Synthesis of the Compatible Solute Ectoine in Virgibacillus pantothenticus Is Triggered by High Salinity and Low Growth Temperature

Abstract

The quantification of the intracellular concentration of ectoine in Virgibacillus pantothenticus revealed that the production of this compatible solute is triggered either by an increase in the external salinity or by a reduction in the growth temperature. This finding reflects increased transcription of the ectoine biosynthetic operon (ectABC) under both environmental conditions.

Increases in the salinity of the environment impose a considerable strain on the water balance of microorganisms (3, 11, 17). To counteract the efflux of water from the cells when they are challenged by high salinity, many microorganisms accumulate large quantities of a particular group of organic compounds, the so-called compatible solutes (3, 11, 17). This accumulation can be achieved either through de novo biosynthesis or through uptake from the environment via dedicated and osmotically controlled transport systems (3, 17). In addition to their osmoprotective function, compatible solutes affect the stability and correct folding of proteins under unfavorable conditions (2, 10, 15). The stabilizing effect of compatible solutes on proteins is generally explained by the preferential exclusion model (27). The protein-stabilizing property of compatible solutes is likely to contribute to the function of these chemical chaperons as heat and chill stress protectants for various microbial species (1, 4, 6, 8, 10, 12, 14, 16, 18, 21, 24).

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is one of the most widely found compatible solutes in the domain of the Bacteria (5, 11, 19, 25). Molecular analyses of its biosynthesis in various gram-negative and gram-positive microorganisms have shown that ectoine production depends on the evolutionarily highly conserved ectABC gene cluster (5, 9, 13, 19, 20, 22, 25, 26). Ectoine synthesis relies on the sequential reactions of the diaminobutyric acid transaminase (EctB), the diaminobutyric acid acetyltransferase (EctA), and the ectoine synthase (EctC) that together convert the precursor l-aspartate-β-semialdehyde into ectoine. Some microorganisms also produce a hydroxylation derivative of ectoine, 5-hydroxyectoine, via an evolutionarily conserved ectoine hydroxylase (5, 12, 23).

Consistent with the proposed function of ectoine as a microbial osmoprotectant, substantial ectoine production occurs in cells that are osmotically challenged. For instance, in Bacillus pasteurii and Salibacillus salexigens, an osmotically mediated induction of the transcription of the ectABC gene cluster occurs and leads to the accumulation of ectoine in accordance with the external salinity (5, 19). However, in certain microorganisms, ectoine biosynthesis seems to be triggered not only by osmotic stress, but also by other environmental cues. For instance, in Streptomyces griseus, increased levels of this compatible solute were found previously when the cells were subjected to high growth temperatures (21), and the ectABC gene cluster of Chromohalobacter salexigens (DSM 3043) is induced when the cells are cultivated at high growth temperatures (7). These findings suggest a role for ectoine as a protectant against the detrimental effects of high temperatures.

By natural-abundance ¹³C nuclear magnetic resonance spectroscopy, it was recently shown that the gram-positive soil bacterium Virgibacillus pantothenticus produces both proline and ectoine when it is exposed to high salinity (19). Here, we have characterized the ectoine biosynthetic genes from V. pantothenticus and demonstrated that their transcription is triggered by two environmental cues: high salinity and a low growth temperature. Chill-induced expression of an ectABC gene cluster has never been observed before.

Ectoine synthesis in V. pantothenticus increases at high salinity.

To monitor proline and ectoine production in V. pantothenticus (DSM 26^T) in response to high salinity, we grew this bacterium at its optimal growth temperature (37°C) to an optical density at 578 nm (OD₅₇₈) of 1 in a chemically defined minimal medium (19) with salinities ranging from 0 M NaCl to 1.9 M NaCl. Cells were then harvested by centrifugation, and the compatible solutes were extracted using a modified Blight and Dyer technique (19). Subsequently, the amounts of proline and ectoine produced were determined by high-performance liquid chromatography (HPLC) analysis (19). There was no proline or ectoine detectable in V. pantothenticus cultures when the cells were grown in a minimal medium without added NaCl (Fig. 1A). As the salinity was raised, there was a concomitant increase in both the proline and ectoine contents of the cells (Fig. 1A). At external salinities of up to 0.7 M NaCl, the proline level rose in proportion to the increase in the external salinity, but then there was no further increase in the proline content despite the fact that the salinity of the medium was raised up to a level of 1.9 M NaCl (Fig. 1A). The ectoine level was initially lower than that of proline (at up to 0.7 M NaCl) but then progressively increased when the salinity of the growth medium was raised up to 1.3 M NaCl (Fig. 1A). A further increase in salinity did not lead to the further enhancement of ectoine production. Consequently, both proline and ectoine seem to serve an osmoregulatory function in V. pantothenticus, but ectoine is clearly the physiologically more important compatible solute (Fig. 1A). It should be noted, however, that the ectoine content of V. pantothenticus cells under high-salinity conditions is about fivefold lower than that of Bacillus pasteurii cells (19). In B. pasteurii cells, the ectoine content is linearly correlated with the external salinity (19), whereas this is clearly not the case for V. pantothenticus (Fig. 1A).

FIG. 1.

Ectoine and proline biosynthesis in V. pantothenticus in response to salt and cold stress. (A) Cultures of V. pantothenticus were grown in minimal medium at 37°C with the indicated concentrations of NaCl to an OD₅₇₈ of approximately 1, and the produced proline (▴) and ectoine (•) were quantitated by HPLC analysis. (B) Cultures of V. pantothenticus were grown in a minimal medium at the indicated temperatures in the absence of salt stress to an OD₅₇₈ of approximately 1, and the proline (▴) and (•) ectoine produced by the cells were quantitated by HPLC analysis. The values given for the ectoine and proline contents are the means of results of two independent measurements each of two independently grown V. pantothenticus cultures.

Ectoine biosynthesis in V. pantothenticus is triggered by low but not by high growth temperatures.

We also monitored both ectoine and proline production in cells of V. pantothenticus that were grown at various temperatures in the absence of salt stress. A reduction of the growth temperature from the optimal growth temperature (37°C) of V. pantothenticus led to a progressive increase in both the ectoine and proline contents of the cells (Fig. 1B). Consequently, for V. pantothenticus, both ectoine and proline biosynthesis can be triggered in the absence of salt stress simply by growing the cells at a low temperature. Cells propagated at 15°C, the lowest growth temperature tested in this series of experiments, exhibited the biosynthesis of approximately 35 μmol of ectoine per g (dry weight) of cells (Fig. 1B). This value is comparable to the ectoine content of V. pantothenticus cells that were grown at 37°C in the presence of 0.3 M NaCl (Fig. 1A). Growth at a low temperature thus triggers a moderate increase in the ectoine content of V. pantothenticus cells in comparison to the ectoine levels found in severely salt-stressed cells (approximately 330 μmol of ectoine per g [dry weight] of cells that were grown in minimal medium with 1.3 M NaCl) (Fig. 1A). While proline production was also triggered by the growth of V. pantothenticus at low temperatures, the level of proline production was considerably lower than that of ectoine production under the same cultivation conditions.

We also tested whether an increase in the growth temperature would trigger ectoine production in V. pantothenticus. However, this was not the case, since there was no ectoine detectable in cells that were continuously grown at 48°C (Fig. 1B), a temperature close to the upper limit for the growth of V. pantothenticus (data not shown). We did not determine the proline content of V. pantothenticus cells at high growth temperatures.

Molecular analysis of the ectoine biosynthetic genes in V. pantothenticus.

We assumed that in V. pantothenticus, ectoine biosynthesis is mediated by the typical ectABC gene cluster and found that this was indeed the case. We initially used a PCR approach to recover a DNA fragment that contained part of the ectB gene and part of the ectC gene. This DNA segment was then used to complete the DNA sequence of the ectABC genes by using chromosomal DNA of V. pantothenticus as a sequencing template (data not shown). We determined in total the sequence of a 2,670-bp DNA segment covering the entire ectABC gene cluster and its 5′ and 3′ regions. The EctABC proteins deduced from the genomic sequence of the V. pantothenticus ectABC gene cluster show a considerable degree of amino acid sequence identity (between 27 and 65%) to the corresponding and functionally characterized EctABC proteins from B. pasteurii (19), Marinococcus halophilus (20), Halomonas elongata (13), C. salexigens (9), S. salexigens (5), “Halobacillus halophilus” (26), Methylomicrobium alcaliphilum (25), and Vibrio cholerae (22) (data not shown). Furthermore, many ectABC genes related to the V. pantothenticus ectABC genes can be found by searching databases containing full or partial sequences of microbial genomes (data not shown). The EctABC proteins from the various microorganisms are all closely related, and their features have already been described in considerable detail (9, 13, 19, 20, 22, 25).

The transcription of the ectABC gene cluster is osmotically controlled.

An intergenic region of 43 bp separates the V. pantothenticus ectA and ectB genes, and the ectB gene is separated from ectC by 30 bp. This tight spacing of the three ect genes suggested that in V. pantothenticus the ectABC gene cluster might be organized as an operon, as it is in B. pasteurii (19), S. salexigens (5), and H. halophilus (26). We used Northern blot analysis to investigate the transcriptional organization of the V. pantothenticus ectABC genes experimentally by using a single-stranded RNA probe (883 bp) that carried part of the ectB gene and part of the ectC gene (Fig. 2A). The isolation of total RNA from V. pantothenticus, the preparation of the single-stranded antisense RNA probe, hybridization conditions, and signal detection were as described previously for the Northern blot analysis of the B. pasteurii ectABC operon (19). No ect-specific RNA signal was detected in the RNA sample isolated from V. pantothenticus cells grown in minimal medium in the absence of salt stress (Fig. 2B). However, a 2.4-kb ect-specific transcript was readily detectable when the V. pantothenticus cells were continuously propagated in a minimal medium that contained 0.7 M NaCl (Fig. 2B). The size of the detected ect transcript matched the calculated distance (2,188 bp) between the start codon of the V. pantothenticus ectA gene and the termination codon of ectC. Hence, the ectABC gene cluster of V. pantothenticus is transcribed as an osmotically regulated operon. This pattern of ectABC transcription was reflected by the amount of ectoine synthesized under both the low- and high-salinity growth conditions that were used for the RNA isolation. There was an increase of about 15-fold in the ectoine content of V. pantothenticus cells that were grown in a minimal medium with 0.7 M NaCl compared to that of the cells that were grown in the absence of NaCl (Table 1).

FIG. 2.

Northern blot analysis of the transcription of the V. pantothenticus ectABC genes in response to high salinity and cold stress. (A) Genetic organization of the ectABC genes in V. pantothenticus and position of the RNA antisense probe used for the transcriptional analysis of the ectABC gene cluster. (B) Northern blot analysis of the ectABC operon. Total RNA was isolated from V. pantothenticus cells grown in a minimal medium under the indicated environmental conditions and hybridized to the ectBC-specific antisense RNA probe.

TABLE 1.

Biosynthesis of ectoine in V. pantothenticus in response to salt and temperature stress

Growth temp (°C)	External salinity (M NaCl)	Ectoine content (μmol per g [dry wt] of cells)a
37	0	6.3 ± 0.2
37	0.7	92.7 ± 2.3
15	0	33.0 ± 1.0
15	0.7	108.4 ± 2.9
48	0	0.3 ± 0.0

^aThe values given are the means ± the standard deviations of results of two independent measurements each of two independently grown V. pantothenticus cultures.

Induction of the transcription of the ectABC operon by cold stress.

The quantification of the ectoine contents of cells that were propagated at suboptimal growth temperatures by HPLC analysis revealed that the V. pantothenticus cells responded to such cold stress with increased ectoine formation (Fig. 1B). To test whether this response was caused by the increased transcription of the ectABC operon, we looked for the ectABC transcript by Northern blot analysis of cells that were grown at 15°C in the absence of salt stress. ectABC transcription in the cold-stressed cells was clearly induced in comparison to that in cells that were propagated at the optimal growth temperature of V. pantothenticus of 37°C (Fig. 2B). Under these growth conditions, there was an increase in the ectoine content of the cold-stressed V. pantothenticus cells of about fivefold in comparison to that of the cells that were grown at 37°C (Table 1).

The highest level of the V. pantothenticus ectABC transcript was detected in cells that were subjected simultaneously to chilling (15°C) and salt stress (0.7 M NaCl) (Fig. 2B), and the highest levels of ectoine were found in the cells under these conditions. No ectABC transcript was detectable in V. pantothenticus cells that were subjected to heat stress (growth at 48°C), and as expected, these cells did not produce any significant amounts of ectoine (Table 1).

Conclusions.

There are two major findings with respect to the data presented in this communication. First, the ectoine content of salt-stressed V. pantothenticus cells gradually increased in response to the amount of NaCl added to the growth medium (Fig. 1A), indicating that V. pantothenticus, like B. pasteurii (19) and S. salexigens (5), can sensitively detect gradual changes in the external salinity. How this detection can be accomplished by the cell at the molecular level is currently unclear. Increased ectoine biosynthesis in salt-stressed V. pantothenticus cells has an obvious function in osmoadaptation.

The most interesting result of our study is the finding that ectoine biosynthesis in V. pantothenticus can be induced at the transcriptional level in the absence of salt stress in cells that are subjected to continued growth at a low temperature (15°C) (Fig. 2B). To the best of our knowledge, such chill stress induction of ectABC transcription in any ectoine-synthesizing microorganism has not been observed before. We consider it likely that the cold-induced ectoine biosynthesis in V. pantothenticus serves a chill protection function for this microorganism. This proposal is fully consistent with previous observations of the protection of the human pathogen Listeria monocytogenes (1, 18) and the soil bacterium Bacillus subtilis (4) from cold stress by exogenously provided compatible solutes (e.g., glycine betaine). Indeed, we have recently found that exogenously provided ectoine or 5-hydroxyectoine serves as an effective chill protectant for V. pantothenticus (J. Bursy, A. Kuhlmann, and E. Bremer, unpublished results).

Nucleotide sequence accession number.

The sequence obtained in this study has been deposited in GenBank under accession number AY585263.