- Volume 99, Issue 2, 1977

Summary: Two methods were used to isolate mitochondria from Crithidia fasciculata. In the first method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the second, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined with the electron microscope and were also shown to possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA.

Analysis by polyacrylamide gel electrophoresis showed two RNA components of molecular weights 0·47 × 106 and 0·22 × 106, in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14·6S, 11·4S, 10·1S and 6·9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H] uridine into the presumed mitochondrial RNA.

Summary: Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 °C and pH 2. The microfluidity of membrane vesicles was determined for the temperature range −2 to 35 °C using spin labels. The temperature at which the low-temperature phase transition occurred in membrane lipids increased with increasing Ca2+ concentration. However, there was no evidence of a low-temperature lipid phase transition for membrane vesicles suspended in a medium containing monovalent instead of bivalent cations.

Summary: A thiosulphate-oxidizing system of Thiobacillus novellus grown on thiosulphate was studied using intact cells, crude cell-free extracts and isolated membrane vesicles. All active preparations oxidized thiosulphate to sulphate, without intermediary accumulation of polythionates, consuming two moles of oxygen for every mole of thiosulphate oxidized. Less active cell-free preparations required reduced glutathione, NADH or sulphite for thiosulphate oxidation. An active membrane-associated thiosulphate-oxidizing system from crude cell-free extracts was isolated by either Sepharose 4B column chromatography or differential centrifugation. The isolated multi-enzyme complex system exhibited high specificity for thiosulphate with an apparent K m value of 0·12 mm. Enzyme activity was optimum at pH 7·5 and 25 °C. The system was sensitive to oxygen, storage at certain temperatures and freezing. Inhibition studies indicated that active sulphydryl groups and an electron transport chain were involved during thiosulphate oxidation. Electron micrographs of active crude extracts and the isolated membrane complex after negative staining showed the presence of spherical structures with a diameter of about 0·1 to 0·4 μm. An ultra-thin section of the membrane complex revealed that the large spherical particles observed in the negatively stained preparations were aggregated structures consisting of smaller vesicles 0·02 to 0·05 μm in diameter.

SUMMARY: The thiosulphate-oxidizing system of Thiobacillus novellus was destroyed by treatment with proteolytic enzymes, phospholipase A, sodium deoxycholate and lysolecithin, indicating essential roles for phospholipids and proteins in the membrane complex. The complex was partially resolved with trypsin and sodium deoxycholate into component enzymes involved in the oxidation of thiosulphate to sulphate: thiosulphate-cleaving enzyme (rhodanese; EC 2.8.1.1), sulphur-oxidizing enzyme (EC 1.13.11.18), sulphite:cytochrome c oxidoreductase (EC 1.8.2.1) and cytochrome c oxidase (EC 1.9.3.1). All attempts to reconstitute the thiosulphate-oxidizing system from these component enzymes were unsuccessful. A possible mechanism for thiosulphate oxidation by the complex is discussed.

Summary: Synchronous cells of the thermosensitive division-defective Escherichia coli strain MACI (divA) divided at the restrictive temperature (42 °C) if they were allowed to grow at 42 °C for a certain period before protein synthesis was inhibited by adding chloramphenicol (CAP) or rifampicin. The completion of chromosome replication was not required for such divA-independent division. Synchronous cells of strain MACI divided in the presence of an inhibitor of DNA synthesis, nalidixic acid, if they were shifted to 42 °C and CAP or rifampicin was added after some time; cells of the parent strain Mc6 (divA+ ) treated in the same way did not divide. These data suggest that coupling of cell division to DNA synthesis depends on the divA function. The ability to divide at 42 °C, whether or not chromosome termination was allowed, was directly proportional to the mean cell volume of cultures at the time of CAP addition, suggesting that cells have to be a certain size to divide under these conditions. The period of growth required for CAP-induced division had to be at the restrictive temperature; when cells were grown at 30 °C, in the presence of nalidixic acid to prevent normal division, they did not divide on subsequent transfer to 42 °C followed, after a period, by protein synthesis inhibition. A model is proposed in which the role of divA as a septation initiator gene is to differentiate surface growth sites by converting a primary unregulated structure, with the capacity to make both peripheral wall and septum, to a secondary structure committed to septum formation.

Summary: Gametangial (sex organ) formation in cultures of a British isolate of Phytophthora infestans, mating type A1, was induced by simultaneous inoculation with both A1 and A2 isolates of P. palmivora. Inoculation with the A2 mating type only was ineffective. Three isolates of P. infestans were examined: two were polyploid, of which one was probably tetraploid; the third had both diploid and polyploid nuclei.

Phytophthora infestans also produced gametangia when inoculated simultaneously with both A1 and A2 types of P. cambivora and P. cinnamomi. The ecological significance of these findings is discussed.

Summary: This study demonstrates that the host range of Pseudomonas plasmid RP1 includes the genus Caulobacter. Caulobacter was shown to acquire three antibiotic resistance markers located in RP1. A fourth plasmid marker, susceptibility to an RNA bacteriophage, was not expressed, but could be transferred from Caulobacter to Escherichia coli. The lack of phenotypic expression of the phage marker was manifested by the inability of the phage to adsorb or to produce plaques on Caulobacter transcipients.

Matings of Pseudomonas aeruginosa and Caulobacter vibrioides cv6 were carried out in the presence of bacteriophage ϕ6, a DNA phage that infects and kills only swarmer cells of Caulobacter. No decrease in plasmid transfer in the presence of phage ϕ6 was detected, suggesting that stalked cells, and not swarmer cells, serve as recipients.

Our evidence suggests that transfer of chromosomal segments from Caulobacter may be mediated by plasmid RP1; such segments are not stably maintained.

Summary: A series of restriction-deficient mutants was isolated from non-lysogenic strains of Staphylococcus aureus belonging to phage groups I and II. Some mutants were sensitive to all phages tested. With one possible exception, all the mutants were unaffected in their modification systems. The breakdown of DNA of phages, restricted in the parental strains, was reduced in both the mutants that were tested. The restriction in propagating strain 3A could be transduced to its restriction-deficient mutant. The transduction efficiency increased after ultraviolet irradiation of the transducing phage suggesting that the gene for restriction is present on the bacterial chromosome.

Summary: Outer membranes were isolated from several strains of Neisseria gonorrhoeae by extraction of whole cells with aqueous lithium acetate. The preparations contained a limited number of components including lipopolysaccharide and two major proteins. One protein was present in all strains examined; the second, which showed anomalous behaviour on sodium dodecyl sulphate-polyacrylamide gel electro-phoresis, was absent from some. Fluorescent labelling of intact N. gonorrhoeae showed that the two proteins and pili were the major surface proteins. Each of the three major outer membrane components was isolated in a homogeneous form by selective extraction followed by gel filtration.

Summary: The K99 antigen of Escherichia coli b41 was isolated by isoelectric precipitation from heated bacterial suspensions. Chromatography and immunoabsorption experiments suggested that the mannose-resistant haemagglutinating activity of partially purified preparations of antigen was K99. The antigen was partially susceptible to bacterial proteases and was inactivated by periodate oxidation. Haemagglutination inhibition experiments with sugars and absorption of K99 with antisera to human blood groups A and B substances suggested that K99 contains a terminal α-linked N-acetylgalactosamine moiety, which is involved in the haemagglutination reaction, and an adjacent terminal α-linked galactose moiety, which plays no part in the reaction.

Summary: Saccharomyces cerevisiae accumulated Co2+ and Cd2+ by two processes: the first, which was metabolism-independent, was presumably cation binding to the cell surface; this was followed by metabolism-dependent, progressive uptake of relatively large amounts of the cations. Two K+ ions were released for each Co2+ ion taken up in freshly prepared cell suspensions whereas extensive loss of cell K+ followed the uptake of Cd2+. Co2+ and Cd2+ appeared to be accumulated via a general cation uptake system, with limited specificity related to the ionic radii of the cations.

Summary: Bacillus megaterium strain ncib7581 grew at 30 °C in a simple chemically defined medium without biotin. At 37 °C the bacteria grew more slowly in the same medium and were deformed, especially during the earlier stages of growth, as filaments or with swollen ends. Addition of biotin restored the normal appearance and rate of growth at 37 °C. The organisms synthesized more biotin at 30 °C (11 ng 1−1 in a stationary phase culture) than at 37 °C (1·4 ng 1−1).

Addition of either aspartate, malate, fumarate or acetate to minimal medium also restored normal morphology and accelerated the growth rate in biotin-deficient medium at 37 °C. Neither acetate nor aspartate increased synthesis of biotin at 37 °C.

After growth in medium containing only 0·2% glucose almost every organism in the culture was able to sporulate at 30 °C whether or not biotin was added. About 50 times fewer spores were formed at 37 °C without biotin; the yield of spores increased about 20-fold at this temperature when biotin, aspartate or dipicolinate was added.

Summary: Fluctuations in cell volume during exponential growth of Escherichia coli k12 changed the effectiveness of the continuous-flow centrifugation method for preparing synchronous cultures. Rates of oxygen uptake in synchronous cultures were measured using an electrode system open to the atmosphere. In synchronous cultures of both the parental strain and an adenosine triphosphatase-deficient mutant, which was incapable of oxidative phosphorylation, respiration rates doubled during the cell cycle but oscillated with a periodicity of approximately half a cycle. Synchronous cultures of the parental strain growing on glycerol and Casamino acids showed a stepwise pattern of oxygen consumption. Continuous flow centrifugation did not markedly affect the increases in the numbers and respiration rates of cells in synchronous cultures. Respiratory oscillations also occurred on inoculation of a late-stationary phase culture into fresh medium, although synchronous division was not observed. The possible mechanisms underlying respiratory fluctuations under different growth conditions are discussed.

Summary: Synchronous cultures of Alcaligenes eutrophus (Hydrogenomonas eutropha h16) were prepared by a size selection method which gave high synchrony indices (0·70 to 0·85). Unlike the smooth exponential increase which was observed in exponentially growing cultures, the respiration rate [μg-atom O min−1 (ml culture)−1] of synchronous cultures exhibited a periodic increase which was composed of two ‘steps’ with rises centred at approximately 0·4 and 0·9 of the cell-cycle. The respiration of exponentially growing and synchronous cultures was stimulated by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone; the degree of stimulation varied during the cell-cycle and was inversely related to the respiration rate. The observed discontinuous patterns of respiration are discussed with reference to the reported respiratory properties of other micro-organisms in synchronous cultures.

Summary: A pseudolysogenic Mycobacterium chelonei and its phage ϕ630 are described. Phage ϕ630 is the first mycobacteriophage reported to be resistant to the non-polar solvents chloroform, dioxan and diethyl ether. The phage had a latent period of 75 min, a rise period of 90 min and a burst size of 51. Evidence is presented for host modification and restriction. Phage ϕ630A, grown on host strain M. chelonei f-630 Rg, plated on the alternative host M. smegmatis atcc607 with an efficiency of plating (e.o.p.) of 10−5. Phage ϕ630B, grown on host M. smegmatis, plated with an e.o.p. of 10−5 on the alternative host f-630 Rg. Phages ϕ630A and ϕ630B adsorbed equally well on their alternative hosts and on their indicator host strains. The progeny of plaques from initial platings on the alternative host, when grown in the alternative host, exhibited a marked reduction in e.o.p. on their original host.