- Volume 131, Issue 11, 1985

Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, a-elastin, fibrinogen, gelatin, haemoglobin and a-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75–9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, l-(l-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 °C, whereas those from virulent strains lost all activity at 60 °C.

Summary: Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The puromycin-inactivating enzyme (puromycin N-acetyltransferase) from S. alboniger also N-acetylates O-demethyl-puromycin. This finding indicates that in certain antibiotic-producing organisms the antibiotic-inactivating enzymes may play a role in self-defence against toxic precursor molecules.

Summary: Mannitol dehydrogenase (EC 1.1.1.138) has been purified to homogeneity from fruit bodies of Agaricus bisporus. M r values of 115000 were determined by gel filtration and 130000 by rate zonal ultracentrifugation. The sedimentation coefficient is 6.5S. The native protein is composed of four subunits of M r 29000. The enzyme is specific for NADP, and shows low activity with sorbitol. Normal Michaelis-Menten kinetics are exhibited for both mannitol and NADP, giving K m values of 16.2 mm and 36μm respectively at pH 7.0. The K m value for NADPH is 38.5μm and that for fructose approximately 1.2 m. The V max is 591 μm min−1 (mg protein)−1 for mannitol synthesis and 5μmol min−1 (mg protein)−1 for fructose synthesis at pH 7.0. Inhibition of fructose synthesis by NADPH is stronger than inhibition of mannitol synthesis by NADP. The results are discussed with respect to the control of enzyme activity under physiological conditions.

Summary: Ornithine carbamoyltransferase and argininosuccinase, two enzymes involved in arginine synthesis, are regulated by cross-pathway amino acid control in Neurospora and show derepression in response to limitation of any one of a number of amino acids. The effects of varying the severity of arginine limitation upon the synthesis of these enzymes, in mycelial cultures of an arginine auxotrophic strain, are reported here. Derepression occurred at arginine concentrations sufficient to allow normal rates of protein accumulation, leading to increases of not more than fourfold in the absolute rate of enzyme synthesis. On the other hand, differential rates of enzyme synthesis increased progressively up to 20-fold or more under extreme conditions of arginine limitation that also limit net protein synthesis. The major part of the derepression response thus occurred at arginine concentrations that allowed low net rates of protein synthesis. The physiological significance of this is not yet understood. Our evidence suggests that these responses were mediated entirely through the cross-pathway control system, and may not be untypical (allowing for variations in magnitude) of derepression resulting through this mechanism in Neurospora.

Summary: Mycobacterium vaccae strain JOB-5 cultured in the presence of propane contained an inducible secondary alcohol dehydrogenase. The enzyme was purified 198-fold using DEAE-cellulose, ω-aminopentyl agarose and NAD-agarose chromatography. The Mr of the enzyme was approximately 136000, with subunits of Mr 37000. The pH optimum for the reaction oxidizing propan-2-ol to propanone was 10–10.5 while the optimum for the reverse reaction was 7.5–8.5. The isoelectric point was 4.9. NAD but not NADP could serve as electron acceptor. The apparent K m values for propan-2-ol and NAD were 4.9 × 10−5M and 2.8 × 10−4 M, respectively. The enzyme was inhibited by thiol reagents and metal chelators. It appears to play an essential role in the metabolism of propane by this bacterium.

Summary: Energy-dispersive X-ray microprobe analysis of electron-dense inclusions in hydrogenosomes isolated from the aerotolerant anaerobic protozoal human parasite Trichomonas vaginalis, Bushby strain, indicated the presence of high levels of Mg, P and Ca. This suggested that divalent cation (e.g. Ca2+ or Mg2+) accumulation by hydrogenosomes may be important in the regulation of intracellular ion concentrations.

Summary: Pigment–protein complexes from Rhodomicrobium vannielii were prepared by detergent solubilization of intra-cytoplasmic membranes followed by gel electrophoresis or sucrose gradient centrifugation. These procedures gave rise to two native pigmented complexes. The major one (designated B800-865) was associated with two polypeptides of M r 11000 and 13000 and was identified with the ‘accessory’ light-harvesting complex II found in other members of the Rhodospirillaceae. The minor complex (designated B885-RC) contained both reaction centre and light-harvesting bacteriochlorophyll. Detergent fractionation and reversible chemical cross-linking, followed by two-dimensional polyacrylamide gel electrophoresis, indicated a specific relationship between a membrane-bound cytochrome c-553 and the M r 31000 subunit of the reaction centre.

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-d-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

Summary: The basic theory of electrolytic conductivity in solutions is described and a model is proposed which allows the direction and relative rates of change of conductivity in microbial cultures to be predicted. Guidelines are presented to enable nutrients to be selected so as to maximize conductivity changes. It is shown that a major consideration in any strategy to maximize conductivity changes in cultures must be to direct as many metabolic activities as possible to act in concert in the production or consumption of protons, and to combine this with use of a pH buffer that exhibits a large change in conductivity on taking up or losing a proton. The ability to predict conductivity changes in microbial systems should permit the rational design of culture media for the selective enumeration of microbes by conductimetric methods and the development of other kinds of conductimetric assays.

Summary: An improved ELISA technique using a new type of enzyme-conjugate - the antibody being linked to β-galactosidase by using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridy1dithio)propionate (SPDP), containing two reactive groups directed towards different functional sites - has been further improved by fine purification of the antibodies. The ELISA was applied in studies of competition among strains of Rhizobium trifolii in three environments: a rhizosphere, a liquid medium and in soil. The strain origin of nodules formed by strain mixtures of different relative composition was investigated, including a study of double strain occupancy. Strain 7612 was found to compete successfully with strain 285 for nodulation. No double strain occupancy occurred. Strain 7612 also competed successfully with strain 285 in soil, but strain 285 dominated when grown together with strain 7612 in a liquid medium in a fermenter.

Summary: Heat evolution during the microbial degradation of glucose in a brown arid andosol soil was studied in a conduction-type calorimeter at 30°C. Reproducibilities of the degradation thermograms in terms of the peak time of thermograms, their peak heights, and the total heat evolution, were within ±0.17%, ±1.1% and ±0·51%, respectively (percentage errors). Changes in the number of viable microbial cells and in the amount of glucose degraded revealed linear relationships both between heat evolution and the amount of glucose degraded, and between heat evolution and the viable cell counts, with correlation factors of 0·987 and 0·968, respectively. The heat evolution per unit glucose was α = 1287 ± 52 kJ (mol glucose)−1. The average heat effect per unit cell was q = 6.7 pW per cell, consistent with values determined for bacterial cells in pure culture. On the basis of these results, we propose a method to evaluate the rate of microbial degradation of organic substances in soil. The apparent rate constant (k d) for microbial degradation of glucose in the soil studied was 0·302 ± 0·002 h−1 at 30°C.

Summary: A novel method for the isolation of Saccharomyces cerevisiae mutants partially defective in haem synthesis is described. Mutant clones were identified by their fluorescence under UV light due to the accumulation of porphyrins in cells, and by their ability to grow on nonfermentable carbon sources due to their preserved haemoprotein synthesis. Thirteen such mutants were obtained by this procedure. The defects in haem synthesis and accumulation of porphyrins in all the mutants were confirmed by spectrophotometric analysis. Complementation tests with biochemically defined, haem-less strains showed that in seven mutants uroporphyrinogen decarboxylase was affected and that in three mutants the defect concerned ferrochelatase. The defects in the remaining three mutants were not defined.

Summary: Cyclic AMP has been shown to be present in 12 different Gram-negative bacteria and the regulation of its concentration, as a function of growth conditions, is similar to that described for Escherichia coli K12. Antibodies raised against catabolite activator protein (CAP) and Rho protein of E. coli K12 were used to check for the occurrence of cross-reactive antigens. Using radioimmunological assays, immunoblotting techniques and biochemical criteria we showed a wide distribution of CAP and Rho, structurally and functionally closely related to the corresponding E. coli K12 proteins. These results suggest that transcription is similarly regulated by these factors in Gram-negative bacteria.

Summary: The use of broad-host-range plasmids derived from RP4 as intermediate vectors for the transfer of narrow-host-range recombinant plasmids from Escherichia coli to Agrobacterium tumefaciens as a preliminary to marker exchange is described. Recombinant plasmids having a ColEl type origin were linked to the RP4 derivative. Cointegrate formation appeared to take place by RecA-independent, homologous recombination within a short piece of DNA derived from the β-lactamase gene of TnllTn3 carried by both vector components, so that it never disrupted the recombinant portion of the construction. pNJ5000 provides an unstable intermediate vector for use in marker exchange experiments, while its stable relative pNJ1020 provides a carrier for use in binary vector systems.

Summary: The fumB gene of Escherichia coli, which complements the fumarase deficiency of a fumA mutant when present in multiple copies, has been located at 93.5 min in the E. coli linkage map and its product has been identified as a polypeptide of 61 kDal. Four overlapping ColEl-fumB + plasmids representing a continuous segment of 23.3 kb of bacterial DNA have been isolated from the Clarke-Carbon E. coli gene bank and the location of the fumB gene relative to the restriction map and the adjacent mel operon has been defined. Hybridization studies have shown that the fumBB gene is homologous to the fumA gene, which complements the fumAl mutation in single and multi-copy situations, and encodes an analogous 61 kDal product formerly regarded as the E. coli fumarase. The hybridization studies also showed that the Bacillus subtilis fumarase gene (citG) is homologous to an independent gene, fumC (formerly g48), which lies adjacent to the fumA gene at 35·5 min in the E. coli linkage map. The N-terminal sequences of the citG and fumC products exhibit a 51% identity over 88 residues. It is possible that the fumC and citG genes are fumarase structural genes of E. coli and B. subtilis, and that the fumA gene may encode a differentially-regulated fumarase or be a positive regulator gene which is essential for the expression of fumC (but not citG). If so, the fumB gene may encode a related enzyme or activator that can replace the fumA function when amplified.

Summary:pDC250 is a 52 kb conjugative cryptic plasmid from Erwinia stewartii strain SW2. This plasmid and its transposon-carrying derivatives, pDC251 and pDC252, were transmissible to E. chrysanthemi, E. amylovora, E. milletiae, E. herbicola, Escherichia coli, Shigella flexneri and Salmonella arizonae. In Esch. coli, E.stewartii and E. chrysanthemi, pDC251 was stable and derepressed for conjugal transfer. Hybridization studies confirmed thatpDC250 is widely distributed among E.stewartii strains and showed that it was also closely related to 34 kb plasmids found in strains lacking a 52kb plasmid. The 34 kb plasmid from E.stewartii strain SSI04, pDC140, was transformed into Esch. coli and compared with pDC250. Restriction mapping of the twoplasmids revealed a nearly identical 20 kb region. In Esch.coli, pDC140 was unstable in the presence of pDC250.

Summary: Bacteriophage Mu pf7701, a KmR derivative of Mu cts62, was inserted into the conjugative Erwinia stewartii plasmid pDC251, which carries Tn10. When pDC251 : : Mu pf7701 plasmids were conjugated from Escherichia coli into derivatives of E. stewartii strain SS104 they were unstable; loss of either or both drug resistance markers occurred. Stable transconjugants resulted from deletion of Mu sequences, integration of the plasmid into the chromosome, or loss of an indigenous 34 kb cryptic plasmid. Among transconjugants selected for KmR, the largest colonies arose from transconjugants in which Mu pf7701 had transposed to the chromosome and the pDC251 : : Mu pf7701 plasmid had been lost; 1300 transconjugants of this type were screened for pathogenicity to corn (Zea mays) seedlings and eight mutants were obtained that did not cause watersoaking symptoms. The insertions of Mu pf7701 in these mutants were in the chromosome.

Summary: The mutant R plasmid pTH10 was used to construct a circular linkage map of the Rhodopseudomonas capsulata B10 chromosome. Mutations affecting nitrogen fixation (nif mutations) were dispersed in several groups on the chromosome. Biochemical analysis of nif mutants allowed identification of the structural gene for the nitrogenase component II or Fe protein (nifH) and a putative regulatory gene, possibly nif A. These two genes appeared closely linked in conjugation experiments, but represented two distinct linkage groups in crosses mediated by gene transfer agent. Other mutants were affected in the synthesis and/or stability of the nitrogenase component I or MoFe protein; synthesis of component II was also affected, but to a lesser extent. In two of these mutants, nitrogenase activity and the content of component I was increased five to sixfold by the addition of 1 mm-molybdate to the growth medium.

Summary: Colloidal gold conjugated goat anti-rabbit IgG and specific antisera raised against purified thioredoxins from Anabaena cylindrica and Anabaena variabilis have been used to determine the localization of thioredoxin in whole filaments of A. cylindrica and A. variabilis. Vegetative cells of A. cylindrica were labelled differently when tested with either of the two anti-thioredoxin sera. The anti-A. cylindrica thioredoxin serum reacted only with A. cylindrica thioredoxin which was located predominantly in the nucleoplasm in vegetative cells; it showed no association with the chromatoplasm. Anti-A. variabilis thioredoxin serum labelled vegetative cells of A. variabilis, particularly in the vicinity of the thylakoid membranes and also labelled A. cylindrica in the vicinity of the thylakoid membranes, indicating that a second thioredoxin or thioredoxin-like protein was present in this cyanobacterium. Heterocysts of A. cylindrica and A. variabilis showed substantially less labelling than vegetative cells when tested with both antisera.

Summary: The properties responsible for the virulence in infant mice of the bovine enterotoxigenic Escherichia coli strain B41 were investigated. A B41K99− variant previously found to be nearly as virulent as the original strain B41 (B41 K99+) possessed F41 antigen and haemagglutinating properties. Two variants that did not haemagglutinate sheep and human erythrocytes were isolated from strain B41K99−. These variants simultaneously lost their ability to agglutinate with F41 antiserum and their haemagglutinating properties. They still produced heat-stable enterotoxin. The first B41K99−F41− variant was much less virulent than strains B41K99+ and B41K99−, the second was not virulent at all. F41 properties were not acquired by other E. coli strains by plasmid transfers. Non-haemagglutinating variants could not be obtained from the original strain B41K99+. However, a B41K99+F41− strain was obtained by a four-step procedure: (i) spontaneous loss of the K99 plasmid, (ii) obtaining a nalidixic acid-resistant mutant, (iii) obtaining a non-haemagglutinating F41− variant, (iv) reacquisition of the K99 plasmid. This B41NalrK99+F41− strain, although producing heat-stable toxin, was not at all virulent, whereas reacquisition of the K99 plasmid by the strain B41NalrF41+ restored virulence. These results show that F41 antigen is an important virulence factor of strain B41 in the infant mouse model.