- Volume 130, Issue 10, 1984

By techniques involving differential centrifugation and specific precipitation with CaCI, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0°C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di-and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaploethanol all inhibited both activities.

A strain of Corynebacterium glutamicum used for industrial production of glutamate had uptake systems for l-glutamate and l-serine. These transport systems were inhibited by a protonophore and by an ionophore, indicating that they were driven by a proton-motive force. Cells grown in the presence of an acylated surfactant used in industry to trigger glutamate excretion are known to have a decreased phospholipid content and highly saturated lipids. These surfactant-treated cells were no longer able to accumulate glutamate, while the serine uptake remained undisturbed. As a working hypothesis, it is proposed that the surfactant-induced membrane modifications could specifically result in an uncoupling of the glutamate uptake system, which could consequently be used as a specific excretion system.

Proteus mirabilis CW977 produced high yields of the bacteriocin proticine 3 upon mitomycin C induction of cultures growing at 30 °C. The proticine was purified and found to have a relative density of 1·299 and to be composed of 10 proteins assembled into structures resembling contractile phage tails. When induction was performed at 41 °C neither proticine particles nor proticine activity was detected, although the growth rate of cells and degree of lysis were indistinguishable from that at 30 °C. Failure in proticine production was due to a 41 °C sensitive stage occurring between 60 and 90 min after the addition of mitomycin C. During this period at 30 °C, two proteins of mol. wt 58000 and 41000 were formed. These proteins were associated with events leading to the formation of proticine particles with biological activity. When the production of both proteins was prevented either by chloramphenicol or as a result of mutation or through sampling before they were formed, no proticine particles were found nor proticine activity detected. The synthesis of both proteins was also inhibited at 41 °C. Co-electrophoresis of the labelled proteins with unlabelled purified proticine confirmed that the protein of mol. wt 58000 was a proticine structural protein. The protein of mol. wt 41000 was not a structural component of proticine and its role, if any, in proticine 3 production is possibly that of an assembly protein.

Continuous N-terminal amino acid sequences between 26 and 64 residues long were determined for the gas vesicle protein (GVP) isolated from four genera of cyanobacteria and two species of halobacteria. Definite homology was established between the GVPsof all six organisms. Within the cyanobacteria the homologies varied from 98% to 85% with the order of decreasing similarity to Anabaena being Aphanizomenon, Oscillatoria, and Dactylococcopsis, in agreement with phylogenetic affinities. (Further homologies were also established between sequences at the C-terminus of GVP from Anabaena and Microcystis.) In the GVP from Halobacterium there was a short non-homologous sequence near the N-terminus followed by 39 residues showing 70% homology with GVP from Anabaena, and 79% with that from the halophilic Dactylo-coccopsis. An antibody raised against the Anabaena gas vesicles was effective in agglutinating gas vesicles from five different cyanobacteria, Microcyclus (a colourless eubacterium), the two halobacteria and Methanosarcina (another archaebacterium). The origin of the gas vesicle is discussed.

To determine whether oral streptococci might exchange genetic information in the oral cavity, paired transformable strains of Streptococcus mutans, Streptococcus sanguis and Streptococcus milleri were grown together. Chromosomal and plasmid-borne antibiotic resistance markers could be readily transferred from S. mutans GS-5 to S. milleri NCTC 10707 or S. sanguis Challis during mixed growth. However, no exchange from the latter two organisms to strain GS-5 could be detected under these conditions. The transfer of genetic information from S. sanguis to S. milleri was also observed.

Plasmid formation by N derivatives of lambdoid phages has been reinvestigated with transducing phages carrying the trp, lac and gal genes of Escherichia coli. Transduction by λN cI derivatives was inefficient and short-lived in each case, under both selective and nonselective conditions. Mutant operators were introduced to relieve possible auto-repression by the cro gene product. Such N-defective phage genomes were able to propagate continually as plasmids, although without selection they were gradually lost from the carrier cells. Plasmid formation remained inefficient, however. The entire chromosome of N phages can be expressed by transcription that leaks through the serially arranged Rho-dependent terminators. Some functions so expressed are deleterious to the plasmid state and cause the instability of λN plasmids.

It was concluded in the preceding paper that λN − cI− genomes probably failed to form plasmids within infected Escherichia coli cells because they leakily express functions that act to destabilize the plasmid state. This prediction was investigated by examining the effect upon plasmid-forming ability of the loss of possible anti-plasmid functions. The loss of Ter function was found to allow long-term plasmid formation, although the efficiency of initial plasmid formation and the heritable stability without selection were low. The combined loss of the int, red and gam gene functions also promoted plasmid growth, although the absence of Ter λ was necessary. In contrast, the presence of Ter80 (due to an h 80 substitution) did not prevent plasmid formation when the int, red and gam genes were absent, indicating that Ter80 does not attack the closed-circular form of the λ chromosome. The combined loss of the ter, int, red and gam gene functions facilitated fully efficient inheritance of the λN − cI− plasmids in the absence of selection, although the efficiency of initial plasmid formation remained low. However, cells harbouring such plasmids suffered a decline in viability, indicating that the plasmids expressed a function (or more than one) that acts to debilitate the host cells - presumably an effect that is increased with this genotype because the modified λN − cI− plasmids are inherently more stable. The possible involvement of the λS and kil functions in destabilization is discussed.

Five of eight strains of Saccharomyces bailii and one of 13 strains of S. bisporus were found to harbour DNA plasmids, pSB1 and pSB2 plasmids were isolated from S. bailii strains IFO 0488 and IFO 1047, respectively, and pSB3 and pSB4 from S. bisporus strain IFO 1730. All four plasmids resemble 2-μm DNA of S. cerevisiae in that (i) their molecular sizes are about 6 kb, (ii) each molecule possesses a pair of inverted repeats, (iii) they exist as a mixture of two isomers and (iv) their copy numbers in the native host are similar. None of them showed homology with 2-μm DNA or with each other by Southern hybridization under moderately stringent conditions, but pSB4 hybridized with the pSR1 DNA, which was found previously in a strain of S. rouxii. Each of the pSB plasmids has DNA sequence(s) effective for autonomous replication in S. cerevisiae. In S. cerevisiae, pSB3 and pSB4 showed intramolecular recombination but neither supported isomerization of 2-μm DNA.

An efficient system for cloning in Bacillus subtilis is described which uses a newly constructed bacteriophage vector, 𝜙105J9. The phage genome contains cloning sites for the enzymes BamHl, Xbal and Sall, and can accommodate inserts of passenger DNA of at least 4 kbp. Recombinant phages, which can both plaque and lysogenize normally, are recovered after direct transfection of protoplasts in the presence of polyethylene glycol. Several fully functional sporulation genes and one biosynthetic gene from B. subtilis have been isolated from genomic libraries that were constructed with the new vector. The system may provide an alternative to some of the cloning methods currently available that use Escherichia coli as host.

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2.4.2.7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase(EC 2.4.2.14). The mutants were recessive and defined a single gene, aptl. They did not excrete purine when combined with ade4+. The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 aptl responded to both adenine and hypoxanthine. Mutants deficient in adenine aminohydrolase (EC 3.5.4.2) activity, aahl, and hypoxanthine: guanine phosphoribosyltransferase (EC 2.4.2.8) activity, hptl, were used to synthesize the genotypes aptl hptl aah+ and aptl hpt+ aahl. The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of aptl mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.

Several lytic and lysogenic actinophages were isolated from soil samples infected with Streptomyces cattleya and S. venezuelae. The morphologies and some biological properties of the phages, and the physico-chemical characteristics of their DNAs, were compared. Electron micrographs indicated that all the phage heads were of an icosahedral form, but head size and length of the tail varied. Two of the phages had a broad host range; the other isolates could lyse only a limited number of species. The molecular sizes of the phage DNAs were between 32·2 and 98·5 kb as estimated by electron microscopy and restriction enzyme analysis. The same study also indicated that one of the DNA species contained cohesive ends. The G + C content of the DNAs ranged between 45·1 and 74·2 mol % as estimated from melting studies. Sedimentation velocity experiments implied that several of the phage DNAs were probably heavily glycosylated or methylated. These modifications might explain the partial or slow digestion of some of the DNAs by several of the 23 restriction enzymes tested. Protoplasts of the appropriate Streptomyces strains could be efficiently transfected with phage DNA in the presence of 25% (w/v) polyethylene glycol (mol. wt 6000).

Nectria haematococca, a fungal pathogen of pea, demethylates the pea isoflavonoid phytoalexin pisatin to yield the less inhibitory product, 3,6a-dihydroxy-8,9-methylenedioxypterocarpan. Among naturally occurring isolates that demethylate pisatin (PDA+ isolates), some can be induced to do so at a high rate by pretreatment with the substrate (PDA1 isolates), while others demethylate pisatin only slowly, regardless of pretreatment (PDAn isolates). Genetic analysis of these pisatin demethylation phenotypes has indicated that one gene confers the PDA1 phenotype and that two distinct genes at different loci each confer the PDAn phenotype. We report here the relationship of these PDA phenotypes to pisatin tolerance and to virulence toward pea. The contribution of each PDA gene to these traits was also evaluated. In initial crosses between tolerant, PDA+ parents, recombinant progeny lacking demethylating activity (PDA- isolates) occurred. These PDA1 progeny were uniformly more sensitive to pisatin than the PDA* parents and all PDA+ progeny. Progeny having PDA1 phenotypes were more tolerant to pisatin than PDAn progeny, which in turn were more tolerant than PDA1 phenotype. Further genetic analyses confirmed that a characteristic pattern of pisatin tolerance was associated with each PDA gene. The gene conferring the PDA1 phenotype was associated with the highest level of tolerance, while the two genes conferring PDAn phenotypes were each associated with an intermediate level of pisatin tolerance. Thus relative tolerance to pisatin appears to depend upon both the presence and amount of pisatin demethylase activity. Virulence toward pea may also be determined by the ability to demethylate pisatin rapidly. In numerous crosses between vV. haematococca isolates with different pisatin demethylation phenotypes, high virulence in progeny was always correlated to the PDA1 phenotype. AH PDAn and PDA1 progeny were low in virulence. Thus the gene that confers high pisatin demethylase activity in N. haematococca is, or is closely linked to, a gene for virulence to pea.

Bacillus subtilis Marburg was found to produce an appreciable amount of an antibiotic in a synthetic medium. Antibiotic activity was produced in parallel with cell growth, and production stopped at the end of exponential growth. When the synthetic medium was supplemented with a small amount of Casamino acids, however, antibiotic was made only at the end of growth and in lesser amounts. The ability of cells to produce the antibiotic increased when stringent (rel + = wild-type) cells underwent a partial stringent response. These conditions also initiated extensive sporulation. An isogenic relaxed (rel) strain produced little antibiotic activity, which decreased under partial amino acid deprivation. In rel + cells, the addition of a low concentration of chloramphenicol, which reduces ppGpp synthesis, also reduced antibiotic synthesis in both normal and amino acid-starved bacteria, without appreciably affecting their growth rate. Guanosine starvation of a gua mutant initiated sporulation, but decreased antibiotic production. The results show that the stringent response initiates both sporulation (differentiation) and antibiotic production (secondary metabolism), but by different mechanisms. It appears that sporulation results from a decrease of GTP, whereas antibiotic synthesis results from a different effect of the stringent response.

Bacillus circulans WL-12 1,3-β-d-glucanases are extracellular enzymes subject to catabolite repression by glucose and synthesized after the depletion of this sugar. Utilization of other complex carbon sources (1,3-β-d-glucan from baker’s yeast, laminarin or xylan) resulted in a 3-to 4-fold increase in the formation of these enzymes, suggesting that they are derepressible and inducible. Under induction conditions four different enzymes were detected by isoelectric focusing that were numbered I, II, III and IV, according to their isoelectric points (3·7, 4·6, 5·5 and 6·5 respectively). Glucanase II was inducible whereas I, III and IV were both derepressible and inducible. In addition, the synthesis of glucanase II was blocked by cyclic AMP. The four enzyme forms displayed an endo- attack on laminarin and yielded similar products, but differed in some physico-chemical parameters such as molecular weight, K m and lytic activity.

Synchronized cultures of Anacystis nidulans (Synechococcus PCC 6301) were induced by a light-dark incubation scheme under moderate light intensity (3·2 J m −2s−1). At incubation temperatures of 32 °C. 35 °C and 38 °C, typical step-wise growth cycles were observed at growth rates of 8, 6 and 4 h doubling times, respectively. Using the same method of inducing cell synchrony but under increased light intensity (4·8 J m −2s−1), cell number increased exponentially and growth rates were twice as high as those at the lower light intensity, at incubation temperatures of 32 °C, 35 °C and 38 °C. At 32 °C under high light intensity, the synthesis of protein, RNA, DNA and cell wall material occurred periodically in a temporal order, although cell number increased exponentially. The data suggested that the growth of Anacystis under these conditions could essentially be characterized as synchronized. The macromolecular synthesis periods (cell cycle events) are apparently controlled by complex sets of genetic and metabolic controls that allow Anacystis to take advantage of changing environmental conditions.