- Volume 122, Issue 2, 1981

The labelling patterns of dihydroxyacetone and hexose phosphate produced after a 2 s incubation of methanol-grown Hansenula polymorpha with [14C]methanol have been determined. In dihydroxyacetone over 90% of the radioactivity was located in C-1 and C-3 and less than 10% in C-2. In glucose over 99% of the radioactivity was evenly distributed between C-1, C-3, C-4 and C-6 with less than 1% in C-2 and C-5. The results are consistent with the operation of a previously proposed pentose phosphate cycle of formaldehyde fixation in which dihydroxyacetone is formed by glycolyl transfer from xylulose 5-phosphate to formaldehyde.

Acinetobacter calcoaceticus grown on ethanol contains an NAD(P)+-independent alcohol dehydrogenase which resembles methanol dehydrogenase from methylotrophic bacteria in many respects. Likewise, the prosthetic group of this enzyme appears to be identical to that of methanol dehydrogenase, namely, pyrrolo quinoline quinone. The organism is unable to grow on methanol, which means that quinoprotein alcohol dehydrogenases are not restricted to methylotrophs. Arguments are presented for the idea that quinoprotein alcohol dehydrogenases exist in other alkane- or alcohol-grown bacteria. Although the enzyme from A. calcoaceticus can be best compared with that from Rhodopseudomonas acidophila in that both have very low affinities for methanol and are activated by aliphatic amines, the two enzymes are immunologically and electrophoretically unrelated. Furthermore, the A. calcoaceticus enzyme shows the broadest substrate specificity hitherto known for this type of enzyme in that it also oxidizes higher aldehydes. The extent of hydration of aldehydes cannot account for the aldehyde substrate specificity of these enzymes but the concept of a dual substrate specificity for alcohols and aldehydes can explain this very well. The different properties of the two enzymes compared with those of methanol dehydrogenases cannot be ascribed to the presence of iron as both enzymes contained a negligible amount of this metal.

Photophosphorylation in vivo by Chlorobium limicola was inhibited by lipophilic cations and the energy-transfer inhibitors diphenylphosphorylazide, Dio-9, 4-chloro-7-nitrobenzofurazan and chlorhexidene. Membrane-bound ATPase activity was also inhibited by these energy-transfer inhibitors. The formation of a membrane potential was stimulated approximately 1.7-fold on illumination, rising to a value between −110 and −150 mV. The sensitivity of the processes producing this membrane potential to uncouplers, energy-transfer inhibitors and 2-heptyl-4-hydroxyquinoline-N-oxide was measured in the light and the dark.

A protein antigen, I/II, was purified from cells and culture supernatants of Streptococcus mutans (serotype c ) by solubilization in urea followed by ion exchange chromatography and gel filtration. Immunological activity was retained after further purification by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The sedimentation coefficient was estimated to be approximately 8·7S by sucrose gradient centrifugation. The use of staining procedures, as well as the linear migration of this protein through different concentrations of acrylamide during SDS-PAGE, indicated that the antigen is probably not a glycoprotein. A lower molecular weight protein containing the free antigen I determinant was shown to have extensive homology with intact antigen I/II which implied that the former was a degradation product of the intact 185000 dalton antigen I/II. Antigen II, although previously defined by its resistance to proteases, could be further digested with trypsin after denaturation by SDS-PAGE. Antigen I/II could not be correlated with a group of glucosyltransferases isolated from whole cells and culture supernatants. The cell surface location of antigen I/II was established by the lactoperoxidase-catalysed iodination of intact cells followed by protein analysis using SDS-PAGE. Previously, the potential importance of antigen I/II has been established by its immunogenicity and capacity to induce a protective immune response against dental caries.

The rate of DNA synthesis in Allomyces arbuscula during differentiation of zoosporangia depends on the stage of development. The first stage of cytoplasmic reorganization in the hyphal apex is accompanied by a rapid decrease in DNA synthesis. The appearance of septa and the development of young zoosporangia are paralleled by a renewal of DNA synthesis which then declines as the zoosporangia mature. The transfer of an actively growing mycelium to the induction medium results in a transient accumulation (up to 1 h post-transfer) of ATP in the cell, which then starts to diminish. Analysis of the pulse-labelled native DNA by neutral CsCl equilibrium density gradient centrifugation indicates that in these early stages, all the DNA is being synthesized at a uniform rate. The amount of rRNA cistrons varies with the developmental stage and, on reorganization of the hyphal apex, the synthesis of such genes is inhibited and subsequently stimulated as zoosporangia mature.

Actinophage active against both neutrophilic and acidophilic streptomycetes were isolated from a variety of soils by a specific enrichment method. No phage were detected in soils with a pH below 6·0, despite the presence of acidophilic streptomycetes in these soils. However, some phage isolated from neutral soils were able to lyse acidophilic streptomycetes at pH 5·5. Selected actinophage were shown to be stable between pH 5·5 and 9·0 in sterile soils and broth, but were rapidly inactivated at lower or higher pH values. Survival of these phage was good in neutral soils, but negligible in acidic soils. This suggested that free phage were unable to remain infective in acidic soils, thus at least partially accounting for the failure to isolate them. Acidity was shown to have variable effects on several stages of phage replication including adsorption, penetration and the length of the latent period. The latter effect was directly related to the metabolic activity of the host.

The growth of the ciliate Tetrahymena pyriformis on non-growing Escherichia coli has been studied by following the time courses of population densities and protozoan mean cell volume in batch cultures. Viable, non-encysted protozoa always stopped feeding before the bacterial density was reduced to zero and non-feeding ciliates tended to swim faster than feeding ciliates. In addition, the number of bacteria and other particles of bacterial size consumed in the formation of one new ciliate, when averaged over the lag and reproductive phases of a culture, declined toward a limiting value of about 1.6 × 104 particles per ciliate as the initial density of such particles was increased.

Phages capable of transducing succinate dehydrogenase mutants (sdh) of Escherichia coli were isolated from pools of artificially constructed recombinant lambda phages using a selective casein digest medium. These phages produced characteristically dense turbid plaques, and as prophages they increased the aerobic growth efficiencies of sdh mutants on complex media but were unable to promote growth with succinate as sole carbon and energy source (an essential feature of sdh + strains). The phages were identified as fumarate reductase transducing phages (λfrdA) by the presence of a characteristic 4·9 kilobase pairs R. HindIII fragment of bacterial DNA, the expression of a polypeptide with a relative molecular mass of 72000 (the frdA gene product) and by comparing their transducing activities with authentic λ frdA phages. In parallel studies a strain containing a ColE1-frd hybrid plasmid (pGS1 = pLC16.43) was characterized. Transfer of pGS1 to sdh mutants was accompanied by increased aerobic growth efficiencies on complex media and the ability to utilize succinate as sole carbon and energy source. It was concluded that fumarate reductase can replace succinate dehydrogenase but the extent of the reversal of the sdh lesion depends on frd gene dosage and the titration of the repressor which normally prevents aerobic synthesis of the reductase.

We have previously described a system of experimental evolution in which many of the mutants of Klebsiella aerogenes selected for faster growth on xylitol (evolvants) synthesized elevated levels of ribitol dehydrogenase and have presented genetic evidence implicating gene duplication in the enzyme superproduction in some of the evolvants. Here we describe a physical approach to the screening for gene duplications and subsequent structure determination. Nick-translated, cloned ribitol operon (rbt) DNA was used as a hybridization probe to identify fragments containing rbt operon sequences in restriction digests of total bacterial DNA. Whilst several of the evolvants probably harbour duplications spanning the entire rbt operon, one of the spontaneously arising evolvants (strain A3) was shown to harbour a small (5·8 kilobase pairs) direct DNA repeat which encodes the dehydrogenase (but not the kinase) of the closely linked d-arabitol operon as well as the dehydrogenase (but not the kinase) of the rbt operon. The hybridization data suggest that there are 4 to 5 copies of the repeat arranged contiguously on the chromosome. The genetic instability of strain A3, the rbt fragment hybridization pattern of an A3 segregant and the activities of the pentitol catabolic enzymes in A3 are all consistent with the proposed gene duplication structure.

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group I) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.

An actinophage designated Bα was isolated from Streptomyces lavendulae S283. Phage Bα produced turbid plaques characteristic of temperate phage on S. lavendulae and other Streptomyces strains. Electron microscopic observation showed that phage Bα belongs to group B of Bradley’s morphological classification. Surviving cells from the plaques were lysogenic: they liberated phages during growth and were not lysed by the phage. Phage Bα was immediately inactivated on dilution into NaCl unless Ca2+ was also present. The cleavage pattern of the phage DNA was studied using restriction endonucleases.

A derivative of Streptococcus cremoris MU0001 (R1) has been isolated which has been cured of an inducible prophage. This isolate, its parent strain and a relysogenized derivative of the cured strain have been compared with respect to their ultraviolet inducibility, production of plaque-forming units, bacteriophage immunity and milk coagulation properties. The bacteriophage (𝜙MU1) harboured by strain MU0001 as a prophage has been characterized by isopycnic density gradient centrifugation, electron microscopy and sodium dodecyl sulphate gel electrophoresis. Antiserum raised against this bacteriophage was used to demonstrate true lysogeny in both the original strain and the relysogenized strain.

Single colony isolates of lactic streptococcal strains, most of which have been utilized for cheese-making in New Zealand, were u.v.-irradiated and their growth response was followed optically. All 45 strains showed either lysis or impairment of growth. Their concentrated, bacteria-free supernatants were examined by electron microscopy for the presence of phage particles. Intact phages were observed in most lysates, while disrupted phage parts were found in the remaining lysates. Plaque-forming ability was demonstrable in a few cases. Lysogenization of a different strain by one of the phages detected by this technique was attempted. Similarities and differences with respect to lytic phages which have been isolated for these same strains in New Zealand cheese factories are discussed in relation to the possible origin and control of phages.

Temporal studies were made of factors associated with increased RNA synthesis in guinea pig liver during Q fever. DNA-dependent RNA polymerase activities increased immediately after infection. The major distribution of RNA polymerase classes shifted from class II to class I during infection. Ornithine decarboxylase activity was induced or stimulated soon after infection and remained elevated throughout the four-day period studied. S-Adenosylmethionine decarboxylase activity increased on the first day after infection and subsequently declined. Concomitantly elevated concentrations of the polyamines putrescine, spermidine and spermine reached a maximum on the first day after infection and then decreased. A model is presented to integrate these and other results to explain how RNA synthesis may be regulated during infection.

Phenotypically determined resistance of gonococci to killing by normal human serum can be generated by growth of susceptible organisms in media containing guinea pig, calf or human serum. However, even in the best medium tested, i.e. defined medium containing 50% (v/v) guinea pig serum, resistance was greatly reduced after 24 h incubation and the maximum number of colony-forming units generated was 107 to 108 ml−1. Resistance was not acquired after incubation in guinea pig serum at low temperature (8 °C), supporting previous indications that metabolic activity was necessary for the generation of resistance. Alteration of the concentration of glutamine, proline, lactate or iron had little or no effect on the generation of serum resistance under the conditions used. Optimum conversion to resistance occurred at pH 6·0 to 6·5 and both non-diffusible and diffusate fractions of dialysed guinea pig serum promoted resistance. Furthermore, resistant organisms could be produced by incubation in defined medium containing the diffusate from guinea pig serum and 0·1% bovine serum albumin, a step which should facilitate identification of the resistance-promoting factor.

A quantitative assay is described for the measurement of cytochrome o in intact cells of E. coli . The procedure involves flash photolysis of the CO-liganded, reduced enzyme in the absence of O2 at temperatures (approx. -100°C) at which the rate of recombination of CO is immeasurably slow. Other CO-binding pigments known to be present, particularly cytochrome d , are excluded from the photodissociation spectrum under these conditions. Measurement of the content of cytochrome o in bacteria separated into size (and thus age) classes by zonal centrifugation shows that the cytochrome accumulates continuously, probably exponentially, throughout the cell cycle and thus constitutes a constant proportion of cell protein during the cycle. The velocity of recombination of CO with cytochrome o at –65 °C is invariant over the cell cycle.

Cyclic AMP phosphodiesterase in Klebsiella aerogene is a soluble cytoplasmic enzyme with an apparent Km of 0·9 mm and a pH optimum of 7·0. It was inhibited by EDTA, Mg2+ and other metal ions. The enzyme activity was inhibited or activated by some nucleotides but not by any metabolite except pyruvate. It was inhibited by the methylxanthines, caffeine, theophylline and methylisobutylxanthine. During starvation or substrate-accelerated death, the enzyme activity remained essentially constant. It is postulated that during substrate-accelerated death the enzyme acts as a drain on the cellular cyclic AMP levels. The cyclic nucleotide concentrations during substrate-accelerated death are proposed to be controlled directly by adenylate cyclase.

Bacterial growth and lethality of Escherichia coli infection of mice were enhanced by X-irradiation but not by treatment with carrageenan. Since carrageenan depletes macrophages but not polymorphonuclear leucocytes, it is concluded that protection against E. coli , at least in the early phases, depends mainly on polymorphonuclear leucocytes.

A number of mutants impaired in penicillin production have previously been isolated from Penicillium chrysogenum and Aspergillus nidulans . During cofermentation of osmotically fragile mycelia derived from these strains, in the presence of inhibitors of cell wall regeneration, intergeneric cosynthesis has been demonstrated between mutants which are probably impaired in different parts of the penicillin biosynthetic pathway.

Transducing particles produced by bacteriophage SPP1 infection of Bacillus subtilis were separated from plaque-forming units by CsCl density-gradient centrifugation. The density in CsCl of DNA isolated from such purified transducing particles was similar to that of bacterial DNA, indicating that the transducing particles probably contain DNA of exclusively bacterial origin. Bacterial DNA synthesized after infection of the donor culture was also encapsulated in the transducing particles. The number of transducing particles was at least 10 times higher than that of the transductants.