- Volume 133, Issue 9, 1987

SUMMARY: cDNA clones representing eight mRNAs expressed abundantly in the dikaryon of Schizophyllum commune at the time of fruiting, but very low in the progenitor monokaryons, were used to estimate the concentrations of these mRNAs in cultures of the dikaryon and monokaryon growing under conditions that suppress the formation of fruit-bodies. Such conditions as high atmospheric carbon dioxide and continuous darkness in surface cultures or growth in suspension cultures generally lower the concentrations of these mRNAs in the dikaryon but do not bring them down to the very low levels of these mRNAs in the monokaryons. Therefore the expression of these mRNAs, which depends on the dikaryotic condition, may be required but is not sufficient for the occurrence of fruiting.

SUMMARY: Escherichia coli strains lacking the terminus region of the chromosome (min 29-36) due to an IS10-promoted deletion did not grow well in rich medium; they also did not grow on fumarate minimal medium because fumAC (min 35·7) is deleted. Strains with secondary mutations that partially suppress the deletion phenotype displayed healthier growth on rich medium and grew on minimal fumarate medium. These suppressor mutants had an IS10 insertion just upstream of the fumB structural gene (min 93·4). A strain with a Tn10 insertion at this location was constructed and used to delete nonessential fumB; fumB deletion mutants grew well on both rich and minimal fumarate media.

SUMMARY: A monoclonal antibody (Tq-1) that interacts with the phosphorylcholine (PC)-bearing antigens of Trichophyton quinckeanum was produced by fusion of myeloma cells (JKAg-8) with spleen cells of BALB/c mice immunized with an alum-precipitated fraction of T. quinckeanum cytoplasmic antigen. It was characterized as an IgM class antibody by immunodiffusion using anti-Ig heavy chain specific reagents, ELISA using immunoglobulin-specific peroxidase-conjugated antibodies, and by gel filtration chromatography; it showed high affinity for Staphylococcus aureus protein-A. Interaction of Tq-1 with PC-like antigens of T. quinckeanum was demonstrated by inhibition studies using ELISA, immunoblotting, immunoprecipitation and immuno electron microscopy techniques. The binding activity of Tq-1 antibody with a range of dermatophyte proteins was completely inhibited by prior incubation with PC hapten. Moreover, dermatophyte antigens reacting with the monoclonal antibody reacted strongly with sera from chronically infected mice. Dermatophyte antigens derived from both young (24 h) and old (20 d) cultures reacted with Tq-1 and this binding was inhibited by PC, suggesting that Tq-1 target antigen PC appears at an early stage during fungal growth and remains throughout its life.

SUMMARY: The presence of circulating antibodies in the sera of patients infected with either Trichophyton concentricum or Trichophyton rubrum, and in the sera of BALB/c mice chronically infected with Trichophyton quinckeanum, was determined by ELISA. High levels of antibody to dermatophyte cytoplasmic antigens were detected both in infected humans and in mice. Partial inhibition of this reaction was observed by pretreatment of the sera with the hapten phosphorylcholine (PC). Moreover, antibodies were shown to have some reactivity with PC when tested by ELISA against PC conjugated to bovine serum albumin. Significant levels of circulating antigen were detected in patients with T. concentricum and T. rubrum infections, but not in uninfected subjects, by an immunoradiometric assay using a monoclonal antibody, Tq-1, which reacts with the PC-like epitopes of dermatophytes. It is possible that this dermatophyte antigen may play a role in modulating the cell-mediated immune responses, which would appear to be defective in most patients with these chronic forms of dermatophytosis.

SUMMARY: Hybrid cell lines were derived which produced monoclonal antibodies directed against gonococcal outer membrane protein IA. One antibody, SM101, recognized 24 P.IA-expressing strains out of 25 tested - the rest exhibited relatively less cross-reactivity. Competitive radioimmunoassays revealed that each antibody could effectively inhibit binding of the others, suggesting close proximity of the epitopes recognized. The antibodies were used in assays in vitro to investigate their potential efficacy in protection against gonococcal infection. In a cytotoxicity assay, the antibodies afforded some protection to epithelial cells challenged with gonococci. They were very effective at bactericidal killing in the presence of complement and, in addition, were opsonic for homologous and heterologous strains. The cross-reacting antibody, SM101, was one of the most effective in both assays. The results show that the conserved epitope on P.IA recognized by antibody SM101 is potentially an effective target on the gonococcal surface for immunoprophylaxis.

SUMMARY: Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 × 102 LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 °C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.

SUMMARY: Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

SUMMARY: The gonococcal chromosome contains a sequence of closely linked genes (for example, sac-1, sac-3, nmp) known or presumed to affect cell envelope structure and which appear to influence susceptibility of gonococci to killing by normal human sera (NHS). Previous work has shown that the serum-resistant isolate FA19, and FA899, a serum-sensitive transformant of FA19, differ in outer membrane protein I (PI) and at the sac-3 genetic locus. However, the sac-3 locus is separable from changes determined by nmp-3, the gene determining PI species. We found that FA 19 and FA899 differ in lipopolysaccharide (LPS) molecular size and in reactivity with a monoclonal antibody which recognizes an LPS (L8) epitope. To address the question of whether the changes in LPS were due to the sac-3 locus, we constructed new transformants of FA 19 using donor DNA prepared from FA899. The new transformants could be divided into three groups: (1) those identical to FA19 in serum resistance (>90% survival at 120 min), in LPS molecular size and in expression of the L8 epitope; (2) those identical to FA899 in serum sensitivity (100% killed at 30 min), in LPS molecular size and in lack of expression of the L8 epitope; (3) those significantly killed by 50% NHS at 120 min, whose LPS molecular size was greater than that of FA 19 but less than that of FA899 and which did not express the L8 epitope. Except for PI there were no differences in other outer-membrane proteins (e.g. PII, PIII, H.8) among these transformants. Studies of PI using monoclonal antibodies which distinguish between PI serovars confirmed that changes in PI were separate from changes in serum resistance or alteration in LPS chemotype. These data indicate that a genetic locus involved in LPS structure appears to be identical with the sac-3 locus, which influences serum susceptibility in Neisseria gonorrhoeae.

SUMMARY: Two chemically different O-polysaccharides, a low molecular mass form of LPS and core LPS produced by chemostat-grown E. coli O157, were analysed by SDS-PAGE, silver staining and immunoblotting. The reactivities of the different O-polysaccharides with antisera prepared against E. coli O157 grown in batch culture, Salmonella O30 or Brucella abortus were very similar, showing that the O-polysaccharides share at least some antigenic determinants. The reactions of the low molecular mass LPS with the antisera indicated it was semi-rough LPS having one repeat unit of the O-polysaccharide attached to core LPS.

SUMMARY: Invertase synthesis, regulation and secretion in the wall-less slime variant of Neurospora crassa was studied. Unlike the wild-type, synthesis of the enzyme was not repressed by glucose. This effect was not related to the os mutation harboured by the slime strain, nor to the phenotypic absence of a cell wall. Three molecular forms of extracellular invertase, which varied in size, were detected in the slime strain. These forms were interconvertible, with the equilibrium in favour of the larger form. Polypeptide analysis of the three separated forms revealed that all contained the same glycoprotein with an M r of 97000. This was completely deglycosylated by treatment with endo-β-N-acetylglucosaminidase H (Endo H) to a polypeptide with an M r of 72000. It was concluded that the three interconvertible forms correspond to the monomeric, dimeric and tetrameric states of the enzyme. Three similar forms of invertase, albeit of slightly different electrophoretic mobility, were found in cell-free extracts, cell walls and spent culture medium of the wild-type strain. After Endo H treatment, analysis showed that these forms contained a polypeptide that was equally reactive against anti-Saccharomyces cerevisiae antibodies, and had the same M r, as the polypeptide produced by the slime strain.

SUMMARY: The distribution of the arginine succinyltransferase pathway was examined in representative strains of Pseudomonas and related bacteria able to use arginine as the sole carbon and nitrogen source for growth. The arginine succinyltransferase pathway was induced in arginine-grown cells. The accumulation of succinylornithine following in vivo inhibition of succinylornithine transaminase activity by aminooxyacetic acid showed that this pathway is responsible for the dissimilation of the carbon skeleton of arginine. Catabolism of citrulline as a carbon source was restricted to relatively few of the organisms tested. In P. putida, P. cepacia and P. indigofera, ornithine was the main product of citrulline degradation. In most strains which possessed the arginine succinyltransferase pathway, the first step of ornithine utilization as a carbon source was the conversion of ornithine into succinylornithine through an ornithine succinyltransferase. However P. cepacia and P. putida used ornithine by a pathway which proceeded via proline as an intermediate and involved an ornithine cyclase activity.

SUMMARY: The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.

SUMMARY: Oscillation of the activities of gluconeogenic enzymes (malate dehydrogenase, phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase) was observed during the cell cycle of chemostat cultures of Saccharomyces cerevisiae. Since ethanol is released by the cells at the beginning of the division cycle, its effect on enzyme expression was determined. Pulsing ethanol to a synchronously dividing yeast culture led to a prolongation of the metabolically active phase as indicated by the course of oxygen uptake and carbon dioxide production rates (concomitant ethanol and glucose assimilation). Enzyme activities also remained elevated as long as ethanol was available to the cells. After a substrate shift from glucose to ethanol during cell division, ethanol was used without a lag phase and enzyme induction increased from the level reached at the point of the substrate change. The data confirmed that the small amount of ethanol produced when the cells begin active reproduction acts as an inducer of gluconeogenic enzymes.

SUMMARY: Pseudomonas putida MT20-3 carrying the Klebsiella pneumoniae nif plasmids pRD1 or pMF250 showed highly O2-sensitive aerobic acetylene reduction on low-N pyruvate or glucose agar. This finding confirms unequivocally that K. pneumoniae nif can be expressed in an obligate aerobe.

SUMMARY: The probe 8-anilino-1-naphthalene sulphonate (ANS) showed increasing fluorescence with Bacillus subtilis metC lyt-2 cells taken from exponentially growing cultures treated with antibiotics that inhibit cell-wall peptidoglycan synthesis. This increase was due to the probe reaching hydrophobic cell constituents, probably membranes, and started within 30 min of the addition of the antibiotics. This corresponded to the time at which membrane function had been shown to be damaged. The increased fluorescence of the cells with ANS persisted after removal of the antibiotics.

SUMMARY: Gametes and zoospores of Allomyces macrogynus and Allomyces arbuscula were repelled by H+, K+, NH4 + or Na+ as well as by Ca2+, Mg2+ and La3+. This negative chemotaxis, which was monitored with a chemotaxis bioassay, occurred no matter what anionic counter-ion was used. The use of a swim-out assay showed that repulsion was similar for all cations and resulted in a band of zoospores that migrated farther into the tube with time. When zoospores allowed to adapt to 10-fold dilutions of 10 mm-KC1 were challenged with 10 mm-KC1 and their motile behaviour monitored in swim-out assays, it was found that the higher the initial concentration of KC1 incubated with the cells, the less repulsion occurred when these cells were challenged with 10 mm-KC1. In addition when zoospores were incubated in a 10 mm solution of one repellent (i.e. KC1, NH4C1 or CaC12) and then challenged in swim-out assays with a 10 mm solution of another repellant (i.e. KC1 or HC1) to determine if the cationic sites of interaction were common or different, the results indicated that the sites were the same since challenging with another cation did not cause zoospore repulsion. The repulsion mechanism was studied further by mixing 10 mm-KC1 and the male pheromone attractant sirenin. These experiments, using the chemotaxis bioassay, showed that in a mixed population of male gametes and zoospores, the male cells were attracted towards the source of the sirenin while all the zoospores were repulsed by KC1. Thus it appears that the mechanism for attraction is different from repulsion or that the attraction mechanism can override the repulsion behaviour.

SUMMARY: A chemically defined medium for growing mass cultures of Tetrahymena was developed. The growth characteristics and the effects of variations in the culture conditions and medium constituents were studied. The results show that Tetrahymena can be grown in a completely defined medium with rates and yields nearly comparable to those obtained in non-defined media. In the defined medium, K+ but not Na+, was indispensable for growth; unlike in media containing proteose peptone, the growth rate was independent of the presence of insoluble Fe3+ complexes, but was more dependent on culture temperature. Although Tetrahymena has an absolute requirement for preformed purine and pyrimidine bases in addition to several amino acids, its proliferation and growth were primarily under amino acid control.

SUMMARY: A method for the production of protoplasts of Clostridium acetobutylicum strain N1-4080 utilizing lysozyme and penicillin G was optimized to yield about 100% protoplasts. Treatment of 109 cells left fewer than 102 that were viable and osmo-resistant. With a mutant strain deficient in autolytic activity, about 1% of the protoplasts regenerated walled cells, whereas most gave rise to stable L-form colonies. Addition of sodium polyanethole sulphonate to the medium improved the regeneration frequency by at least a factor of 10, probably by somehow inhibiting the residual autolytic activity of the strain.

SUMMARY: The fatty acid composition of Streptococcus sanguis NCTC 7865 was not altered by changing the cation composition (Na+/K+) of the growth medium; glucosyltransferase (GTF; EC 2.4.1.5) also remained constant. In contrast, fructosyltransferase (FTF-S; EC 2.4.1.10) production was reduced by at least 50% in medium with a high Na+ concentration. Growth in the presence of ionophores (gramicidin, nigericin or valinomycin) resulted in an increased proportion of saturated fatty acids, principally octadecanoic acid (C18:0), while the proportion of unsaturated fatty acids, predominantly octadecenoic (C18:1) and hexadecenoic (C16:1) acids, decreased. GTF-S production was reduced in the presence of ionophores whereas FTF-S production was completely abolished. Tween 80 significantly increased both GTF-S production and the proportion of unsaturated fatty acids in the cytoplasmic membrane; FTF-S production was unaltered by Tween 80. The production of GTF-S was inversely proportional to the C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane. It was concluded that FTF-S production is directly influenced by protonmotive force (pmf), whereas GTF-S production is affected more by the physical properties of the cytoplasmic membrane, in particular its fatty acid composition. However, as perturbations in pmf generation can lead to variations in membrane fatty acid composition it can be argued that pmf indirectly influences GTF production by changing the saturated:unsaturated or C18:0:C18:1 fatty acid ratio of the cytoplasmic membrane.

SUMMARY: The growth rate of Dunaliella salina was optimal in the presence of 2·5 to 5% (w/v) NaCl and growth continued up to 30% NaCl. The carotene to chlorophyll ratio gradually increased from about 1·1 at 2·5% NaCl to 7·7 at 30% NaCl and the chloroplast showed some degeneration; however, with 30% NaCl, intact thylakoids were still recognized. The total lipid content of the cells decreased with increasing salinity but the major lipid classes in all extracts were monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols and phosphatidylglycerols. Considerable proportions of diacylglycerotrimethylhomoserines were also present, whereas the proportions of phosphatidylcholines and phosphatidylethanolamines were relatively low. Increasing the salinity of the medium resulted in a large increase in the proportions of constituent linolenic acid (18:3) both in total lipids and in the galactolipids and of palmitoleic acid (16:1) in phosphatidylglycerols. The results are discussed in view of the probable relation of chloroplast lipids to photosynthetic activity, and the fact that the osmoregulator glycerol is produced via photosynthesis.