- Volume 110, Issue 2, 1979

SUMMARY: Mutants of Escherichia coli K12 blocked in the oxidation of pyridoxine 5′-phosphate (‘Oxidase’ mutants) excreted pyridoxine at an initial rate of 19 pmol h−1 (108 bacteria)−1, i.e. 0·6 nmol h−1 (mg dry wt)−1, when starved for pyridoxal. Glycolaldehyde, l-phosphoserine, dl-serine and, to a lesser extent, l-leucine stimulated the rate of pyridoxine excretion, but there was no significant stimulation by 2′-hydroxypyridoxine. 4′-Deoxypyridoxine inhibited or stimulated growth of the ‘Oxidase’ mutant, depending on the relative concentrations of added pyridoxal and 4′-deoxypyridoxine. It was concluded that stimulation of growth by 4′-deoxypyridoxine was due to its conversion to pyridoxal.

SUMMARY: Trehalose, a storage sugar of baker's yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5·5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, α-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative analysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of α-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of α-glucoside-transporting systems being five.

SUMMARY: In mouse peritoneal macrophages infected in vitro with Salmonella typhimurium the number of viable bacteria and the number of stainable bacteria detected by light microscopy both increased at similar rates with a doubling time of more than 1 h. Antibiotics were not present; instead extracellular bacteria were removed by frequently rinsing the cells. The bacterial doubling time in the same medium in the absence of macrophages was about 20 min. Penicillin added to macrophage monolayers rapidly entered the macrophages, reaching a diffusion equilibrium. The penicillin-induced bacterial death rate appeared to depend on the bacterial division rate as well as on the penicillin concentration. These properties of penicillin were used to monitor intracellular bacterial division and death rates. The results indicated that intracellular killing, with the disappearance of stainable bacteria, did not contribute to the extended doubling time in macrophages. It was concluded that the intracellular environment of the bacteria was probably growth inhibitory but not bactericidal.

SUMMARY: When exponentially growing Dictyostelium discoideum amoebae were harvested from growth medium and suspended in a nutrient-free phosphate buffer, the cells released 20 to 30% of their total β-N-acetylglucosaminidase and α-mannosidase activities. This release was inhibited by sodium azide but stimulated (to a level of 90% release) by various saccharides when added to the phosphate buffer at millimolar concentrations. Various salts, amino acids and nucleotides were unable to stimulate the release process. Non-metabolizable sugars were more stimulatory than metabolizable sugars, but no clear distinction related to structure, ability to be transported or ability to be metabolized was found to account directly for the varying effects of the sugars on glycosidase release. The rate and amount of glycosidase release was dependent on the sugar concentration but not on the cell density. The results presented suggest that sugars stimulate a specific release of a variety of lysosomal enzymes and the relationship of this release to D. discoideum morphogenesis is discussed.

SUMMARY: Salmonella typhimurium did not inhibit fusion of lysosomes with the phagocytic vacuoles in infected macrophages and caused no increase in cyclic adenosine 3′: 5′-monophosphate. Glutaraldehyde-killed bacteria showed rapid ultrastructural degeneration within the phagolysosomes. In contrast, untreated bacteria were resistant to digestion by lysosomal enzymes. Intracellular survival of this species appears to depend on resistance to, and not evasion of, lysosomal enzymes.

SUMMARY: When ingested by mouse peritoneal macrophage monolayers, live Mycobacterium microti caused a sustained increase in monolayer cyclic AMP content and fusion of lysosomes with the bacterium-containing phagosomes was impaired. Ingested live M. bovis BCG caused a transient increase in cyclic AMP and the defect in phagolysosome formation was less pronounced. Dead mycobacteria and live M. lepraemurium neither enhanced monolayer cyclic AMP content nor inhibited phagolysosome formation. Mycobacterium microti and BCG exceeded M. lepraemurium in cyclic AMP-synthesizing activity in vitro but the question of whether bacterial cyclic AMP contributed substantially to the increments in infected macrophages was not resolved. Antibody-coated BCG retained the ability to synthesize cyclic AMP and to enhance monolayer cyclic AMP but lost the ability to inhibit phagolysosome formation in macrophages. The observations are discussed in terms of possible control of phagolysosome formation by cyclic nucleotides.

SUMMARY: R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 × 106. It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1·26 g cm−3. These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium, Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.

SUMMARY: Two serine proteases in extracts of Escherichia coli grown to stationary phase were purified to homogeneity using affinity chromatography on gramicidin S-Sepharose 4B. One enzyme was closely related to, if not identical with, the ‘trypsin-like’ protease II of E. coli. The other was capable of cleaving the subtilisin chromogenic substrate N-carbobenzoxy-l-alanyl-l-alanyl-l-leucine-p-nitroanilide and resembled the intracellular serine proteases of Bacillus spp. The amino acid composition of this E. coli protease was similar to that of the Bacillus licheniformis enzyme. These data indicate a relationship between proteolytic enzymes of evolutionary distant Gram-negative Enterobacteriaceae and Gram-positive spore-forming Bacillus.

SUMMARY: The ultrastructure of the wall of Candida albicans strain 6406 was examined in polyene-resistant organisms obtained by continued incubation after the cessation of growth. The walls of organisms harvested either during the exponential phase of growth or after 24 h starvation, when examined in situ, showed the typical layered appearance. After 72 h starvation, when the resistance to amphotericin B methyl ester (AME) was 60 times greater than that of exponentially growing organisms, both the periplasmic material and the distinct electron-dense layers were absent from the wall. At this stage there was no increase in the thickness of the wall. After 144 h starvation the thickness of the wall had increased from 143 ± 22 nm (exponential phase organisms) to 211 ± 58 nm. If after 144 h starvation the organisms were incubated for 1 h in fresh nutrient medium they regained their sensitivity to AME and the wall regained the periplasmic material and its characteristic multilayered appearance. During the first 24 h starvation there was a considerable fall in the soluble glucan fraction, but on continued incubation there was little change in the relative proportions of the major carbohydrate constituents of the cell. Thin sections of purified walls isolated from organisms harvested either during exponential growth or after 144 h starvation were identical in appearance and characterized by the absence of the electron-dense layers observed in sections of intact cells and by a reduction in thickness to 100 ± 20 nm.

SUMMARY: While lomofungin at 20 μg ml−1 effectively halted the synthesis of ribonucleic acids in protoplasts of Saccharomyces cerevisiae, the onset of biogenesis of new wall on the protoplast surface was not impaired. The synthesis of mannan-protein wall matrix continued for about 30 min after addition of lomofungin while the formation of 1,3-β-glucan networks was not influenced even after prolonged incubation. These results suggest that the life-span of limiting mRNA molecules coding for the proteins of the wall matrix is about 30 min while the turnover of 1,3-β-glucan synthases is relatively low.

SUMMARY: Ubiquinone was extracted from free-living Rhizobium japonicum, grown aerobically or anaerobically, and from the symbiotic bacteroid form; it was tentatively identified as the Q-10 homologue. The ubiquinone concentration was highest in symbiotically grown R. japonicum but the ratio ubiquinone:total cytochrome was about 1·5:1 in membrane particles from organisms grown under all three conditions. The ubiquinone was reduced 75% by NADH, completely oxidized by oxygen but not oxidized by nitrate. NADH oxidase activity and nitrate reductase activity in membrane particles from organisms grown under the different conditions were similar except that nitrate reductase activity was low in aerobically grown organisms. It is concluded that ubiquinone functions in electron transport to oxygen but not to nitrate.

SUMMARY: The effect of dissolved oxygen concentration on glucose metabolism, poly-β-hydroxybutyr-ate (PHB) synthesis and respiration of nitrogen-fixing Azotobacter beijerinckii was investigated over a range of values spanning oxygen limitation and sufficiency (nitrogen limitation) in continuous culture. The activities of the Entner-Doudoroff enzymes decreased with increasing oxygen supply while glucose-6-phosphate dehydrogenase was unaffected; β-ketothiolase and acetoacetyl-CoA reductase decreased more markedly, reflecting the fall in PHB content. The metabolic quotients for O2 and CO2 increased rapidly with increasing oxygen supply, in keeping with the respiratory protection of nitrogenase. The specific activities of all these enzymes, and also of fructose-1,6-bisphosphate aldolase, increased with increasing dilution rate under either oxygen or nitrogen limitation, except that both β-ketothiolase and acetoacetyl-CoA reductase activities fell at the highest dilution rate (0·23 h−1) under oxygen limitation. It was concluded that the role of the Entner-Doudoroff sequence as the major route of glucose metabolism in A. beijerinckii was unaffected by oxygen concentration and growth rate.

SUMMARY: Six mutants of Bacillus subtilis 168 that are temperature-sensitive in spore outgrowth were isolated. The outgrowth process proceeds normally at 35 °C, but at the non-permissive temperature (47 °C) it is arrested at a specific stage characteristic for each mutant strain. The mutants are not altered in vegetative growth whether at 35 °C or at 47 °C. They were characterized for their ability to synthesize RNA, proteins and DNA during outgrowth. A mutant defective in spore germination was also isolated; less than 5% of its spores can germinate at any of the temperatures tested. The mutations were mapped by means of transduction and transformation. The isolation of a number of outgrowth mutants which map at different loci and which affect outgrowth at different times is discussed in relation to the regulation of this process.

SUMMARY: Bacillus megaterium NCIB 7581, when growing exponentially in a simple chemically defined medium (without 2,6-diaminopimelic acid), contained only 4 mm-diaminopimelate in the free amino acid pool; this diaminopimelate was 84% (w/w) meso-isomer and 16% ll-isomer. Growing organisms could take up any of the three isomers of diaminopimelate from the medium, though the dd-isomer was taken up at only half the rate of the other two isomers (meso- and ll-). When lysine was also present in the medium, most (70%, w/w) of the diaminopimelate that was taken up entered peptidoglycan; only about 5% of the total uptake was into the free amino acid pool. When dd-diamino[14C]pimelate was supplied in the medium, 86% of the diamino[14C]pimelate incorporated into peptidoglycan was present as the meso-isomer and 14% was the dd-isomer.

SUMMARY: The influence of zinc, lead and cadmium contamination on the phylloplane microflora of Lolium perenne growing at various distances from a smelting complex was investigated. Bacteria, yeasts and filamentous fungi were isolated using a leaf washing method, and relative metal tolerance was assessed by the incorporation of zinc, lead and cadmium salts into the growth media. Numbers of fungal propagules were also assessed by direct observation of leaf segments. Non-pigmented yeasts of the genus Cryptococcus were particularly tolerant to the metals, both in vitro and in vivo. Bacteria were less tolerant in vitro, but populations in the field were found even under the highest levels of metal contamination. Filamentous fungi were more tolerant than bacteria to heavy metals in vitro but population levels appeared to be particularly influenced by lead contamination on the leaf surface.

SUMMARY: L-Arginine was required for growth by a high percentage of strains of Staphylococcus species that were niche-specific and/or host-specific, but was usually not required for growth by species showing a wide host range. Growth stimulation patterns with arginine intermediates indicated that most of the auxotrophic strains had blocks in an early step(s) in arginine biosynthesis. These strains were designated phenotypically as Arg (CHG) according to the Salmonella typhimurium classification scheme. Staphylococcus simulans strains appeared to be either ArgA or ArgI. The ArgI strains of S. sirnulans and S. capitis had moderate to high ornithine carbamoyltransferase (EC 2. 1. 3, 3) activities and therefore could not be designated as argl mutants. ArgI strains in other species had no or very low ornithine carbamoyltransferase activities. All of the natural Staphylococcus auxotrophs tested grew in the presence of L-citrulline and had moderate to high argininosuccinase (EC 4. 3. 2. 1) activities. Arginine auxotrophs of species with a wide host range were often capable of reverting to arginine-independent or complete prototrophic growth, whereas auxotrophs of species that tended to be niche-specific and/or host-specific were incapable of reversion to arginine-independence, even in the presence of various mutagens. A relationship between the nature of arginine auxotrophy and habitat is suggested.

SUMMARY: A Bacillus subtilis mutant is described which forms heat-resistant spores only in the presence of external dipicolinic acid (DPA). The mutation, dpa-1, is localized in a new sporulation locus, linked to pyrA. The dpa-1 strain is unable to synthesize DPA but can incorporate external DPA. The amount of DPA incorporated, the frequency of heat-resistant spores and their degree of resistance are all dependent on the concentration of external DPA. Spores of dpa-1 strains exhibit normal resistance to most chemicals, including octanol and chloroform, but not to ethanol, pyridine, phenol and trichloroacetic acid. Complete resistance to the latter group depends on DPA. DPA incorporation is slow and apparently requires an energy supply but not protein synthesis. Direct involvement of DPA in the heat-resistance of the spores is suggested. Thin sections of DPA-less spores exhibit clearly visible cytoplasmic membranes and ribosomes. These structures are absent or less visible in the core of spores obtained with added DPA.