da Costa et al., 1998
; Galinski, 1995
; Galinski & Trüper, 1994
; Kuhlmann & Bremer, 2002
; Severin et al., 1992
; Ventosa et al., 1998
; Wohlfarth et al., 1990
). With the known exception of the
-proteobacteria Rhodovulum sulfidophilum (formerly Rhodobacter sulfidophilum; Hiraishi & Ueda, 1994
) and Rhodovibrio salinarum (formerly Rhodospirillum salinarum; Nissen & Dundas, 1984
) all ectoine-producing proteobacteria found so far belong to the gamma subdivision of this bacterial lineage. Ectoine has not been reported, however, in cyanobacteria, archaea or eukaryotes.
Among the -proteobacteria, most members of the Halomonadaceae (Halomonas and Chromohalobacter) are moderate halophiles that display one of the broadest salinity ranges found in prokaryotes (Ventosa et al., 1998
). Chromohalobacter salexigens 3043 (formerly Halomonas elongata DSM 3043; Arahal et al., 2001
) is able to grow from 0·15 M to 4·3 M NaCl in complex medium, and from 0·5 M to 3 M NaCl in minimal medium (Arahal et al., 2001
; Cánovas et al., 1996
). Recently, O'Connor & Csonka (2003)
characterized the ion requirements of C. salexigens and made the unexpected finding that while this organism needs moderate concentrations of Na+ and Cl ions, its growth rate was stimulated by a number of other salts, indicating that C. salexigens requires a combination of NaCl and high ionic strength for optimal growth. Osmoadaptation in C. salexigens is mainly achieved by de novo synthesis of ectoine and its hydroxy derivative, hydroxyectoine (Cánovas et al., 1997
). In addition, when they are provided externally, C. salexigens accumulates osmoprotectants such as glycine betaine, which is taken up from the medium or synthesized from choline (Cánovas et al., 1996
, 1998b
). In a previous work, we isolated and characterized the C. salexigens ectABC genes, encoding the main route for ectoine synthesis in this micro-organism. The ectB gene encodes the enzyme diaminobutyric acid transaminase, which catalyses the conversion of aspartate semialdeyde into diaminobutyric acid; ectA encodes diaminobutyric acid acetyltransferase, responsible for the acetylation of diaminobutyric acid to N
-acetyldiaminobutyric acid, and ectC specifies ectoine synthase, which catalyses the cyclic condensation of N
-acetyldiaminobutyric acid into ectoine (Cánovas et al., 1998a
). The C. salexigens EctA, EctB and EctC proteins show the same organization as well as a significant degree of sequence identity to the enzymes of ectoine synthesis in Halomonas elongata (Göller et al., 1998
), Marinococcus halophilus (Louis & Galinski, 1997
) and Sporosarcina pasteurii (Kuhlmann & Bremer, 2002
). Thus, it seems that the ectoine biosynthetic route, which was first elucidated at the biochemical level in the closely related H. elongata (Ono et al., 1999
; Peters et al., 1990
), is evolutionarily well conserved in all ectoine-producing bacteria characterized so far.
Knowledge on how ectoine synthesis is regulated is important for several reasons. This compatible solute has biotechnological applications as a stabilizing agent for enzymes, DNA and whole cells (Galinski, 1995; Ventosa et al., 1998
). Therefore, the dissection of the region involved in the transcriptional control of ectoine synthesis, and the elucidation of post-transcriptional control mechanisms, are necessary to generate modified strains improved in ectoine production for prospective industrial use. In addition, the synthesis of this compatible solute is widespread in the bacterial world as a means to cope with changes in external osmolarity. In the absence of external osmoprotectants, C. salexigens synthesizes ectoine as its main osmoadaptation mechanism. Because C. salexigens can grow in a very broad salinity range, it must be able to rapidly and finely adjust its cytoplasmic solute concentration in response to variable osmotic stress. In order to achieve this, this micro-organism must presumably have a powerful regulatory mechanism controlling the synthesis of ectoine. Within the Halomonadaceae, the transcriptional regulation of ectoine synthesis has not been investigated. In Halomonas elongata, Kraegeloh & Kunte (2002)
reported that, under a moderate upshock (from 0·5 M to 1 M NaCl), cells treated with chloramphenicol synthesized ectoine, suggesting that, at least under these experimental conditions, ectoine synthesis is mainly regulated at the level of enzyme activity. However, there is an important difference in the salinity requirements of Halomonas elongata and C. salexigens (Cánovas et al., 1996
; Vreeland & Martin, 1980
) and also a different pattern of ectoine accumulation in the presence of betaine (Cánovas et al., 1996
; Wohlfarth et al., 1990
), suggesting that regulation of the ectoine pathway might be different in these two micro-organisms. In this study we investigated the long-term regulation of ectoine synthesis in C. salexigens, with emphasis on the transcriptional regulation.
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METHODS |
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DNA manipulation and plasmid construction.
Plasmid DNA was isolated from E. coli by the alkaline lysis method (Sambrook & Russell, 2001). Restriction enzyme digestion and ligations were performed as recommended by the manufacturer (Promega). DNA regions upstream of each ect gene were obtained by PCR amplification by using as a template plasmid pME2, which contains the C. salexigens ectABC region (GenBank/EMBL accession no. AJ11103), Pfu DNA polymerase (Stratagene), and specific oligonucleotides that in some cases were modified to include appropriate restriction sites (modifications are shown underlined). A 296 bp region containing the promoter region located upstream of ectA was amplified by using the oligonucleotide pair MA1 (5'-CGCTCTAGAACTAGTGGAT-3'; this sequence belongs to the vector polylinker located upstream of the EcoRI site defining the 5'-end of the ectABC region cloned in pME2,) and MA2 (5'-GTTCTCGGCTGCAGGCGTCAT-3'), digested with EcoRIPstI (resulting in a 288 bp insert) and cloned into pMP220 to give plasmid pME7. A 442 bp region containing the promoter located upstream of ectB was synthesized by using the oligonucleotides MB1 (5'-CGGTACCCCTATCTGCTGCT-3') and MB2 (5'-GAATCTGGGTCTGCAGAGAT-3'), digested with KpnIPstI (resulting in a 431 bp insert) and inserted into pMP220 to generate pME8. A 678 bp region was amplified by using the oligonucleotides MC3 (5'-CCGTGTGGTCTAGAATCGCT-3') and MC4 (5'-TGCTGATCCTGCAGGTACAT-3'), digested with XbaIPstI (resulting in a 662 bp insert containing 395 bp upstream of ectC) and cloned into pMP220. The plasmid constructed in this way was named pME9.
S1 nuclease analyses of mRNAs.
C. salexigens cells were grown in minimal medium M63 to OD600 0·5, and total RNA was isolated as previously described (Monsalve et al., 1995). S1 nuclease reactions were performed as described by Ausubel et al. (1989)
, using 25 µg total RNA and an excess of a 32P-end-labelled single-stranded DNA (ssDNA) hybridizing to the 5' region of the mRNA. The ssDNA probes were generated by linear PCR, using as a substrate the plasmid pME2, which contains a 3 kb EcoRI fragment including the ectABC region. Prior to the linear amplification reaction, this plasmid was digested with EcoRI for ectA S1 assay (the EcoRI recognition sequence is located 275 bp upstream of ectA start codon), XmnI for ectB S1 assay (the XmnI recognition sequence is located 629 bp upstream of ectB start codon) or HindIII for ectC S1 assay (the HindIII recognition sequence is located 378 bp upstream of ectC start codon). The primers used to generate the probes were MS-A2 (5'-TCGTCGGTCGTGGGCTTAC-3'), MS-B1 (5'-AACGGTAGGAAAAGAACG-3') and MS-C1 (5'-TGCGGGTGATGTTGAACGAG-3') for mapping the 5'-end of putative transcripts starting at ectA, ectB and ectC, respectively.
Extraction of intracellular solutes and 13C-NMR spectroscopy.
Cytoplasmic solutes were extracted essentially as described by Cánovas et al. (1997), but with the following modifications. C. salexigens was grown in 200 ml minimal medium M63 until late-exponential phase (OD600 11·2). Cells were collected by centrifugation and washed with the same medium with no carbon source. To extract the cytoplasmic compatible solutes, pellets were resuspended in 10 ml double-distilled water and, after 10 min incubation at room temperature, cell debris was removed by centrifugation. Supernatants were extracted twice with chloroform and freeze-dried. Cell extracts were resuspended in 0·5 ml D2O. Natural 13C-NMR abundance spectra were recorded on a Bruker ac200 spectrometer at 50 MHz with a probe temperature of 2022 °C. Signals were identified by comparison with spectra of pure compounds or with spectra previously described (Cánovas et al., 1997
, 1999
).
Assay for -galactosidase activity.
-Galactosidase activity was measured basically as described by Miller (1992)
. Overnight cultures of Lac E. coli cells (in LB with Tc), or C. salexigens DSM 3043 (lacks intrinsic
-galactosidase activity) cells (in SW-2 with Tc), harbouring each lacZ fusion or the plasmid vector pMP220, were diluted (1 : 100) in fresh minimal medium M63. Cultures were grown at 37 or 40 °C and 0·8 ml aliquots were taken at different time intervals. Cells were washed with buffer Z (without
-mercaptoethanol) to remove salts. Absorbance at 420 nm was determined with a Perkin Elmer 551S UV/Vis spectrophotometer. All assays were performed three times from two parallel cultures, and the standard deviation was calculated. The residual
-galactosidase activity derived from the plasmid vector pMP220 was subtracted in all determinations.
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RESULTS |
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Construction of PectAlacZ and PectBlacZ transcriptional fusions
To further characterize the expression of the ectABC genes, transcriptional fusions of the promoter regions PectA (containing putative promoters PectA1 to PectA4) and PectB (containing putative promoter PectA5) were constructed to the E. coli reporter gene lacZ (Fig. 2). A DNA region of 288 bp spanning positions 231 to +56 relative to the PectA1 transcription start site was placed upstream of the promoterless lacZ gene in the low-copy vector pMP220, yielding the plasmid pME7. Similarly, a 431 bp fragment spanning positions 400 to +30 relative to the PectB transcription start site was cloned in pMP220 to give the plasmid pME8. To confirm the absence of a monocistronic ectC transcript, a 662 bp region containing 395 bp upstream of ectC was also cloned in pMP220, giving the plasmid pME9. The constructs were transferred from E. coli DH5
to wild-type C. salexigens by conjugation, and expression of the three fusions in both host strains was preliminarily tested on plates of minimal medium M63 supplemented with the
-galactosidase substrate X-Gal (Fig. 2c
). Whereas colonies of E. coli and C. salexigens carrying plasmids pME7 and pME8 showed a Lac+ phenotype, transconjugants harbouring pME9 were Lac. These results revealed that the ectABC genes are transcribed in E. coli and confirmed the existence of promoter(s) upstream of ectA (in pME7) and ectB (in pME8), but not upstream of ectC (in pME9).
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Because the ectABC genes were expressed in E. coli, we measured the response of the PectA promoter region to increasing osmolarity in this host (Fig. 3). Basal transcription levels in cells grown in minimal medium M63 to the stationary phase (see below) in the absence of NaCl were quite high (aproximately 2000 Miller units). In addition, the activity of the PectAlacZ fusion was linearly correlated with the osmotic strength of the growth medium, with a 2·5-fold increase in expression from 0 M to 0·5 M NaCl. These results indicate that, in E. coli, the PectA promoter region is not only expressed at a high basal level, but is also osmoregulated. In addition, the basal expression level of the PectAlacZ fusion appeared to be as efficient in E. coli as in C. salexigens (see below).
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DISCUSSION |
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Kuhlmann & Bremer (2002) reported that the ectABC genes of Salibacillus pasteurii are expressed from a single osmoregulated promoter. The 10 and 35 sequences of this promoter resemble the consensus sequences of promoters recognized by the main vegetative sigma factor
A (equivalent to
70 of Gram-negative bacteria). This situation differs from what we have found upstream of ectA in C. salexigens. Nevertheless, it has been firmly established that
S controls the expression of several osmoregulated genes. Thus, the E. coli genes proP, encoding the proline uptake system (Xu & Johnson, 1997
), and cfa, encoding the cyclopropane fatty acid synthase (Wang & Cronan, 1994
), are transcribed from two promoters, one depending on
70, the other one being recognized by
S. There are some osmoregulated genes whose transcription is entirely dependent on
S. Some examples are the E. coli genes otsAB, involved in trehalose synthesis (Hengge-Aronis et al., 1991
), and osmY, encoding a periplasmic protein of unknown function (Yim et al., 1994
), among others. Thus, in E. coli and other enteric bacteria
S is not only involved in stationary-phase induction of transcription, but functions as the master regulator of the general stress response, coordinating an emergency reaction to stress as well as long-term adaptation to many different stresses including starvation, high osmolarity, high or low temperature, acidic pH, or oxidative agents (Hengge-Aronis, 1999
, 2000
). Similarly,
B, controlling the general stress response in Gram-positive bacteria, is partially responsible for the transcriptional control of some osmoregulated genes, such as opuE and opuD from Bacillus subtilis, encoding the proline and glycine betaine uptake systems, respectively (Bremer & Krämer, 2000
). In agreement with this, computer-assisted analysis of the Marinococcus halophilus DNA upstream of ectABC revealed the presence of a putative binding site for
B as the most promising candidate for transcription regulation in this bacillus-like micro-organism (Louis & Galinski, 1997
). Very interestingly, a transcriptional fusion of this promoter region (referred to as ectUp) with the reporter gene gfp was not only expressed but also osmotically induced in E. coli (Bestvater & Galinski, 2002
). The presence of sequences at the promoter region that might be potentially recognized by the E. coli sigma factors
70 or
S may explain this heterologous expression.
As C. salexigens is a truly halophilic bacterium, unable to grow below 0·5 M NaCl, transcriptional analysis of the ectABC genes in C. salexigens could not be performed in the absence of salt. Since at 0·5 M NaCl the growth is extremely slow, 0·75 M NaCl was selected as the lowest salt concentration in this study. It is remarkable that, at this low salinity, expression of the PectA and PectB promoters was quite high (Fig. 4a), suggesting that ectABC may be a partially constitutive system, and cells accumulated high amounts of ectoine and hydroxyectoine (Fig. 7a
), indicating that even at this salinity they are under osmotic stress. These findings agree with the fact that addition of osmoprotectants such as choline or glycine betaine stimulates growth of C. salexigens at all salinities, including 0·5 M NaCl (Cánovas et al., 1996
). In contrast, the halotolerant bacillus S. pasteurii (Kuhlmann & Bremer, 2002
) produced ectoine in significant amounts only at elevated osmolarities. Fully consistent with this is the finding that the ectABC genes of S. pasteurii are not expressed at 0 M NaCl. Consequently, it has been suggested that ectoine synthesis in S. pasteurii must depend primarily on the stimulation of gene transcription under hypertonic conditions (Kuhlmann & Bremer, 2002
).
The technical problems related to the failure of the E. coli -galactosidase to function above 0·75 M NaCl prevented us from quantitatively monitoring the expression of the ectABC at higher salinities. Nevertheless, the S1 protection assay performed with RNA from cells grown at 2·5 M NaCl (Fig. 1a
) clearly indicated that the putative PectA1 (
70-dependent), PectA3 (
S-dependent) and PectA4 might be osmoregulated promoters. These results suggest that transcriptional activation of ectoine synthesis plays a role in the long-term response to osmotic stress in C. salexigens. In agreement with this, a linear relationship was found between the expression of the PectAlacZ fusion and increasing salinity in E. coli. This induction by hyperosmolarity in E. coli may be attributed, at least in part, to an increased intracellular concentration of
S. It has been demonstrated that high osmolarity increases the rate of
S translation of already existing E. coli rpoS mRNA, and reduces
S proteolysis by the RssB recognition factor-ClpXP protease system (Hengge-Aronis, 2002b
). However, the nature of the intracellular signals that are triggered by osmotic stress and their interactions with the post-transcriptional control mechanisms in E. coli are completely unknown at present.
Temperature induction of PectA and PectB suggests that ectoine might have a physiological role in thermoprotection of C. salexigens, in addition to osmoprotection. This hypothesis, which has yet to be tested experimentally, correlates with the emerging evidence that some compatible solutes, such as the disaccharide trehalose, function in vivo as general stress protectants. Cánovas et al. (2001) reported that trehalose accumulation by Salmonella typhimurium is thermoregulated, and found evidence that this disaccharide is crucial for growth at high temperature. In fact, ectoine and its derivative hydroxyectoine were shown in vitro to function as powerful stabilizing agents of enzymes against a number of stresses, including thermal denaturation (Cánovas et al., 1999
, Lippert & Galinski, 1992
). In E. coli cells exposed to high temperature,
S proteolysis is reduced (Hengge-Aronis, 2002b
). Moreover, temperature-triggered translational induction, as well as stabilization of otherwise labile
32, occurs (Yura et al., 2000
). Although it is very tempting to speculate that induction of PectAlacZ and PectBlacZ transcription in C. salexigens cells grown at 40 °C might be attributed to an increased cellular level of sigma factors orthologous to E. coli
S and
32 as a consequence of the heat stress, this needs experimental evidence.
As expected, expression of PectAlacZ and PectBlacZ fusions was reduced in the presence of the DNA gyrase inhibitor nalidixic acid. The S1 protection assays (Fig. 1a) suggest that when C. salexigens is grown at 0·75 M or 2·5 M NaCl, active
70 and
S bind to the PectA promoter region. The 10 sequences of putative PectA2 (
70- dependent) and PectA3 (
S- dependent) are separated by 30 nt, whereas the association RNA polymerase core-sigma factor is estimated to cover a DNA region of about 80 nt. Therefore,
70- and
S-RNA polymerase holoenzymes must compete for binding to these two promoters. Similar to the situation in E. coli (Hengge-Aronis 1999
, 2002a
), local DNA topology, as well as the formation of specific nucleoprotein structures that involve additional regulatory factors and/or histone-like protein, might modulate ectABC expression in response to environmental conditions.
The presence of external osmoprotectants (ectoine or glycine betaine) reduced the basal transcription level of PectAlacZ and PectBlacZ in C. salexigens (Figs 1 and 6). The same transcriptional response was found by Bestvater & Galinski (2002)
for the M. halophilus ectUpgfp fusion in E. coli. Likewise, relief of osmotic stress by glycine betaine resulted in a reduced expression of the E. coli betA and betB, and B. subtilis opuE loci, encoding osmoregulated glycine betaine synthesis from choline, and proline transport, respectively (Eshoo, 1988
; Spiegelhalter & Bremer, 1998
). The decreased transcription of these osmoregulated genes upon addition of external osmoprotectants does not seem to be due to a direct effect of the solute on the transcription machinery. Instead, it may be that osmoprotectant accumulation attenuates the (unknown) activation signal for the former systems (Wood et al., 2001
). Our data indicate that in cells continuously grown at low or high salinity, transcriptional control by glycine betaine cannot be the only mechanism responsible for the absence of cytoplasmic ectoine. This suggests the existence of a post-transcriptional control mechanism that might operate at the level of enzyme activity. However, alternative mechanisms such as an increased efflux of ectoine in the presence of betaine cannot be ruled out. This is in agreement with the general assumption that transport of compatible solutes is preferred over their synthesis, because the latter is energetically less favourable (Oren, 1999
).
Bacterial response to environmental stresses is complex, and the involvement of multiple promoters and transcription factors belonging to different regulatory pathways is a rather common strategy that ensures an appropriate expression to the changing environment (Vicente et al., 1999). For some osmoregulated systems, the general stress response and the specific osmostress response are linked through the general stress factors
S or
B that control systems such as trehalose synthesis in E. coli (Hengge-Aronis et al., 1991
), osmoprotectant uptake in B. subtilis (Bremer & Krämer, 2000
), and possibly ectoine synthesis in C. salexigens (this study). In C. salexigens, the response to heat and osmotic stress might also overlap since PectB might be dependent on
32. Some osmoregulated systems, such as the E. coli and C. salexigens betBA genes for glycine betaine synthesis, are clustered with their transcriptional regulator (i.e. betI) within the chromosome (Lamark et al., 1991
; Cánovas et al., 2000
). However, no sequences encoding regulatory proteins are found upstream or downstream of the C. salexigens ectABC cluster (unpublished results). In conclusion, a number of as yet undetermined signal transduction pathways might operate in C. salexigens leading to osmoregulated ectoine synthesis. Identifying and characterizing these routes will be some of our main experimental aims in the near future.
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ACKNOWLEDGEMENTS |
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Received 27 February 2004;
revised 14 May 2004;
accepted 2 June 2004.
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