The cytoplasm of bacterial cells is a highly crowded cellular compartment that possesses considerable osmotic potential. As a result, and owing to the semipermeable nature of the cytoplasmic membrane and the semielastic properties of the cell wall, osmotically driven water influx will generate turgor, a hydrostatic pressure considered critical for growth and viability. Both increases and decreases in the external osmolarity inevitably trigger water fluxes across the cytoplasmic membrane, thus impinging on the degree of cellular hydration, molecular crowding, magnitude of turgor, and cellular integrity. Here, we assess mechanisms that permit the perception of osmotic stress by bacterial cells and provide an overview of the systems that allow them to genetically and physiologically cope with this ubiquitous environmental cue. We highlight recent developments implicating the secondary messenger c-di-AMP in cellular adjustment to osmotic stress and the role of osmotic forces in the life of bacteria-assembled in biofilms.

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients.

The second messenger cyclic di–adenosine monophosphate (c-di-AMP) is essential in the Gram-positive model organism Bacillus subtilis and in related pathogenic bacteria. It controls the activity of the conserved ydaO riboswitch and of several proteins involved in potassium (K⁺) uptake. We found that the YdaO protein was conserved among several different bacteria and provide evidence that YdaO functions as a K⁺ transporter. Thus, we renamed the gene and protein KimA (K⁺ importer A). Reporter activity assays indicated that expression beyond the c-di-AMP–responsive riboswitch of the kimA upstream regulatory region occurred only in bacteria grown in medium containing low K⁺ concentrations. Furthermore, mass spectrometry analysis indicated that c-di-AMP accumulated in bacteria grown in the presence of high K⁺ concentrations but not in low concentrations. A bacterial strain lacking all genes encoding c-di-AMP–synthesizing enzymes was viable when grown in medium containing low K⁺ concentrations, but not at higher K⁺ concentrations unless it acquired suppressor mutations in the gene encoding the cation exporter NhaK. Thus, our results indicated that the control of potassium homeostasis is an essential function of c-di-AMP.

Streptomyces coelicolor A3(2) synthesizes ectoine and 5-hydroxyectoine upon the imposition of either salt (0.5 M NaCl) or heat stress (39°C). The cells produced the highest cellular levels of these compatible solutes when both stress conditions were simultaneously imposed. Protection against either severe salt (1.2 M NaCl) or heat stress (39°C) or a combination of both environmental cues could be accomplished by adding low concentrations (1 mM) of either ectoine or 5-hydroxyectoine to S. coelicolor A3(2) cultures. The best salt and heat stress protection was observed when a mixture of ectoine and 5-hydroxyectoine (0.5 mM each) was provided to the growth medium. Transport assays with radiolabeled ectoine demonstrated that uptake was triggered by either salt or heat stress. The most effective transport and accumulation of [ ¹⁴ C]ectoine by S. coelicolor A3(2) were achieved when both environmental cues were simultaneously applied. Our results demonstrate that the accumulation of the compatible solutes ectoine and 5-hydroxyectoine allows S. coelicolor A3(2) to fend off the detrimental effects of both high salinity and high temperature on cell physiology. We also characterized the enzyme (EctD) required for the synthesis of 5-hydroxyectoine from ectoine, a hydroxylase of the superfamily of the non-heme-containing iron(II)- and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). The gene cluster ( ectABCD ) encoding the enzymes for ectoine and 5-hydroxyectoine biosynthesis can be found in the genome of S. coelicolor A3(2), Streptomyces avermitilis , Streptomyces griseus , Streptomyces scabiei , and Streptomyces chrysomallus , suggesting that these compatible solutes play an important role as stress protectants in the genus Streptomyces .

Recently, a new type of K⁺transporter, Ktr, has been identified in the bacteriumVibrio alginolyticus(T. Nakamura, R. Yuda, T. Unemoto, and E. P. Bakker, J. Bacteriol.180:3491-3494, 1998). The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane. The genome sequence ofBacillus subtiliscontains two genes for each of these subunits:yuaA(ktrA) andykqB(ktrC) encode homologues to theV. alginolyticusKtrA protein, andyubG(ktrB) andykrM(ktrD) encode homologues to theV. alginolyticusKtrB protein. We constructed gene disruption mutations in each of the fourB. subtilis ktrgenes and used this isogenic set of mutants for K⁺uptake experiments. Preliminary K⁺transport assays revealed that the KtrAB system has a moderate affinity with aK_mvalue of approximately 1 mM for K⁺, while KtrCD has a low affinity with aK_mvalue of approximately 10 mM for this ion. A strain defective in both KtrAB and KtrCD exhibited only a residual K⁺uptake activity, demonstrating that KtrAB and KtrCD systems are the major K⁺transporters ofB. subtilis. Northern blot analyses revealed thatktrAandktrBare cotranscribed as an operon, whereasktrCandktrD, which occupy different locations on theB. subtilischromosome, are expressed as single transcriptional units. The amount of K⁺in the environment or the salinity of the growth medium did not influence the amounts of the variousktrtranscripts. A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective. In thektrABstrain, the osmotically mediated growth defect was associated with a rapid loss of K⁺ions from the cells. Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically inducedproHJ,opuA, andgsiBgenes was not impaired, demonstrating that a high cytoplasmic K⁺concentration is not essential for the transcriptional activation of these genes at high osmolarity. Taken together, our data suggest that K⁺uptake via KtrAB and KtrCD is an important facet in the cellular defense ofB. subtilisagainst both suddenly imposed and prolonged osmotic stress.

The gram-positive soil bacteriumBacillus subtilisoften faces increases in the salinity in its natural habitats. A transcriptional profiling approach was utilized to investigate both the initial reaction to a sudden increase in salinity elicited by the addition of 0.4 M NaCl and the cellular adaptation reactions to prolonged growth at high salinity (1.2 M NaCl). Following salt shock, asigBmutant displayed immediate and transient induction and repression of 75 and 51 genes, respectively. Continuous propagation of this strain in the presence of 1.2 M NaCl triggered the induction of 123 genes and led to the repression of 101 genes. In summary, our studies revealed (i) an immediate and transient induction of the SigW regulon following salt shock, (ii) a role of the DegS/DegU two-component system in sensing high salinity, (iii) a high-salinity-mediated iron limitation, and (iv) a repression of chemotaxis and motility genes by high salinity, causing severe impairment of the swarming capability ofB. subtiliscells. Initial adaptation to salt shock and continuous growth at high salinity share only a limited set of induced and repressed genes. This finding strongly suggests that these two phases of adaptation require distinctively different physiological adaptation reactions by theB. subtiliscell. The large portion of genes with unassigned functions among the high-salinity-induced or -repressed genes demonstrates that major aspects of the cellular adaptation ofB. subtilisto high salinity are unexplored so far.