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Journal of Bacteriology, July 2005, p. 4752-4759, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4752-4759.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chill Activation of Compatible Solute Transporters in Corynebacterium glutamicum at the Level of Transport Activity

Nuran Özcan, Reinhard Krämer,^* and Susanne Morbach

Institute of Biochemistry, University of Cologne, Cologne, Germany

Received 29 November 2004/ Accepted 15 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K⁺ and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10°C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K⁺ in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K⁺ concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K⁺, was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K⁺-mediated process.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Temperature sensing in general and chill sensing in particular by membrane-embedded proteins is a common phenomenon which is not well understood. Physiological responses and adaptation phenomena to chill stress have been reported for a number of prokaryotes, e.g., Bacillus subtilis, Listeria monocytogenes, and cyanobacteria (3, 9, 12, 18, 19, 22, 25, 39, 46); related systems have also been studied in plants and in humans (17, 44).

Chill stress response in bacteria includes at least three different mechanisms, synthesis of so-called cold shock proteins (6, 7), synthesis of branched-chain and unsaturated fatty acids (2, 21, 26, 45, 47), and uptake and synthesis of compatible solutes (4, 23). The first two types of response necessarily involve regulation at the level of expression and have been studied in detail in Escherichia coli, Bacillus subtilis, and Listeria monocytogenes (1, 2, 11, 15, 18, 19, 20, 32, 33, 41, 45, 46, 50). The third response may also involve regulation at the level of protein activity, at least as far as uptake of compatible solutes is concerned. These phenomena have been studied in great detail in L. monocytogenes (4, 5, 48). To date, three uptake systems for compatible solutes are known in this bacterium, both a primary (Gbu) and a secondary (BetL) betaine carrier as well as the primary carnitine transporter OpuC (4). Under chill stress, Gbu-mediated betaine uptake seems to be of major importance, and its action results in a significant growth stimulation of chill-stressed cells. Gbu is directly activated upon exposure to low temperatures down to 1°C. The response of Gbu to chill stress becomes apparent only in the absence of osmotic stress, since this transporter is also responsible for betaine uptake upon onset of hyperosmotic stress, which actually leads to higher values of transport activity than those observed in response to chill stress (14). Although experimental evidence has been provided for a correlation between osmotic stress and the physical state of the membrane (14), the nature of the stimulus related to chill stress which is perceived by Gbu is not clear.

We are interested in the activity control of transport proteins from the soil bacterium Corynebacterium glutamicum in response to external stimuli. The focus of the present work is the mechanisms of stimulus perception and signal transduction involved in the response to external stress. We have previously characterized in detail the response of BetP, a secondary betaine uptake system of C. glutamicum, to hyperosmotic stress. A sudden increase in the external osmolality leads to a concomitant loss of cytoplasmic water and, consequently, an increase in internal osmolality. Betaine uptake by BetP is instantly activated upon application of hyperosmotic stress after addition of either external ionic or nonionic solutes (28, 30). The response to osmotic stress is observed not only in C. glutamicum but also to a similar extent when BetP is heterologously expressed in Escherichia coli or when the carrier is isolated, purified, and reconstituted in functionally active form in proteoliposomes (28, 35, 36). Using the reconstituted system, the primary signal related to osmotic stimulation was identified as being the increase in the cytoplasmic or luminal K⁺ (or Rb⁺ and Cs⁺) concentration (36, 37). Furthermore, experimental evidence has been provided that the C-terminal domain of BetP is directly involved in K⁺ sensing (38). Besides the increase in internal K⁺, we have recently provided experimental evidence for alternative pathways of stimulus perception by BetP concerning activity downregulation after osmotic adaptation, probably via a change in the membrane surroundings (8). This work on chill sensing by BetP reveals an alternative way of stimulus perception and signal transduction by BetP of C. glutamicum.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids are described in Table 1. C. glutamicum cells were grown in either brain heart infusion (BHI) medium (Difco, Detroit, Mich.) or minimal medium (27) at 30°C, and E. coli cells were cultivated in Luria-Bertani (LB) medium at 37°C.

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TABLE 1. Bacterial strains and plasmids used in this study

Chemicals. The enzymatic synthesis of labeled glycine betaine was performed according to Landfald and Strom (24) and has been described previously (28). All other chemicals were of analytical grade.

Transport assays. For measuring uptake of [¹⁴C]glycine betaine in C. glutamicum expressing the genes of the transport proteins from pEKEX2, cells were grown overnight in BHI medium at 30°C with kanamycin (50 µg/ml). Gene expression was induced with 0.2 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). If not indicated, cells were washed in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl. After suspension in the same buffer, cells were energized with 10 mM glucose and incubated on ice. After incubation at various osmolalities and temperatures for 3 min, the reaction was started by the addition of 250 to 750 µM [¹⁴C]glycine betaine. At time intervals, samples were filtered through glass fiber filters (GF; Schleicher & Schuell GmbH, Dassel, Germany) and washed twice with 2.5 ml of 100 mM LiCl. The radioactivity on the filters was determined by liquid scintillation counting.

For measuring uptake of [¹⁴C]glycine betaine in E. coli MKH13 cells expressing the betP gene from the E. coli/C. glutamicum shuttle vector pEKEX2 (13), cells were grown at 37°C in LB medium supplemented with kanamycin (50 µg/ml) overnight and inoculated in fresh medium to an optical density at 600 nm of 0.5. Synthesis of the carrier was initiated by addition of 0.2 mM IPTG. After 2 h, the cells were harvested and washed in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl. After suspension in the same buffer, the cells were energized with 10 mM glucose and incubated on ice. After incubation at various osmolalities and temperatures for 3 min, the reaction was started by the addition of 250 µM [¹⁴C]glycine betaine. At given time intervals, samples were taken, filtered through glass fiber filters (GF; Schleicher & Schuell GmbH), and washed twice with 2.5 ml of 500 mM sucrose and 50 mM MgCl₂. The radioactivity on the filters was determined by liquid scintillation counting.

Measurements of the internal potassium concentration. Cultivation and preparation of C. glutamicum DHPF pbetP cells for potassium measurements was performed according to the conditions for transport assays. The cells were incubated in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl at various temperatures for 3 min, centrifuged, and washed twice in 0.4 M sorbitol. The internal solutes were released by treatment of the cells with by 0.1% cetyltrimethylammonium bromide (CTAB). The potassium concentration was detected via flame photometry (ELEX 6361, Eppendorf, Hamburg, Germany). Potassium levels present in the cell-free control were subtracted from the detected values of internal potassium of the cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
To avoid the possible influence of chill and other environmental stress on the expression of the transporters to be studied, we used the C. glutamicum strain DHPF, which is devoid of all five known carriers for compatible solutes (40) and expressed the genes of single transporters individually from the IPTG-inducible pEKEX2 plasmid (13).

Temperature dependence of transport activity of BetP, EctP, and LcoP under hyperosmotic conditions. The carriers for compatible solutes in C. glutamicum belonging to the betaine-carnitine-choline transporter (BCCT) family, namely, BetP, EctP, and LcoP, differ in their substrate specificity, but they are all effectively and instantaneously activated in response to hyperosmotic stress (28, 31, 40). Within seconds after addition of osmotically active solutes to C. glutamicum cells, these carriers reach their V_max values of transport activity, typically at an external osmolality around 1.2 osmol/kg (31). Here we study another external stimulus, namely, chill stress. We first tested the temperature dependence of the three transport proteins for compatible solutes under V_max conditions, i.e., under hyperosmotic stimulation at an external osmolality of 1.25 osmol/kg in the range between 5 and 30°C (Fig. 1). Cells were grown in BHI medium and harvested at 30°C and thus were not adapted to low temperatures. After briefly washing at 4°C, the respective assay temperature and the shift to hyperosmotic conditions were established 3 min before start of the transport assay. The transport rates shown in Fig. 1A are presented as relative values for two reasons. First, the absolute transport activities of the three proteins are very different (average V_max values at 25°C were 255, 17, and 14 µmol/[g of cell dry mass {cdm} · min] for BetP, EctP, and LcoP, respectively), and second, the expression levels of the corresponding plasmid-encoded genes varied significantly between the experiments (around ±40%). Although the absolute rates differed in these experiments, the regulation pattern of the three transporters was highly reproducible. In Fig. 1B, the temperature dependence of the three proteins is plotted as an Arrhenius diagram. In this figure, the logarithms of average values of absolute transport rates at each temperature are plotted.

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FIG. 1. Temperature-dependent activity profiles of BetP (squares), EctP (triangles), and LcoP (circles) in C. glutamicum DHPF pEKEX2 under hyperosmotic conditions. (A) The cells were grown at 30°C in BHI medium, washed in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl, suspended in the same buffer, and energized with 10 mM glucose. For the transport assays, cells were subjected to hyperosmotic conditions (1.25 osmol/kg) by the addition of NaCl for 3 min at the indicated temperatures before the reaction was started by addition of 750 µM [14C]glycine betaine. For better comparison, relative rates are given (normalized to the maximum values at higher temperatures [100%]); the absolute values are mentioned in the text. (B) Arrhenius plot of the transport activity of BetP (squares), EctP (triangles), and LcoP (circles) under hyperosmotic conditions (absolute values of transport activity).

Although all three carrier proteins are characterized by an increase of activity with increasing temperature, the data in Fig. 1 demonstrate that they respond rather differently to changing temperatures. Whereas both BetP and LcoP show a "normal" behavior, as indicated by a linear (BetP) or nearly linear (LcoP) dependence in the Arrhenius plot between 5 and 30°C, EctP has a complex temperature dependence. The activation energy which can be derived from Fig. 1B amounts to about 50 kJ/mol for BetP (5 to 25°C) and about 60 and 120 kJ/mol for LcoP (20 to 30°C and 10 to 20°C, respectively). For EctP, no straight lines are obtained in the Arrhenius plot, and a temperature optimum around 25°C is observed, which is close to the optimum growth temperature of 30°C for C. glutamicum.

Temperature dependence of transport activity of BetP, EctP, and LcoP at low osmolality. When solute uptake by the three transport proteins BetP, EctP, and LcoP was measured in relation to temperature at an external osmolality of 0.2 mosmol/kg, i.e., at low osmolality, a pattern completely different from that seen at high osmolality was observed. The difference of BetP transport activities in response to temperature with respect to osmotic conditions is demonstrated in Fig. 2. In Fig. 3, relative betaine uptake activities catalyzed by BetP, EctP, and LcoP between 5 and 30°C are compared. Obviously, the temperature dependence at low osmolality does not represent "normal" Arrhenius behavior. In view of their largely different absolute values of uptake activity, for a direct comparison of the three carrier proteins the relative transport rates were combined in Fig. 3A. None of the three transport proteins shows a "typical" temperature dependence, as would be expected from a straight line in an Arrhenius plot (not shown). All three carriers display comparable profiles, with activity optima at different temperatures, ranging from 25°C (EctP) and around 20°C (LcoP) down to around 10°C (BetP). The average absolute values for the measured transport activity at the respective optimum temperatures were 90 µmol/(g cdm · min) for BetP at 10°C, 0.6 µmol/(g cdm · min) for LcoP at 20°C, and 1.5 µmol/(g cdm · min) for EctP at 25°C. Because of the fact that the three transporters were synthesized from plasmid-encoded genes, the measured transport rates differ from those observed in the wild type (31).

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FIG. 2. Temperature-dependent activity profiles of BetP in C. glutamicum DHPF pEKEX2 at low (squares) and high (circles) osmolality. Cells were prepared as described in the legend to Fig. 1. After incubation either in the basic buffer (0.2 osmol/kg) or in the same buffer additionally supplemented with 600 mM NaCl (1.25 osmol/kg) at various temperatures for 3 min, the reaction was started by addition of 750 µM [14C]glycine betaine. The activity of BetP at low osmolality was normalized to the average maximum activity at high osmolality at 30°C, which was 290 µmol/(g cdm · min).

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FIG. 3. Temperature-dependent activity profiles of BetP (squares), EctP (triangles), and LcoP (circles) in C. glutamicum DHPF pEKEX2 at low external osmolality. (A) Cells were prepared as described in the legend to Fig. 1. After incubation in this buffer (0.2 osmol/kg) at various temperatures for 3 min, the reaction was started by addition of 750 µM [14C]glycine betaine. (B) Normalization of the activity of BetP (squares), EctP (triangles), and LcoP (circles) at low osmolality to the activity at high osmolality of the respective transporters.

As seen from the measured values of transport activity, and closely similar to what had been published previously (28, 29, 31, 40), the absolute activity of all three carrier proteins at 25 to 30°C was much lower in the absence of osmotic stimulation than in its presence. To be able to evaluate how significant the observed chill-dependent stimulation is in comparison to the osmostress-dependent activation, in Fig. 3B the relative activity at low osmolality is shown as the percentage of the activity at high osmolality; consequently, the data shown are ratios for each temperature. The lower the data points in this figure, the lower the relative activity at low osmolality. Identical temperature dependences at both low and high osmolality would result in a straight line parallel to the abscissa. As a matter of fact, this situation was found for EctP, which thus turns out to be not stimulated by chill at all. LcoP was sensitive to chill, with an optimum around 15°C; however, the extent of activation was rather low. In contrast, BetP was effectively activated at temperatures around 10°C and reached nearly the same value as measured in response to osmotic stress at that temperature.

Osmotic and chill activation of BetP do not overlap. Since BetP was shown to be activated to similar extents at low temperature by osmotic and chill stress, we were interested in whether the two different stimuli functionally overlap. Thus, transport activity of BetP was determined at low (10°C) and optimal (30°C) growth temperatures as a function of increasing external NaCl concentrations, i.e., increasing osmotic stress (Fig. 4). At high temperature, the profile obtained closely resembled previously published results (28). Betaine uptake at 10°C, however, was not significantly activated by increasing osmotic stress, i.e., BetP seems to be already maximally activated under these conditions.

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FIG. 4. Activity regulation of BetP in C. glutamicum DHPF pbetP at 30°C (circles) and 10°C (triangles) in relation to external osmolality. The cells were grown at 30°C, washed in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl, suspended in the same buffer, and energized with 10 mM glucose. For the transport assays, cells were subjected to hyperosmotic conditions by the addition of NaCl for 3 min at the indicated temperatures before the reaction was started by addition of 750 µM [14C]glycine betaine.

It has recently been shown using a reconstituted system that an increase in the cytoplasmic K⁺ concentration is a major trigger in osmostress-related activation of BetP (36, 37). Since activation by chill stress led to the same maximum activity as that reached by osmotic stimulation, we investigated whether chill activation may be related to an increase in cytoplasmic K⁺. For this purpose, the possible influence of temperature variation on the cytoplasmic K⁺ content was studied. We found that the content of cytoplasmic K⁺ remained at around 1.1 mmol/g cdm with only little variation when measured in C. glutamicum cells in the temperature range from 5°C to 30°C.

It may be argued that we actually determined the total amount of K⁺ in this experiment, whereas it may be the concentration of free K⁺ which matters for BetP activation. In addition, although internal K⁺ was shown to be a major trigger activating BetP in proteoliposomes from E. coli lipids, it was not possible so far to unequivocally prove the direct dependence on K⁺ in intact cells because of the inability to change cytoplasmic K⁺ without significantly changing the physiological state of the cell. Consequently, we used another experimental approach to resolve the question of a possible overlap of osmotic and chill-related activation of BetP. Recently, we showed a functional correlation of the mechanism of osmostress-dependent BetP activation by an increase in the luminal K⁺ concentration with the previous finding that the C-terminal extension of BetP is involved in osmostress-dependent activity regulation (38). In E. coli lipid proteoliposomes, activation of BetP by increasing luminal K⁺ concentration requires the presence of an intact C-terminal domain even in the absence of an osmotic gradient (38). A mutant form of BetP lacking the C-terminal 25 amino acids was unable to be activated by either osmotic stress or an increase in cytoplasmic K⁺ and displayed a more or less constant betaine uptake activity. It was therefore interesting to test whether the C-terminal domain is involved in sensing chill stress.

There is an inherent problem with this type of experiment, since the recombinant form of BetP with a truncated C-terminal domain exhibits a drastically decreased external affinity for the cotransport ion Na⁺ (37). To avoid any influence of an increasing NaCl concentration on BetP activity as a consequence of increasing saturation of the Na⁺ binding site, osmotic stimulation was induced by adding KCl instead of NaCl at a fixed low NaCl concentration. By this approach, the concentration of external Na⁺, which, because of the extremely high K_m, is present at nonsaturating levels, was kept constant and therefore did not influence the actual transport activity. The betaine uptake activity of BetP25, the C-terminally truncated form of BetP, was measured upon expression in C. glutamicum DHPF (Fig. 5). A distinct activity optimum was observed at around 15°C, independent of whether osmotic stress imposed at 500 mM KCl was present or not. A difference between chill activation of the wild-type and truncated proteins is observed, in particular with respect to a shift in the optimum temperature of activation from 10 to 15°C. The fact, however, that a distinct chill activation which is independent from osmotic stimulation is also observed in the case of BetP25 indicates that the C-terminal domain of BetP is not of core importance for chill-dependent activation. This is in accordance with the observation described above that luminal K⁺ also does not seem to be relevant in this respect.

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FIG. 5. Temperature-dependent activity profiles of plasmid-encoded BetP25 at low and high osmolality in C. glutamicum DHPF pbetP. The cells were grown at 30°C in BHI medium, washed in buffer containing 25 mM potassium phosphate (pH 7.5) and 100 mM NaCl, suspended in the same buffer, and energized with 10 mM glucose. After incubation in the basic buffer (squares) or in the same buffer supplemented with 500 mM KCl (circles) at various temperatures for 3 min, the reaction was started by addition of 250 µM [14C]glycine betaine.

Chill sensing by BetP probably depends on the membrane surroundings. Since the results described above indicate that chill activation of BetP is most probably independent of cytoplasmic K⁺, we investigated other possible signal input pathways. An alternative signal for membrane-embedded proteins (42), in particular in relation to osmosensing (10, 34, 49) and chill sensing (14), is an alteration of the lipid surrounding of membrane proteins. Consequently, we studied chill sensing and activation of BetP when it was inserted into a different membrane, namely, that of E. coli. For this purpose, betP was heterologously expressed in E. coli MKH13 (16), a strain which is devoid of known uptake systems for glycine betaine, and the temperature dependence of BetP activity was measured in the presence of both high and low osmolality (Fig. 6). We have previously shown that BetP, when inserted into membranes of intact E. coli cells, still responds to osmotic stress with a rapid increase in betaine uptake, although the optimum of activation was shifted to lower osmotic stress (28). In the experiment whose results are shown in Fig. 6, the "normal" temperature dependence of betaine uptake activity was observed under full stimulation with NaCl, and no particular optimum at low temperature was found in the absence of osmotic stress. This result shows that chill activation of BetP is not an intrinsic structural or functional property of this carrier protein independent of surrounding conditions. As a plausible explanation for the different responses of BetP observed in the two different membranes, BetP may be assumed to be influenced by its actual membrane surroundings. In any case, BetP thus depends on additional signal input mechanisms from the surrounding hydrophilic or hydrophobic phases.

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FIG. 6. Temperature-dependent activity profiles of BetP in MKH13 pbetP at low and high osmolality. The cells were grown at 37°C in LB medium and washed in buffer containing 50 mM potassium phosphate (pH 7.5) and 50 mM NaCl. After suspension in the same buffer, the cells were energized with 10 mM glucose and incubated on ice. After incubation in the basic buffer (squares) or in the same buffer supplemented with 600 mM NaCl (circles) at various temperatures for 3 min, the reaction was started by the addition of 250 µM [14C]glycine betaine.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The three transport proteins BetP, EctP, and LcoP have been previously shown to be closely similar in terms of activation by hyperosmotic stress. They are all members of the BCCT family and share highly conserved residues, in particular in the eighth transmembrane segment and the adjacent cytoplasmic loop. In spite of this similarity in structural and functional aspects, they responded quite differently to chill stress. The aim of this approach was to elucidate whether the regulation of these transporters by osmotic and temperature stress is based on identical or different mechanisms.

When the temperature dependence of the three carrier proteins was studied in the presence of high osmolality, i.e., under full osmotic stimulation, BetP and LcoP showed a more or less "normal" temperature profile, i.e., a continuously increasing activity at increasing temperature. The Arrhenius plot revealed that in particular BetP had a constant activation energy. The situation was more complicated in the case of EctP, which also showed a temperature optimum close to the optimal growth temperature of C. glutamicum.

The situation changed completely if the temperature dependence of these carrier proteins was studied in the absence of osmotic stimulation. Interestingly, the three transporters showed distinctly different responses to variation of temperature at low osmolality. Except for having the activity optimum at different temperatures, however, the profiles of the three carriers were quite similar in shape. EctP had identical temperature profiles at low and high osmolalities although not a "normal" temperature dependence, showing a distinct temperature optimum at around 25°C irrespective of the state of osmotic stimulation. Consequently, this carrier can be classified as osmotically but not chill activated. LcoP showed an activity optimum at low osmolality at a somewhat lower temperature than EctP, i.e., near 20°C. A comparison with its transport activity in the presence of hyperosmotic conditions revealed a small but significant chill stimulation around 15°C. The most interesting situation was found for BetP. We observed a pronounced chill stimulation at low temperature (near 10°C). At low temperature, BetP reached the same activity in the presence and in the absence of hyperosmotic stress.

A comparison of these results in terms of a combined influence of osmotic and chill stress is instructive (Fig. 3). In the case of EctP, a distinct temperature dependence with an optimum around 25°C was observed, and on the other hand, EctP could be strongly stimulated by osmotic stress. Osmotic stimulation simply caused a parallel shift to higher activities by a factor of 12. We thus conclude that these two stimuli affecting the transport activity of EctP act independently and are additive for EctP. In spite of its structural similarity to EctP, BetP responded completely differently. Osmotic stimulation and temperature dependence are independent and not additive. At any given temperature, full stimulation was reached by hyperosmotic stress. Near the temperature optimum of chill activation, the same maximum activity may also be achieved by chill stimulation. Interestingly, this result also indicates that BetP, which is strongly regulated by osmotic stress at 30°C, does not respond to this influence at all at 10°C. LcoP represents an intermediate case with respect to its regulation behavior. A small extent of chill stimulation independent of osmotic stimulation was observed, but at any given temperature full stimulation was reached only by osmotic stress. Near the temperature optimum of chill stimulation, this type of activation reached 25% of maximum transport activity.

The pronounced temperature-dependent activity profile of BetP prompted us to study possible stimuli which may be responsible for the observed activation. For BetP, an increase in the cytoplasmic K⁺ concentration has been shown to trigger osmodependent activation (36). This parameter does not seem to be relevant for activation of BetP by chill. Two different arguments in favor of this statement were provided, namely, (i) the fact that no significant change in internal K⁺ concentration related to changing temperatures was observed and, more important, (ii) the observation that a mutant BetP lacking the C-terminal 25 amino acids revealed a chill response similar to that of wild-type BetP. Because this mutant responds neither to hyperosmotic stress nor to an increase in the internal K⁺ concentration (38) but is still able to become activated at low temperatures, we conclude that chill activation of BetP is triggered by a stimulus different from that related to osmotic stress.

The nature of this alternate type of stimulus modulating BetP activity is not clear. Changes in the physical properties of the membrane are a relevant candidate. We and others have shown that addition of membrane-active compounds, such as local anesthetics, changes the regulatory behavior of this type of carrier (35, 40, 43), and recently we have demonstrated that these membrane-active compounds in fact act differently on BetP, EctP, and LcoP (40). Whereas different local anesthetics all activate BetP, EctP was inactivated by the same compounds. Again, LcoP showed an intermediate response in being activated by tetracaine only. Another interesting observation was seen for the activity of BetP during adaptation to osmotic change (8). Under these conditions, BetP activity was independent of both changes in cytoplasmic K⁺ and the presence of the regulatory C-terminal domain of BetP. Here we show that BetP, synthesized in E. coli, did not respond to chill stress, although osmodependent activation of BetP was fully retained in this heterologous host (28). The results in E. coli do not exclude the participation of an additional (proteinaceous) factor in C. glutamicum responsible for chill activation.

Adaptation and response to chill stress have been studied in detail in Bacillus subtilis (9, 15, 45, 46) and in Listeria monocytogenes (3, 22, 39). In B. subtilis, global responses at the level of the transcriptome and its contribution to low-temperature adaptation (7, 9) have been described, as well as the response at the level of fatty acid synthesis (21, 46). The primary focus of the investigations in B. subtilis was stress adaptation and not the immediate response of cells to chill stress at the level of protein activity; these results are thus not directly comparable to the present work. The extensive studies on chill stress and its impact on transport of compatible solutes in L. monocytogenes, however, are of particular relevance for the results obtained in C. glutamicum. It has been shown in this food-borne pathogen that the primary, ATP-dependent Gbu transporter is mainly responsible for chill stress-induced betaine accumulation, resulting in a significant growth stimulation at low temperatures (4, 5, 14). Suggestions concerning the mechanism of how a temperature-dependent alteration of the membrane state could affect Gbu activity have been put forward; these, however, do not seem to be relevant for BetP, since Gbu, in contrast to BetP, is a multisubunit protein. On the other hand, it is highly interesting that, similar to the results obtained for BetP from C. glutamicum, the betaine transporter Gbu in L. monocytogenes is characterized by a "normal" Arrhenius plot in the presence of hyperosmotic stress and by activation at low temperatures in its absence (14). In contrast to BetP from C. glutamicum, however, chill activation of Gbu reaches only about 20% of the value observed for full stimulation by osmotic stress at the same temperature. In this case, it is thus not easy to decide whether the activation by two different types of stress is additive or not. For Gbu, there is in fact experimental evidence indicating a correlation of the observed state of activity of the transporter with the physical state of the membrane, which was analyzed by Fourier transform infrared spectroscopy measurements of the CH₂ bands of membrane phospholipids (14).

In contrast to L. monocytogenes (4), it is not yet clear for C. glutamicum whether chill activation of BetP is of physiological relevance for this soil bacterium. Although C. glutamicum in general appears to be highly tolerant to chill with respect to growth (not shown), we have so far no experimental evidence that the presence of BetP and external betaine results in a significant growth stimulation at low temperature. It is interesting, however, that both the activity of BetP, leading to a significantly increased accumulation of betaine, and transcription of betP, resulting in a higher content of BetP in the membrane, were in fact found to be stimulated under these conditions (unpublished results).

 
This work was partially financed by a grant from the Deutsche Forschungsgemeinschaft within the Priority Program 1070.

 
* Corresponding author. Mailing address: Institute of Biochemistry, University of Cologne, Zülpicher Str. 47, 50674 Köln, Germany. Phone: 49 221 470 6461. Fax: 49 221 470 5091. E-mail: r.kraemer{at}uni-koeln.de.

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Journal of Bacteriology, July 2005, p. 4752-4759, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4752-4759.2005
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