- Volume 132, Issue 1, 1986

SUMMARY: The fatty acid content of Bordetella pertussis endotoxin has been estimated by several methods. Expressed as 3-hydroxytetradecanoic acid, it was 0·74 μmol (mg lyophilized material)-1, 0·38 μmol being ester-bound, and 0·32 μmol in amide linkage. Reported molar ratios of ester-bound to amide-bound fatty acids in endotoxins of various bacterial species range from 2·4 to 2 in B. pertussis, to 5 to 2 in Salmonella minnesota; according to these figures large differences must exist in the degree of substitution, and the substitution pattern of the glucosaminyl-β-1,6-glucosamine unit present in the hydrophobic region of endotoxins. When fatty acids, released by acid and alkaline hydrolyses of the B. pertussis endotoxin, were extracted into chloroform, unidentified chromogenic substances appearing in the extract interfered with their colorimetric estimation; no interference was observed when hexane was used instead of chloroform.

SUMMARY: The changes in morphology of Penicillium expansum during freezing and thawing were observed under a light microscope fitted with a temperature controlled stage. At slow rates of cooling (< 15°C min-1) extensive shrinkage of the hyphae was observed. Cooling at rates greater than 50°C min-1 induced intracellular ice formation in all hyphae. At rates intermediate between 15 and 50°C min-1 shrinkage and intracellular nucleation were observed, occasionally within the same hyphae. These data are examined in terms of the osmotic stresses that cells are exposed to at different temperatures during freezing and the biophysics of injury at different rates of cooling is discussed.

SUMMARY: Before fusion, hyphae of the basidiomycete Phanerochaete velutina responded similarly to one another when grown on a cellophane membrane, regardless of whether they were genetically the same or different. Long-range (up to 250 μm) curvature (homing) to specific sites in the lateral wall of recipient compartments often occurred in fusions involving main hyphal apices. Induction of tip outgrowth from lateral walls was most evident before short-range, tip-to-tip fusions resulting in H-connections between main hyphae. Spitzenkörper (apical bodies) became aligned with receptive sites before directed growth. A period (about 5–20 min) of expansion of the contact region preceded formation of a fusion pore. Fusions were abundant in the vicinity of septa, but never observed between tips of main hyphae which repelled one another. In fusions involving hyphae from the same thallus or of mating-incompatible homokaryons, the fusion pore usually enlarged until it occupied virtually the entire contact area. Except in the case of clamp-connexions, nuclear interchange was followed by aggregation and division in the pore region before septum formation. Between different heterokaryons, the fusion pore never expanded fully, nuclei were rarely exchanged, and rapid cytoplasmic lysis and vacuolation occurred. Lysis also occurred sooner or later between sexually compatible homokaryons; only in a few cases was dolipore dissolution and nuclear migration observed.

SUMMARY: We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.

SUMMARY: Deletion mutants of recombinant plasmids encoding the KS71B fimbrial antigens of the uropathogenic Escherichia coli strain KS71 (O4:K12) were constructed. The effects of these mutations were tested by transforming the mutated plasmids into non-fimbriated E. coli HB101 cells and testing the transformants for fimbriation and haemagglutination. A deletion transcriptionally upstream from the fimbrial subunit gene increased the expression of KS71B fimbriae. Deletion of the fimbrial subunit gene resulted in non-fimbriated but haemagglutinating transformants, whereas a deletion 6 kb transcriptionally downstream from the subunit gene resulted in non-haemagglutinating but fimbriate transformants, indicating that fimbriation and haemagglutination were genetically separable. We also present evidence suggesting that the fimbrillin and haemagglutinin are physically associated in the wild-type KS71 strain.

SUMMARY: A general method is presented that enables viral mutants to be isolated in any required region of their genome. An application of the method is reported relating to the isolation of conditional lethal mutants in the ‘semi-essential’ region of phage Mu. Some properties are described of one mutant found in this way, Mucts62 SEEam8 Apl, which is typical of a class of mutants that were isolated.

SUMMARY: We have developed a convenient system for genetic analysis of Salmonella typhi exploiting the properties of the mutator phage Mu. In spite of the fact that wild-type Salmonella typhi strains do not allow Mu to form plaques on them, we have shown that these strains are actually sensitive to the phage. It proved possible to use Mu to induce mutations and to promote intra- and interspecific genetic transfer, without having to introduce the phage into the bacteria by means other than infection. Furthermore, we isolated Salmonella typhi derivatives on which Mu formed plaques, and studied the behaviour of Mu in these and wild-type strains.

SUMMARY: A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage λ1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4·7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660–700 units (mg protein)-1], pI value (8·5), and subunit molecular weight (32 x 103). Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2–4 fold higher than the current production strain).

SUMMARY: The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and fdp-74 on the B. subtilis chromosome (a tentative map order of gnt-10, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-10 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4·5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage ϕ 105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2·4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon.

SUMMARY: lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA + and pho regulatory genes, or a transducing ϕ 80 phoA + phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with ϕ 80 phoBin phoA + phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS + lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.

SUMMARY: Surface charge and hydrophobicity of Treponema pallidum have been investigated in relation to phagocytosis by human polymorphonuclear leucocytes (PMNs) in vitro. The treponemal surface was relatively hydrophobic and negatively charged but despite these properties, phagocytosis, as assessed by luminol-enhanced chemiluminescence, was minimal in the absence of serum. Preopsonization of bacteria with serum reduced surface hydrophobicity but promoted phagocytosis, suggesting that specific immune mechanisms may be more important in controlling phagocytosis of T. pallidum in vitro than non-specific surface properties. T. pallidum evoked a much weaker chemiluminescence response from PMNs than the non-pathogenic treponeme Treponema phagedenis biotype Reiterii even though similar numbers of bacteria were phagocytosed, suggesting differences in the reactivity of the surface components of the two organisms toward PMNs. The reactivity of T. pallidum towards PMNs could be increased by removal of the bacterial outer membrane by Triton X-100 treatment. These observations reinforce the suggestion that the outer surface of T. pallidum is inherently inert.

SUMMARY: Indicator plates containing eosin, methylene blue, glucosamine and proline were used to select mutants of Candida albicans impaired in the utilization of glucosamine. One such mutant, strain hOG298, grew on glucosamine at a slower rate than the parent and was severely impaired in growth on N-acetylglucosamine. The mutant was unable to express the first three steps in the N-acetylglucosamine pathway: viz the permease, N-acetylglucosamine kinase and N-acetylglucos-amine-6-phosphate deacetylase. Glucosamine-6-phosphate deaminase was, however, induced by N-acetylglucosamine. The mutant still possessed a constitutive uptake system and kinase activity for glucosamine but glucosamine neither increased the glucosamine kinase activity nor induced N-acetylglucosamine kinase. These findings accounted for the decreased growth rate on glucosamine. The parent strain formed germ-tubes in N-acetylglucosamine or 4% (v/v) serum but the mutant formed germ-tubes only in serum.

SUMMARY: Pseudomonas sp. isolate UV3, when added to conidial suspensions of Colletotrichum acutatum, stimulated germination and appressorium formation on glass slides and on host surfaces. A siderophore (S a ), purified from low-iron culture of the bacteria, stimulated germination and appressorium formation to a significantly greater extent than the bacterial cells alone. The fully chelated siderophore (S a Fe) and free ferric iron were inhibitory in vitro to germination and appressorium formation but inhibited only the latter in vivo. The S a is compared with nutrients at similar concentrations, and the role of the phytoplane microflora and associated siderophores is discussed in relation to fungal infection.

SUMMARY: The permeability parameter (C) for the movement of cephalosporin C across the outer membrane of Pseudomonas aeruginosa was measured using the widely accepted method of Zimmermann & Rosselet. In one experiment, the value of C varied continuously from 4∙2 to 10∙8 cm3 min−1 (mg dry wt cells)−1 over a range of concentrations of the test substrate, cephalosporin C, from 50 to 5 μm. Dependence of C on the concentration of test substrate was still observed when the effect of a possible electric potential difference across the outer membrane was corrected for. In quantitative studies of β-lactam permeation the dependence of C on the concentration of β-lactam should be taken into account.

SUMMARY: The capacity of the detergent-degrading bacterium Pseudomonas C12B to grow on, and oxidize, a range of primary and secondary alcohols of chain lengths from C2 to C16 was examined. The organism was able to grow on most primary alcohols tested, and especially well on the C10–C12 homologues. Of several secondary alcohols, C11–C13 homologues of alkan-2-ols supported good growth, as did some long-chain symmetrical or near symmetrical alcohols. There was constitutive synthesis of primary and alkan-2-ol dehydrogenase activities, enabling the organism to oxidize primary alcohols and alkan-2-ols up to at least C11. Experiments with resolved d- and l-octan-2-ols suggested that the constitutive alkan-2-ol dehydrogenase activity was specific for d-isomers. Growth on secondary alcohols induced synthesis of l-alkan-2-ol dehydrogenase(s) and also greatly increased the capacity of extracts to oxidize racemic alkan-2-ols in the range C6–C11. An enzyme with high activity towards symmetrical alcohols was also induced by growth on secondary (especially symmetrical) alcohols. Collectively, the various alcohol dehydrogenase activities detected would enable Pseudomonas C12B to oxidize all the alcohols liberated from mixed alkyl sulphate detergents by the organism’s complement of two primary and three secondary alkylsulphatases.