- Volume 103, Issue 1, 1977

Proline utilization during growth of Saccharomyces cerevisiae is inhibited by the presence of other amino acids. Steady-state simultaneous utilization of proline and glutamate is possible, however, in a nitrogen-limited continuous culture. A switch to steady-state inhibition of proline utilization may be effected by a change in growth rate. Under the conditions studied, inhibition occurred on the catabolic route from proline to glutamate and was not mediated through the concentration of pool amino acids. In batch culture, the control site was shifted to proline permease by increasing the extracellular concentration of glutamate.

Specific activity of benzyl alcohol dehydrogenase in carbon-limited continuous cultures was at a maximum at a specific growth rate of 0·2 h−1, but fell off at lower and higher growth rates. The specific activity in nitrogen-limited cultures was always lower and was inversely proportional to growth rate. There was severe repression of benzyl alcohol dehydrogenase during metabolism of l(+)-mandelate or phenylglyoxylate in batch cultures. Synthesis of benzyl alcohol dehydrogenase was followed in experiments where various compounds, including a gratuitous inducer and an anti-inducer of the mandelate enzymes, were added to uninduced or pre-induced cultures and to constitutive and blocked mutants. The results led to the conclusion that there were at least two types of repression. One was caused by phenylglyoxylate carboxy-lyase (or a compound synthesized co-ordinately with it), but not by the other mandelate enzymes or by l(+)-mandelate, phenylglyoxylate, benzaldehyde or benzoate. A second type of repression was observed during rapid growth or after the addition of compounds such as succinate which are rapidly and completely metabolized.

Changes in adenine nucleotide composition of suspensions of starved Beneckea natriegens on aeration and anaerobiosis have been studied using succinate as the respiratory substrate. The anaerobic steady-state adenylate pool is only about 50 to 60 % of the aerobic steady-state pool, and the energy charge is also much lower under anaerobic conditions. Aeration of anaerobic suspensions causes the ATP and total adenylate concentrations and the energy charge to rise rapidly with an overshoot in the ATP concentration before the aerobic steady-state is attained. Shifts to anaerobic conditions result in oscillations of the energy charge and adenine nucleotide concentration and the anaerobic steady-state is only attained relatively slowly. Sequential aeration and anaerobiosis stabilizes the adenine nucleotide pool size, permitting estimates of P/O ratios. The P/O ratio, with succinate as the respiratory substrate, has been assayed by pulsing anaerobic suspensions with small amounts of oxygen (generated from H2O2 and catalase) and quenching metabolic activity before anaerobiosis had been regained, then analysing the adenine nucleotide composition. P/O ratios of 0·2 decreasing to 0·02 with increasing oxygen pulse size have been observed. Extrapolation to zero pulse size, when little turnover of the adenylates occurs, gives a P/O ratio of about 0·4. This is much lower than the efficiency of energy transduction as indicated from H+/O experiments. Therefore, either the observed and extrapolated P/O ratios are unrealistically low and further correction is required, or only a small proportion of the energy transduction is involved in phosphorylation under these conditions.

Arthrobacter globiformis NCIBIO683 was grown under different conditions in continuous and batch culture to produce rods, cocci and cystites. All forms were resistant to lysozyme and enzyme L1 and did not autolyse readily. Walls contained polysaccharide, made up of glucose, galactose and rhamnose, as well as peptidoglycan and phosphorus. The polysaccharide constituted about 60 % of the wall of each form, although the molar ratios of glucose, galactose and rhamnose varied from 1:1:1 in the cystites to 1:3:2 in rods and 1:3:5:3 in cocci. The molar ratios of alanine, glutamic acid, threonine and lysine in the peptidoglycan were similar in all three preparations (4:1:1:1). Electron microscopy showed that the wall was structurally homogeneous but was thicker in cocci (10 to 27 nm) than in rods (6 to 12 nm). The wall in cystites varied in thickness (6 to 40 nm) and was sometimes distorted due to the accumulation of large amounts of glycogen which expanded the organism and displaced the ribosomes from most parts of the organism. Glycogen granules were also seen in rods but never in cocci. Division of Arthrobacter was by binary fission and budding. Buds could usually be distinguished from parent organisms since the latter contained less ribosomes and mesosomes. Large periplasmic spaces were found, especially in budding cells. Cross-wall formation differed in binary fission and budding.

During the transition from cocci to rods of Arthrobacter globiformis grown under constant conditions in perfusion microculture, the shape of the original coccus was maintained for many hours during a sequence of multiple bud formation. Buds matured into rods in which the tips were phase-dark. Cross-wall formation, which cut off the buds from the parent cells, occurred by centripetal growth. V-forms were produced by angular growth, germination of adjacent cocci and slow bending division. Rods could be transformed into cystites by omitting fixed nitrogen from the medium. Cystites were capable of budding on restoration to complete media. Budding, originally occurring at the poles of the cystite, also occurred at the middle of the cystite, following the growth of a cross-wall at this point.

The effect of 5-fluoro-2-deoxyuridine (FdUrd), 6-azauridine, miracil D, 5-fluorouracil, cycloheximide, actinomycin D and puromycin on light-induced synchronous conidiation in Botrytis cinerea Pers. ex Fr. is described. FdUrd, 6-azauridine, miracil D, 5-fluorouracil, and cycloheximide inhibited photoinduced conidiation at the appropriate concentrations. Suppression of conidiation was greater when these inhibitors were added just before, rather than after, the photoinduction period. Actinomycin D and puromycin, both at 10−4 to 10−6 m, were ineffective. Incorporation of labelled precursors into nucleic acids and protein in the presence of FdUrd, miracil D, 5-fluorouracil and cycloheximide was greatly suppressed. The results suggest the involvement of nucleic acids and proteins in light-induced conidiation in B. cinerea.

A mutant (strain g311) of Bacillus subtilis w23, which requires abnormally large amounts of glycerol, was isolated and characterized. Glycerol consumption is high in the mutant even in the presence of glucose. With limiting glycerol, growth stops in mid-exponential phase and autolysis occurs (glycerol-deprival autolysis). During this autolysis the cell wall lytic enzyme seems to be more active. Inhibitors of protein synthesis or energy supply prevented both autolysis and increase in the activity of the wall lytic enzyme. Synthesis of glycerol-catabolizing enzymes in g311 was less sensitive to catabolite repression than in the parent strain. The results suggest that g311 has a defect in the regulation of the glp system and that exhaustion of glycerol causes an increase in autolysis.

A converting phage for ampicillin resistance - phage 5006Mpa - was used to transduce Proteus mirabilis strain pm5006 to ampicillin resistance. The infecting phage carried TnI as the source of its phenotype. The recipient had the conjugative plasmid P-lac as well as a non-self-transmissible plasmid as residents. The latter was a recombinant between a morganocinogenic plasmid Mor174 and R plasmid R772 which coded for kanamycin resistance. This recombinant plasmid had possibly undergone transductional shortening as a result of previous uptake by transducing phage 5006M. From these transductants, three transducing systems for transfer of morganocinogeny were obtained. The first consisted of the complete transduction of a plasmid expressing markers of Mor174 and ampicillin resistance at frequencies of about 1 × 10−5 per phage particle adsorbed. This frequency could have equalled that of the adsorption of transducing phage to recipient cells. The transduced plasmid was formed by translocation of Tn1 to the Mor174R772 complex with inactivation of the kanamycin resistance marker of the latter. The transducing phage was named phage 5006Mmora. The second - a high frequency transducing system for morganocinogeny, kanamycin and ampicillin resistances - was the result of markers of the Mor174R772 complex inserting contiguously to Tn1 in the 5006Mpa-pm5006 cryptic prophage sequence. The insertion converted the terminally redundant, circularly permuted phages into deficient phages lacking some (not always the same) genes and being unable to circularize (and hence lysogenize) due to lack of terminal repetition. The recombination of two defective genomes resulted in doublets which could reduce to the prophage state. This mutual aid explained the phage multiplicity dependence phenomenon encountered with this system. The phage was named phage 5006 The third system of transduction resulted from deletion of non-essential genes from the oversized genomes just described. This restored terminal redundancy and consequently allowed individual genomes to circularize and thus transduce the three markers. This phage was named phage 5006Mdpmorka. These phages also transduced their markers to P. mirabilis pmoxk, a strain to which only slight and variable adsorption of the phage could be demonstrated. Properties of the systems are described.

The properties of a Bacteroides thetaiotaomicron phage, β 1, are described. Phenol-extracted phage β 1 DNA transfects Ca2+-treated B. thetaiotaomicron cells and transfection is not limited to a particular stage in the growth cycle. An obligatory step in the transfection procedure is a 33-fold dilution in broth, allowing replication of the infected cells. Prolonged incubation of treated cells with DNA prior to dilution in broth results in a large decrease in phage titre. The application of this system to the investigation of the genetics of obligate anaerobes is discussed.

Rates of oxygen uptake and heat dissipation were measured in cultures of Schizosaccharomyces pombe that had been induced to divide synchronously by adding 2 mm-2′-deoxyadenosine and then removing the inhibitor after 4 h. Respiratory oscillations occurred during the last 1·5h of treatment with deoxyadenosine and throughout the subsequent period of synchronous growth. Before completion of the first synchronous division three peaks of oxygen uptake occurred, the third peak being coincident with cell division. These peaks were less sensitive to the rate-stimulating effect of the uncoupler, carbonyl cyanide m-chlorophenylhydrazone, than were the troughs, so that in the presence of the uncoupler the oscillations were attenuated. In the absence of uncoupler, heat dissipation of the culture increased linearly during and after deoxyadenosine treatment, with sharp increases (approximate doublings) in the rate of dissipation occurring at intervals similar to the mean generation time of an exponential culture. Heat dissipation also increased continuously in samples removed from such a culture and incubated with the uncoupler. The possible modulation of oxygen uptake rates by respiratory control, and the implications of linear increases in heat dissipation are discussed.

The effect of adding dibutyryl cyclic AMP to five species of Mucorales was tested. With Mucor hiemalis and M. mucedo a rapid halt of apical extension and a slower rate of resumed growth were observed together with marked morphological changes, including hyphal dilation, apical swelling, septation and multiple branching. Sporangiospore development, and the mating reaction in M. mucedo were both inhibited; carotene pigmentation was increased. Phycomyces blakesleeanus, Blakeslea trispora and Zygorhynchus moelleri were insensitive to dibutyryl cyclic AMP.

Sporulation kinetics and spore heat resistance data were compared for a lysogenic strain of Clostridium perfringens, s9, before and after curing with ultraviolet irradiation. The cured strain showed the same growth rate in broth media as the lysogenic strain but took 6 h longer to form refractile spores. For lysogenized and cured strains the percentages of refractile spores produced that were heat-resistant (80 ° C for 15 min) were 50 and 0·2, respectively. When reinfected with the temperate phage, the cured strain produced spores in 2 to 3 h, like the original lysogenic culture, and 10 % of the spores produced were heat-resistant.

The expression of phenomena associated with the bacterial function controlling RNA synthesis was studied in leucine-deprived or 5-methyltryptophan-treated cultures of Anacystis nidulans. Both procedures retarded cell growth, RNA and protein accumulation, elicited the accumulation of high intracellular concentrations of guanosine 5′-diphosphate-3′-diphosphate (ppGpp) and guanosine 5′-triphosphate-3′-diphosphate (pppGpp), and promoted a regime of non-coordinate synthesis of stable and messenger RNA. The rate of polymerization of nascent RNA chains did not appear to be retarded in the growth-limited cultures.

Nitrogen fixation (Nif)-derepressed mutants of Klebsiella pneumoniae consumed, under optimum conditions, 7·5 to 8.5 mol glucose per mol N2 fixed. The nitrogenase system of these mutants catalysed the production of about 1·3 mol H2 per mol N2 reduced. Almost one-third of the energy as ATP and reductant used by nitrogenase in vivo may be lost in H2 production, since an ATP/2e ratio of approximately 4 was obtained. Nitrogenase-catalysed H2 production was not substantially suppressed by increasing the partial pressure of N2 from 0·2 atm (20 kPa) to 1 atm (101 kPa). In the absence of N2, H2 production catalysed by nitrogenase increased about threefold. It is concluded that nitrogenase-catalysed H2 production is of major importance in the overall efficiency of biological N2 fixation in vivo.