- Volume 126, Issue 2, 1981

Mitochondrial adenosine triphosphatase (ATPase) of the ciliate protozoon Tetrahymena pyriformis ST is completely inhibited by antiserum prepared against F1-ATPase purified from Schizosaccharomyces pombe, and by naturally occurring inhibitor proteins from this yeast and from bovine heart mitochondria. An ATPase inhibitor protein is also present in extracts of T. pyriformis. Mitochondrial ATPase of T. pyriformis is only partially inhibited by the F0-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, oligomycin, leucinostatin, triethyltin sulphate and venturicidin, and (at high titres) by the F1-ATPase inhibitors Dio-9, efrapeptin, 4-chloro-7-nitrobenzofurazan and spegazzinine. Aurovertin, citreoviridin and quercetin were not inhibitory. Resistance to inhibitors distinguishes this mitochondrial ATPase from all those previously examined.

Fungal and bacterial oxidation of steroid hydrocarbons has been examined. A rapid assay method for detection of metabolites derived from 5α-[4-14C]cholestane is described. Out of 87 organisms tested, 16 gave isolable oxidation products, while others degraded the hydrocarbon without producing stable intermediates. Preliminary characterization of these compounds is based on chromatographic properties.

Intact cells harvested from O2-limited batch cultures of Escherichia coli K12 contained high levels of the CO-binding cytochromes d, o and a 1. In photodissociation difference spectra (i.e. photolysed minus reduced + CO), a peak at 436 nm and a trough at 415 nm have been assigned to an o-type cytochrome, and not cytochrome d, by photolysis with white light and an He-Ne laser. The reaction of reduced cytochrome o 436 with O2 at sub-zero temperatures involved O2 binding to give intermediate(s) with spectral characteristics similar to those of the reduced oxidase-CO complex. The reaction with O2 at successively higher temperatures (range − 98 to − 59 °C) was accompanied by the formation of a trough (with reference to the CO-liganded state) at 436 nm which eventually shifted to 432 nm, indicative of the oxidized form. The apparent energy of activation at low temperatures was 446; kJ mol−1 (107; kcal mol−1). There was a linear relationship between the rate of formation of the oxygen compound and the O2 concentration up to about 0·5; mm. The second-order constant for this reaction was 109; m−1 s−1 at -100C, at least 10-fold greater than for the reaction of cytochrome o 432 with O2 in cells from vigorously aerated cultures. The reaction of both types of cytochrome o with O2 was not readily reversible in the light or in the dark and was further distinguished from the reaction with CO by the markedly lower velocity of the CO reaction. Comparisons are drawn between the reactions with O2 of cytochrome(s) o in E. coli from O2-sufficient and O2-limited cultures and of mitochondrial cytochrome a 3. It is proposed that, like the synthesis of cytochrome d, the formation of cytochrome o 436 represents an adaptation of the organism to reduced O2 availability.

The buoyant density of isolated mitochondria and glyoxysomes obtained from Neurospora crassa grown under different conditions was investigated by density-gradient centrifugation on Percoll. Mitochondria isolated from N. crassa in the exponential phase of growth with sucrose as the carbon source were more dense (1·077; g ml−1) than those isolated from hyphae in the stationary phase (1·069; to 1·073; g ml−1). A second population of mitochondria (density 1·059; to 1·061; g ml−1) was also present in the stationary phase. When N. crassa was grown with acetate as the carbon source, mitochondria isolated from hyphae in the exponential phase of growth were found to be less dense (1·079; g ml−1) than those present in the stationary phase (1·089; g ml−1). The glyoxysomes were found to be less dense than the mitochondria and their density decreased from 1·076; to 1·055; g ml−1 as the hyphae aged.

Mitochondria isolated from Neurospora crassa grown with sucrose as the sole source of carbon and energy oxidized citrate and cis-aconitate at high rates, and showed good respiratory control and high ADP/O ratios. The oxidation of both substrates was inhibited strongly by rotenone but only slightly by malonate. In contrast, isocitrate was oxidized slowly, with poor respiratory control. Treatment with detergent showed that both NAD+- and NADP+-isocitrate dehydrogenases were situated within the mitochondrial matrix. It thus appeared that the rate of oxidation of exogenous isocitrate was limited by a slow rate of translocation across the inner mitochondrial membrane.

When N. crassa was grown in an acetate-containing medium, the mitochondrial pellet, prepared by differential centrifugation, catalysed a rapid oxidation of isocitrate and contained a high activity of isocitrate lyase. The oxidation of isocitrate and succinate was strongly inhibited by malonate but only slightly by rotenone. Density-gradient centrifugation revealed that the apparent oxidation of isocitrate by mitochondrial pellets was due to contamination by glyoxysomes. Isocitrate was converted into glyoxylate and succinate in the glyoxysomes, then succinate was translocated across the inner mitochondrial membrane and oxidized by the respiratory chain.

A protease associated with spore coats of Bacillus cereus was extracted with 25 mM-dithioerythritol plus 2 M-urea at pH 9·0; and purified by ammonium sulphate fractionation and affinity chromatography. The spore protease differed from two intracellular proteases in pH optimum, pH stability, heat stability, stability in the presence of dithioerythritol and urea, molecular weight and in lack of reactivity with antibody to one of the intracellular proteases. The distinctive nature of the spore protease was also suggested by the fact that in a mutant with an altered intracellular protease, the properties of the spore protease remained unchanged. Inhibitor studies suggested that the spore protease was of the serine type with some similarities to trypsin. Two mutants were isolated with altered heat and pH stabilities of the spore protease; the intracellular proteases were unaltered. Spores produced by the mutants contained the same amount of coat protein per spore as the wild-type and germinated as well as the wild-type.

Agaricus bisporus and other fungi (Basidiomycetes, Ascomycetes, Fungi Imperfecti and Phycomycetes) grew and produced transparent zones in opaque media containing killed Bacillus subtilis as sole carbon and nitrogen source. Other Gram-positive and Gram-negative bacteria were also degraded by A. bisporus. Degradation of B. subtilis by A. bisporus was examined by light and electron microscopy. Liquid cultures were assayed for extracellular fungal enzymes which included β-N-acetylglucosaminidase, lipase, nucleases and acid, neutral and alkaline proteases.

Microbial biomass in compost may serve as a source of nitrogen, carbon and minerals, and as a water reserve for the growth of A. bisporus.

Sexual hybridization of two divergent lines of Aspergillus nidulans, which had been selected for increased penicillin titre through successive cycles of mutagenesis, released considerable variation for this character. The recovery of segregants with titres equivalent to that of the unselected ancestor suggested that mutations in different genes had been selected in the two lines. However, complementary segregants with substantially improved titres were not found, indicating interactions, probably of a duplicate nature, among the induced mutations. All the genetic variation released by hybridization was fixed following two generations of selection for high titre, but only a small gain over the initial selection lines was achieved. Hybridization of divergent strains has been widely advocated as a means of strain development. The failure to achieve the anticipated gains in this programme is attributed primarily to the unfavourable interactions amongst the induced mutations. Whether similar interactions occur generally in crosses between strains selected by mutagenesis remains to be established and will be an important factor in determining the contribution of recombinational approaches to yield improvement.

Phage j2, a lysogenic phage in Salmonella typhi J2, was shown to produce tiny plaques on various Vi type strains of S. typhi, to be a generalized transducing phage, and to have many characteristics including a serological one in common with phage P1 of Escherichia coli. Lysogenization of various S. typhi type strains with j2 or P1-group phages usually resulted in the alteration of the phage types of the S. typhi strains, except that phage j2 did not cause alteration of type 53. Phage j2 transduced, at high frequencies, much larger DNA molecules (up to at least 70 megadaltons) than those known to be transduced by Salmonella phage P22: this should prove useful for the genetic analysis of S. typhi.

A mating system to transfer the ability of growing autotrophically as a hydrogen bacterium (aut + marker) has been developed in Nocardia opaca strain 1b. Following filter mating between an aut + trp str strain and an aut strain, transconjugants of the phenotype Aut+Trp+Strs were obtained. The number of recombinant colonies exceeded the number of revertants by a factor of 105. This and other crosses indicated that only the aut + marker is transferred. Among other markers tested (growth on lactate and benzoate) no new combinations were found. A few strains of the Rhodococcus taxon were tested for their ability to act as recipients. Transconjugants of Rhodococcus erythropolis and Corynebacterium hydrocarboclastus able to grow autotrophically were isolated. Most transconjugants were able to act as aut + donors, but with different recipient spectra. As indicated by the observation of sectored colonies after autotrophic growth, by the measurement of the frequency of loss of autotrophy compared to auxotrophic markers, and by the effect of mitomycin C treatment, the aut + marker does not become integrated in the recipient chromosome in a stable state. It is probably located on a plasmid.

Phage X was isolated from sewage as plating on Escherichia coli or Salmonella typhimurium strains harbouring the incompatibility group X plasmid R6K. It also plated on a strain of Serratia marcescens carrying this plasmid. It failed to form plaques on Proteus mirabilis, P. morganii or Providencia alcalifaciens harbouring R6K, but did multiply on them. No phage increase occurred with homologous R− strains. Phage X also plated or registered an increase in titre on E. coli or S. typhimurium strains carrying various plasmids of incompatibility groups M, N, P-1, U or W as well as the unassigned plasmid R775. It adsorbed to pili determined by a group P-10 plasmid in a Pseudomonas aeruginosa strain but did not multiply on this organism. The phage was filamentous and curly, resistant to ribonuclease and diethyl ether and sensitive to chloroform. It adsorbed to the tips of pili.

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F0 lac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F0 lac; it had a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F0 lac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F0 lac was found to be serologically related to phage UA-6. another isolate specific for EDP208.

The 23 kilobase multiple drug resistance r-determinant (r-det) of the R plasmid NR1 is an IS1-mediated transposon, Tn2671. Drug-resistant Escherichia coli transductants isolated after infection with bacteriophage P1::Tn2671 derivatives carry the intact r-det in their chromosomes. Independently isolated transductants carry the r-det at different locations on the chromosome. From the E. coli chromosome, Tn2671 can transpose to various locations on the phage P7 genome. Throughout these processes, r-det is maintained as a stable unit. Various possible molecular mechanisms, which all might contribute with characteristic frequencies to the transposition of Tn2671, are discussed. The results presented are relevant to the understanding of mechanisms for a wide spreading of drug resistance genes.

The plasmid SCP2, initially discovered through the occurrence of a high fertility variant, SCP2*, is a self-transmissible fertility factor capable of promoting chromosomal recombination within Streptomyces coelicolor A3(2). Further high fertility variants of SCP2, similar to SCP2*, were isolated from amongst the recombinants produced in matings involving SCP2, and their genetic properties were compared. SCP2 and its derivatives elicit lethal zygosis on transfer into an SCP2− recipient; this plasmid-determined phenotype allowed the isolation of SCP2− strains and the detection of the interspecific transfer of SCP2* by mating from S. coelicolor to Streptomyces parvulus and Streptomyces lividans, whereupon it underwent stable maintenance. The transfer genes of SCP2 and SCP2*, which are not normally fully expressed, were shown to undergo transient derepression on entry into an SCP2− strain. An ‘entry disadvantage’ system determined by SCP2 and SCP2* was recognized.

The production by the fungus Nectria haematococca of antibiotic pigments related to fusarubin can be increased by (i) culture conditions unfavourable for good growth, (ii) multiplication of two cytoplasmic factors of unknown molecular nature, and (iii) mutation of nuclear genes. Six nuclear genes involved in the regulation of naphthoquinone release have been characterized and mapped. Some have three allelic forms: the wild allele, one or several mutated alleles determining the hyperproduction of pigments, and one or several intragenic suppressors of the latter mutations. These genes also seem to be involved in developmental events, especially in sexual reproduction.

An oligosporogenic strain of Bacillus cereus T was found to carry an inducible phage. CsCl-ethidium bromide gradients of lysates of this strain contained satellite DNA of the same density as the phage genome. Neither the phage nor the satellite DNA could be detected in the sporogenic parental strain. Purified phage preparations increased sporulation from 10-50-fold when added to cultures of the oligosporogenic strain.