- Volume 133, Issue 12, 1987

SUMMARY: Monoclonal antibodies (mAbs) have been raised to gonococcal outer membranes. A panel of six mAbs was identified by several criteria as reacting with outer membrane protein III (P. III). Competitive radioimmunoassays showed that the mAbs could be grouped into three pairs recognizing different epitopes on P. III. These epitopes are equally present on all pathogenic Neisseria. The mAbs demonstrated differing protective effects in model systems. Those directed against one epitope were particularly effective in protecting Chang conjunctiva epithelial cells against gonococcal challenge. mAbs against this epitope and another promoted complement-mediated bactericidal activity, while those directed against the third epitope were ineffective. Thus the biological effects of mAbs directed against P. III vary according to the epitope recognized.

SUMMARY: We have previously cloned a 44 kb fragment from the virulence plasmid of Shigella flexneri serotype 5 strain M90T which is capable of restoring invasiveness to an avirulent, plasmidless mutant. This report presents a genetic and physical analysis of Tn5 mutations in recombinant clone pHS4108. Tn5 mutagenesis allowed identification of at least five regions implicated in the entry phenotype. These regions were located on a 20 kb portion of pHS4108. Expression of the insertion mutants was studied by immunoblots using the serum of a convalescent monkey infected by S. flexneri 2a, which recognized four plasmid-associated polypeptides. We propose that the four immunogenic polypeptides, a, b, c and d, are encoded by an operon.

SUMMARY: Twenty-five strains of Renibacterium salmoninarum, from diverse geographical locations, were all hydrophobic when tested by ammonium sulphate aggregation and adherence to hydrocarbons. In addition, all strains caused haemagglutination of rabbit erythrocytes, but not of erythrocytes from salmonid fish. The hydrophobic and haemagglutinating properties were further characterized for R. salmoninarum ATCC 33209 (the type strain). Treating the bacteria with protease K or trypsin decreased hydrophobicity but had no effect on the ability to haemagglutinate rabbit erythrocytes. Heating the bacteria to 62 or 100 °C reduced hydrophobicity and removed the haemagglutinin from the cell surface. The haemagglutinin may be the heat-stable bacterial antigen extracted from the tissue of infected fish in serodiagnostic procedures.

SUMMARY: Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, λ), P5-1 (IgG2, λ), P7-1 (IgG2, λ), P8-1 (IgG2, λ) and P10-1 (IgG2, λ), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 μg per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 μg per 106 cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.

SUMMARY: The activity of extracellular polygalacturonase (PG) was strongly induced in Fusarium moniliforme by growing the fungus in a minimal medium containing pectin as sole carbon source. PG was the major protein component of the extracellular fluid whereas no detectable PG activity was found in culture filtrates of the fungus grown in a medium containing glucose as carbon source. F. moniliforme PG was purified to homogeneity by a procedure involving ammonium sulphate precipitation, carboxymethyl cellulose chromatography and preparative isoelectric focusing. The purified protein showed one protein band in non-denaturating PAGE and two major bands (molecular mass 41.5 and 45.kDa) plus two minor bands (38.0 and 48.5 kDa) in SDS-PAGE. The bands consisted of glycosylated polypeptide chains. Poly(A)-containing RNA, purified from total RNA extracted from F. moniliforme grown in PG-inducing conditions and translated in vitro in a reticulocyte cell-free translation system, produced two polypeptide chains (47 and 51 kDa) which were not present in the translation products of the non-induced poly(A)-containing RNA. The two polypeptide chains were immunoprecipitated with an IgG against F. moniliforme homogeneous PG.

SUMMARY: Evidence is presented that in Acinetobacter calcoaceticus oxidation of glucose to gluconate by the periplasmic quinoprotein glucose dehydrogenase (EC 1.1.99.17) leads to energy conservation. Membrane vesicles prepared from cells grown in carbon-limited chemostat culture exhibited (1) a high rate of glucose-dependent oxygen consumption and gluconate production, (2) glucose-mediated cytochrome reduction, (3) uncoupler sensitive, glucose-dependent generation of a membrane potential and (4) glucose-driven accumulation of amino acids. Furthermore, oxidation of glucose to gluconate by whole cells was associated with ATP synthesis. These results confirm and extend previous observations that periplasmic glucose oxidation can act as a driving force for energy-requiring processes. It is therefore concluded that the incomplete oxidation of glucose by bacteria may serve as an auxiliary energy-generating system.

SUMMARY: Streptomyces G26, isolated from compost, is a moderately thermophilic actinomycete and a facultative autotroph capable of aerobic growth on CO or on CO2/H2. Soluble extracts of CO-grown mycelium contained enzymes of the Calvin cycle together with a CO oxidoreductase; the latter was present in heterotrophically grown mycelium at only 1% of the level found in CO-grown cultures. The CO oxidoreductase of Streptomyces G26 was unique in its ability to rapidly reduce low-potential acceptors such as the viologen dyes in addition to acceptors with E o’ values between + 11 mV and +429 mV. Although complete purification of the enzyme proved difficult, evidence is presented that extracts contained only one CO oxidoreductase. The partially purified enzyme had a very low apparent K m for CO, comparable with the CO oxidoreductase from Pseudomonas thermocarboxydovorans. Highest activities were observed at pH 7.2 and 60–65 °C. The enzyme was inactivated by methanol, suggesting that it is a molybdenum hydroxylase like the CO oxidoreductases from other aerobic bacteria.

SUMMARY: The existence of energy-dependent Mn2+ influx in Aspergillus niger ATCC 11414 was demonstrated both with mycelia grown for production of citric acid and in resting mycelia suspended in ‘Good’-buffers. A specific high-affinity transport system was detected at submicromolar concentrations of 54Mn2+ which functioned independently of the transport of Mg2+ and Ca2+, but was preferentially inhibited by Zn2+, Cu2+, Cd2+. The rate of Mn2+ transport was suboptimal in the citric acid production medium (pH 3.8 or lower) as the pH optimum was found to be 5.6 in the buffer experiments.

SUMMARY: Alternaria alternata synthesizes the polyketides alternariol (AOH) and alternariol monomethyl ether (AME) during the stationary growth phase when it is grown in the dark. In the present study it is reported that these polyketides are also found in the conidia of this fungus as well as the vegetative hyphae. Young conidia were able to synthesize AOH de novo as shown by the incorporation into AOH of [3H]acetate. Since AOH synthesis in the conidia showed no need for de novo synthesis of proteins, it is proposed that hyphal cytoplasm that is able to synthesize AOH is translocated into the developing conidia.

SUMMARY: Treatment of male hyphae of the water mould Achlya ambisexualis with the female sex pheromone, antheridiol, caused them to stop extending. This response was accompanied by a marked decline in the characteristic inward current just behind the hyphal apex. After a hypha had stopped, a site of inward current developed some way behind the apex. Then an antheridial branch developed at this site. These responses only occurred in the absence of nutrients; in the presence of nutrients the hyphal extension rate and current pattern were unaffected by the addition of antheridiol.

SUMMARY: The expression of three putative terminal oxidases (cytochromes aa 3, o and d) of Bacillus cereus was studied in response to oxygen tension and the nature of the carbon source used for growth. High levels of cytochromes b and o were expressed irrespective of culture conditions. Oxygen deprivation caused a large drop in the levels of cytochromes c and aa 3. Cytochrome d appeared at low oxygen tensions, in anaerobic cultures and during slow aerobic growth on poorly utilized carbon sources (casein hydrolysate). No cytochrome d was detected during fast aerobic growth in fermentable (sucrose-containing) media or when NaNO3 was added to anaerobic cultures. Cytochromes aa 3, o and d were reduced by physiological substrates (NADH and succinate) and by ascorbate plus tetramethyl-p-phenylenediamine (TMPD). Oxidase activity using ascorbate-TMPD as electron donor was significantly more resistant to cyanide inhibition in cells containing none or trace amounts of cytochrome aa 3. Thus, the involvement of cytochromes o and d in the cyanide-resistant respiration of Bacillus cereus is suggested.