Lysis of Vibrio cholerae cells: direct isolation of the outer membrane from whole cells by treatment with urea

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.     

Porins of Vibrio cholerae: purification and characterization of OmpU.

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity. The synthesis of all the three proteins is regulated by the osmolarity of the growth medium. The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded. The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units. Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit. Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein. The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium. The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that of OmpU. Southern blot hybridization of V. cholerae genomic DNA digested with several restriction endonucleases with nick-translated E. coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes. OmpU is also not antigenically related to OmpF. Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V. cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium. OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the possible virulence determinants in V. cholerae (V. L. Miller and J. J. Mekalanos, J. Bacteriol. 170:2575-2583, 1988).     

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.     

Membrane-bound enterotoxin of Vibrio cholerae

The mode of transport of the complex toxin molecule of Vibrio cholerae (which has a mol. wt of 84000 and consists of several subunits) across the inner and outer membranes of V. cholerae is not known. In this study we found two peptides in the outer and inner membranes of V. cholerae which may be the form in which the toxin subunits are transported across the membrane. We examined two growth conditions: aerobic growth at 37 degrees C, when most of the synthesized toxin is membrane-bound; and anaerobic growth at 37 degrees C, when little toxin remains membrane-bound, the toxin being released into the growth medium. When V. cholerae was grown aerobically at 37 degrees C, the outer and the inner membranes contained two peptides with mol. wts of approximately 22000 and 6000 which were not found in the outer or the inner membrane of anaerobically grown cells. Sodium deoxycholate, which releases membrane-bound toxin, released several peptides including the 22000 and the 6000 mol. wt peptides. Trypsin also released the 22000 and 6000 mol. wt peptides. Purified cholera toxin had three kinds of peptides, of mol. wt 21000 (A1 peptide), 11000 (B subunit) and 5000 (A2 peptide). We postulate that the membrane peptides may be precursors of the A subunit of the toxin molecule.     

Effect of bile on the cell surface permeability barrier and efflux system of Vibrio cholerae

Gram-negative bacteria are inherently impermeable to hydrophobic compounds, due to the synergistic activity of the permeability barrier imposed by the outer membrane and energy dependent efflux systems. The gram-negative, enteric pathogen Vibrio cholerae appears to be deficient in both these activities; the outer membrane is not an effective barrier to hydrophobic permeants, presumably due to the presence of exposed phospholipids on the outer leaflet of the outer membrane, and efflux systems are at best only partially active. When V. cholerae was grown in the presence of bile, entry of hydrophobic compounds into the cells was significantly reduced. No difference was detected in the extent of exposed phospholipids on the outer leaflet of the outer membrane between cells grown in the presence or absence of bile. However, in the presence of energy uncouplers, uptake of hydrophobic probes was comparable between cells grown in the presence or absence of bile, indicating that energy-dependent efflux processes may be involved in restricting the entry of hydrophobic permeants into bile grown cells. Indeed, an efflux system(s) is essential for survival of V. cholerae in the presence of bile. Expression of acrAB, encoding an RND family efflux pump, was significantly increased in V. cholerae cells grown in vitro in the presence of bile and also in cells grown in rabbit intestine.     

Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli.

Supplementation of the growth medium with high concentrations of NaCl, KCl, or sucrose caused a drastic change in the ratio of the two peptidoglycan-associated major outer membrane proteins of Escherichia coli K-12 in that the amounts of proteins b and c present in cell envelope preparations decreased and increased, respectively. Kinetic studies showed that, after the osmolarity of the medium was changed, one protein was hardly incorporated into the membrane, whereas the other was incorporated with an increased rate. After about 1.5 to 2 generations, the cell envelopes obtained the b/c ratio characteristic for the new medium, and both proteins were subsequently incorporated in the cell ensured this new ratio. Once proteins b and c were incorporated in the cell envelope, they were not converted into each other by changes in osmolarity of the growth medium.     

Role of ectoine in Vibrio cholerae osmoadaptation

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.     

Strain-specific variation in the protein and lipopolysaccharide composition of the group B meningococcal outer membrane.

Variation in the protein and lipopolysaccharide composition of the meningococcal outer membrane may be due to either serotype differences or to changes in cultural conditions. There are 12 antigenically distinct serotypes of group B meningococci, and these are associated with distinct major outer membrane protein patterns on sodium dodecyl sulfate-polyacrylamide gels. In most strains the predominant outer membrane protein carries the serotype-specific determinant. Certain strains, when grown under similar conditions in different media showed an altered membrane composition. The type 2 strain, M986, grown in modified Frantz medium-A, had a reduced amount of the major 41,000-dalton protein while a 28,000-dalton protein predominated. The altered protein composition may be related to changes in cell metabolism as reflected by the pH of the medium after growth. Growth of the organism in Frantz medium-B caused a negligible drop in pH and the 41,000-dalton protein remained predominant. There was also variation associated with changes in the growth rate. Increasing the aeration caused a concomitant increase in growth rate and cell yield. We observed two quantitative changes in outer membrane proteins in four of seven strains examined: (i) where only a single major protein changed (three strains), and (ii) where an increase in one protein component was associated with a decrease in another protein (one strain). When the strains were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) with either high or low aeration, the total protein in the outer membrane remained constant. In contrast, with high aeration there was a significant increase in lipopolysaccharide. These studies suggest that the cell surface proteins may be altered by the organism to meet a variety of environmental conditions.     

Influence of growth rate and iron limitation on the expression of outer membrane proteins and enterobactin by Klebsiella pneumoniae grown in continuous culture.

The influence of the growth rate on outer membrane protein composition and enterobactin production was studied with Klebsiella pneumoniae grown under conditions of iron limitation in chemostats. More enterobactin was produced at fast (D = 0.4 h-1) and slow (D = 0.1 h-1) growth rates in continuous cultures than in either logarithmic- or stationary-phase batch cultures. When the growth rate was controlled under conditions of carbon limitation and the iron level was reduced to 0.5 microM, the iron-regulated outer membrane proteins and enterobactin were induced at the fast growth rate. At the slow growth rate, although the iron-regulated outer membrane proteins were barely visible, a significant level of enterobactin was still produced. These results suggest that under conditions of either carbon or iron limitation, the growth rate can influence the induction of the high-affinity iron uptake system of K. pneumoniae. Other outer membrane proteins, including a 39-kilodalton peptidoglycan-associated protein, were found to vary with the growth rate and nutrient limitation.     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Characterization of cell surface material removed by water and NaCl washing ofParacoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency

Paracoccus denitrificans grown on complex medium deficient in Mg²⁺ and Ca²⁺ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg²⁺ and Ca²⁺ sufficient) or complex medium supplemented with Mg²⁺+Ca²⁺ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg²⁺ and Ca²⁺ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.     

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membranes of animal cells. II. The metabolism and turnover of the surface membrane

Turnover studies of the surface membrane and of cell particulate matter of L cells in tissue culture in logarithmic and plateau phase of growth have been made. The rate of incorporation of isotope into these fractions and the rate of fall of specific activities of labeled L-cell fractions have been observed. The following interpretation of the data appears most likely although other interpretations are possible. Growing and nongrowing cells synthesize approximately similar amounts of surface membrane and particulate material. In the growing cell the material is incorporated with net increases in substance. There is relatively little turnover. In the nongrowing cell newly synthesized material is incorporated, but a corresponding amount of material is eliminated so that there is turnover without net increase of substance. Our results suggest that there is no gross differential turnover between the protein, lipid, and carbohydrate of the surface membrane under the conditions of our experiments. Metabolic inhibitors or omission of amino acids in the culture medium lead to a decrease in synthesis of surface membrane and cell particulates and cause an equivalent decrease in the rate of degradation of surface membrane and of particulates; therefore the synthetic and degradative aspects of turnover appear to be coupled. As cultures of nongrowing cells in suspension or on a glass surface age, their synthetic and turnover capacities diminish. Our results suggest that the cell may exist in a nongrowing state with a level of synthesis similar to that of a growing cell. It can exist in this state with a high level of turnover.     

Effect of iron limitation on growth, siderophore production, and expression of outer membrane proteins of Vibrio cholerae.

Vibrio cholerae strains secrete a phenolate-type siderophore when grown in low-iron medium. The siderophore was detected as early as 3.5 h after downshift to iron-poor medium, and it continued to accumulate in the medium as the cells entered stationary phase. Two clinical isolates and an environmental isolate were examined for the amount of siderophore produced. The environmental isolate produced more siderophore and continued to secrete it at concentrations of iron that repressed synthesis in the clinical isolates. Concomitant with production of siderophore, at least six new proteins were seen in the outer membranes of iron starved cells. One of the proteins was large (200,000 Mr [220K]) and appeared to be loosely associated with the outer membrane. The other five proteins had approximate Mr values of 77K, 76K, 75K, 73K, and 62K. The 62K protein, like the 40K major outer membrane protein, was heat modifiable. One or more of these proteins may be a component of the receptor for the iron-siderophore complex.     

Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins

IrgA is an iron-regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the El Tor biotype, three non-O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to irgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron-regulated outer membrane protein of V. cholerae, we determined the amino-terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two El Tor strains of V. cholerae had a major iron-regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in irgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB-dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol-substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB-like protein in V. cholerae remain to be determined.