- Volume 130, Issue 11, 1984

Superoxide dismutase (SOD, superoxide: superoxide reductase, EC 1.15.1.1) activity was studied in 23 strains of Campylobacter spp. using disc polyacrylamide gel electrophoresis. Different enzyme patterns were observed with extracts of different species of Campylobacter; three migration bands were found in all strains of C. sputorum subsp. sputorum and C. sputorum subsp. bubulus (relative mobilities, Rm, 0·57, 0·76 and 0·85), and C. fetus subsp. fetus (Rm 0·60, 0·72 and 0·81), while four migration bands (Rm 0·52, 0·57, 0·73 and 0·82) were found in C. fetus subsp. venerealis. One band (Rm 0.73) was found in C. coli CIP 7080 and two bands (Rm 0·59, 0·73) in C. jejuni CIP 702. Superoxide dismutase activities were very high in the Campylobacter strains, especially in C. fetus subsp. fetus [specific activity 7·8–55·7 U (mg protein)−1] compared with those in Escherichia coli (1·5 U mg−1), Propionibacterium acnes (1·6 U mg−1) and Veillonella alcalescens (0·2 U mg−1).

Subcellular fractionations using metrizamide density gradients revealed intermediary stages of respiratory adaptation of Saccharomyces uvarum grown anaerobically in the presence of the density label 16-bromo-9-hexadecenoic acid. Prior to adaptation, activities of malate dehydrogenase and oligomycin-sensitive ATPase were contained within a membrane population at ρ = 1·20 g ml−1. After 10 min adaptation cytochrome c oxidase activity was associated with these membranes, with ATPase-containing membranes at lower densities (ρ = 1·05 to 1·14 g ml−1) and with membranes containing malate dehydrogenase at higher density (ρ = 1·24 g ml−1). After further adaptation these enzymes were associated firstly with two distinct membrane populations at ρ = 1·17 and 1·20 g ml−1 and finally with a single population of mitochondria at ρ = 1·16 g ml−1. The significance of these changes is discussed in terms of mitochondrial differentiation. Peroxisomes were evident even in early stages of respiratory adaptation and were well separated from mitochondria in later stages.

2-Keto-3-deoxygluconate : NAD 5-oxidoreductase (EC 1.1.1.127) is an enzyme used in pectinolysis by some phytopathogenic bacteria such as Pseudomonas and Erwinia. It converts 2,5-diketo-3-deoxygluconate (DKII) into 2-keto-3-deoxygluconate (KDG) with a concomitant oxidation of NADH. The reaction is reversible in the presence of NAD. The oxidoreductase isolated from Erwinia chrysanthemi was purified 40-fold by protamine treatment, (NH4)2SO4 fractionation, chromatography on DEAE-Sephadex and filtration on Sephadex G-200. The enzyme appeared relatively stable. There was no effect of EDTA or of added ions except Ag+. This ion and the thiol reagent p-chloromercuribenzoate inhibited the enzyme. Other uronic acids, sugars and organic acids tested were neither substrates nor inhibitors of its activity. The optimum pH was 10 for the oxidation of KDG and 7·5 for the reduction of DKII. Both oxidation and reduction reactions showed saturation kinetics with apparent K m values of 7·7 10−3 m for KDG and 4 10−4 m for NAD (at 37 °C, pH 8·6), and of 3·4 10−4 M for DKII and 0·3 10− m for NADH (at 37 °C and pH 7·0). The enzyme reaction was strongly inhibited by NADH at concentrations higher than 2 × 10−4 m.

The location of the catalytic site of the membrane-bound respiratory fumarate reductase of Escherichia coli was investigated using mutants and inhibitors of dicarboxylic acid transport. Comparison of apparent K m and V max values for fumarate in intact cells and in inverted membrane vesicles showed that externally added fumarate was required to be transported across the cytoplasmic membrane prior to reduction. The catalytic site of fumarate reductase must therefore be located on the cytoplasmic face of the membrane.

Reduction of cells or extracts of Nitrosomonas europaea with dithionite leads to a peak in absorption spectra at 460 nm. Most of this chromophore is bound to hydroxylamine oxidase, but a small fraction exists as a separate protein, namely cytochrome P-460. An improved purification of both these proteins is described. The mobility of cytochrome P-460 on gel electrophoresis in the presence of SDS corresponded to a molecular weight of 18000, whereas a molecular weight of 52000 was determined by gel filtration. The band obtained by electrophoresis gave a yellow-green fluorescence in UV light. Electrophoresis in the presence of 8 m-urea, or after acid-treatment, resulted in red fluorescence from cytochrome P-460.

We previously reported that extracts from N. europaea gave eight fluorescence bands in SDS-polyacrylamide gels; cytochrome P-460 was found to correspond to the previously unassigned band VI. Electrophoresis of purified hydroxylamine oxidase did not give a band in common with cytochrome P-460. Highly purified cytochrome P-460 gave no detectable flavin-type fluorescence. There was no antigenic similarity between hydroxylamine oxidase and cytochrome P-460.

The way in which the dolipore apparatus contains hyphal damage, and the process of septal sealing have been studied in Coriolus versicolor using combined light and electron microscopy. The technique used allows the structure of septa in adjacent damaged and undamaged hyphae to be compared. The results show that septal sealing, following damage, is a two stage process. The first is the instantaneous plugging of the pore channel with electron-dense material. The second, beginning several minutes later, involves the detachment of the septal apparatus present in the ruptured compartment and a re-modelling of the septal swelling on the other side of the wall to give a permanent seal. The parenthesomes play no part in the plugging response.

Capsular development and soybean lectin binding properties of Rhizobium japonicum 3I1B110 were examined by the glutaraldehyde/ruthenium red/uranyl acetate method permitting observation of whole bacteria and their polar capsules by transmission electron microscopy. Ultrastructurally, the polar capsule consisted of (i) an electron-dense aggregated material in close contact with the cell surface which stained with ruthenium red and intensively bound soybean lectin, and (ii) a less electron-dense fibrillar material at the periphery of the capsule which stained with ruthenium red but did not bind soybean lectin. The latter material interconnected with the aggregated capsular material at the cell surface. As the culture advanced into stationary phase, these acidic capsular materials were shed into the culture medium where they kept their respective lectin-binding activity. The cells correspondingly decreased in their lectin-binding activity and encapsulation with culture age. However, the remaining encapsulated cells in late stationary phase were still able to bind the lectin in a hapten-specific way.

Measurement of the nitrate-reducing potential by bacteria in saltmarsh sediment, using a thermal gradient block incubator, revealed seasonal physiological changes in the community. A mesophilic part of the nitrate-reducing community was always present, although it achieved maximum development at the end of the summer and minimum development at the end of the winter. In contrast, a distinct psychrotrophic part of the community achieved maximum development at the end of the winter but disappeared during summer. Chemostat enrichment of nitrate-reducing bacteria at 10 °C isolated predominantly Pseudomonas spp., but Vibrio spp. predominated in enrichments at 25 °C. The observed seasonal changes in situ might reflect differential seasonal selection of these two groups of bacteria.

Resistance to the endogenous antibiotic was studied in three actinomycetes that produce inhibitors of RNA polymerase. The three producers, Nocardia mediterranei (rifamycin producer), Streptomyces spectabilis (streptovaricin producer) and Streptomyces lydicus (streptolydigin producer), were each highly resistant to the antibiotic they produce (MIC >200 μg ml−1) and in vivo RNA synthesis was also resistant. However, cross-resistance to the other RNA polymerase inhibitors was not found. Resistance to these antibiotics was due to target site modification, since the RNA polymerase enzymes of the three producing organisms were highly resistant in vitro to the corresponding antibiotic, and no antibiotic-inactivating enzymes were detected. A mutant was isolated from S. spectabilis which was sensitive to steptovaricin (its own product) and also showed an increased sensitivity to rifamycin and streptolydigin. This mutant had RNA polymerase which was extremely sensitive to the three antibiotics.

Mutants of Acinetobacter calcoaceticus EBF 65/65 that could not grow on intermediates of the mandelate or benzyl alcohol pathways were isolated and in some cases the enzymic lesions were identified. Several catabolic markers were mapped using the plasmid pAVl. The mandelate genes appeared to be clustered near the auxotrophic marker phe-1 but were not all contiguous with each other. The gene responsible for the appearance of the novel l(+)-mandelate dehydrogenase appeared to be close to a gene responsible for the activity of the original d(−)-mandelate dehydrogenase.

Purified mitochondria were obtained from the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici by mechanical disruption of protoplasts, followed by differential and density gradient centrifugation. DNA, extracted from the mitochondria, was shown by electron microscopy and restriction endonuclease analysis to be a 46·5 kilobase pair circular molecule.

The fumarase (citG) gene of Bacillus subtilis 168 has been identified in a collection of λ phages carrying EcoRI-generated fragments of B. subtilis DNA. Regions of the cloned DNA have been subcloned into plasmid vectors, and the ability of prophages and multicopy plasmids to complement Escherichia coli and B. subtilis fumarase mutations has been examined. Two EcoRI fragments of 1·5 and 5·1 kb are both required for fumarase expression in E. coli and B. subtilis. The level of fumarase activity from a single copy of the B. subtilis citG gene expressed in E. coli is approximately one-tenth of that from the normal E. coli gene; the level is increased by expression from a pBR322-derived multicopy plasmid. The citG gene has been located within the cloned DNA by transposon mutagenesis and by expression studies, which have also identified a polypeptide of M r 49000 as the product of the citG gene. The properties of a truncated derivative of this polypeptide have indicated the direction of transcription of the citG gene.

Pseudomonas putida NCIB 9869 carries a transmissible plasmid pRA500 of approximately 500 kb which encodes the degradation of 3,5-xylenol via a gentisate pathway. Several mutant strains which were unable to utilize 3,5-xylenol were isolated and these strains either carried deleted derivatives of pRA500 or lacked plasmid DNA. Biochemical and restriction endonuclease analysis of the wild-type and mutant strains showed that the structural and/or regulatory genes for 3,5-xylenol metabolism were encoded within a 130–140 kb region of pRA500 and that, with the exception of the first enzyme of the pathway, 3,5-xylenol methylhydroxylase, all the enzymes were encoded within a 50–70 kb segment of that region. pRA500 also encoded for resistance to inorganic mercuric ions; the genes for this phenotype were located separately from those for the degradation of 3,5-xylenol.

The staphylococcin-like peptide Pep 5 is produced by the penicillin resistant strain Staphylococcus epidermidis 5. This strain is immune to the peptide. Plasmid analysis of S. epidermidis 5 by agarose gel electrophoresis and electron microscopy demonstrated five plasmids with molecular weights ranging from 5·8 × 106 to 29 × 106. Variants of S. epidermidis 5 not producing Pep 5 or which had become penicillin sensitive were induced by various curing treatments. Strains lacking the 13·9 × 106 mol. wt plasmid (pED502) had lost penicillin resistance, and those lacking the 12·3 × 106 mol. wt plasmid (pED503) failed to produce Pep 5. pED503 is also responsible for the immunity of the producer cell to Pep 5. Plasmid pED502 could be transformed into S. aureus RN 981 which then became resistant to penicillin. pED503 could not be transformed into S. aureus RN 981, but could be transformed into S. epidermidis 5 variants previously cured of this plasmid; the transformants then regained the properties of Pep 5 production and immunity.