- Volume 129, Issue 10, 1983

When a mutant of Azotobacter vinelandii was grown in continuous culture the amount of exocellular polysaccharide produced was dependent on both the dissolved oxygen tension (d.o.t.) and the carbon source: sucrose supported alginate synthesis in phosphate-limited medium whereas sorbitol did not. Changes in the specific activities of two of the key enzymes of alginate biosynthesis (phosphomannose isomerase and GDPmannose pyrophosphorylase), measured in extracts of cells grown with sucrose under a range of d.o.t. values, were reflected by the observed changes in alginate production; the activity of GDPmannose dehydrogenase was unchanged. A similar correlation between the specific activities of these enzymes and the rate of alginate production was observed during a transition from sorbitol to sucrose as the sole carbon ource, but in this experiment the activity of GDPmannose dehydrogenase also increased with increasing alginate production. After prolonged continuous cultivation on sucrose the mutant gradually lost the ability to produce alginate. The key enzymes of alginate biosynthesis could not be detected in extracts of this non-alginate-producing strain, which had also lost the ability to encyst. These results support the suggestions that alginate formation is controlled by derepression of key biosynthetic enzymes and that alginate plays an important role in encystment.

Glucan synthetases from cell-free extracts of Saprolegnia monoica synthesize (1 → 3)-β or (1 → 4)-β-glucans according to the assay conditions. Bacitracin and flavomycin, inhibitors of the synthesis of lipid-linked saccharides, were efficient inhibitors of glucan synthesis [90% inhibition for (1 → 3)-β-glucans, 60% inhibition for (1 → 4)-β-glucans]. These antibiotics also inhibit glycosyl transfer to different lipids. The glucan synthesis pathway does not seem to involve glycolipid intermediates and the inhibition produced by the antibiotics is probably due to their effect on enzymes rather than to depletion of lipid acceptors.

Isoelectric focusing of extracts of carrot root and apple fruit parenchyma infected by each of three isolates of Botrytis cinerea showed the presence of forms of endopolygalacturonase (PG) with isoelectric points (pI) of about 4·5 (a minor peak) and 8·3–8·8. One isolate gave the hitherto unreported peak of pI 8·8 in both tissues, whereas the other two isolates gave a peak of 8·3. The molecular weights of the PGs of pI 8·3–8·8 were estimated to be about 30000. The variation in pi probably reflects the variability of B. cinerea. No evidence of specific enzymic adaptation to host tissues was shown by the ability of PGs to cause changes in permeability of carrot and apple parenchyma.

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5′-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10–13%) and a considerable activity (38–4%) of 5′-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human eiythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.

Rhizobium trifolii lipopolysaccharide (LPS) preparations lost their receptor properties for phage 1P after deacetylation by alkali. LPS treated with acetylesterase isolated from Lactobacillus plantarum showed a simultaneous decrease in the amount of acetyl groups and phage receptor activity. The content of O-acetyl groups in the LPS of a phage-resistant mutant was significantly decreased when compared to that in LPS from the parent phage-sensitive strain 24SM. Phage 1P could cleave acetyl groups from the extracted LPS without any detectable formation of reducing groups. Binding of phage 1P to the LPS receptor was irreversible. It was demonstrated by electron microscopy that phage DNA was ejected in vitro in the presence of LPS from R. trifolii 24SM but not of that from the phage-resistant mutant.

The genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23·3 kilobases of genomic DNA. proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA. The pro operon is contiguous with the gene phoE. Hybridization experiments showed no homology between proAB of E. coli and the other regions of the E. coli genome or with the DNA of several other bacterial species.

Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing α-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 ± 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8·6: the apparent K m for leucine was 7·0 mm. Activity was totally inhibited by 1 mm-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated α-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography en DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the K m for α-ketoisocaproic acid was 4·2 mM and activity was optimal around pH 8·0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group. FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mm.

Extracellular glucosyltransferases (sucrose: 1,6-α-d-glucan 3-α-and 6-α-glucosyltransferase) of Streptococcus mutans HS6 (serotype a) were purified from the culture supernatant by DEAE-Sepharose chromatography, ConA-Sepharose chromatography and chromatofocusing. The enzymes I and II with specific activities of 6·20 and 5·86 i.u. mg−1, respectively, exhibited slightly different isoelectric points (pI 4·5 and 4·2) and the molecular weights were estimated to be 161000 and 174000, respectively, by SDS-PAGE. The enzymes had the same optimum pH of 5·5 and the same K m values of 1·3 mm for sucrose and of 83 μm-glucose equivalent for dextran T10. By double immunodiffusion test on agar, these enzymes were immunologically identical to each other. Analysis by GLC of the glucans synthesized de novo from sucrose by the enzymes (I and II) established that they were 1,6-α-d-glucans with 20 and 24·5 mol% 1,3,6-branch points, respectively. Both are therefore Afunctional enzymes.

The phosphofructokinase in crude extracts of Phycomyces blakesleeanus required the presence of ammonium salts, AMP or fructose 2,6-bisphosphate for its activity. The enzyme had slightly sigmoidal kinetics with respect to fructose 6-phosphate as substrate. It was slightly inhibited by high ATP concentrations and by citrate. Fructose 2,6-bisphosphate stimulated the Phycomyces blakesleeanus phosphofructokinase; the K m for fructose 6-phosphate decreased, the inhibition by ATP was completely relieved and the affinity for the activator ammonia was increased. AMP also stimulated the catalytic activity but there was poor co-operation with fructose 2,6-bisphosphate. Preliminary experiments showed that fructose 2,6-bisphosphate also stimulated the phosphofructokinase from Mucor rouxii, Neurospora tetrasperma and Agaricus bisporus.

When Erwinia chrysanthemi was grown in a sodium polypectate/yeast extract/salts medium adjusted with sorbitol to water activity (aw ) values of 0·990 and 0·980, extracellular pectate lyase (PL) production was repressed, whereas intracellular PL levels were not affected. Inorganic solutes (NaCl. LiCl, KCl, Na2SO4 and a NaCl/KCl/Na2SO4 mixture) at 0·990 aw strongly stimulated the intracellular levels of PL (6-to 17-fold greater than the control), whereas extracellular levels were only slightly increased. Intra-and extracellular PL levels were not affected by LiCl, NaCl and KCl at 0·995 a w. A NaCl/sorbitol mixture (0·990 a w) completely inhibited extracellular but not intracellular PL production. Lowering of the aw of cultures during mid-exponential growth phase with NaCl (0·990 a w) resulted within 30 min in a 70-fold increase in intracellular PL activity. When sorbitol was used in similar experiments, extracellular PL production was inhibited to a greater extent than intracellular levels.

Three mutations impairing penicillin production in Aspergillus nidulans, npeB, npeC and npeD, have been located on linkage groups III, IV and II, respectively, and positioned relative to other loci on these chromosomes.

Heterokaryons were formed in intra- and interspecific crosses between Fusarium sporotrichioides and F. tricinctum auxotrophs. Segregant homokaryons were evaluated for trichothecene toxin production in culture. Results were consistent with nuclear control of toxin synthesis. The sexual compatibility of auxotrophs and 30 additional F. tricinctum sensu Snyder & Hansen strains was tested. Perithecial production was restricted to crosses between Florida isolates pathogenic to English ivy (Hedera helix). The linkage of several auxotrophic markers was determined by analysis of progeny of certain crosses. No T-2 toxin was produced by sexually compatible F. tricinctum isolates.

Interspecific and intraspecific heterokaryons of Verticillium albo-atrum and Verticillium dahliae were formed by protoplast fusion or microinjection. Diploid formation was increased 1000-fold following either treatment. Combinations of auxotrophic strains which normally never form heterokaryons were obtained with both techniques. The results suggest that a variety of incompatibility factors exist in Verticillium. Compatibility and incompatibility in their strict sense did not apply to the strains used in this work, and the terms ‘heterokaryon-former’ and ‘heterokaryon non-former’ have been introduced instead. The compatibility functions are assumed to be nuclear and somehow associated with cell wall formation or components affecting the cell wall. Temperature had a profound effect on both the morphology and the nutritional marker ratio of interspecific heterokaryons. Although the frequencies of diploid formation for intc specific and intraspecific diploids were very similar, the corresponding haploidization frequencies for these diploids were markedly different. An examination of random aneuploid conidia showed that spontaneous haploidization and mitotic crossing-over occurs in interspecific diploids of Verticillium. The genetic segregation of interspecific diploids grown in the presence of haploidizing agents suggested that the two fungi are closely related and that substantial chromosomal homology may exist between them.

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.

Prolonged culturing in the laboratory has resulted in the formation of a stable derivative of the smooth Group E bacterial strain, Salmonella anatum A1, that is sensitive to both the R-core-specific bacteriophage Felix 01 and O-polysaccharide-specific bacteriophage ε15. The variant strain, designated S. anatum A1-1, exhibits a normal number of irreversible binding sites for ε15 but the relative quality and/or accessibility of those sites appears to be diminished. Infectious ε15 phage particles are released more rapidly from S. anatum A1-1 than from its parent under acidic pH conditions known to interfere with the phage DNA ejection step.

The purified lipopolysaccharide (LPS) of S. anatum A1-1 exhibits a reduced rhamnose/hep-tose ratio in chemical assays. Fractionation of this LPS on SDS-urea-polyacrylamide gels followed by silver staining reveals a narrower range of O-polysaccharide chain lengths relative to that of the parent (0 to, 20 vs. 0 to 40 repeating units, respectively).

Plasmid pHM2 contains a Bacillus subtilis spoIIA gene and is able to replicate in both Escherichia coli and B. subtilis. The plasmid was mobilized at low frequency by the E. coli F plasmid. Nearly 30 % of the mobilized plasmids contained an insert of Tn1000 (γδ). Fourteen of the inserts were in the B. subtilis DNA portion of pHM2. Of these, two adjacent inserts abolished expression of the plasmid spoIIA gene in B. subtilis. From the map positions of flanking inserts that do not abolish spoIIA gene expression, it is estimated that the gene is probably not more than 700 bp long.

Two spoIVC mutants of Bacillus subtilis were labelled with [35S]methionine either at the time of resuspension in sporulation medium or 1, 2 or 3 h later, and radioactive proteins were detected after cell extracts had been subjected to two-dimensional gel electrophoresis. The mutants completed almost all of the changes in protein synthesis that occur in the wild-type in these conditions. A heavily labelled protein was found in the mutants that has also been observed in a spoO mutant but does not occur in the wild-type.