- Volume 102, Issue 2, 1977

Mutants of Escherichia coli k12 have been isolated that grow on media containing pyruvate or proline as sole carbon sources despite the presence of 10 or 50 mm-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP:acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA:orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta− and Ack− mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate:CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.

Phosphofructokinase was not detected in extracts of Candida 107 prepared in a variety of ways but was highly active in cells treated with toluene. Disruption of these cells destroyed activity of phosphofructokinase indicating that the enzyme is extremely labile. As patterns of labelling from [1-14C]glucose and [6-14C]glucose showed that 60% of glucose was metabolized via the pentose cycle, augmentation of this cycle is necessary to account for the high molar growth yields of this yeast. Phosphoketolases, reacting with xylulose 5-phosphate and fructose 6-phosphate, were found but the extent to which they contribute to glucose metabolism was not assessed.

Phosphoketolase(s), catalysing the cleavage of xylulose 5-phosphate and fructose 6-phosphate, were found in Rhodotorula graminis, R. glutinis, Candida 107 and C. tropicalis. Activity towards the latter substrate only was found in C. humicola. Neither activity was found in Saccharomyces carlsbergensis. The enzyme(s) were isolated and purified eightfold from R. graminis. Inorganic phosphate, thiamin pyrophosphate and Mg2+ were required for activity. The enzyme had a K m of 1·25 mm for xylulose 5-phosphate and was markedly sensitive to NADH, NADPH, ATP and acetyl-CoA and less sensitive to phosphoenol-pyruvate, citrate and dodecanoyl-CoA. The activity of phosphoketolase was halved in the presence of an active transketolase which competes with it for the same substrate. The contribution of phosphoketolase to glucose catabolism may be only slight.

Polynucleotide phosphorylase activity was demonstrated in free-living and the symbiotic bacteroid forms of Rhizobium meliloti and in free-living Rhizobium japonicum. Crude extracts of R. meliloti f-28 catalysed the polymerization of 3·0 μmol ADP (mg protein)−1 h−1. The polynucleotide phosphorylase activity of symbiotic R. meliloti was about 50 % of that of free-living R. meliloti and remained almost constant throughout the development of the nodules. Partially-purified fractions of free-living R. meliloti f-28 catalysed the polymerization of ribonucleoside diphosphates, the phosphorolysis of synthetic homopolymers and natural heteropolymers, and the β-phosphate exchange reaction. The enzyme had a pH optimum of 8·0 and had an obligatory requirement for Mg2+ for maximum activity. The phosphorylase was not highly primer-dependent, had low specificity for guanosine-containing substrates, and exhibited cooperative-type kinetics.

The DNA of 13 yellow-pigmented strains of the genus Thermus, isolated from hot springs in Iceland, had a mean base composition of 60·5 to 65·1 mol % GC. In their growth characteristics and sensitivity to antibiotics, these strains resembled Thermus aquaticus strain yti. Glutamate, acetate and sucrose, with ammonium ions as a nitrogen source, supported growth. The enzymes of the tricarboxylic acid cycle were present and the glyoxylate cycle was constitutive. The stability of malate dehydrogenase in crude extracts was similar to that of strain yti.

An auxotroph enrichment procedure has been developed for Phycomyces blakesleeanus which has proven useful in obtaining both auxotrophs and drug-sensitive mutants. The technique is based on the differential heat sensitivity between ungerminated auxotrophic spores and germinated prototrophic spores. Germinated spores and mycelia die when left at temperatures higher than about 35 °C for 16 to 24 h but ungerminated spores survive this treatment and subsequently germinate if transferred to complete medium. With the dwarf colonial strain, replica plating permits the rapid characterization of these newly selected auxotrophs.

A series of mutants of Escherichia coli k12 resistant to lipopolysaccharide (LPS)-specific bacteriophages were isolated, and examined with regard to their general properties, phage typing, chemical analysis of their LPS, and genetic analysis. Fourteen classes of mutants were distinguished on the basis of phage typing and sensitivity to bile salts. Three of the mutant classes are sensitive to phages to which the parent is resistant. Mutants which are sensitive to bile salts generally lack heptose in their LPS, but two mutant classes are exceptions to this rule. Analyses of the sugars in the purified LPS of all mutant classes indicated that mutants were obtained which are blocked at most stages in core polysaccharide synthesis. On the basis of the chemical analysis, in conjunction with phage typing data and other known properties of the mutants, it is deduced which residue(s) is involved as a receptor for each of the phages used and which residues hinder these receptors. Some of the mutant classes do not seem to be changed in their LPS structure. Many of the mutations map in or near the rfa locus, but some are far removed from this region.

Three classes of mutants of Escherichia coli k12, isolated by selection for resistance to lipopolysaccharide-specific bacteriophages, were agglutinated by Concanavalin A which is presumed to interact with the lipopolysaccharide component of the outer membrane. Wheat germ and soy bean agglutinins did not agglutinate the parent or mutant strains. The adsorption of certain bacteriophages was also inhibited by Concanavalin A. The pattern of inhibition of adsorption of bacteriophages suggests that non-specific masking of receptors may occur, as well as specific masking of terminal glucose residues. Although bacteria were agglutinated by Concanavalin A, the permeability of the outer membrane seemed unaffected.

The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically. Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly. However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages. Pili determined by the only IncFV plasmid F0 lac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili. It is concluded that F, F0 lac and 711 pili have basic differences in spite of a morphological resemblance.

Summary: The relative rates of the glycolytic pathways were compared in vegetative mycelium and sporophore tissue of Agaricus bisporus. Simple radiorespirometry gave a C-1/C-6 ratio of 8·3 for pileus slices and 4·6 for vegetative mycelium. Glucose-6-phosphate dehydrogenase activity was greater in sporophores than in vegetative mycelium; glucosephosphate isomerase activity was higher in the mycelium. There was no significant difference between mannitol dehydrogenase activities in sporophore and mycelium. The results were consistent with a greater proportion of glucose oxidation occurring via the hexose monophosphate pathway in the sporophore than in the mycelium.

Treatment of the dimorphic fungus Aureobasidium pullulans with ethidium bromide resulted in the production of several mutants, cultures of which produce an increased proportion of yeast-like cells. A comparison of several mutant isolates with the wild type suggested that the yeast-like cell is responsible for the production of the polysaccharide, pullulan. Mutation was also shown to affect other metabolic patterns, e.g. the reduced accumulation of extracellular ethanol by the mutant. The cytochrome components of the respiratory chain in the wild type were examined by whole-cell spectrophotometry and compared with those of the mutants; the latter had a decreased cytochrome content.

The polyene antibiotic, amphotericin, inhibited phosphate uptake in Candida albicans more strongly than it inhibited growth. Cultures grown from an inoculum of young (2 h) cells were more affected than those inoculated with old (24 h) cells. Thus, the polyene displays a double effect on C. albicans (and presumably on other eukaryotic cells): it interferes with membrane sterols and also inhibits synthesis of a factor (or factors) during growth. Whether this factor(s) interferes with the uptake of the polyene antibiotic or neutralizes its effect by reacting with it remains unsolved.

Elongation of the stipe was investigated in Agaricus bisporus. The cell lengths in premarked regions of the stipe from base to top were compared. They were also compared with total stipe expansion and with the expansion occurring within the premarked regions. Cell elongation accounted for the elongation of the lower and mid-regions of the stipe but could not account for the elongation of the upper stipe region. Using autoradiography it was shown that there was a non-polar addition of new wall materials in the elongating cells of the stipe and, particularly in the upper stipe, cell division could be seen to occur by the formation of new septa. A model for cell growth in the elongating stipe of A. bisporus is described.

The ‘Phofil’ mutant of Rhodopseudomonas sphaeroides showed a disturbed adaptation with respect to a change from aerobic to photosynthetic conditions. The mutation was a pleiotropic one: firstly, the cells formed multinucleate filaments with occasional septa, and secondly, they integrated only a fraction of the synthetized pigments. The phenotypes appeared in photosynthetic conditions during a period of unbalanced growth (unadapted state) followed by balanced exponential growth where the phenotypes were corrected (adapted state). Ultraviolet light irradiation or exposure to low temperature, but not partial or total depigmentation, caused adapted cells to return to the unadapted state. Except for those associated with pigments, no appreciable defect in the polypeptide composition of the cell envelope was detected by SDS-polyacrylamide gel electrophoresis. However, the protein/phospholipid ratio of the envelope fraction was approximately 50 % higher, in the mutant than in the parent. These findings, and others described in the text, suggested the existence of defects in the cell wall and in the inner membrane.