Janet M. Wood

Osmosensing and osmoregulatory compatible solute accumulation by bacteria.

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.

Bacterial Osmoregulation: A Paradigm for the Study of Cellular Homeostasis

To thrive, cells must control their own physical and chemical properties. This process is known as cellular homeostasis. The dilute solutions traditionally favored by experimenters do not simulate the cytoplasm, where macromolecular crowding and preferential interactions among constituents may dominate critical processes. Solutions that do simulate cytoplasmic conditions are now being characterized. Corresponding cytoplasmic properties can be varied systematically by imposing osmotic stress. This osmotic stress approach is revealing how cytoplasmic properties modulate protein folding and protein?nucleic acid interactions. Results suggest that cytoplasmic homeostasis may require adjustments to multiple, interwoven cytoplasmic properties. Osmosensory transporters with diverse structures and bioenergetic mechanisms activate in response to osmotic stress as other proteins inactivate. These transporters are serving as paradigms for the study of in vivo protein-solvent interactions. Experimenters have proposed three different osmosensory mechanisms. Distinct mechanisms may exist, or these proposals may reflect different perceptions of a single, unifying mechanism.

Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli

The osmolality required to activate osmosensory transporter ProP and the proportion of cardiolipin (CL) among the phospholipids of Escherichia coli rise with growth medium osmolality. Most CL synthesis has been attributed to the cls gene product. Transcription of cls increased with osmolality. The proportion of CL was low and osmolality‐independent in cls– bacteria. It increased more dramatically on the transition to stationary phase in cls– than cls+ bacteria. Thus, Cls is responsible for osmoregulated CL synthesis and other enzymes may contribute to CL accumulation during stationary phase. The proportion of phosphatidylglycerol (PG) was elevated and it increased with medium osmolality in cls– bacteria. A cls defect impaired growth of E. coli on solid and in liquid media at low and, more strongly, at high osmolality. Bacteria cultured at high osmolality without osmoprotectant were shorter and rounder than those cultured at low osmolality or with glycine betaine. Fluorescence microscopy showed that CL and ProP colocalize at the poles and near the septa of dividing E. coli cells. The polar localization of ProP was independent of its expression level but correlated with the proportion and polar localization of CL. Association with CL (and not PG) may be required for polar ProP localization.

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls(-) bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H(+)-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.

Proline porters effect the utilization of proline as nutrient or osmoprotectant for bacteria

Proline is utilized by all organisms as a protein constituent. It may also serve as a source of carbon, energy and nitrogen for growth or as an osmoprotectant. The molecular characteristics of the proline transport systems which mediate the multiple functions of proline in the Gram negative enteric bacteria, Escherichia coli and Salmonella typhimurium, are now becoming apparent. Recent research on those organisms has provided both protocols for the genetic and biochemical characterization of the enzymes mediating proline transport and molecular probes with which the degree of homology among the proline transport systems of archaebacteria, eubacteria and eukaryotes can be assessed. This review has provided a detailed summary of recent research on proline transport in E. coli and S. typhimurium; the properties of other organisms are cited primarily to illustrate the generality of those observations and to show where homologous proline transport systems might be expected to occur. The characteristics of proline transport in eukaryotic microorganisms have recently been reviewed (Horak, 1986).

The ion coupling and organic substrate specificities of osmoregulatory transporter ProP in Escherichia coli.

Transporter ProP of Escherichia coli, a member of the major facilitator superfamily, mediates osmoprotective proline or glycine betaine accumulation by bacteria exposed to high osmolality environments. Morpholinopropane sulfonic acid, a common constituent of microbiological media, accumulates in osmoadapting E. coli cells but it is not osmoprotective and it did not influence proP transcription or ProP activity. The apparent K(m) for proline uptake via ProP increased with decreasing pH in the range 7.5-4. ProP-dependent proline uptake by de-energized bacteria was associated with alkalinization of the external medium. Thus ProP mediates cotransport of H(+) and zwitterionic proline and a transporter functional group with a pK(a) of 5-6 is implicated in catalysis. Exogenous proline or glycine betaine elicits K(+) release from osmoadapting E. coli cells and ProP activity is stimulated by exogenous K(+). However, uptake of proline or glycine betaine stimulated K(+) efflux from K(+)-loaded bacteria which expressed either ProP or alternative, osmoregulatory transporter ProU. This indicated that ProP was unlikely to mediate K(+) efflux. Zwitterions ectoine, pipecolate, proline betaine, N,N-dimethylglycine, carnitine and 1-carboxymethylpyridinium were identified as alternative ProP substrates. Choline, a cation and a structural analogue of glycine betaine, was a low affinity inhibitor but not a substrate of ProP.

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP, LacY, ProW, AqpZ, MscS, and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).

Osmosensing by bacteria.

Osmosensors are proteins that sense environmental osmotic pressure. They mediate or direct osmoregulatory responses that allow cells to survive osmotic changes and extremes. Bacterial osmosensing transporters sense high external osmotic pressure and respond by mediating organic osmolyte uptake, hence cellular rehydration. Detailed studies of osmosensing transporters OpuA, BetP, and ProP suggest that they sense and respond to different osmotic pressure–dependent cellular properties. These studies also suggest that each protein has a cytoplasmic osmosensory or osmoregulatory domain, but that these domains differ in structure and function. It is not yet clear whether each transporter represents a distinct osmosensory mechanism or whether different research groups are approaching the same mechanism by way of different paths. Principles emerging from this research will apply to other osmosensors, including those that initiate signal transduction cascades in prokaryotes and eukaryotes.

Na+ (Li+)-proline cotransport in Escherichia coli.

SummaryNa+ and Li+ were found to stimulate the transport ofl-proline by cells ofEscherichia coli induced for proline utilization. The gene product of the put P gene is involved in the expression of this transport activity since the put P+ strains CSH 4 and WG 148 show activity and the put P− strain RM 2 fails to show this cation coupled transport. The addition of proline was found to stimulate the uptake of Li+ and of Na+. Attempts to demonstrate proline stimulated H+ uptake were unsuccessful. It is concluded that the proline carrier (coded by the put P gene) is responsible for Na+ (or Li+)-proline cotransport.

Cardiolipin Controls the Osmotic Stress Response and the Subcellular Location of Transporter ProP in Escherichia coli

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.