High Osmolarity Conditions Research Articles

Environmental regulation of Salmonella typhi invasion-defective mutants.

Salmonella typhi is the etiologic agent of human typhoid. During infection, S. typhi adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host, S. typhi must overcome numerous complex extracellular and intracellular environments. Since relatively little is known about S. typhi pathogenesis, studies were initiated to identify S. typhi genes involved in the early steps of interaction with the host and to evaluate the environmental regulation of these genes. In the present study, TnphoA mutagenesis was used to study these early steps. We isolated 16 Salmonella typhi TnphoA mutants that were defective for both adherence and invasion of the human small intestinal epithelial cell line Int407. Twelve of sixteen mutations were identified in genes homologous to the S. typhimurium invG and prgH genes, which are known to be involved in the type III secretion pathway of virulence proteins. Two additional insertions were identified in genes sharing homology with the cpxA and damX genes from Escherichia coli K-12, and two uncharacterized invasion-deficient mutants were nonmotile. Gene expression of TnphoA fusions was examined in response to environmental stimuli. We found that the cpxA, invG, and prgH genes were induced when grown under conditions of high osmolarity (0.3 M NaCl). Expression of invG and prgH genes was optimal at pH 6.5 and strongly reduced at low pH (5.0). Transcription of both invG and prgH TnphoA gene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both invG and prgH genes appear to be regulated by DNA supercoiling, a mechanism influenced by environmental factors. These results are the first to demonstrate that in S. typhi, (i) the prgH and cpxA genes are osmoregulated, (ii) the invG gene is induced under low oxygen conditions, (iii) the invG gene is pH regulated and growth phase dependent, and (iv) the prgH gene appears to be regulated by DNA supercoiling. Since our experimental conditions were designed to mimic the in vivo environmental milieu, our results suggest that specific environmental conditions act as signals to induce the expression of S. typhi invasion genes.

Trehalose induces antagonism towards Pythium debaryanum in Pseudomonas fluorescens ATCC 17400

Pseudomonas fluorescens ATCC 17400 shows in vitro activity against Pythium debaryanum under conditions of iron limitation. A lacZ reporter gene introduced by transposon mutagenesis into the P. fluorescens ATCC 17400 trehalase gene (treA) was induced by a factor released by the phytopathogen Pythium debaryanum. The induction of the lacZ gene was lost upon treatment of the Pythium supernatant with commercial trehalase. A trehalose concentration as low as 1 microM could induce the expression of treA. The mutation did not affect the wild-type potential for fungus antagonism but drastically decreased the osmotolerance of the mutant in liquid culture and suppressed the ability of P. fluorescens ATCC 17400 to utilize trehalose as a carbon source. A subsequent transposon insertion in treP, one of the trehalose phosphotransferase genes upstream of treA, silenced the lacZ gene. This double mutant restricted fungal growth only under conditions of high osmolarity, which probably results in internal trehalose accumulation. These data confirm the role of the disaccharide trehalose in osmotolerance, and they indicate its additional role as an initiator of or a signal for fungal antagonism.

Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation

Serratia marcescens is a nosocomial pathogen with a high incidence of beta-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to beta-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.

Functional interaction of Escherichia coli RNA polymerase with inorganic polyphosphate.

RNA polymerase from the stationary growth phase cells of Escherichia coli is tightly associated with an acidic compound(s) and exhibits altered promoter selectivity, with reduced transcriptional activity of the genes highly expressed in the exponentially growing cells. Here we have examined the nature of the RNA polymerase-associated acidic compound(s). RNA polymerase isolated from stationary-phase cells of E. coli was found to be tightly associated with inorganic polyphosphates (poly P), and cannot be dissociated even after chromatography on phosphocellulose or DNA-cellulose columns. Since RNA polymerase-poly P complexes reconstituted in vitro showed similar properties, poly P was not binding covalently. The inhibitory effects of poly P on transcription were examined using two forms of the holoenzyme, one containing sigma70, the major sigma for transcription of the genes expressed during exponential cell growth and the other containing sigma38, the principal sigma in the stationary growth phase. At low salt concentrations, poly P inhibited transcription by both Esigma70 and Esigma38 holoenzymes. With an increase in the concentration of potassium glutamate, the poly P inhibition was relieved. At high salt concentrations, the Esigma70 holoenzyme is not able to function, but the Esigma38 holoenzyme is however activated. These results show that poly P may play a role in the promoter selectivity control of RNA polymerase in E. coli which is growing under conditions of high osmolarity and in the stationary growth phase.

Osmo-stress-induced changes in neutral trehalase activity of the fission yeast Schizosaccharomyces pombe

Exposure of repressed growing cultures of Schizosaccharomyces pombe to various extracellular concentrations of NaCl, sorbitol or glycerol resulted in a reversible increase in neutral trehalase activity which was maintained while the cells were in the presence of high environmental osmolarity. Treatment of osmo-stress-induced trehalase by phosphatase lead to a decreased activity indicating that the active enzyme is phosphorylated. The stress response following the osmotic shock required protein synthesis and was independent of the cAMP-dependent protein kinase pathway. Cells disrupted for wis1 or phh1 (identical to sty1 and spc1), which encode members of the mitogen-activated protein kinase (MAPK) cascade, showed that the osmo-stress-induced increase in trehalase markedly diminished. In contrast, the heat shock-induced increase in trehalase remained unchanged in these cells. Taken together, the data suggest that the elevation of trehalase activity in Schiz. pombe under conditions of high osmolarity is due to de novo synthesis of the enzyme and that this process is modulated through a MAPK signal transduction pathway as part of the physiological response to the osmotic stress. The wis1-phh1 MAPK cascade, however, does not appear to form part of the mechanism underlaying the increase in trehalase after heat stress. © 1997 Elsevier Science B.V. All rights reserved.

Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine

Corynebacterium glutamicum accumulates glycine betaine under conditions of high osmolarity. Previous work revealed the existence of a high-affinity glycine betaine permease which is osmotically regulated. In the present study, the corresponding gene was cloned. The betP gene, encoding the glycine betaine uptake carrier, was isolated by heterologous complementation of mutant strain Escherichia coli MKH13. From sequence analysis it is predicted to encode a protein of 595 amino acids. This protein shares 36% identity with the choline transport system BetT and 28% identity with the carnitine transport system CaiT of E. coli, as well as 38% identity with a protein with an unknown function from Haemophilus influenzae. Analysis of hydropathy indicated a common structure for all four transport proteins. After heterologous expression of betP in E. coli MKH13, the measured Km values for glycine betaine and the cotransported Na+ were similar to those found in C. glutamicum, whereas the modulation of activity by osmotic gradients was shifted to lower osmotic values.

Activation and regulation of the Spc1 stress-activated protein kinase in Schizosaccharomyces pombe.

Spc1, an osmotic-stress-stimulated mitogen-activated protein kinase (MAPK) homolog in the fission yeast Schizosaccharomyces pombe, is required for the induction of mitosis and survival in high-osmolarity conditions. Spc1, also known as Sty1, is activated by Wis1 MAPK kinase and inhibited by Pyp1 tyrosine phosphatase. Spc1 is most closely related to Saccharomyces cerevisiae Hog1 and mammalian p38 kinases. Whereas Hog1 is specifically responsive to osmotic stress, we report here that Spc1 is activated by multiple forms of stress, including high temperature and oxidative stress. In this regard Spc1 is more similar to mammalian p38. Activation of Spc1 is crucial for survival of various forms of stress. Spc1 regulates expression of genes encoding stress-related proteins such as glycerol-3-phosphate dehydrogenase (gpd1+) and trehalose-6-phosphate synthase (tps1+). Spc1 also promotes expression of pyp2+, which encodes a tyrosine phosphatase postulated as a negative regulator of Spc1. This proposal is supported by the finding that Spc1 associates with Pyp2 in vivo and that the amount of Spc1 tyrosine phosphorylation is lower in a Pyp2-overproducing strain than in the wild type. Moreover, the level of stress-stimulated gpd1+ expression is higher in delta pyp2 mutants than in the wild type. These findings demonstrate that Spc1 promotes expression of genes involved in stress survival and that of regulation may be commonly employed to modulate MAPK signal transduction pathways in eukaryotic species.

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.

Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa.

Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

MicF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli.

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.

The H-NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp, the leucine regulatory protein.

The H-NS protein, the product of the hns gene, plays a central role in the cellular response of bacteria to environmental stresses such as modification of osmolarity and temperature. The leucine regulatory protein (Lrp) controls a wide array of operons both as an activator (e.g. ilvIH) and as a repressor. We demonstrate that H-NS can decrease the activity of Lrp in stationary phase and under conditions of high osmolarity. Strains containing hns mutations have higher levels of Lrp-activated ilvIH transcription, while strains carrying the hns+ allele on a pBR322 plasmid have lower activity of Lrp-directed ilvIH gene expression.

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli.

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42 degrees C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpR-EnvZ two-component system.

Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells

The study of the effects of osmolarity and growth phase on Salmonella typhi adherence to and invasion of Henle 407 epithelial cells provides the first evidence of a clear overlap between these two environmental stimuli. High-osmolarity conditions are required in the late-log phase for optimum induction of the adherent and invasive phenotypes.

Modification des caractères culturaux et biochimiques du Salmonella paratyphi B après incubation dans l'eau de mer

After incubation in seawater Salmonella paratyphi B cells rapidly became unable to grow on bacteriological media. Previous adaptation to high osmolarity conditions greatly slowed down this process. Strains isolated from seawater microcosms after varying incubation periods were qualitatively different and showed changes in some of their growth (colony shape and size) and biochemical properties (acidification of some sugars, gelatinase activity, acetoin production, nitrate reduction). Because of these modifications, the bacteria showed atypical profiles and could not be identified as members of the Salmonella genus. The alteration of the phenotype, although reversible, could explain some of the false-negative results obtained upon isolation and identification of these bacteria in seawater samples.

Purification of the regulatory protein AlgR1 and its binding in the far upstream region of the algD promoter in Pseudomonas aeruginosa.

A regulatory protein AlgR1, previously suggested to be a member of a two-component sensory transduction system because of its homology to OmpR and NtrC and its ability to allow activation of the algD promoter under conditions of high osmolarity, has been hyperproduced in Escherichia coli after deletion of the upstream region including part of the Shine-Dalgarno sequence of the algR1 gene and its subsequent cloning under the tac promoter. The AlgR1 protein is purified as a monomer, and the sequence of the nine N-terminal amino acids of the monomer matches with that predicted from the DNA sequence of the algR1 gene. The purified AlgR1 protein binds to two separate DNA fragments of the algD upstream region. DNase protection experiments identify these two DNA segments as 14-mer sequences centered at -382 and -458 regions, which contain a common CCGT-TCGTC sequence in them. While the presence of at least one AlgR1 binding site is important for the activation of the algD promoter, the presence of both of the binding sites in the upstream region leads to a higher level of activation.

The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri

In Shigella flexneri, the ompB locus (containing the ompR and envZ genes) was found to modulate expression of the vir genes, which are responsible for invasion of epithelial cells. vir gene expression was markedly enhanced under conditions of high osmolarity (300 mosM), similar to that encountered in tissues both extra- and intracellularly. Two ompB mutants were constructed and tested for virulence and for osmotic regulation of vir genes. An envZ::Tn10 mutant remained invasive, although its virulence was significantly decreased as a result of its inability to survive intracellularly. By using a vir::lac operon fusion, this mutation was shown to decrease beta-galactosidase expression both in low- and high-osmolarity conditions but did not affect vir expression in response to changes in osmolarity. A delta ompB deletion mutant was also constructed via allelic exchange with an in vitro-mutagenized ompB locus of Escherichia coli. This mutation severely impaired virulence and abolished expression of the vir::lac fusion in both low- and high-osmolarity conditions. Therefore, a two-component regulatory system modulates virulence according to environmental conditions. In addition, the mutation affecting a spontaneous avirulent variant of S. flexneri serotype 5, M90T, has been mapped at the ompB locus and was complemented by the cloned E. coli ompB locus. Introduction of the vir::lac fusion into this mutant did not result in the expression of beta-galactosidase (Lac-).

Osmotic regulation of PhoE porin synthesis in Escherichia coli

In Escherichia coli, adaptation to hyperosmotic conditions alters the expression of the outer membrane porins OmpF and OmpC. The amount of PhoE porin, which is normally induced by phosphate deprivation, was greatly reduced in cells adapted to high-osmolarity conditions. Osmoregulation of PhoE operated independently of the activity of the PhoR phosphate sensor and did not involve cross-talk from the homologous osmosensor EnvZ. PhoE synthesis was partially restored by additional copies of the positive regulator phoB+ and by the osmoprotectant glycine betaine.

High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains

Chronic lung infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF) patients. Transcriptional activation of the P. aeruginosa algD gene, which encodes GDPmannose dehydrogenase, is essential for alginate synthesis. Activation of algD is dependent on the product of the algR gene. Sequence homology between the P. aeruginosa algR gene and the Escherichia coli ompR gene, which regulates the cellular response to changes in osmolarity of the growth medium, together with the abnormally high levels of Na+ and Cl- in respiratory tract fluid in CF patients suggested that high osmolarity in the lung of the CF patient might be a signal contributing to the induction of alginate synthesis (mucoidy) in infecting P. aeruginosa. In both mucoid and nonmucoid P. aeruginosa strains (containing a functional algR gene), transcriptional activation of algD increased as the osmolarity of the culture medium increased. The increased activation of algD at high osmolarity was not in itself sufficient to induce alginate synthesis in nonmucoid strains, however, suggesting that other environmental factors are involved in full activation of the alginate genes. The targets of AlgR and OmpR, the algD promoter and the ompC and ompF promoters, respectively, were found to have appreciable sequence homology in the -60 to -110 regions. In E. coli, OmpR was capable of activating the algD promoter nearly as well as AlgR, but in both cases, activation occurred only under conditions of high osmolarity.

Role of micF in the tolC-mediated regulation of OmpF, a major outer membrane protein of Escherichia coli K-12

Mutation in the tolC locus greatly reduces normal synthesis of OmpF, a major porin protein of Escherichia coli K-12. Experiments that use ompF-ompC chimeric genes demonstrate that a tolC mutation exerts its effect at either the promoter or the amino-terminal end of the ompF gene. Direct analysis of ompF mRNA from tolC+ and tolC strains showed that the amount of ompF transcript in the latter was greatly reduced. We have also observed that, in addition to reducing the amount of OmpF, a tolC mutation increases the level of OmpC protein to a much greater extent than occurs in an OmpF mutant and also increases micF RNA synthesis as shown by increased beta-galactosidase synthesis in a micF-lacZ fusion strain. Based on these observations, we suggest that an increased expression of the micF gene in a tolC mutant results in the reduced expression of ompF and that a major effect of the tolC mutation may be to push the porin-regulating system to favor ompC and micF to a greater extent than under high-osmolarity conditions.

Osmoregulation in a Proline-producing Strain of Serratia marcescens

Summary: A proline-producing strain of Serratia marcescens grew more rapidly than the wild-type strain in a medium of high osmolarity due to high concentrations of NaCl, KCl, Na2SO4, (NH4)2 HPO4, sodium glutamate, glucose or sucrose. Growth inhibition by NaCl was partially reversed by proline in the wild-type strain, and by glutamate and proline in the proline-producing strain. Intracellular proline and glutamate concentrations under conditions of high osmolarity were studied. The wild-type strain accumulated endogenously synthesized glutamate, and concentrated proline taken up from the external medium. In contrast, the proline-producing strain accumulated a large amount of endogenously synthesized proline. This increased proline content contributes to the osmotolerance of the proline-producing strain. The growth inhibition by NaCl was also reversed by glycinebetaine in S. marcescens wild-type and proline-producing strains.