- Volume 129, Issue 7, 1983

A strain of Claviceps purpurea, which has consistently failed to elaborate ergot alkaloids when growing as a parasite, has been shown to perform only the first step of the ergoline biosynthetic pathway catalysed by dimethylallyltryptophan (DMAT) synthetase. The next step, involving N-methylation of DMAT, did not operate. [14C]Agroclavine and lysergic acid, normally intermediates in alkaloid biosynthesis, were accepted by parasitic sclerotial tissue as substrates and were metabolized to lysergic acid amide (LAA). This amide therefore constituted a previously unreported end product for C. purpurea. It is concluded that the mutant has a metabolic block in the pathway following DMAT and, while enzymes for several subsequent steps are present, the fungus seems unable to form the usual cyclic tripeptide ergot alkaloids. The steps involved in metabolizing agroclavine to LAA were insensitive to 1 m-phosphate, while this concentration of phosphate completely inhibited DMAT synthetase. This double mutant therefore has unique potential for exploring control mechanisms in ergot alkaloid biosynthesis.

Cytochromes of the a, b and c types in membranes from the thermophilic bacterium PS3 have been characterized with respect to their spectral, potentiometric and ligand-binding properties. The integrated approach used (a) conventional potentiometric analysis with non-linear least-square analysis, (b) incorporation of the potentiometric procedures into a spectral decomposition protocol at 298 and 77 K, (c) fourth-order finite difference and fourth derivative analysis, and (d) photodissociation studies of the CO-liganded components. Either one c-type cytochrome with a split α-band (547, 552 nm at 77 K) or two cytochromes c with similar redox potentials (approx. +230 mV) were found, together with at least three b-type cytochromes (554, 557 and 561 nm at 77 K). On the basis of its high redox potential (+104mV), cytochrome b 557 was tentatively equated with the o-type oxidase of this organism. A c-type cytochrome that formed a photodissociable complex with CO was also detected. The α-band of the previously purified cytochrome c oxidase was resolved into two components, having spectral and potentiometric properties remarkably similar to those of the analogous mitochondrial oxidase.

A NAD-dependent malate dehydrogenase is the principal enzyme for malate oxidation by Mycobacterium leprae, FAD-dependent malate-vitamin K reductase was detected at about 1% the level of the NAD-dependent activity. Both enzyme activities were detected in extracts from M. leprae treated with NaOH to abolish host-derived activities which might be adsorbed to the bacteria and the NAD-dependent enzyme was shown to be electrophoretically distinct from the host-tissue enzyme, thus establishing that these were both authentic bacterial enzymes. Mycobacterium leprae does not possess malic enzyme.

Plasmid R68.44 was transferred to Pseudomonas putida PP3 at a frequency of approximately 10−6. In the new strain, P. putida PPW1, the plasmid, designated pUU1, lost a 2·0 kb fragment corresponding to IS21 which had previously been implicated in enhanced chromosome mobilization, but gained between 3·4 and 5·7 kb of DNA. Plasmid pUU1 was used to mobilize the fraction I dehalogenase gene at a low frequency of approximately 10−1 into another strain of P. putida. The R-prime plasmid, designated pUU2, contained an additional 9·2 to 11·0kb fragment which carried the fraction I dehalogenase.

Treatment of Escherichia coli with the alkylating agents diethyl sulphate, ethyl methane-sulphonate and N-methyl-N′-nitro-N-nitrosoguanidine produces a different pattern of expression of SOS functions. There is a full induction of recA-dependent inhibition of cell respiration, a slight induction of lambda prophage, and no inhibition of cellular division. In a comparative study with bleomycin; an agent which is able to induce these three SOS functions, we have also shown that the differences in expression of SOS functions are not due to any variation in the pattern of DNA synthesis, or DNA degradation after treatment with alkylating agents. These results suggest that the kind of damage induced in the DNA may be important in determining which SOS function is expressed.

Closely linked mutations in either of the two putative genes of the sporulation locus spoIIA can affect, in quite diverse ways, spore incidence, the production of alkaline phosphatase and DNAase, and the stability of the cells in sporulation medium. It is concluded that the locus has a regulatory function affecting the activation or induction of at least two, and possibly more, sporulation-associated operons.

Ten genetic markers were located on six linkage groups in Aspergillus parasiticus by means of the parasexual cycle. Diploids, selected by complementation of spore colour and auxotrophic markers, were subjected to induced haploidization by treatment with benlate. Haploid segregants, including recombinants resulting from mitotic crossing over, were distinguished from diploids by their stability and growth in the presence of benlate. Evidence is presented for the linkage of two genetic markers, norA and verA, which affect the biosynthesis of aflatoxin.

Mutations at 18 loci which map to five linkage groups in Dictyostelium discoideum are shown to affect resistance to antimicrotubule agents (including coumarin). The resistance or sensitivity mutations are classified according to whether their effects are limited to antimicrotubule agents, or whether cross-resistance or sensitivity to other compounds (notably acriflavin and cycloheximide) is observed. The latter class is likely to include mutations affecting permeability and is probably not of interest in the search for mutations directly affecting the cytoskeleton. All four acriflavin-resistance loci (acrA to acrD), many coumarin-sensitivity loci (couB to couE, couI), and both arsenate-resistance loci (arsA, arsB) fall into this category. Many mutants isolated on the basis of resistance to antimicrotubule agents (e.g. benlate, thiabendazole) in fact map at acrA or acrC. Some mutations affecting resistance to microtubule inhibitors also affect spore shape. Analysis of mutations such as couG370 and couH361, which affect coumarin sensitivity, spore shape, temperature sensitivity and, in the case of couH361, thiabendazole sensitivity provides the possibility of an ultrastructural approach to the study of the cytoskeleton in D. discoideum.

Bacteriophage T4 wild-type is not sensitive to heating at 60 °C. Trypsin at this temperature quickly inactivates the bacteriophage, with first-order kinetics for about the first 60 min. The half-inactivation period is around 15 min. The characteristics of this inactivation reaction have been studied. Inactivation of T4 particles is paralleled by a loss in ability to adsorb to bacteria. SDS-PAGE reveals a ‘clipping’ of gp 37, the protein of the distal part of the long tail fibres, during the inactivation reaction, gp 37 is the only protein to be modified under these conditions. Mutants have been isolated which resist this modification; they map in gene 37.

The lys gene of Bacillus subtilis was inserted into prophage 𝜙105. The recombinant phage (𝜙105dlys) contained DNA which was about 2 MDal smaller than the wild-type phage DNA, and the phage particles had no tails. The phage did not plaque but, when provided with tails in vitro, it transduced both lys-1 and lys-3 strains of B. subtilis to Lys +. The lys + gene was located on a 2·5 MDal EcoRI restriction fragment. Subsequently this phage was used to clone, using a similar technique, the spoIIIB gene(s). The second recombinant phage, 𝜙105dspoIIIB, was also defective, i.e. without tails. The DNA was 1.5 MDal smaller than the wild-type phage DNA and the spoIIIB2 + gene was located on a 3 MDal EcoRI fragment. When provided with tails in vitro, phage 𝜙105dspoIIIB transduced cells of a spoIIIB2 recipient to Spo+. In these transductants the spoIIIB2 mutation was complemented, and the cells sporulated normally.

The outer membrane (OM) proteins of a Pseudomonas syringae pv. syringae strain (HS191), capable of causing holcus leaf spot of corn and other grasses, and two plasmidless derivatives of HS191, one avirulent (AO111) and one with reduced virulence (PSG100), were isolated by selective solubilization of the cytoplasmic membrane (CM) with 0·4 % (w/v) SDS or by centrifugation on sucrose density gradients. OM preparations were enriched in hexosamine and 2-keto-3-deoxyoctonate and contained little of the CM enzymes NADH oxidase and malate dehydrogenase. Characterization of OM preparations by SDS-PAGE and isoelectric focusing indicated a minimum of 20 OM proteins; protein bands 8 and 9 quantitatively predominated. In addition, some of the proteins were modified by heat and others by 2-mercaptoethanol. Although protein 5 was absent in both plasmidless strains, AO111 differed from PSG100 in lacking protein 5a and having two proteins (8b′ and 8c′) with different pi values. It is suggested that plasmid pCG131 coded for protein 5 (mol. wt 68000) and repressed the synthesis of proteins 4a (78000) and 5a (63000). The relationship of specific changes in OM protein composition to virulence is discussed.

Replication of the antibiotic resistance plasmids pI258, pT10501 and pC221 has been investigated in a mutant of Staphylococcus aureus NCTC 8325, which is temperature-sensitive for the initiation of chromosome replication. Replication of pI258 stopped rapidly at the non-permissive temperature, whilst replication of pT10501 and pC221 continued (although at a lower rate than in the wild-type). It is proposed that the product of the mutant gene may be required directly for pI258 replication, but not for replication of pT10501 or pC221.

Streptomyces lividans 66 was shown to harbour two self-transmissible plasmids: SLP2, which acts as a sex factor, and SLP3. Derivatives of this strain which had lost both plasmids were used as host strains to study a range of Streptomyces plasmids for their ability to promote their own transfer and to mobilize chromosomal markers. A linkage map of the S. lividans chromosome containing ten markers was derived from the results of matings using several different sex plasmids, and protoplast fusions. SLP2 was transferred interspecifically to S. parvulus ATCC 12434 and to S. coelicolor A3(2); in the latter it acted as a fertility factor. Interspecific crosses also led to the discovery of a further plasmid, SLP4, from S. coelicolor. SLP2, SLP3 and SLP4 could not be visualized on agarose gels using standard plasmid isolation procedures, but their presence was detected by transformation into S. lividans.

Phage M was specific for bacterial strains, of various genera, harbouring plasmids of the M incompatibility group. It formed turbid plaques which varied from pin point to more than 2 mm in diameter on all hosts where plaques were detected. The phage had an hexagonal outline with a diameter of 27 nm. It contained RNA but differed from other plasmid-dependent RNA phages in being sensitive to chloroform. It adsorbed along the length of shafts of M pili.

The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC :: Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA :: Tn802) was rapidly lost from strain SU51 both at 30 °C and 42 °C; (2) the IncFIII plasmid pSU397 (ColB-K98 :: Tn802) was lost from strain SU51 at 42 °C but not at 30 °C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316.