- Volume 130, Issue 4, 1984

By varying the composition of the growth medium and the genotype of the bacterial strain, five isoenzymes of CoA-dependent aldehyde dehydrogenase could be detected in Escherichia coli. Two isoenzymes (A, mol. wt 520000; and B, mol. wt 370000) were produced only in the presence of ethanolamine and vitamin (or coenzyme) B12 (‘inducible isoenzyme’), whereas the other three isoenzymes (C, mol. wt 900000; D, mol. wt 120000; and E, mol. wt 720000) were produced only in the absence of ethanolamine and vitamin B12 (‘repressible isoenzymes’). Partial purification and characterization of these isoenzymes revealed strong similarities, with respect to pH optima and substrate affinities, between isoenzymes within either of the two classes, but significant differences between the two classes. Mutant studies demonstrated that the relationships between the isoenzymes and between CoA-dependent aldehyde dehydrogenase and ethanolamine ammonia-lyase are both structural and regulatory in nature, and a two-operon model is proposed to account for the common control of the enzymes of ethanolamine catabolism in E. coli.

The incubation of an enzyme preparation of Agrobacterium tumefaciens with UDP-[14C]Glc led to the formation of radioactive substances soluble in chloroform/methanol/water (1 : 1 : 0·3, by vol.). These substances had properties similar to the polyprenol diphosphate saccharides. The lipid moiety appears to be an unsaturated polyprenol. Mild acid hydrolysis of the substances liberated five water-soluble products which were separated by TLC and characterized by borohydride reduction-acid hydrolysis, partial acid hydrolysis, methylation analysis, TLC and paper chromatography and paper electrophoresis. The products behaved like: galactose; the disaccharide Glc(β1-3)Gal; the tetrasaccharide Glc(β1-4)Glc(β1-4)Glc(β1-3)Gal; an octasaccharide and a pyruvylated octasaccharide. The last two substances were compared by paper electrophoresis, TLC and methylation analysis with an octasaccharide obtained by the action of a hydrolytic enzyme on the exopolysaccharide of Agrobacterium tumefaciens. The octasaccharide and the pyruvylated octasaccharide from the lipid behaved like those obtained by enzyme action on the A. tumefaciens exopolysaccharide. These results suggest that the lipid-bound saccharides are involved in the biosynthesis of the extracellular polysaccharides.

Glutamine synthetase (EC 6·3·1·2) was purified about 430-fold from the nitrifying bacterium Nitrobacter agilis by affinity chromatography on Blue Sepharose CL-6B and gel filtration on Sepharose 4B. The enzyme (apparent mol. wt 700000), which consisted of 12 subunits, each of mol. wt 58000, required a divalent cation for both biosynthetic and transferase activities. Regulation of glutamine synthetase both by a feedback inhibition involving amino acids and by an adenylylation/deadenylylation mechanism was studied. The enzyme was highly adenylylated (75-90%) in cell free extracts from cells grown on nitrite as the sole source of nitrogen. The adenylylated form of the enzyme could be deadenylylated by treatment with snake venom phos-phodiesterase. An isoactivity pH of 7·4 was recorded for glutamine synthetase.

Two isoenzymes of glutamate dehydrogenase located in the cytoplasmic fraction of Nitrobacter agilis were specific for NAD+ and NADP+, respectively. The NAD+-dependent enzyme functioned in both directions, i.e. amination and deamination, whereas the NADP+ enzyme was primarily for the amination of 2-oxoglutarate to glutamate. The NADP+ enzyme was purified 52-fold (free of the NAD+ enzyme) by affinity chromatography on 2′,5′-ADP Sepharose 4B, and some of its properties studied. Substrate activation of the amination reaction of the NADP+ enzyme was observed with NH+ 4 and NADPH. A comparison is made of the properties of the purified NADP+ enzyme from Nitrobacter agilis and Nitrosomonas europaea. The possible roles of two isoenzymes of glutamate dehydrogenase in Nitrobacter agilis are discussed.

Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme involved in fixed carbon dissimilation in photosynthetic micro-organisms; in heterocystous cyanobacteria it may also be implicated in the supply of reductant to nitrogenase. In crude cell-free extracts of the N2-fixing cyanobacterium Anabaena variabilis G6PDH activity was reversibly deactivated by the thiol agent dithiothreitol in the presence of a low molecular weight protein (12000mol. wt). Glucose 6-phosphate reversed deactivation when added at high concentration, or prevented deactivation if added with the thiol. NADP+, which, like glucose 6-phosphate, is a G6PDH substrate, also deactivated the enzyme; deactivation was reversed or prevented by adding glucose 6-phosphate or glutamine. Purified thioredoxin from Anabaena cylindrica, at very low concentrations (2 nm), deactivated purified G6PDH in a manner identical to that observed when crude extracts were used in the presence of dithiothreitol. Glutathione did not affect the enzyme.

Phosphoribulokinase (ATP:d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was purified from the cyanobacterium Chlorogloeopsis fritschii. The enzyme had a molecular weight of 230000, as determined by gel filtration, and a pH optimum of 8·6. Divalent cations were essential for activity, maximal activity being supported by Mg2+, while Mn2+, Ca2+ and Co2+ were less effective. AMP, ADP, phosphoenolpyruvate, aspartate and malate inhibited enzyme activity completely at 1 mm. No effects on phosphoribulokinase activity were observed with NAD, NADH, NADP or NADPH at up to 10 m m or with glyoxylate at up to 20 mm. The enzyme was activated in semi-purified extracts by the addition of dithiothreitol and reduced glutathione. SDS-PAGE of SDS-dissociated enzyme revealed only one polypeptide band of molecular weight 40000. This suggests that C. fritschii phosphoribulokinase is a hexamer consisting of six subunits of identical size.

Acidification observed in cultures of Penicillium cyclopium was mostly due to the extrusion of protons into the medium. In the absence of NH+ 4 protons were extruded together with citrate. The citric acid thus produced underwent a dynamic equilibrium of continuous efflux and uptake, which was shifted towards the latter at decreasing glucose concentrations. In cultures supplied with NH+ 4 and glucose a stoichiometric coupling of H+ excretion with NH+ 4 uptake was observed. If the protons excreted were not absorbed by external buffers (e.g. tartrate ions), the pH of the medium dropped to below 2, thereby exceeding the capacity of the cells to maintain their internal pH. The decrease of intracellular pH was accompanied by the cessation of NH+ 4 uptake, H+ extrusion and growth. In the presence of tartrate ions the NH+ 4/H+ exchange proceeded until the glucose concentration of the medium dropped below 25 mm. Here, the external pH was kept above 3, without seriously affecting the intracellular pH. After termination of H+ extrusion an appreciable amount of NH+ 4 was taken up together with hydrogentartrate anions, thereby causing a further acidification of the medium, which was finally reversed due to the uptake of tartaric acid in the late phase of growth.

Sporulation of Bacillus sphaericus strain 2297 in a synchronous liquid culture was studied by electron microscopy. The t 0 of sporulation occurred 7 h after the beginning of the lag phase. Crystal-like inclusions first appeared at t 2 and reached their final size between t 5 and t 6. The release of the spore/inclusion complex occurred at about t 15 (22 h after inoculation).

Toxicity against Culex pipiens larvae was related to sporulation and appeared during the early stages of sporulation. The LC50 (24 h) decreased about 105-fold between t 0–2 and t 7, in correlation with the formation of crystalline inclusions. Heat resistance of spores appeared later than toxicity.

Techniques of antigenic analysis were used to examine the synthesis of the exosporium during sporulation of Bacillus cereus. An antiserum to a soluble extract of exosporium from mature spores was used to analyse extracts of cells from various stages of the Bacillus life cycle by the technique of one-dimensional immunoelectrophoresis. Exosporium antigens were absent from vegetative cell extracts and were first detected in extracts of cells at stage III of sporulation, where they appeared simultaneously in the soluble and particulate cellular fractions. The results indicate that the exosporium contains antigens which are spore specific.

A method allowing isolation and microbiological analysis of individual pilosebaceous units (follicles) was used to study biopsies of back skin obtained from volunteer acne vulgaris patients. The main microbial groups isolated were members of the genera Propionibacterium, Staphylococcus and Pityrosporum. The incidence (and mean density) of these organisms in 140 normal follicles was 12% (2·6 × 105 per follicle), 4% (5·5 ×103 per follicle) and 13% (102 per follicle) respectively. Colonized follicles were not distributed evenly amongst the subjects studied. The results are analysed and discussed from an ecological standpoint.

The distribution and abundance of mat-forming Oscillatoriaceae were investigated at a siltdepositing freshwater site in England over a period of 4 years. Maximum growth occurred during April and May and experiments showed that mat buried by fine sediment could regain the surface by gliding movements. The number of trichomes reaching the surface was light-dependent but chemotactic responses were also apparent. Gliding rates of 1–2 μs−1 were recorded and the trichomes moved through the sediment as a left-handed screw with a pitch of 60°. A surface illumination of 1 klx (4 W m−2) was optimal for mat formation. Light intensities exceeding 2·5 klx produced a photophobic response resulting in lateral trichome migration to regions of lower surface illumination when these were provided. Migration into the sediment was not observed in the surface intensity range 0–10 klx. The onset of light saturation measured by the 14CO2 method was 3 klx for Microcoleus lyngbyaceus, close to the optimum for mat development. Experiments employing 14CO2 in the presence of sulphide and absence of oxygen suggested that the organisms were adapted to an aerobic environment.

Specialized transducing phages pllspoC and ϕ105spoIIC, carrying the Bacillus subtilis sporulation gene spoIIC, were constructed by the prophage transformation method. An EcoRI fragment (2·4 MDal) carrying the spoIIC gene was isolated from the ϕ105spoIIC genome and recloned into the EcoRI site of plasmid pUB110. The recombinant plasmids corrected the sporulation defect of a Spo− Rec− host, but slightly inhibited the sporulation of a Spo+ Rec− host.

Irradiation of Bacteroides fragilis cells with far-UV light resulted in the immediate, rapid and extensive degradation of DNA which continued for 40 to 60 min after irradiation. During the degradation phase, DNA synthesis was decreased but was never totally inhibited. DNA degradation after irradiation was inhibited by chloramphenicol and caffeine. DNA synthesis in irradiated cells was reduced by chloramphenicol but resumed after 100 min at the same exponential rate as in irradiated cells without chloramphenicol. Irradiated cells continued to synthesize DNA for 40 min in the presence of caffeine but after this time DNA synthesis was completely inhibited and never recovered. RNA and protein synthesis were decreased by UV irradiation and the degree of inhibition was proportional to the UV dose. Colony formation was not affected immediately by UV irradiation and continued for a dose-dependent period before inhibition. There was an inverse relationship between UV dose and inhibition of colony formation which occurred sooner in cells irradiated with lower doses of UV light. The characteristics of DNA synthesis in B. fragilis cells after UV irradiation differ from those in wild-type Escherichia coli cells, where DNA synthesis is stopped immediately by UV irradiation, but resemble those in E. coli recA mutant cells where extensive degradation occurs following UV irradiation.

492a and 492c were two strains of Pseudomonas aeruginosa isolated from the sputum of a patient with cystic fibrosis. The strains were closely related but expressed different antibiograms. 492c was hypersensitive (10-100 times more sensitive than 492a) to the β-lactam antibiotics carbenicillin, methicillin, flucloxacillin, mecillinam and cefuroxime and the non-β-lactam, nalidixic acid. 492c also showed enhanced sensitivity (48 times more sensitive than 492a) to chloramphenicol, trimethoprim and novobiocin. 492a and PAO8 expressed similar levels of antibiotic resistance, except for trimethoprim, to which 492a was five times more sensitive than PAO8. Two genes associated with antibiotic hypersensitivity were mapped in the 30 min region of the chromosome, by means of R68.45-mediated plate matings between a Leu− mutant of 492c and PAO8, followed by transductional analysis using phage F116L. The first of these genes, blsA1 was closely linked to nalBand in a PAO background, was associated with hypersensitivity to the β-lactams and a moderate increase in sensitivity to chloramphenicol, trimethoprim, nalidixic acid and novobiocin. A further increase in sensitivity to the latter three antibiotics was associated with the second gene, tpsA1, which mapped between ser-3 and hisV. This gene could also be transferred to PAO from 492a, thus 492c could have arisen from 492a in vivo following a single chromosomal mutation at the blsA locus. Isolation of a blsA mutant of PAO969 provided further evidence for this theory.

Recombinant plasmids were constructed that carried various fragments of the DNA specifying the Escherichia coli genes ptsH and (part of) ptsI, the genes for the common components of the phosphoenolpyruvate : sugar phosphotransferase. Expression of plasmid-specified functions in minicells showed that ptsH and ptsI were transcribed clockwise. Most of the transcription of ptsI was from the ptsH promoter, but some was from a second site within or after ptsH.

Various streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss. The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations. The frequency of instability can be enhanced by UV irradiation. Two major repair systems have been found in Escherichia coli: the ‘error-free’ system which is inhibited by caffeine and the ‘error-prone’ system which is inhibited by arsenite. Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development. Some evidence was also found for the presence of an arsenite inhibitable UV repair system. The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S. cattleya. The arsenite system may be implicated in the repair of such events. Genetic instability was also induced by single strand breaks in DNA caused by 32P.