- Volume 133, Issue 7, 1987

SUMMARY: Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of α-glucosidase, α-amylase and β-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.

SUMMARY: The life cycle of an anaerobic fungus isolated from the rumen of sheep was studied and was found to be similar to that of the chytrids. The organism was monocentric. At 39 °C the duration of the life cycle varied from about 26 to 32 h. The zoospores were spherical or oval in shape and were polyflagellate (8 to 17 flagella per zoospore). During the first 6·5 h of growth there was a rapid development of an extensive, non-septate, highly branched rhizoidal system; during this period the ‘main’ rhizoid increased in length exponentially with a doubling time of 2·49 h. Between 6·5 to 9·5 h after inoculation, the rate of extension of the main rhizoid declined, and no further extension occurred after 9·5 h. The main rhizoid increased in width at its base from 2·2 to 15·0 μm during the first 13 h after inoculation, indicating that intercalary wall growth occurred. Nuclei were occasionally observed in the rhizomycelium using DAPI (4′,6-diamidino-2-phenylindole) staining. Zoosporangia varied in shape from spherical to columnar, and some columnar zoosporangia were observed to become spherical. The zoosporangium initially increased in volume at an exponential rate with a doubling time of 1·56 h. Between 14 to 20 h after inoculation, growth of the zoosporangium decelerated and little growth occurred after 20 h: the zoosporangium had a final volume of 2·5 × 105μm3. At about 21 h after inoculation, a septum was formed at the base of the zoosporangium, delimiting it from the rhizoidal system. The formation of this septum was correlated with the cessation of zoosporangial growth and the onset of zoosporogenesis. After zoosporogenesis, zoospores (about 88 zoospores per zoosporangium) were liberated through a pore formed in the zoosporangial wall opposite the main rhizoid. About 3 h after zoospore release the rhizoidal system became less refractile, suggesting that autolysis had occurred. Growth of the isolate was inhibited by nikkomycin (an inhibitor of chitin synthase), but not by amphotericin B or nystatin (antibiotics which bind to sterols).

SUMMARY: The establishment of different bacterial populations and fungi in the rumen was investigated in lambs reared under different conditions of diet and management. The rumen was rapidly colonized by an abundant microflora after birth. By day 2 strictly anaerobic bacteria predominated (109c.f.u. ml−1); their population increased slightly during the first week of life and again when the animals began to ingest solid feed (3 weeks). The composition of the microflora in the 2-10-d-old lambs was quite different from that of adult sheep. The aerobic and facultatively anaerobic bacterial count was 10-100-fold lower than the strictly anaerobic count during the first week, and decreased steadily afterwards. In flock-reared lambs, cellulolytic and methanogenic bacteria appeared very early after birth (3-4 d). At the end of the first week the population of these bacteria reached a level close to that generally observed in a mature rumen. The cellulolytic bacteria were also able to survive in the rumen of lambs fed cow's milk exclusively. Anaerobic fungi appeared later (8-10 d). They were present in all lambs studied until 3 weeks of age, and then disappeared in most of them when a solid diet was given.

SUMMARY: The Bacillus subtilis α-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis α-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.

SUMMARY: The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated. Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion. Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid. m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate. Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate. Similar results were obtained with P. putida strains MT20 and MT84, which contain different TOL plasmids. We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.

SUMMARY: Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for the tetracycline-resistance plasmid pT 181.

SUMMARY: Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.

SUMMARY: Strains carrying only one species of pock-forming plasmid, designated as pSK3*, were isolated from two different derivative strains of Streptomyces kasugaensis MB273 which contained three species of plasmids, pSK1, pSK2 and pSK3. Single and double digestion of pSK3*with seven restriction endonucleases yielded fragments identical with those of pSK3 and assignable to those obtained from pSK1*and pSK2*. In particular, digestion with Bg/II alone or in combination with other restriction endonucleases afforded the same size fragments as those of pSK1*and pSK2*. Strains containing pSK3*induced pocks on lawns of strains carrying pSK1*or pSK2*and resisted pock formation by the latter strains. Therefore, it was concluded that pSK3*was a pSK3 derivative with elevated pock-forming ability and represented a composite plasmid consisting of two elements, pSK1*and pSK2*, without any loss of their plasmid functions. Deletion derivative plasmids constructed from the Bg/II fragments of pSK3*provided evidence supporting the above conclusion. Pock formation by a pSK3*-containing strain against strains carrying pSK1*, or pSK2*or no plasmid accompanied the transfer of pSK3*from the former to the latter. Segregation of pSK1*and pSK2*from pSK3*was observed in mycelium from pocks caused by pSK3*-containing strains and on subculture of pSK3*-containing strains.

SUMMARY: Deletion derivatives and recombinants of the plasmid pSK3*, which is a cointegrate of pSK1*and pSK2*in Streptomyces kasugaensis, were constructed and analysed for their ability to transfer and ‘pock’ on strains carrying pSK1*or pSK2*. Various deletions in the pSK1*and/or pSK2*regions of pSK3*were grouped into nine classes on the basis of their pock-forming ability and pock resistance. Analysis of these deletions and insertions provided tentative locations of DNA regions for two pock-resistance determinants (Porl and Por2), two pock-forming determinants (Poc1 and Poc2) consisting of plasmid transfer and spread determinants (Tra/Spr), and two replication determinants (Rep1 and Rep2) corresponding to the pSK1*and pSK2*regions of pSK3*. In particular, the Por2 function in the pSK2*region was determined to be located in a 1·35 kb segment.

SUMMARY: The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6·8 to 8·0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.

SUMMARY: Murine monoclonal antibodies (Mabs) were raised against two outer-membrane-associated polypeptides of Treponema pallidum (47 and 44 kDa). Three Mabs against each polypeptide were investigated further and only those directed against the 44 kDa polypeptide were demonstrated to have immobilizing activity. The specificity of the Mabs for T. pallidum was determined by Western blotting procedures and the surface association of the antigens was inferred by immunogold electron microscopy. The clear distinction between these two polypeptides in their biological activity could help to explain the pathobiology of syphilis infections as the 47 kDa antigen has been shown to be associated with the outer membrane of this organism. Inactivity of such a surface-located protein in antibody-mediated anti-treponemal mechanisms could account for the observed ability of this organism to survive in the face of strong antibody responses in infection.

SUMMARY: A suitable immunization protocol for the stimulation of a murine antibody response to the axial filament polypeptides of Treponema pallidum was established. A range of monoclonal antibodies (Mabs) specific for different epitopes of the major axial filament polypeptide (37 kDa) was generated which demonstrated diversity in their ability to react with other treponemal species. Immunogold electron microscopy located the 37 kDa antigen on the surface of the axial filament structure. The early appearance of specific antibody to this polypeptide in infected man and rabbit indicates that such Mabs are potentially useful for the diagnosis of early syphilis.

SUMMARY: Binary suspensions of bacteria isolated from the gastric juice of achlorhydric patients were used to determine conditions which favour nitrite accumulation during nitrate reduction. Suspensions of Veillonella parvula and Haemophilus parainfluenzae accumulated nitrite during nitrate reduction in the absence of nitrite-reducing Neisseria subflava or Streptococcus sanguis. The maximum concentration of nitrite that transiently accumulated decreased predictably as the ratio of nitrite-removing bacteria to nitrite-accumulating bacteria increased. This ratio, but more importantly the bacterial density, determined the duration of nitrite accumulation. These results are correlated with the previously reported tendency of nitrite to accumulate in the gastric juice of hypogammaglobulinaemic and pernicious anaemic patients, and with the extremely high incidence of gastric cancer in the two groups.