- Volume 134, Issue 9, 1988

Enzymes involved in pyruvate metabolism were assayed in crude extracts of Rhodobacter capsulatus cells grown photosynthetically with different carbon sources. Pyruvate dehydrogenase, pyruvate carboxylase, pyruvate kinase and phosphoenolpyruvate carboxykinase (ATP) were all present in extracts of cells grown on dl-lactate, whereas phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase and pyruvate, orthophosphate dikinase were undetectable in extracts of cells grown on either dl-lactate, dl-malate or acetate. Malate-grown cells and acetate-grown cells contained lower levels of pyruvate carboxylase and higher levels of pyruvate kinase than did lactate-grown cells, and malate-grown cells also contained lower levels of phosphoenolpyruvate carboxykinase. Pyruvate dehydrogenase activity was undetectable in extracts of acetate-grown cells. Two mutant strains, which were unable to grow on pyruvate, lactate or any other compound metabolized via pyruvate, were found to be deficient in pyruvate carboxylase activity, indicating that this anaplerotic enzyme is essential for growth on pyruvate or related compounds as sole added carbon source. This requirement for pyruvate carboxylase can be bypassed to some extent, however, since the mutants grew on acetate, albeit more slowly than the wild-type and after a long lag period. The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present at high levels in extracts of acetate-grown cells, both of the mutants and of several wild-type strains tested, whereas isocitrate lyase activity was undetectable in three different strains of Rhodobacter sphaeroides. This is consistent with previous suggestions that acetate is assimilated via the glyoxylate cycle in R. capsulatus but not in R. sphaeroides.

Changes in the mycolic acid composition of three cutaneous strains of corynebacteria were caused by the addition of Tween 80 to the culture medium. Gas chromatography-mass spectrometry showed that the carbon chain length and the degree of unsaturation had been affected: the levels of corynomycolic acid with 36 carbon atoms and two double bonds increased significantly.

Autophosphorylation of hexokinase PII was studied using an enzyme purified from Saccharomyces cerevisiae. Incubation of this enzyme preparation with [γ-32P]ATP and Mn2+ or Mg2+ gave a phosphoprotein of molecular mass 58000 which corresponded to hexokinase PII. d-Xylose stimulated autophosphorylation of hexokinase PII. Dilution of hexokinase PII over a 10-fold concentration range did not change the specific activity of hexokinase PII autophosphorylation suggesting that it may occur by an intramolecular mechanism.

Incorporation of 32P into Saccharomyces cerevisiae isocitrate lyase was observed after addition of glucose to a culture incubated with [32P]orthophosphoric acid. A band of 32P-labelled protein was coincident with the enzyme band when immunoprecipitates were subjected to SDS-PAGE and autoradiography. No label was found in the band corresponding to the isocitrate lyase when immunoprecipitation was done with a control pre-immune serum or in the presence of excess pure unlabelled enzyme. The incorporation of phosphate was associated with a decrease in enzyme activity. Phosphorylated isocitrate lyase was not proteolytically degraded when cells were cultured in mineral medium. The loss of protein antigenicity only took place when the yeast was grown in a complex medium containing glucose.

Bacillus thuringiensis var. israelensis crystal proteins were purified by FPLC on a Mono Q column to yield 130, 65, 28, 53, 30-35 and 25 kDa proteins. All the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. A precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. In assays against a range of insect cell lines, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LC50 values in vitro. By contrast, the activated forms of the 130 kDa and 65 kDa protoxins (53 kDa and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells, and each showed a unique toxicity profile which, in the case of the 130 kDa preparation, was restricted to Anopheles and Culex cell lines.

Summary: A role for one of many exocellular enzymes produced by Pseudomonas aeruginosa - phospholipase C (PLC) - as a prime candidate virulence factor in fleecerot dermatitis has been examined. The addition of Tween 80 in tryptose minimal medium effectively perturbed the membrane system of a field isolate of P. aeruginosa, resulting in increased production and release of a periplasmic enzyme marker, alkaline phosphatase (AP), and also of PLC. PLC activity levels in the culture supernatant were 10- to 15-fold higher in the presence of Tween than in its absence. Apart from AP, the culture medium contained little or no detectable proteolytic enzyme activity, thereby facilitating the partial purification of a haemolytic form of PLC by anion-exchange chromatography. This enzyme, when injected intradermally into the skin of sheep, elicited histopathological lesions virtually identical to those seen in naturally occurring fleecerot. In addition, serum from each of eight sheep afflicted with fleecerot contained high levels of circulating anti-PLC antibody activity when assayed by ELISA. Since these antibodies did not affect the enzymic function of PLC, it is likely that they do not bind to, or are incapable of conformational modification of, the active site.

The pigments of Corynebacterium poinsettiae were shown to exist as a carotenoprotein complex in the cell membrane and could be completely extracted by detergents. The pigments in such extracts were unstable. Sodium ascorbate (0·2%) was effective in stabilizing the pigment in the complex in Triton X-100 extracts (greater than 90% retention) and was superior to 2-mercaptoethanol, dithiothreitol, butylated hydroxytoluene, dithioerythritol and cysteine. Electrophoresis of Triton X-100 extracts in a Triton X-100-PAGE system separated the intact carotenoprotein from other membrane proteins whereas in an SDS-PAGE system the pigment was separated from the protein. All the pigments were bound to a single protein with an apparent M r of 15000. The protein was present in colourless and pigment mutants.

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4·0–5·0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.

Spontaneous revertants of the cdc30 mutation in Saccharomyces cerevisiae simultaneously regained the ability to grow and divide at 36·5 °C on glucose-containing media along with a more thermostable phosphoglucose isomerase (PGI). An independently isolated allele of cdc30 gave a similar phenotype to that previously described including temperature-sensitivity of PGI. Isoelectric focussing allowed the separation of two isoenzymes of PGI. These results all support the idea that two genes - PGI1 and CDC30 - are responsible for PGI activity in yeast. Diploid strains homozygous for the cdc30 mutation sporulated poorly in potassium acetate irrespective of whether the cells had previously been cultured at a temperature that was permissive or restrictive for cell cycle progression. This was not surprising because a strain defective in PGI would not be expected to be able to complete the gluconeogenic events of sporulation.

Phosphoprotein patterns in two mutants of Saccharomyces cerevisiae, cdc25-20(ts) and cdc25-20(ts) bcy 1, were analysed by two-dimensional polyacrylamide gel electrophoresis. Comparison with the phosphoprotein patterns of the mutants cyrl-2(ts) and bcy 1, analysed in a previous study, demonstrated not only that the CDC25 gene product is a positive element in the regulation of adenylyl cyclase activity, as suggested by recent studies, but that it is also a negative element in the phosphorylation of a 31 kDa protein (p31c and p31d), a protein whose phosphorylation is correlated with cell cycle arrest, and dephosphorylation with cell cycle initiation, respectively. Moreover, the phosphorylation phenotype of p31c and p31d suggests that the activity of the CDC25 protein is subject to feedback regulation by cAMP-dependent protein kinase, and that the CDC25 protein is a key element in an ammonium (NH+ 4) signal-response system.

Summary: A partial Sau3 AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R′ hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.

A novel klebicin, klebicin B, produced by an isolate of Klebsiella pneumoniae has been identified. It is encoded by a 5.5 kb plasmid, pKlebB-K 17/80, which is mobilized into K. pneumoniae UNF5023 by a large plasmid found in the same strain. The 5.5 kb plasmid has been cloned into the high-copy-number vector pUC19 and the restriction map of the resulting recombinant plasmid pRJ180 has been determined. Using sub-cloning and transposon mutagenesis, the klebicin B structural gene, the klebicin B immunity gene and the mitomycin C (MC) sensitivity gene (lys) present on pRJ180 have been localized. Transposon inserts which inactivated klebicin production also abolished lysis protein production encoded by pRJ180, but did not affect klebicin B immunity. Using SDS-PAGE an MC-induced polypeptide of 85 kDa was observed in cultures of K. pneumoniae UNF5023(pRJ180). This polypeptide was absent in cultures carrying plasmid pRJ180 with a Tn1000 insert which inactivated klebicin production. Analysis of the polypeptides present in the medium of Escherichia coli JM83 hsdR(pRJ180) or K. pneumoniae UNF5023(pRJ180) indicated that the 85 kDa polypeptide is specifically secreted from the producing cell. Klebicin B has been purified, using gel filtration, from a cell-free extract of K. pneumoniae UNF5023(pRJ180) which had been induced with MC. After boiling in sample buffer the purified klebicin B gave rise to two peptides on SDS-PAGE, one of 85 kDa and the other of 11 kDa. Klebicin B-resistant mutants of K. pneumoniae UNF5023 were sensitive to klebicin A, colicin B and colicin D.

Phage X-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of Escherichia coli, Salmonella typhimurium or Serratia marcescens carrying either the IncX plasmid R6K, or the unique plasmid R775. Phage X-2 differs morphologically from a previously described very broad host range filamentous phage X which also lyses plasmid R6K-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. Phage X-2 is serologically unrelated to phage X and the X-like phages IKe and I2-2. The adsorption site of the phage on the plasmid-bearing strains could not be determined but evidence implicating conjugative pili is presented.

Analysis of 213 field isolates of Botrytis cinerea and 240 ascospore isolates of its sexual form Botryotinia fuckeliana indicated that sexual compatibility of this fungus is controlled by a single mating type gene with two alleles. Most isolates were heterothallic, that is, they were self-sterile and able to produce ascospore progeny when crossed with reference strains carrying the mating type gene MAT1-1 or MAT1-2. About 16% of the field isolates and 6% of the ascospore progeny were homothallic, that is, self fertile and compatible with both MAT1-1 and MAT1-2 strains. Both mating types are widespread in nature. The close association of MAT1-1 and MAT1-2 field isolates on various hosts in several regions of Italy shows that sexual reproduction and meiotic recombination might be an important source of genetic variation in this pathogenic fungus.

Listeria monocytogenes serotypes 4a, 4b and 7, and L. ivanovii, all grown at 20 °C, were negatively stained and examined by electron microscopy. Crude extracts of the cell surface of L. monocytogenes serotypes 1/2b, 3b, 3c, 4a, 4b, 4d and 7 and of L. ivanovii (all grown at 20 °C) were examined by SDS-PAGE and Western blotting using (i) affinity-purified polyclonal monospecific antibody, and (ii) monoclonal antibody, each raised against 29 kDa flagellin of serotype 4b. No flagella were seen on serotype 7 by electron microscopy and no flagellin was detected in crude cell surface extracts of serotype 7 either in silver-stained gels or in Western blots. The monospecific polyclonal antibody detected flagellins of approximate molecular mass 29 kDa in each of the seven flagellate strains including L. ivanovii. The monoclonal antibody detected 29 kDa flagellin in serotypes 1/2b, 3b, 4a, 4b and 4d, but not the flagellins of serotype 3c or L. ivanovii, which had a slightly lower molecular mass. Following prolonged electrophoresis of crude flagellar extracts the 29 kDa complex was resolved into three closely migrating bands. In a heterologous system using serotype 1/2b crude flagellar extract, all three bands were detected using the polyclonal antibody whereas only two bands were detected by the monoclonal antibody. It is concluded that polyclonal anti-flagellin antibodies are not useful tools with which to distinguish serotypes of L. monocytogenes sensu lato in immunoblotting, but that differences can be determined using a monoclonal antibody directed against particular components of the flagellar complex. These differences did not fully correspond to those anticipated from results of agglutination tests.

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.