- Volume 93, Issue 2, 1976

Summary: Pseudomonas am1 is a facultative methylotroph which grows on a wide range of carbon compounds. A mutant of Pseudomonas am1 (ict41) grew only on C1 compounds and is thus an artificial obligate methylotroph. Measurements of activities of the components of the 2-oxoglutarate dehydrogenase complex suggest that the E2 component (dihydrolipoamide transsuccinylase) is not functional. All other tricarboxylic acid cycle enzymes were present with activities comparable to those in wild-type Pseudomonas am1 and cytochrome levels were unchanged in the mutant. Suspensions of the mutant oxidized pyruvate, lactate, β-hydroxybutyrate, acetoacetate and 2-oxoglutarate at very low rates. By contrast, C1 compounds were oxidized at the same rate as in wild-type bacteria. Two revertants of ict41 which regained 2-oxoglutarate dehydrogenase activity also regained the ability to oxidize and grow on the same substrates as wild-type bacteria. It is concluded that lack of 2-oxoglutarate dehydrogenase may well be the basis of obligate methylotrophy in some bacteria.

Summary: In Pseudomonas aeruginosa, the synthesis of histidase, urocanase and amidase is severely repressed when succinate is added to a culture growing in pyruvate + ammonium salts medium. When growth is nitrogen-limited, catabolite repression by succinate of histidase and urocanase synthesis does not occur but succinate repression of amidase synthesis persists. Amidase synthesis is not regulated in the same way as histidase synthesis by the availability of other nitrogen compounds for growth.

Growth of P. acruginosa strain PACI in succinate + histidine media is nitrogen-limited since this strain is defective in a histidine transport system. When methyl-ammonium chloride is added to succinate + histidine media, growth inhibition occurs. Mutants isolated from succinate + histidine + methylammonium chloride plates were found to be resistant to catabolite repression by succinate even in ammonium salts media. It is suggested that the hut genes of P. aeruginosa may be regulated in the same way as in Klebsiella aerogenes, by induction by urocanate and activation by either the cyclic AMP-dependent activator protein or by glutamine synthetase.

Summary: A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a+a3 ) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyes carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mm-MgCl2 or 0·4 mm-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH: cytochrome c oxidoreductase sedimented with mitochondrial whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.

Summary: Botrytis cinerea Pers. ex Fr. in stationary liquid cultures conidiated asynchronously in darkness after 4 days’ growth. Synchronous conidiation was induced by irradiating dark-grown cultures with near-ultraviolet light for 12 h. The number of conidia increased very rapidly 10 h after the end of the photo-induction period, and conidiation was completed by the 14th hour. Filter paper cultures of the fungus also showed synchronous conidiation upon irradiation with near-ultraviolet light, but the rapid increase in the number of conidia took place 2 h earlier, conidiation being completed by the 12th hour. Cultures irradiated with blue light, however, produced sterile mycelia and showed complete suppression of conidiation.

SUMMARY: The growth form of a strain of Claviceps purpurea in axenic culture has been controlled by the amino nitrogen source. Within the pairs asparagine/aspartic acid or glutamine/glutamic acid the amide promoted sphacelial growth of the colony whereas the acid supported differentiation of plectenchymatic sclerotial tissue and synthesis of ergot alkaloids. Sclerotial colonies showed purple pigmentation. The mycelium had a greater lipid content, rich in ricinoleic acid, and sporulation was much less than in sphacelial colonies. Changes in the relative distribution of amino acids between the free and peptidyl components of the cells was most marked with respect to lysine; growth on asparagine resulted in more than half remaining free, whereas less than 10 % remained free on aspartic acid. Lysine supplied exogenously as a nitrogen source did not promote sclerotial differentiation. Frequent transfers to fresh medium of colonies grown on dialysis membrane accentuated the extent of sphacelial or sclerotial growth; four transfers during an 18-day growth period yielded mycelia with an alkaloid content (0·4 %, w/w) similar to that of parasitic ergot sclerotia. Asparagine and glutamine were taken up more rapidly from liquid media than their corresponding acids, but each acid exerted a dominant effect over the amide, in a mixture providing equivalent nitrogen, resulting in differentiation from sphacelial to sclerotial growth analogous to that occurring during parasitism. The apparently greater (w/w) proportion of total amino acids in sphacelial mycelia than in sclerotial mycelia mainly reflected the lower lipid content of these tissues, but this factor was insufficient to account for the persistence of a significant proportion of the total lysine amongst the free amino acids. The promotion of sclerotial growth by aspartic and glutamic acids was not confined solely to the experimental strain of the fungus. Although some isolates failed to differentiate into plectenchymatic mycelia on these nitrogen sources, the extent of their sphacelial growth, as indicated by the degree of sporulation, was always much reduced with respect to that promoted by asparagine.

SUMMARY: By suitable choice of medium, Rhodospirillum rubrum has been grown both in normal (length 2 μm) and filamentous (length up to 60 μm) forms. Both forms were highly motile, and negatively-stained preparations showed bipolar flagellated cells, with an average of seven flagella at each pole. Motion consisted of a series of runs and tumbles, the distribution of run time-lengths being Poissonian. Both forms tumbled in response to dark shock and showed negative chemotaxis to oxygen. The observation that the motility pattern was very similar in normal and filamentous forms makes chemical control of tumbling unlikely and favours a system involving membrane potentials.

Summary: Spores of Clostridium bifermentans were treated with hydrogen peroxide until their peripheries had lost refractility. The centres of such spores only retained refractility at acid pH. Adding monovalent cations or increasing the pH caused the treated spores to lose their remaining refractility and decreased the turbidity of spore suspensions. Divalent cations prevented or reversed this loss of central refractility and decreased the fall in turbidity. Calcium ions also prevented but did not reverse the loss of central refractility which occurred on drying or applying pressure. Electron micrographs of spores treated with hydrogen peroxide showed that the cortex was depleted or absent and that the loss of central refractility was accompanied by protoplast swelling. It is suggested that divalent cations make spores resistant to drying and pressure by cross-linking negatively charged groups within the protoplast, and that together with hydrogen ions they neutralize the negatively charged groups, thus preventing the swelling of the protoplast, loss of refractility and fall in extinction which occur when divalent cations are replaced by monovalent cations.

Summary: The measurement of the time at which normal colony-forming ability returns in irradiated cultures of Micrococcus radiodurans tsI held at 30 °C can be used to estimate the time of completion of recombination repair. By comparing the times to complete such repair in populations given increasing radiation doses it is possible to calculate the rate of recombination repair. The rate was independent of the radiation dose; recombination could repair in one minute the damage caused either by 1·2 krad gamma radiation or 4 × 10−6 J mm−2 u.v. radiation.

The time taken for the normal rate of DNA synthesis to return in irradiated M. radiodurans tsI was measured under conditions identical to those used to measure recombination repair. The delay in DNA synthesis was 1·0 min per 1·2 krad gamma radiation and 1·0 min per 5·6 × 10−6 J mm−2 u.v. radiation. The data suggest that the normal rate of DNA synthesis resumes immediately after the completion of recombination repair of gamma-induced damage, but before the completion of recombination repair of u.v.-induced damage. It is postulated that cell death at the lethal dose of u.v. radiation is caused by a second round of replication of DNA which is still being repaired by recombination.

SUMMARY: The P class R factor RP4 was transferred from Escherichia coli k12 to Acinetobacter calcoaceticus. RP4 conferred similar levels of antibiotic resistance in A. calcoaceticus to those in the E. coli K12 donor. Only slight instability of RP4 in A. calcoaceticus was detected. Transfer of RP4 between strains of A. calcoaceticus was by conjugation and was accompanied by transfer of chromosomal genes. Possible polarity of marker transfer was observed and linkage between a number of chromosomal markers was demonstrated.

Summary: Post-fusion incompatibility among plasmodia derived from Physarum polycephalum strain 29 is controlled by interactions between alleles at three loci, two of which are linked. A reaction will occur between a plasmodium which is heterozygous or a homozygous dominant at a locus, and one which is a homozygous recessive at the same locus. Depending on genotype with respect to the three loci, incompatibility reactions among plasmodia can be absent, unilateral or bilateral.

SUMMARY: Leucocidin from Pseudomonas aeruginosa strain 158 was released from bacteria by autolysis and purified 19-fold by ammonium sulphate precipitation (20% saturation) and combined ‘tandem’ gel filtration on Sephadex G-100 superfine and Bio Gel P-100. The product gave a single band (mol. wt. 27000) after poly-acrylamide gel electrophoresis with sodium dodecyl sulphate (SDS). However, it was separated into two active peaks (pI 5·0 and 5·2) by isoelectric focusing, and into five bands by disc electrophoresis without SDS. All bands contained leucocidic activity of about the same specific activity and retained their homogeneity.

The purified toxin was thermolabile and was inactivated by pronase, but not by several other proteases. The ultraviolet light absorbancy was typical of proteins. Antibodies directed against leucocidin were detected by passive haemagglutination and by toxin-neutralization. These antibodies inhibited the cytotoxic action of leucocidin bound to granuloyctes. The toxin damaged all tested leucocytes (granulocytes of various animal species and lymphocytes of humans) and a number of tissue cultures, but was ineffective against erythrocytes, thrombocytes and isolated granules from polymorphonuclear leucocytes. The intravenous lethal dose for mice was about 1 μg

SUMMARY: Human polymorphonuclear leucocytes treated in vitro with leucocidin from Pseudomonas aeruginosa underwent characteristic morphological alterations as shown by phase-contrast and scanning electron microscopy. Within a few minutes of exposure to leucocidin the granulocytes became round, and protoplasmic extrusions appeared on the cell membrane, were withdrawn again and put out at another point of the cell. The final stage of the leucocidin-treated leucocyte was an enlarged, rounded vesicle with apparently intact plasma membrane. Omission of calcium ions from the diluting buffer caused certain differences in the morphologic appearance of the damaged leucocytes.

Summary: The addition of complex supplements (particularly amino acids) to cultures of Pseudomonas putida growing on a good carbon source did not result in a substantial increase in the growth rate. Amino acids entered the cells within 30 s of addition and reached significant internal pool concentrations. Endogenous amino acid biosynthesis was quickly inhibited (about 75 %), with a substantial sparing of the original carbon source. Within 20 min of supplementation significant respiration of added amino acids was detected, yet the ATP pool size did not increase and the bacteria did not grow faster.

The RNA content of p. putida growing in complex medium differed from that of enteric bacteria in that, although it varied with growth rate, it was not substantially larger than the RNA content of bacteria grown in a minimal medium with a good carbon and energy source. The rate of RNA accumulation on shift-up remained substantially unchanged on supplementation if the minimal medium had a carbon source producing fast growth, and did not increase for about 30 min if the carbon source was relatively poor. In other respects RNA synthesis was similar to that of the enteric bacteria, being stringently controlled, inhibited by trimethoprim and continuing in the presence of chloramphenicol. It is proposed that growth of P. putida in complex media is limited by the rate of synthesis of stable RNA.

SUMMARY: Changes in the ethanol-soluble carbohydrate content of Agaricus bisporus mycelium and sporophores grown on semi-defined media and commercial compost were studied. The accumulation of mannitol in the sporophore during its growth was not accompanied by an increase in mycelial mannitol. The other major soluble carbohydrate of the sporophore, trehalose, decreased throughout the growth of the sporophore; a parallel decrease was observed in the mycelium. The main accumulation of mannitol was in the pileus and stipe of the sporophore and was accompanied by a decrease in the soluble protein content of these tissues. Before fruiting, glucose and sucrose were present in the mycelial samples in similar quantities to mannitol, but their levels decreased during fruiting. Small quantities of glucose were present in the sporophore. The results are discussed in relation to the possible functions of the soluble carbohydrates.

SUMMARY: Oxygen-limited (N2-fixing) chemostat cultures of Klebsiella pneumoniae supplied with a N-free medium were established by introducing low atmospheric O2 concentrations into the gas supply of anaerobic glucose-limited N2-fixing chemostat cultures; the molar growth yield for glucose and the efficiency of N2 fixation (µg N fixed/mg glucose consumed) were increased (by up to 82%) from the anaerobic values.

Acetylene-reducing activity was inhibited reversibly by O2 in samples from O2-limited and anaerobic glucose-limited chemostat cultures. Oxygen uptake rates in samples from these chemostat cultures were similar, but C2H2-reducing activity in samples from O2-limited chemostat cultures was more tolerant of low atmospheric O2 concentrations, in part because of a higher population density. In the absence of glucose, O2 was required at a low atmospheric concentration for C2H2 reduction in samples from either O2-limited or anaerobic glucose-limited chemostat cultures. The possibility is discussed that ATP generated from oxidative phosphorylation can be used for N2 fixation in K. pneumoniae.