Molecular and biochemical characterization of mechanosensitive channels in Corynebacterium glutamicum

Database searches in the Corynebacterium glutamicum genome sequence revealed homologs of the mechanosensitive channels MscL and YggB of Escherichia coli. To elucidate the physiological role of these putative channels deletion mutants were constructed. Betaine efflux induced by osmotic downshock of the mscL deletion mutant was nearly identical to that of the wild-type, whereas the yggB deletion mutant showed a reduced efflux rate. Interestingly, the double deletion strain, which was expected to have an even more decreased capability of betaine excretion, had only a slightly reduced efflux rate compared to the wild-type and did not show an increased mortality after osmotic downshift. These results led to the hypothesis that C. glutamicum may possess a third type of mechanosensitive channel not related to the MscL and YggB/KefA families. Furthermore it is unlikely that an MscM-like activity is responsible for the betaine efflux, because of the high transport capacity detected in the double deletion mutant.     

Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity

Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well-defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

Effect of Mutation of Potassium-Efflux System,KefA, on Mechanosensitive Channels in the Cytoplasmic Membrane of Escherichia coli

The effect of a kefA mutation on the mechanosensitive channels in the cytoplasmic membrane of Escherichia coli was established by introducing a mutation of the kefA gene into wild-type E. coli by P1 transduction. The mutation of the kefA gene not only made the cells sensitive to K⁺ in the medium but also changed the mechanosensitive channel activity. The kefA mutation did not change the conductances of the two mechanosensitive channels in the cytoplasmic membrane of E. coli, but it prolonged the channel open time. Also, the kefA mutation made the cells more sensitive to pressure in comparison to wild-type cells. The high sensitivity to pressure of the kefA mutant was not modulated by betaine or by the potassium gradient across the membrane. The effect of the kefA mutation on mechanosensitive channels was not due to a membrane fluidity change. KefA might be a regulator for mechanosensitive channels. Received: 6 September 1995/Revised: 13 December 1995     

Lactococcus lactis uses MscL as its principal mechanosensitive channel

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.     

BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.     

Ionic regulation of MscK,a mechanosensitive channel from Escherichia coli

Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D-glucamine (NMDG). An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.     

Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity

Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at > 2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.     

Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.     

Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin

Abstract The addition of 1 mM glycine betaine to the growth medium of Chromatium sp. NCIMB 8379 relieved growth inhibition caused by exposure to supra-optimal Nad concentrations. Intracellular glycine betaine concentrations were dependent upon the NaCl concentration of the growth medium up to 3 M exogenous Nad. Kinetic data for the accumulation of [methyl-¹⁴C]-glycine betaine demonstrated that Chromatium sp. NCIMB 8379 possesses a constitutively expressed active transport system for glycine betaine. The transport system was saturable with respect to glycine betaine concentration and exhibited typical Michaelis-Menten type kinetics: K _m= 24 μ M, V _max= 306 nmol min⁻¹ mg protein⁻¹ at an external NaCl concentration of 1 M. The rate of glycine betaine transport decreased progressively with increasing growth medium NaCl concentration. This transport system may represent an adaptive response to growth in high osmolarity environments in this halotolerant isolate, allowing accumulation of glycine betaine from the external cell environment or recycling synthesised glycine betaine which has passively diffused from the cell.     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

Responses of Bacillus subtilis to hypotonic challenges: physiological contributions of mechanosensitive channels to cellular survival

Mechanosensitive channels are thought to function as safety valves for the release of cytoplasmic solutes from cells that have to manage a rapid transition from high- to low-osmolarity environments. Subsequent to an osmotic down-shock of cells grown at high osmolarity, Bacillus subtilis rapidly releases the previously accumulated compatible solute glycine betaine in accordance with the degree of the osmotic downshift. Database searches suggest that B. subtilis possesses one copy of a gene for a mechanosensitive channel of large conductance (mscL) and three copies of genes encoding proteins that putatively form mechanosensitive channels of small conductance (yhdY, yfkC, and ykuT). Detailed mutational analysis of all potential channel-forming genes revealed that a quadruple mutant (mscL yhdY yfkC ykuT) has no growth disadvantage in high-osmolarity media in comparison to the wild type. Osmotic down-shock experiments demonstrated that the MscL channel is the principal solute release system of B. subtilis, and strains with a gene disruption in mscL exhibited a severe survival defect upon an osmotic down-shock. We also detected a minor contribution of the SigB-controlled putative MscS-type channel-forming protein YkuT to cellular survival in an mscL mutant. Taken together, our data revealed that mechanosensitive channels of both the MscL and MscS types play pivotal roles in managing the transition of B. subtilis from hyper- to hypo-osmotic environments.     

Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12.

The amounts of cytoplasmic water and of all osmotically significant cytoplasmic solutes were determined for Escherichia coli K-12 grown in 3-(N-morpholino)propane sulfonate (MOPS)-buffered glucose-minimal medium containing 0.5 M NaCl in the presence and absence of the osmoprotectants betaine and proline. The goal of this work is to correlate the effects of osmoprotectants on the composition of the cytoplasm with their ability to increase the growth rate of osmotically stressed cells. At a concentration of 1 mM in the growth medium, betaine increases the growth rate more than does proline; choline, which is converted to betaine by E. coli, appears to have an intermediate effect on growth rate. The accumulation of either betaine or proline reduces the cytoplasmic amounts of K+, glutamate, trehalose, and MOPS (the major cytoplasmic osmolytes accumulated in the absence of osmoprotectants), so that at this external osmolarity the total amount of cytoplasmic solutes is essentially the same in the presence or absence of either osmoprotectant. More betaine than proline is accumulated, so the extent of replacement of cytoplasmic solutes is greater for betaine than for proline. Accumulation of these osmoprotectants is accompanied by a large (20 to 50%) increase in the volume of cytoplasmic water per unit of cell dry weight (Vcyto). This effect, which appears to result from an increase in the volume of free water, Vf (as opposed to water of hydration, or bound water), is greater for betaine than for proline. Taken together, these results indicate that the molar effects of betaine and proline on water activity and on the osmotic pressure of the cytoplasm must be significantly larger than those of the solutes they replace. Cayley et al. (S. Cayley, B. A. Lewis, H. J. Guttman, and M. T. Record, Jr., J. Mol. Biol. 222:281-300, 1991) observed that, in cells grown in the absence of osmoprotectants, both growth rate and Vcyto decreased, whereas the amount of cytoplasmic K+ (nK+) increased, with increasing external osmolarity. We predicted that the observed changes in nK+ and Vcyto would have large and approximately compensating effects on key protein-nucleic acid interactions of gene expression, and we proposed that Vf was the fundamental determinant of growth rate in osmotically stressed cells. The properties of cells cultured in the presence of betaine and proline appear completely consistent with our previous work and proposals. Accumulation of betaine and, to a lesser extent, proline shifts the set of linked physiological parameters (nK+, Vcyto, growth rate) to those characteristic of growth at lower osmolarity in the absence of osmoprotectants. Models for the thermodynamic basis and physiological consequences of the effect of osmoprotectants on Vcyto and Vf are discussed.     

Osmotic regulation of intracellular solute pools in Lactobacillus plantarum.

Bacteria respond to changes in medium osmolarity by varying the concentrations of specific solutes in order to maintain constant turgor pressure. The cytoplasmic pools of K+, proline, glutamate, alanine, and glycine of Lactobacillus plantarum ATCC 14917 increased when the osmolarity of the growth media was raised from 0.20 to 1.51 osmol/kg by KCL. When glycine-betaine was present in a high-osmolarity chemically defined medium, it was accumulated to a high cytoplasmic concentration, while the concentrations of most other osmotically important solutes decreased. These observations, together with the effects of glycine-betaine on the specific growth rate under high-osmolarity conditions, suggest that glycine-betaine is preferentially accumulated in L. plantarum. Uptake of glycine-betaine, proline, glutamate, and alanine was studied in cells that were alternately exposed to hyper- and hypo-osmotic stresses. The rate of uptake of proline and glycine-betaine increased instantaneously upon increasing the osmolarity, whereas that of other amino acids did not. This activation occurred also under conditions in which protein synthesis was inhibited was most pronounced when cells were pregrown at high osmolarity. The duration of net transport was a function of the osmotic strength of the assay medium. Glutamate uptake was not activated by an osmotic upshock, and the uptake of alanine was low under all conditions tested. When cells were subjected to osmotic downshock, a rapid efflux of accumulated glycine-betaine, proline, and alanine occurred whereas the pools of other amin acids remained unaffected. The results indicate that osmolyte efflux is, at least to some extent, mediated via specific osmotically regulated efflux systems and not via nonspecific mechanisms as has been suggested previously.     

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.     

Glycine betaine uptake by the ProXVWZ ABC transporter contributes to the ability of Mycobacterium tuberculosis to initiate growth in human macrophages

Mycobacterium tuberculosis maintains a large genetic capacity necessary for growth in different environments during infection and survival upon aerosol transmission to new hosts. Screening for bacterial RNAs produced in response to host interactions produced candidate lists where we noted proXVWZ, annotated as encoding a putative glycine betaine or proline transporter. As high surface-to-volume ratios make bacterial cells particularly vulnerable to changes in water availability, we investigated the contributions of this transporter to the ability of M. tuberculosis to colonize macrophages. An H37Rv proXVWZ mutant was impaired for initial survival and intracellular growth and exhibited reduced growth at elevated medium osmolarity. This defect could be complemented by restoring proXVWZ and was attributable to a failure to accumulate the compatible solute glycine betaine. We then demonstrated that ProXVWZ allows M. tuberculosis to obtain betaine from host macrophages and thereby contributes to early steps in colonizing this niche.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.