Transformation efficiency of EMS-induced mutants of Streptococcus mutans of altered cell shape.

Some Streptococcus mutans strains change shape from bacillary to coccal or ellipsoid form in response to the ratio of bicarbonate to potassium or of borate to potassium in growth media. So that insight into determinants of shape of these streptococci could be gained, and future genetic studies facilitated, the shapes of a series of transformable and nontransformable strains of S. mutans were studied and attempts made to isolate a mutant of augmented transformability. Several strains were mutagenized by ethylmethane sulfonate and mutants with altered colonial and cellular morphologies isolated. Cell shapes were studied by Gram stain and Nomarski interference microscopy, and by scanning and transmission electron microscopy. Diverse shape-altered mutants were isolated from seven transformable and two nontransformable strains of S. mutans. Among these, length-to-width ratios ranged from > 10 to about 0.25. Regulation of timing of cell division, septum formation, or septum completion events may have been altered in these mutants. While most mutants substantially or completely lost transformability, mutant LT11 had transformation efficiency of 1.3 x 10(-4) to 2.3 x 10(-3), more than two to three orders of magnitude greater than its parental UA159 and the well-known transformable strain GS5(HK), respectively. There was no evidence of production of competence factor by LT11. Competence of LT11 was maintained for at least six months upon storage at -70 degrees C, facilitating its use for genetic studies. While the morphologies of several shape-altered mutants were no longer responsive to changes of the bicarbonate/potassium, unlike those of their parentals, the morphology of LT11 persisted in its response to this condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction and intracellular localization of the 80-kilodalton heat-shock protein of Neurospora crassa.

The most abundant heat-shock protein of Neurospora crassa is a multimeric glycoprotein of 80-kilodaltons (i.e., HSP80), induced strongly by hyperthermia and at a lower level by sodium arsenite, ethanol, and carbon source depletion. Immunoelectron microscopy, using indirect immunogold labelling demonstrated that HSP80 was undetectable in mycelium cultured at the normal growth temperature of 28 degrees C, but it appeared rapidly following the commencement of heat-shock treatment at 48 degrees C. HSP80, visualized by the gold label, was observed almost exclusively in the cytoplasm, exhibiting a uniform distribution. Association of this protein with cellular membranes and (or) targeting to a particular subcellular compartment or organelle was not apparent.

Role of cytoskeleton in isotonic cell volume control of rabbit proximal tubules.

Stability of mammalian cell volume depends primarily on the sodium pump. When active cation transport of rabbit renal proximal tubules is blocked by ouabain, cells swell, but their size is limited by residual volume control mechanisms. This "ouabain-resistant" volume control is not an active process, as it operates in the presence of cyanide and dinitrophenol and in the absence of exogenous energy. Nevertheless, it remains incompletely explained by known transmembrane oncotic and hydrostatic forces. We tested the hypothesis that the cytoskeleton contributes to isotonic cell volume control. Isolated, collapsed rabbit proximal convoluted tubules (PCT) were crimped at both ends with micropipettes and had their volume assessed optically. PCT in ouabain (1 mM) swelled to 1.40 above control with protein, 1.62 without protein, and 1.89 with the cytoskeleton inhibitors vincristine (5 microM) and cytochalasin B (50 microM) and without protein. Tubulozole-C and cytochalasin D gave similar results. A hydrostatic pressure of 50 cmH2O increased tubule volume to 1.93 before the tubule basement membrane (TBM) prevented further volume increase. We conclude that volume of renal tubule cells in ouabain is limited partly by external protein, but primarily by the cytoskeleton. The TBM prevents massive swelling and tubule disaggregation.

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.

Factors influencing cell shape in the mutans group of streptococci.

Electron and light microscopic and growth studies of representatives of the diverse species of mutans streptococci revealed the cells to be either bacillary or coccoid in shape. Some strains changed from bacillary to coccoid if the HCO3-/K+ ratio of the media was increased and from coccoid to bacillary if the ratio was decreased. Doubling times of rods and cocci were the same despite an HCO3-/K+ ratio change between 0.008 and 2.84. For strain 10449S, no tested anions or cations substituted for HCO3- or K+ to produce this effect, except for B4O7(2-). Strain 10449S grown at a high B4O7(2-)/K+ ratio became ellipsoid, and this phenomenon was associated with slower doubling times. Up to three incomplete septa could be observed in one rod, but no more than one incomplete septum could be observed in either ellipsoid or spherical cells. Interseptal distances were greatest in rods, shorter in spheres, and shortest in ellipses. All of the above differences were statistically significant (P less than 0.001).

Proposed mechanism for generation and localization of new cell division sites during the division cycle of Escherichia coli.

The earliest detectable event at future sites of cell division in Escherichia coli is the appearance of paired periseptal annuli that flank the site of formation of the division septum. The development and localization of these structures were followed as the cell progressed through the division cycle. The data suggest that (i) new periseptal annuli are generated from annuli already in position at the midpoint of the newborn cell; (ii) the nascent annuli are then displaced laterally during cell elongation to positions at 1/4 and 3/4 cell length; and (iii) the annuli at 1/4 and 3/4 cell length are retained during division, becoming the midpoint annuli of the newborn cells at the sites of the forthcoming division septum. The results indicate that the sites of future divisions can be identified and committed to the division process prior to the division cycle in which these sites are utilized for septum formation, and they suggest a model in which preexisting sites of cell division generate future division sites by a replication/displacement mechanism.

Membrane-murein attachment at the leading edge of the division septum: a second membrane-murein structure associated with morphogenesis of the gram-negative bacterial division septum.

Electron microscopy of plasmolyzed cells of Salmonella typhimurium revealed a continuous zone of membrane-murein attachment at the leading edge of the division septum at all stages of septal invagination. The membrane-murein attachment site had a characteristic ultrastructural appearance and remained as a bacterial birth scar at the new pole of each of the two daughter cells after cell separation. The continuous zone of membrane-murein attachment at the leading septal edge represents the second organelle based on a topologically ordered domain of membrane-murein adhesion to be described at the site of cell division.

Bicarbonate and potassium regulation of the shape of Streptococcus mutans NCTC 10449S.

Morphological changes of S. mutans NCTC 10449S associated with growth in modified Jordan medium and FMC medium (Terleckyj et al., Infect. Immun. 11:649-655, 1975) were studied by scanning electron microscopy. The cells were bacillary in Jordan medium, but coccoid and of unequal size in FMC. Transfer of the cells from Jordan medium to FMC and vice versa reversed their shapes, as did salt exchange between these media. Morphological changes could not be ascribed to either medium pH, concentration of P, or Na+/K+ ratio. However, they were growth dependent, since the changes did not occur when the cells were suspended in salt components alone or in media supplemented with protein synthesis inhibitors. Only a high bicarbonate/K+ ratio, as in FMC, produced spherical cells, whereas cells remained bacillary in medium with a low bicarbonate/K+ ratio, as in Jordan medium. Manipulating this ratio in other media resulted in similar shape changes. Thus, the shape of S. mutans 10449S can be dictated by the ratio of bicarbonate to K+ in the growth medium.

Source of the hepatic microsomal trans-2-enoyl CoA hydratase bifunctional protein: endoplasmic reticulum or peroxisomes.

The present study was designed to investigate the hepatic localization of the microsomal bifunctional trans-2-enoyl CoA hydratase. Despite the low activity (less than 10%) of peroxisomal marker enzymes in isolated hepatic microsomes (acyl CoA oxidase (this study), catalase, and urate oxidase (L. Cook, M. N. Nagi, J. Piscatelli, T. Joseph, M. R. Prasad, D. Ghesquier, and D. L. Cinti, 1986, Arch. Biochem. Biophys. 245, 24-26), additional evidence in this study suggests that the microsomal enzyme is derived from peroxisomes. For example, the microsomal hydratase activity was associated with the ribosomal fractions but not with the smooth endoplasmic reticulum. In addition, when an extract of the peroxisomal enzyme was incubated with either free ribosomes or membrane-bound ribosomes, marked binding was observed with each of the fractions. Furthermore, the ease of release of the bifunctional enzyme from both free ribosomes and membrane-bound ribosomes by only KCl suggests that the bound enzyme is not a nascent protein. Labeling of liver tissue from DEHP-treated rats with rabbit immune IgG made to the purified microsomal hydratase followed by gold conjugated goat anti-rabbit IgG suggested a single subcellular site for the bifunctional hydratase--the peroxisomal organelle.

Compartmentalization of the periplasmic space at division sites in gram-negative bacteria.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.

Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope.

Cell envelopes of Salmonella typhimurium and Escherichia coli were disrupted in a French pressure cell and fractionated by successive cycles of sedimentation and floatation density gradient centrifugation. This permitted the identification and isolation of several membrane fractions in addition to the major inner membrane and murein-outer membrane fractions. One of these fractions (fraction OML) accounted for about 10% of the total cell envelope protein, and is likely to include the murein-membrane adhesion zones that are seen in electron micrographs of plasmolyzed cells. Fraction OML contained inner membrane, murein, and outer membrane in an apparently normal configuration, was capable of synthesizing murein from UDP-[3H]N-acetylglucosamine and UDP-N-acetylmuramylpentapeptide and covalently linking it to the endogenous murein of the preparation, and showed a labeling pattern in [3H]galactose pulse-chase experiments that was consistent with its acting as an intermediate in the movement of newly synthesized lipopolysaccharide from inner membrane to outer membrane. The fractionation procedure also identified two new minor membrane fractions, with characteristic protein patterns, that are usually included in the region of the major inner membrane peak in other fractionation procedures but can be separated from the major inner membrane fraction and from contaminating flagellar fragments by the subsequent floatation centrifugation steps.

Effects of fatty acids on Na/Ca exchange in cardiac sarcolemmal membranes.

Three structurally distinct amphiphiles palmitic acid, oleic acid, and palmityl carnitine were studied to determine their effects on sodium dependent calcium uptake by purified cardiac sarcolemmal vesicles (PSL). Sodium dependent calcium uptake by PSL when studied over a 20 min reaction period was composed of an initial rapid uptake (20.9 +/- 0.93 nmol/mg X 30 s, mean +/- S.E. n = 20) a plateau in calcium content (42.4 +/- 3.2 nmol/mg, mean +/- S.E. n = 20) and a slow spontaneous release characterized by a first order rate constant of 0.68 +/- 0.08/h (mean +/- S.E. n = 18). Both palmityl carnitine and palmitic acid inhibited, whereas oleic acid stimulated initial calcium uptake. All three amphiphiles shortened the time to peak calcium content, inhibited peak calcium content and increased the rate constant for calcium release. All these effects were observed at fatty acid: membrane phospholipid mole ratios of 0.67 : 1 to 1.67 : 1 for oleic acid and palmityl carnitine and 0.02 : 1 to 0.42 : 1 for palmitic acid. These effects do not reflect disruption of membrane vesicle structure and may be explained, at least in part, by amphiphile induced increases in sarcolemmal membrane ion permeability. Although amphiphile accumulation has been implicated in the pathogenesis of cellular abnormalities in the ischemic myocardium, this study has shown that large amounts of amphiphile relative to membrane lipid are required to alter sarcolemmal membrane function in vitro.

The aetiological agent of cat scratch disease.

A highly pleomorphic, gram-positive bacterium was cultured from an excised lymph node of a patient with cat scratch disease (CSD). The organism had morphological forms similar to those of the bacterium observed in Warthin-Starry stains of lymph node sections from CSD patients and may be the aetiological agent of this disease. Electron microscopic examination of lymph node sections from another patient with CSD showed organisms with morphological forms similar to those of the isolated bacterium. Biochemical and physiological analyses of this isolate suggested that it is not a commonly recognised contaminant or human pathogen and that it may be a member of the genus Rothia. This organism appears to resemble the bacterium that was identified as the aetiological agent of Parinaud's oculoglandular syndrome, a specific form of CSD, over 70 years ago.

Structure-function studies of canine cardiac sarcolemmal membranes. II. Structural organization of the sarcolemmal membrane as determined by electron microscopy and lamellar X-ray diffraction.

The morphological and ultrastructural properties of highly purified canine cardiac sarcolemmal vesicles, prepared by a modification (Colvin, R.A., Ashavaid, T.F. and Herbette, L.G. (1985) Biochim. Biophys. Acta 812, 601-608) of the method of Jones et al. (Jones L.R., Madlock, S.W. and Besch, H.R. (1980) J. Biol. Chem. 255, 9971-9980), were examined by several techniques. Thin-section electron microscopy showed predominantly intact unilamellar vesicles with little staining beyond the lipid bilayer boundaries. Freeze-fracture electron microscopy demonstrated that the majority of particles are approx. 90 A diameter and present at a density of 780 +/- 190 micrometers-2 (+/- S.D.). If it is assumed that some of these particles represent the (Na+ + K+)-ATPase, the finding that they are largely confined to the convex fracture face suggests a predominant right-side-out orientation of these sarcolemmal vesicles that is consistent with biochemical assays. The sarcolemmal membrane width measured by electron microscopy (unhydrated membrane width of 50-70 A) is consistent with the unit cell dimensions of 56-77 A determined by lamellar X-ray diffraction (hydrated membrane width). A unit cell dimension of 56-62 A was also found by X-ray diffraction for sarcolemmal lipids extracted from these preparations, indicating that the isolated sarcolemmal preparations do not contain a significant surface coat (glycocalyx). As both cardiac and skeletal sarcoplasmic reticulum membranes have a 80-100 A membrane width, these findings demonstrate that the purified sarcolemmal membrane is structurally distinct from both cardiac and skeletal sarcoplasmic reticulum. In contrast to the protein-rich skeletal sarcoplasmic reticulum membrane, which contains a single essential protein responsible for the regulation of cytosolic Ca2+ concentration, the sarcolemma is a lipid-rich membrane that contains a variety of proteins associated with many regulatory functions served by this membrane in cardiac muscle.

Spontaneous in vitro transformation of human fibroblasts.

The establishment of a spontaneously transformed tumorigenic human fibroblast line, VIP-F:T, is described. This line was developed from a primary culture of normal skin from a donor from whom a separate nontransformed fibroblast line, Pen-F2, also was established. The transformed line VIP-F:T exhibited aneuploid karyotype with a marker chromosome, showed anchorage-independent growth, and produced progressively growing tumors with morphologic characteristics of sarcoma in CD-1 (nu/nu) nude mice. The normal fibroblast line Pen-F2 exhibited diploid karyotype, showed no anchorage-independent growth, and produced no tumors in the nude mice. The spontaneously transformed fibroblast line VIP-F:T and its normal counterpart Pen-F2 will be valuable in studies of oncogene expression and in other investigations relevant to neoplasia.

Clonal analysis of cytotoxic T cell response against human melanoma.

We investigated the feasibility of generating cytotoxic T cell clones against autologous human melanoma cells using a melanoma cell line (VIP) and a spontaneously transformed autologous fibroblast line (VIP-F:T). Cytotoxic lymphocytes (CL) generated against the VIP melanoma cells in one-way mixed lymphocyte-tumor cell interactions were expanded in interleukin 2 for 2 wk. The expanded CL were cloned in limiting dilution. Two phenotypically homogeneous clones (3:1 and E.5) were obtained bearing OKT3 phenotype. Both clones expressed cytotoxicity selectively only against the sensitizing autologous target VIP. cytotoxicity assays performed with clone E.5 against the VIP target cells in the presence of autologous unfractionated lymphocytes or serum showed no modulation of autoreactivity of clone E.5. These results indicate that analysis of cellular immune response against autologous tumor cells might be feasible using autoreactive clones generated by the currently available in vitro cloning technology.

The periseptal annulus: An organelle associated with cell division in Gram-negative bacteria.

Evidence is presented that the site of cell division in Salmonella typhimurium is flanked by two circumferential zones of cell envelope differentiation, the periseptal annuli, which separate the division site from the remainder of the cell envelope. Each annulus is composed of a continuous structure in which the membranous elements of the cell envelope are closely associated with the murein cytoskeleton. The paired annuli appear early in the division process and the region between them defines a new cellular domain, the periseptal compartment, within which the division septum is formed.

Tris(hydroxymethyl)aminomethane buffer modification of Escherichia coli outer membrane permeability.

The effect of tris(hydroxymethyl)aminomethane (Tris) buffer on outer membrane permeability was examined in a smooth strain (D280) and in a heptose-deficient lipopolysaccharide strain (F515) of Escherichia coli O8. Tris buffer (pH 8.00) was found to increase outer membrane permeability on the basis of an increased Vo of whole-cell alkaline phosphatase activity and on the basis of sensitivity to lysozyme and altered localization pattern of alkaline phosphatase. The Tris buffer-mediated increase in outer membrane permeability was found to be dependent upon the extent of exposure to and concentration of the Tris buffer. The Tris buffer effects were demonstrated not to be due to allosteric activation of cell-associated alkaline phosphatase and were specific for Tris buffer. Exposure of cells to Tris resulted in the release of a limited amount of cell envelope component. Investigators utilizing Tris buffer are cautioned that Tris is not physiologically inert and that it may interact with the system under investigation.

Citrate-tris(hydroxymethyl)aminomethane-mediated release of outer membrane sections from the cell envelope of a deep-rough (heptose-deficient lipopolysaccharide) strain of Escherichia coli O8.

A heptose-deficient lipopolysaccharide strain of Escherichia coli O8, strain F515, was found to release portions of its outer membrane when cells were exposed to 10 mM citrate buffer (pH 2.75) for 30 min and subsequently exposed to 100 mM tris(hydroxymethyl)aminomethane buffer (pH 8.00). The outer membrane component release was found to be composed of protein, lipopolysaccharide, phospholipid (cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol), and alkaline phosphatase. The outer membrane component was released from the cell envelope in the absence of cell lysis, as no glucose-6-phosphate dehydrogenase activity or succinic dehydrogenase activity was detected. Morphologically, the outer membrane component appeared to consist of laminar fragments and vesicles which had an associated alkaline phosphatase activity.