Heterologous ectoine production in Escherichia coli: By-passing the metabolic bottle-neck
© Bestvater et al; licensee BioMed Central Ltd. 2008
Received: 19 February 2008
Accepted: 29 August 2008
Published: 29 August 2008
Transcription of the ectoine biosynthesis genes ectA, ectB and ectC from Marinococcus halophilus in recombinant Escherichia coli DH5α is probably initiated from three individual σ70/σA-dependent promoter sequences, upstream of each gene. Consequently, mRNA-fragments containing the single genes and combinations of the genes ectA and ectB or ectB and ectC, respectively, could be detected by Northern blot analysis. Under the control of its own regulatory promoter region (ectUp) a seemingly osmoregulated ectoine production was observed. In addition, aspartate kinases were identified as the main limiting factor for ectoine production in recombinant E. coli DH5α. Co-expression of the ectoine biosynthesis genes and of the gene of the feedback-resistant aspartate kinase from Corynebacterium glutamicum MH20-22B (lysC) led to markedly increased production of ectoine in E. coli DH5α, resulting in cytoplasmic ectoine concentrations comparable to those reached via ectoine accumulation from the medium.
To master the osmotic stress of saline environments, halophilic organisms accumulate highly water-soluble organic osmolytes, so-called compatible solutes [1–3]. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), the compatible solute that was first discovered in Ectothiorhodospira halochloris , is one of the most commonly found osmolytes in nature [5–9]. Besides their osmotic effect, ectoines as well as other compatible solutes have been found to improve protein folding and to protect biomolecules such as enzymes, nucleic acids, antibodies and even whole cells against heating, freeze-thawing, drying or chemical treatment [10–14]. Additional applications of ectoine include use as protective additive, modulator of proinflammatory response and moisturizer for skin care products [15–17], and potentially also for treatment of diseases related to protein misfolding [18–21]. In view of its potential as a stabilizing, protective and pharmaceutical agent, a bioprocess for ectoine production named "bacterial milking" has been developed for commercial exploitation using the halophilic eubacterium Halomonas elongata .
In this study, we report the transcription initiation sites of the ectoine gene cluster as determined by RACE (rapid amplification of cDNA ends) in both, the donor Marinococcus halophilus as well as the genetically engineered E. coli DH5α. In addition, we report on potential metabolic limitions for heterologous ectoine production and the generation of a new recombinant production strain freed from one such metabolic "bottle-neck" which limits substrate supply of the ectoine biosynthetic pathway.
Determination of transcription start points by rapid amplification of cDNA ends (RACE)
In the donor (M. halophilus) single gene transcription of ectA and ectB appears to be under control of σA-dependent promoters (Fig. 3A and 3B). The initiation sites are located 114 nucleotides and 44 nucleotides upstream of the start codon for the genes ectA and ectB, respectively. In addition to the transcription initiation sites for the single gene products, in M. halophilus, a site for the transcription of ectAB and/or ectABC could be found 34 bp upstream of the start codon of ectA. These results were obtained when the mRNA in the RACE procedure was transcribed to cDNA over two consecutive genes (approx. 0.9 kb), starting from an ectB-specific primer. Unfortunately, we were unable to detect an analogous transcription initiation site for ectBC, probably because of the length of the potential RACE product (1.6 kb for ectBC compared to only 0.9 kb in the case of ectAB). In contrast to the promoters for single gene transcription, the transcription initiation site of ectAB and/or ectABC is under the control of a putative σB-dependent promoter (Fig. 3A). Taken into consideration that the primary sigma factor σA is responsible for the expression of the essential genes for cell survival, whereas σB mediates the general stress response, this could point towards an interplay of various mechanisms regulating the expression of the ectoine genes in M. halophilus. The RACE reverse transcription product of ectC mRNA appears to stop 83 nucleotides upstream of the start codon (|◀◀ in Fig. 3C), but no corresponding promoter consensus sequence could be assigned to this initiation site. Therefore, the possibility remains that this initiation site is an artefact, possibly due to the formation of a secondary structure of mRNA (terminating loop), or the result of mRNA processing. Downstream of this hypothetical transcription initiation site a potential σA-dependent promoter site, comprising a -10 and a -35 region, could be found (Fig. 3C) with a potential transcription initiation point 29 bp upstream of ectC. This promoter site is apparently not used in M. halophilus under the conditions employed, it is however recognized by the genetically engineered host, E. coli DH5α.
In E. coli, transcription of each single gene ectA, ectB and ectC is under control of a σ70/σA-dependent promoter with both -10 and -35 region. Transcription is initiated 29 bp upstream of the start codon for ectC, 44 bp for ectB and probably 114 bp for ectA (Fig 3). In the case of ectA, experimental proof of the predicted transcription initiation site posed a problem as resulting cDNA fragments were terminated 89 bp upstream of the start codon (|◀◀ in Fig. 3A). No suitable promoter sequence was found in this region. Even though the experimental characterization of a transcription initiation site upstream of ectA was not successful, we were able to reveal the likely initiation site shown in Fig. 3A from the data available. Louis and Galinski  were able to demonstrate that a deletion 146 bp upstream of the start codon (shaded area in Fig. 3) resulted in unregulated ectoine production in E. coli XL1-Blue pOSM16. In this work we demonstrated, using DH5α pOSM2, that a deletion 100 bp upstream of the start codon (↱ pOSM2 in Fig. 3A) completely disabled transcription of ectA. From this we concluded a promoter region beyond 100 bp and somewhere around 150 bp upstream of ectA, probably identical to the σA-dependent site identified for M. halophilus. (Fig. 3A). This proposal is corroborated by the finding that the deletion in pOSM16 changes the first two thymidine nucleotides (TT) of the sequence of the potential σ70-dependent -35 region TTGAAA (Fig. 3A). The consensus sequence for this -35 region is TTGACA .
Growth and ectoine production of genetically engineered E. coli
The comparatively low ectoine levels in recombinant E. coli DH5α pOSM12 could not be explained by limitation of the cells' capacity because the ectoine-accumulating control strain tolerated higher intracellular ectoine concentrations nor by leakage because ectoine was not detectable in the medium (HPLC sensitivity limit: 10 μM). In addition, higher cytoplasmatic ectoine levels caused a significant growth-promotion of the control strain and appeared to have no negative effect on the cells. We therefore assumed that regulatory mechanisms in the metabolism of E. coli caused the limitation of ectoine production in the recombinant strains. To determine potential candidates for the limiting steps, the ectoine biosynthetic pathway had to be analyzed in context with E. coli's metabolic network, where L-aspartate β-semialdehyde, the substrate of the first enzyme of the ectoine biosynthetic pathway, is an intermediate of the biosynthetic pathway of the amino acids of the aspartate family (Fig. 1A). As E. coli has three aspartate kinase activities (I-III), which are regulated by feedback inhibition and/or transcriptional repression , we suspected a bottle-neck for the supply of this metabolic precursor in E. coli DH5α pOSM12.
Role of aspartate kinases in ectoine synthesis
Opening the bottle-neck with plasmid pAKECT1
The results obtained from the supplementation experiments provide strong evidence that the aspartate kinases, which are the key regulatory enzymes for the biosynthetic pathway of aspartate family amino acids in E. coli, represent a bottle-neck for ectoine production in the non-halophilic host DH5α pOSM12 because of stringent feed-back inhibition and/or transcriptional repression. Our strategy to relieve this metabolic restriction was to alter the regulation of the biosynthetic pathway by co-expression of the ectoine biosynthetic genes together with the feedback-insensitive aspartate kinase (lysC) from C. glutamicum MH20-22B, which had already been successfully expressed in E. coli [27, 28].
Enzymatic activity of the aspartate kinases
Aspartate kinase activity
specific activity [nmol (min mg protein)-1]
E. coli DH5α pOSM12
5.4 ± 0.2
2.4 ± 0.1
E. coli DH5α pOSM2
5.2 ± 0.2
2.2 ± 0.1
E. coli DH5α pAKECT1
10.0 ± 0.4
10.1 ± 0.5
Transcription of ectoine genes in M. halophilus and recombinant E. coliDH5α
Data on transcription regulation are still rare for halophilic eubacteria, but have been investigated in detail for the Gram-positive non-halophilic model organism Bacillus subtilis, and, of course, E. coli. Transcription of genes essential for cell survival during exponential growth is controlled by the primary sigma factors σA and σ70, respectively, which recognize a promoter consensus sequence comprising a -35 region TTGACA, a spacer of 16–18 nucleotides and a -10 region TATAAT . Of the three σA/σ70 promoters identified in this study, only the one upstream of ectB (ATGACA-N18-TATTAT) had previously been identified by Louis & Galinski , because the other two (TTGAAA-N17-TAAGCT and TCGACT-N17-TATGAT) deviated by 4 and 3 nucleotides, respectively, from the consensus sequence.
General stress response, on the other hand, is mediated by the alternative sigma factor σB in Bacillus subtilis (among other Gram-positive bacteria) and σS in E. coli [29, 30]. For σB-dependent promoters a -35 region GTTTAA and a -10 region GGGTAT, separated by a spacer of 12–14 nucleotides, have been proposed . For σS-dependent promoters in E. coli Espinosa-Urgel et al.  proposed a -10 region CTATACT, which is only slightly different from the -10 region recognized by σ70. A conserved -35 region could not be defined so far, but an intrinsic curvature in this region is proposed to compensate for it. Due to these marked differences it is rather unlikely that a genuine σB-dependent promoter of a Gram-positive bacterium should be recognized by the Gram-negative E. coli.
In earlier work Louis and Galinski  could not resolve the question whether the three ectoine biosynthetic genes (ectA, ectB, ectC) are transcribed separately or as a single operon. Using the RACE method we have now shown that they are transcribed both as single genes and as mRNA's comprising ectAB, ectBC and possibly ectABC in M. halophilus (not shown). Heterologous expression of the ectoine gene cluster in E. coli, also resulted in single and double gene mRNA products.
We have shown here the successful expression of the ectoine biosynthesis genes ectA, ectB and ectC (as well as ectAB and ectBC) from the Gram-positive M. halophilus in the Gram-negative E. coli. This is explained by recognition of all three σA-dependent promoters preceeding individual genes of the ectoine biosynthesis gene cluster of M. halophilus. Due to the conformity of the consensus sequence of σA- and σ70-dependent promoters this result is not surprising. In addition to the σA-dependent promoters, a σB-dependent promoter for the transcription of ectAB (and possibly ectABC) could be characterized upstream of ectA, suggesting that transcription of the single and the multiple gene products is initiated via different regulatory mechanisms in the donor M. halophilus. This promoter recognition sequence (GTTTGT-N13-AGGTAT) deviates by 3 nucleotides from the consensus sequence and had, therefore, previously not been recognized by Louis and Galinski . A potential σB-dependent promoter 280 bp upstream of ectA, which was proposed by Louis and Galinski , is apparently not involved in the regulation of transcription of ectoine genes under the experimental conditions employed. Use of the σB-dependent promoter by recombinant E. coli was neither demonstrated nor to be expected. Due to the similarity of the σS-dependent -10 region CTATACT to a σ70-dependent promoter, it cannot be stated without further investigation, e.g. by sigma-factor binding studies, whether transcription of ectoine biosynthetic genes in E. coli is under control of σ70, σS or an interplay of both. In addition it has already been shown in several studies that σ70-dependent promoters could also be recognized by σS [33, 34], and a regulatory interplay of σ70 and σS, based on changes in binding affinity affected by global regulatory factors, was proposed [35, 36].
Recent work on Bacillus pasteurii has shown that the ectABC genes are organised in a single operon in this organism. Expression of ectoine genes was only observed when cells were grown at elevated osmolarity and a single gene transcript (2.6 kb) and a typical σA-dependent promotor region were identified . Unfortunately upshock experiments were not conducted, hence the possibility still remains that an additional stress response promoter (σB-dependent) may also be involved in the organism's short-term adaptation. In contrast to B. pasteurii (a halotolerant species) M. halophilus is a true halophile with a growth optimum at around 2 M salt. It is therefore not surprising that its salt stress response strategies are apparently more complex.
In a comprehensive promoter analysis of the ectoine gene cluster in Chromohalobacter salexigens (member of the Halomonadaceae) four putative transcription initiation sites were identified, at 44, 96, 134 and 149 bp upstream of the ectA start codon. Two of these were of the σ70-type, one probably σS and a fourth promoter with no similarity to known sigma factors. Consequently, the authors concluded the existence of a complex regulation pattern of ectoine synthesis in this true halophile .
The observation of osmoregulated ectoine production in E. coli DH5α pOSM12 could of course result from posttranscriptional regulation, including for example controlled uptake and/or excretion. However, as both transport systems for ectoine (ProP and ProU) are functional in E. coli DH5α pOSM12 and ectoine was not detected in the medium (at 10 μM sensitivity), we propose that ectoine synthesis is, at least partly, controlled at the level of enzyme activity. Still, the inability of E. coli DH5α pOSM2 to transcribe ectA and ectAB in the absence of IPTG and ectAB even under IPTG-induction, as well its impaired ectoine production, stresses the importance of the DNA region upstream of ectA (ectUp) for controlled expression of the ectoine biosynthesis genes. A transcriptional fusion of this promoter region (ectUp) with the reporter gene gfp was shown to be osmotically induced in E. coli and, more importantly, down-regulated in the presence of externally supplied compatible solutes . The promoter region upstream of ectA, therefore, appears to sense a regulatory signal, apparently common for both the Gram-positive M. halophilus and the Gram-negative E. coli.
Opening up of a metabolic bottle-neck for ectoine production
Successful heterologous expression of the ectoine biosynthetic genes from the halophilic M. halophilus in E. coli XL1-Blue by Louis and Galinski  enabled us for the first time to utilize genetically engineered strains for ectoine production. However, as E. coli XL1-Blue in medium MM63 displayed a narrow salinity range (1–3% NaCl) and growth rates were by a factor of 2–5 lower than with DH5α, experiments in this study were conducted with E. coli DH5α. The pivotal role of E. coli aspartate kinases as a limiting metabolic bottle-neck had been realised before, as shown by the use of E. coli feedback-insensitive aspartate kinase to enhance threonine production in transgenic alfalfa (Medicago sativa L.) . The data presented here prove that stringent feedback-regulation and/or transcriptional repression of the aspartate kinases in E. coli is in fact also the main limiting factor for recombinant ectoine production in this host.
When growth rates of E. coli DH5α pAKECT1 are compared to those of the control which accumulates ectoine from the medium (Fig. 5), one can see that the ectoine-synthesizing construct has a 40% lower growth rate at 2% and 3% NaCl. This difference may be explained by energy requirements and side-effects of a deregulated amino acid metabolism within the aspartate family. However, as this difference is diminished at higher salinities and completely abolished at 5% NaCl, one may conclude that, at the upper range of salt tolerance, ectoine production in the genetically engineered strain is equally efficient as ectoine uptake and that growth-limitations caused by overexpression of foreign genes and overproduction of aspartate family amino acids become less important.
As highest cytoplasmic ectoine concentrations (0.4 mmol (g dry weight)-1) were already achieved at a medium salinity of only 3% NaCl, this could be seen as an opportunity for ectoine production at comparatively low salt concentrations and relatively high growth rate (μ = 0.21, td = 3.3 h) . Louis and Galinski  have previously reported similar cytoplasmic ectoine levels (0.38 mmol (g dry weight)-1) in recombinant E. coli XL1-Blue (containing plasmid pOSM11) at 3% NaCl. This strain however has a much lower growth rate (approx. 0.1) under the same conditions and appears to experience other growth-limiting restrictions. In order to improve the space-time yield of heterologous ectoine production even further, future work will address the option to combine the ectoine biosynthetic gene cluster with its corresponding genuine aspartate kinase from M. halophilus. The chances are that this enzyme will be feed-back regulated and/or transcriptionally repressed when osmotic equilibrium is achieved. Unfortunately, this postulated gene has so far not been identified in M. halophilus.
Co-expression of ectoine biosynthetic genes from M. halophilus and feedback-insensitive aspartate kinase from C. glutamicum MH20-22B in E. coli DH5α pAKECT1 resulted in strongly elevated ectoine levels, which correlated with the levels obtained when ectoine was accumulated from the growth medium (0.4 mmol (g dry weight)-1). A most important observation during all our studies with E. coli DH5α pAKECT1 was that ectoine levels increased only up to 3% NaCl (as in accumulating cells) and that ectoine was not detected in the growth medium at the end of the experiments. In case of unregulated synthesis an efflux of the overproduced ectoine via mechanosensitive channels (Msc) would have been conceivable [42–44]. The above conclusion appears to stand in contrast to the findings by Schubert et al.  who demonstrated continuous excretion of ectoine from a transgenic E. coli. The authors introduced the ectoine gene cluster from Chromohalobacter salexigens (devoid of the promoter region) into E. coli DH5α under the control of a tet promoter. Following a high-cell density fermentation to 20 g L-1 (cell dry weight) and subsequent induction, they observed continuous excretion of ectoine at a rate of 2 mg g-1 h-1, while the cellular level of ectoine stayed low (5 mg (g dry weight)-1). Such a low leakage rate would not have been detected under the low-cell density conditions employed here. Another experimental difference of the work reported here, besides the different origin of the genes, is transcriptional control by the orginal promoter region (ectUp) and use of growth conditions, under which compatible solute uptake systems are activated.
It is intriguing that ectoine levels were nearly identical in accumulating cells with ectoine in the growth medium, and in synthesizing cells harbouring pAKECT1. The only viable conclusion seems to be that heterologously expressed ectoine biosynthetic enzymes of M. halophilus are, according to osmotic needs, tuned and regulated in the phylogenetically distant host E. coli. This phenomenon may be explained by allosteric regulation of gene products at the level of enzyme activity, caused by yet unkown general osmotic response mechanisms shared by a large range of different bacteria.
In conclusion, we demonstrated that a metabolic bottle-neck for ectoine production in the non-halophilic recombinant E. coli DH5α can be relieved by coexpression of a deregulated aspartate kinase from C. glutamicum, and in doing so we paved the way for alternative, economically viable production methods. The surprising observation, however, that heterologous expression of the ectoine biosynthetic genes does not lead to overproduction in the host under the conditions employed, stresses the need to investigate regulatory mechanisms at enzyme level in order to disclose the biochemical signal which indicates osmotic balance to the cell.
Organisms, growth conditions and plasmids
Marinococcus halophilus DSM 20408T was grown at 37°C in complex medium FP5 or FP10 consisting of 1.47% (w/v) liquid fish peptone S490 (Primex AS, Norway), 10 g L-1 glucose · H2O, 2 g L-1 NH4Cl, 0.5 g L-1 K2HPO4 and either 45 g L-1 NaCl and 5 g L-1 artificial sea salt (FP5) or 90 g L-1 NaCl and 10 g L-1 artificial sea salt (FP10). Glucose and K2HPO4 were autoclaved separately and added to the medium after cooling.
E. coli DH5α (F- ø80dlacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(rK- mK+) deoR thi-1 supE44 λ-gyrA96 relA1) and XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac(F+ proAB lacIq ZΔM15 Tn10) were grown aerobically at 37°C either in Antibiotic Medium No.3 (Oxoid, Wesel, Germany) or in minimal medium MM63  with 3.0 ml L-1 vitamin solution  and 1–5% NaCl. For selection of cells harbouring the plasmid pHSG575  or derivatives, chloramphenicol was added to the medium at a final concentration of 25 μg ml-1. For supplementation experiments, the medium MM63 with 3% NaCl contained 1 mM L-lysine, L-threonine, L-methionine, L-aspartic acid or fumaric acid. For induction of the lac and tac promoter on plasmid pOSM2 and pAKECT1, respectively, the medium contained 1 mM IPTG. The cells were harvested by centrifugation (5000 g; 4°C) and freeze-dried.
Plasmid pRK1 containing the gene lysC from Corynebacterium glutamicum MH20-22B was kindly provided by Lothar Eggeling (FZ Jülich, Germany). Plasmids pOSM2 and pOSM12, comprising vector pHSG575 and DNA fragments encoding the ectoine gene cluster, were isolated from E. coli XL1-Blue pOSM2 and pOSM12 [7, 24, 48].
Total RNA was isolated from exponentially growing cells using the High Pure RNA Isolation Kit (Boehringer, Mannheim, Germany) according to the recommendation of the manufacturer. Northern blots were performed following standard methods , except for using DIG-labeled RNA probes, produced with the DIG RNA Labeling Kit (Boehringer, Mannheim, Germany), at 68°C for prehybridisation and hybridisation. After blocking the membrane and binding of anti-DIG-alkaline phosphatase conjugate (Boehringer, Mannheim, Germany) to the DIG-labeled RNA, chemoluminescence of CDP-Star™ (Boehringer, Mannheim, Germany) was detected by exposure of the membrane to a chemiluminescence film.
Rapid amplification of cDNA ends (RACE)
Primers used for the rapid amplification of cDNA ends (RACE)
ectA (RT primer)
ectA (PCR primer)
ectB (RT primer)
ectB (PCR primer)
ectC (RT primer)
ectC (PCR primer)
Plasmid isolation from E. coli was performed using the GFX™ Micro Plasmid Prep Kit (Amersham, Braunschweig, Germany). DNA fragments were isolated from agarose gels with the Silica Spin Fragment DNA Kit (Biometra, Göttingen, Germany). Restriction digests, ligations and PCR reactions were performed according to the recommendations of the enzyme manufacturer (New England Biolabs, Schwalbach, Germany). Transformation of E. coli was carried out by the calcium chloride/rubidium chloride method [51, 52]. Sequencing was performed by GATC (Konstanz, Germany).
Construction of the plasmid pAKECT1
The construction of plasmid pAKECT1 (Fig. 7) was performed in three steps. First the ectC DNA sequence was amplified from the plasmid pOSM12 using PCR primers, which created an additional XmaI restriction site upstream and additional BamHI, ClaI and SalI restriction sites downstream of ectC. After subcloning into the vector pGEM-T® (Promega, Mannheim, Germany) and sequencing, XmaI- and SalI-digested ectC was ligated to XmaI- and SalI-digested pHSG575 . In a second step the DNA fragment of plasmid pRK1 (Fig. 7), containing lysC under the control of a tac promoter and lacI q , was ligated to this plasmid downstream of ectC, using ClaI and BamHI restriction sites. From the resulting intermediate plasmid a DNA fragment, containing the genes lysC and lacI q , was re-cut by BamHI- and SpeI-digest and ligated to BamHI- and SpeI-digested pOSM12 (Fig. 7).
The ectoine gene cluster in pAKECT1 is under the control of its own regulatory promoter region (ectUp), whereas lysC is under the control of a tac promoter. Consequently, we were able to separately induce ectoine synthesis via osmotic stress and deregulated aspartate kinase activity via isopropyl-β-D-thiogalactopyranoside (IPTG).
Approx. 30 mg of freeze-dried cell material from an exponentially growing shaking culture (medium MM63) was used for the extraction of intracellular solutes according to a modification of the method of Bligh and Dyer  with methanol/chloroform/water (10:5:4) as described previously . The cell extracts were analysed by isocratic HPLC using a GromSil® aminopropyl column (Grom, Herrenberg, Germany) and acetonitrile/water (75:25 v/v) at a flow rate of 1 ml min-1 as the mobile phase. For13C-NMR analysis of cell extracts 1.5 g freeze-dried cell material was processed as above. The polar phase was evaporated to dryness at 70°C and dissolved in 1 ml D2O supplemented with 10 mg 3-(trimethylsilyl) propionic acid sodium salt (TMSP) as an internal reference and 30 μl acetonitrile as internal standard.1H -decoupled 13C-NMR spectra relative to TMSP were recorded in pulsed Fourier-transform (FT) mode on a Bruker ARX 400 spectrometer operating at 100.62 MHz (13C) and 400 MHz for the proton channel.
Aspartate kinase was assayed in extracts of cells resuspended in 50 mM (NH4)2SO4, 50 mM triethanolamine, 5 mM dithioerythritol, 1 mM EDTA pH 7.5 according to the method described by Black and Wright . The assay mixture was composed of 100 mM Tris/HCl pH 7.5, 14.5 mM ATP, 42 mM MgCl2, 431 mM (NH4)2SO4, 613 mM NH2OH·HCl, 95 mM sodium L-aspartate and 125 μl of extract in a total volume of 1.2 ml. After incubation at 30°C for 30 minutes the reaction was stopped by the addition of 600 μl 3.8% (w/v) FeCl3. 6H2O and 5.8% trichloroacetic acid in 1.4 M HCl. After centrifugation the absorbance of the assay mixture at 546 nm was measured and compared to a calibration curve obtained with aspartyl hydroxamate. Total protein concentrations were determined using the BCA (bicinchoninic acid) Protein Assay Kit (Pierce, Rockford, USA) according to the recommendations of the manufacturer.
We would like to thank Lothar Eggeling for providing plasmid pRK1. This work was supported by the Deutsche Forschungsgesellschaft (GA 393/3-2) and the European Union (BIO4-CT96-0488).
- Brown AD: Bacterial water stress. Bact Rev. 1990, 40: 803-846.Google Scholar
- da Costa MS, Santos H, Galinski EA: An overview of the role and diversity of compatible solutes in bacteria and archaea. Adv Biochem Eng Biotechnol. 1998, 61: 117-153.Google Scholar
- Bremer E, Krämer R: Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes. Bacterial Stress Responses. Edited by: Storz G, Hengge-Aronis R. 2000, Washington D.C.: ASM Press, 79-97.Google Scholar
- Galinski EA, Pfeiffer HP, Trüper HG: 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidine carboxylic acid: A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem. 1985, 149: 135-139.View ArticleGoogle Scholar
- Severin J, Wohlfahrt A, Galinski EA: The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J Gen Microbiol. 1992, 138: 1629-1638.View ArticleGoogle Scholar
- Galinski EA: Osmoadaptation in bacteria. Adv Microb Physiol. 1995, 37: 273-328.View ArticleGoogle Scholar
- Louis P, Galinski EA: Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology. 1997, 143: 1141-1149.View ArticleGoogle Scholar
- Khmelenina VN, Kalyuzhnaya MG, Sakharovsky VG, Suzina NE, Trotsenko YA, Gottschalk G: Osmoadaptation in halophilic and alkaliphilic methanotrophs. Arch Microbiol. 1999, 172: 321-329.View ArticleGoogle Scholar
- Lentzen G, Schwarz T: Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol. 2006, 72: 623-634.View ArticleGoogle Scholar
- Louis P, Trüper HG, Galinski EA: Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Appl Microbiol Biotechnol. 1994, 41: 684-688.View ArticleGoogle Scholar
- Göller K, Galinski EA: Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives. J Mol Catal B: Enzymatic. 1999, 7: 37-45.View ArticleGoogle Scholar
- Knapp S, Ladenstein R, Galinski EA: Extrinsic protein stabilization by the naturally occurring osmolytes β-hydroxyectoine and betaine. Extremophiles. 1999, 3: 191-198.View ArticleGoogle Scholar
- Malin G, Iakobashvili R, Lapidot A: Effect of tetrahydropyrimidine derivatives on protein-nucleic acids interactions. J Biol Chem. 1999, 274: 6920-6929.View ArticleGoogle Scholar
- Barth S, Huhn M, Matthey B, Klimka A, Galinski EA, Engert A: Compatible solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Appl Environ Microbiol. 2000, 66: 1572-1579.View ArticleGoogle Scholar
- Bünger J: Ectoine added protection and care for the skin. Eurocosm. 1999, 7: 22-24.Google Scholar
- Beyer N, Driller H, Bünger J: Ectoine – a innovative multi-functional active substance for the cosmetic industry. SÖFW Journal. 2000, 126: 27-29.Google Scholar
- Buommino E, Schiraldi C, Baroni A, Paoletti I, Lamberti M, de Rosa M, Tufano MA: Ectoine from halophilic microorganisms induces the expression of hsp70 and hsp70' in human keratinocytes modulating the proinflammatory response. Cell Stress Chaperones. 2005, 10 (3): 197-203.View ArticleGoogle Scholar
- Tatzelt J, Prusinger SB, Welch WJ: Chemical chaperones interfere with the formation of scrapie prion protein. EMBO Journal. 1996, 15: 6363-6373.Google Scholar
- Kolter T, Wendeler M: Chemical chaperones – a new concept in drug research. Chem Bio Chem. 2003, 4: 260-264.View ArticleGoogle Scholar
- Furusho K, Yoshizawa T, Shoji S: Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado-Joseph disease gene product with an expanded polyglutamine stretch. Neurobiology of Disease. 2005, 20: 170-178.View ArticleGoogle Scholar
- Kanapathipillai M, Ku SH, Girigoswami K, Park CB: Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106–126. Biochem Biophys Res Comm. 2008, 365: 808-813.View ArticleGoogle Scholar
- Sauer T, Galinski EA: Bacterial milking: A novel bioprocess for production of compatible solutes. Biotechnol Bioeng. 1998, 57: 306-13.View ArticleGoogle Scholar
- Jebbar M, Talibart R, Gloux K, Bernard T, Blanco C: Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics. J Bacteriol. 1992, 174: 5027-35.Google Scholar
- Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Gotoh T: High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987, 61: 63-74.View ArticleGoogle Scholar
- Wösten MMSM: Eubacterial sigma-factors. FEMS Microbiol Rev. 1998, 22: 127-150.View ArticleGoogle Scholar
- Patte JC: Biosynthesis of Threonine and Lysine. Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology. Edited by: Neidhardt FC, Curtis R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE. 1996, Washington DC: ASM Press, 528-541.Google Scholar
- Menkel E, Thierbach G, Eggeling L, Sahm H: Influence of increased aspartate availability on lysine formation by a recombinant stain of Corynebacterium glutamicum and utilization of fumarate. Appl Environ Mirobiol. 1989, 55: 684-688.Google Scholar
- Cremer J, Eggeling L, Sahm H: Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl Environ Microbiol. 1991, 57: 1746-1752.Google Scholar
- Völker U, Völker A, Maul B, Hecker M, Dufour A, Haldenwang WG: Separate mechanisms activate σB of Bacillus subtilis in response to environmental and metabolic stresses. J Bacteriol. 1995, 177: 3771-3780.Google Scholar
- Wise AA, Price CW: Four additional genes in the sig B operon of Bacillus subtilis that control activity of the general stress factor σB in response to environmental signals. J Bacteriol. 1995, 177: 123-133.Google Scholar
- Hecker M, Schumann W, Völker U: Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol. 1996, 19: 417-428.View ArticleGoogle Scholar
- Espinosa-Urgel M, Chamizo C, Tormo A: A consensus structure for σS-dependent promotors. Mol Microbiol. 1996, 21: 657-659.View ArticleGoogle Scholar
- Nguyen L, Jensen D, Thompson N, Gentry D, Burgess R: In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product. Biochemistry. 1993, 32: 11112-11117.View ArticleGoogle Scholar
- Tanaka K, Takayanagi Y, Fujita N, Ishihama A, Takahashi H: Heterogeneity of the principal σ factor in Escherichia coli: the rpoS gene product, σS, is a second principal σ factor of RNA polymerase in stationary-phase Escherichia coli. Proc Natl Acad Sci USA. 1993, 90: 3511-3515.View ArticleGoogle Scholar
- Hengge-Aronis R: Interplay of global regulators and cell physiology in the general stress response of Escherichia coli. Curr Opin Microbiol. 1999, 2: 148-152.View ArticleGoogle Scholar
- Ishihama A: Functional modulation of Escherichia coli RNA polymerase. Annu Rev Microbiol. 2000, 54: 499-518.View ArticleGoogle Scholar
- Kuhlmann AU, Bremer E: Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus ssp. Appl Environ Microbiol. 2002, 68: 772-783.View ArticleGoogle Scholar
- Calderón MI, Vargas C, Rojo F, Iglesias-Guerra F, Csonka LN, Ventosa A, Nieto JJ: Complex regulation of the synthesis of the compatible solute ectoine in the halophilic bacterium Chromohalobacter salexigens DSM 3043T. Microbiology. 2004, 150: 3051-3063.View ArticleGoogle Scholar
- Bestvater T, Galinski EA: Investigation into a stress-inducible promoter region from Marinococcus halophilus using green fluorescent protein. Extremophiles. 2000, 6 (1): 15-20.View ArticleGoogle Scholar
- Galili S, Guenoune D, Wininger S, Hana B, Schupper A, Ben-Dor B, Kapulnik Y: Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene. Transgenic Res. 2000, 9 (2): 137-144.View ArticleGoogle Scholar
- Galinski EA, Bestvater T, Louis P, Eggeling L: Verfahren zur Produktion von Ectoinen (1,4,5,6-Tetrahydro-2-methyl-4-pyrimidin-carbonsäure und 1,4,5,6-Tetrahydro-2-methyl-5-hydroxy-4-pyrimidin-carbonsäure) in nicht-halophilen Organismen unter Verwendung salzarmer Medien. German patent application: DE 199 25 615 A1. 1999Google Scholar
- Lamark T, Styrvold OB, Strøm AR: Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli. FEMS Microbiol Lett. 1992, 75: 149-154.View ArticleGoogle Scholar
- Sukharev SI, Blount P, Martinac B, Kung C: Mechanosensitive channels of Escherichia coli: The MscL gene, protein, and activities. Annu Rev Physiol. 1997, 59: 633-657.View ArticleGoogle Scholar
- Csonka LN, Epstein W: Osmoregulation. Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology. Edited by: Neidhardt FC, Curtis R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE. 1996, Washington DC: ASM Press, 1210-1224.Google Scholar
- Schubert T, Maskow T, Benndorf D, Harms H, Breuer U: Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium. Appl Environ Microbiol. 2007, 73: 3343-3347.View ArticleGoogle Scholar
- Larsen PI, Sydne LK, Landfald B, Strøm AR: Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch Microbiol. 1987, 147: 1-7.View ArticleGoogle Scholar
- Imhoff JF, Trüper HG: Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototrophic bacterium containing bacteriochlorophyll b. Arch Microbiol. 1977, 114: 115-121.View ArticleGoogle Scholar
- Louis P: Molekularbiologische Untersuchungen an Marinococcus halophilus – Identifizierung der Gene zur Ectoinbiosynthese und deren osmoregulierte Expression in Escherichia coli. PhD thesis. 1996, University of Bonn, Institute of Microbiology & Biotechnology, GermanyGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidmann JG, Smith JA, Struhl K: Short Protocols in Molecular Biology. 1992, New York: John Wiley and Sons Inc, 2Google Scholar
- Bertoli DJ, Burrows PR: A simple RACE method based on CTAB precipitation. Meth Mol Cell Biol. 1994, 5: 118-121.Google Scholar
- Hanahan D: Studies on transformation of Escherichia coli with Plasmids. J Mol Biol. 1983, 166: 557-580.View ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis TE: Molecular Cloning. A Laboratory Manual. 1989, Cold Spring Harbor: Cold Spring Harbor PressGoogle Scholar
- Bligh EG, Dyer WJ: A rapid method of lipid extraction and purification. Can J Biochem Physiol. 1959, 37: 911-917.View ArticleGoogle Scholar
- Galinski EA, Herzog RM: The role of trehalose as a substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris). Arch Microbiol. 1990, 153: 607-613.View ArticleGoogle Scholar
- Black S, Wright NG: β-Aspartokinase and β-aspartyl phosphate. J Biol Chem. 1955, 213: 27-38.Google Scholar
- Peters P, Galinski EA, Trüper HG: The biosynthesis of ectoine. FEMS Microbiol Lett. 1990, 71: 157-162.View ArticleGoogle Scholar
- Tao T, Yasuda N, Ono H, Shinmyo A, Takano M: Purification and characterization of 2,4-diaminobutyric acid transaminase from Halomonas sp. Annual Reports of International Centre of Cooperative Research in Biotechnology, Japan. 1992, 15: 187-199.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.