- Volume 130, Issue 3, 1984

Chromobacterium violaceum forms cyanide as a secondary metabolite with glycine as the precursor and methionine as a stimulator. As the metabolism of glycine and methionine are interrelated by the provision of carbon-one units for the tetrahydrofolate pool by the activities of serine hydroxymethyltransferase (SHMT) and glycine cleavage enzyme (GCE), we have investigated the regulation of formation and activity of these enzymes by C. violaceum. The synthesis of SHMT was unaffected by growth under conditions of high cyanogenesis (on a medium containing glutamate, glycine and methionine) or low cyanogenesis (on glutamate alone), nor was its synthesis affected by addition of potential end-products of carbon-one metabolism to the growth medium. The activity of GCE was very low, less than 1% of the activity of SHMT, irrespective of the growth conditions. This suggests that nearly all the carbon-one units for the tetrahydrofolate pool are supplied by the activity of SHMT; this could lead to excess formation of glycine and might explain the role of cyanogenesis. The activity of SHMT was competitively inhibited by glycine and cysteine.

The hydrogenation of α-linolenic acid to stearic acid which occurs with the mixed bacteria of the sheep rumen can be demonstrated in vitro by mixing exponential phase cultures of only two species of rumen bacteria and incubating for a further period with α-linolenic acid. One bacterium must be able to hydrogenate α-linolenic acid to predominantly trans-octadec-11-enoic acid which is then used 11 substrate by a second bacterium. Cultures grown from small mixed inocula failed, with the exception of one pair of bacteria, to hydrogenate α-linolenic acid to stearic acid. The products from these cultures showed that one of the pair of bacteria had outgrown the other. For stearate production it was necessary to use inocula with a minimum number of cells rather than cells in a particular phase of growth. Two of the bacteria used, P2/2 and T344, after several years in pure culture show an increased isomerization of the octadecenoic acid products.

A high molecular weight polysaccharide was isolated from Bacillus megaterium ATCC 19213 cultured on a fructose mineral salts medium. This polymer was a heteropolysaccharide composed of d-glucose, d-xylose, d-galactose and l-arabinose in molar concentrations of 7.7:1.1:1.0:0.6, respectively. The polysaccharide neither bound concanavalin A nor formed a coloured complex with iodine, distinguishing it from many of the glucans isolated from other sporeformers. The concentration of the polymer decreased during forespore development, suggesting that it served as a carbon storage compound for use during formation of the spore.

The synthesis of propanediol oxidoreductase, an enzyme permitting the anaerobic metabolism of fucose and rhamnose, has been described as being controlled by the prd locus closely linked to the fuc locus in wild-type cells of Escherichia coli. However, strain AA-787, deleted in the fuc and prd loci, grew anaerobically on rhamnose, displaying propanediol oxidoreductase activity. From the deleted strain we derived a constitutive producer of propanediol oxidoreductase able to grow on 1,2-propanediol by oxidizing the diol to lactaldehyde which was further metabolized to lactate. Transduction experiments showed that this ability to use propanediol was closely linked to the rha locus. Peptide mapping of fucose- and rhamnose-induced propanediol oxidoreductase of wild-type cells established structural differences between the two enzymes, indicating two structural genes, one for each sugar metabolizing system.

Cells of the yeast Metschnikowia reukaufii were transformed to true protoplasts by means of snail gut enzyme. The plasma membrane of purified protoplasts was labelled either by [3H]dansyl chloride or by Na125 I. The crude lysate of osmotically ruptured protoplasts was fractionated by two subsequent centrifugations on sucrose density gradients. The two protein peaks obtained at densities of 1·16 and 1·13 g cm−3 were identified according to their characteristic markers (radioactivity, cytochrome c oxidase, oligomycin-sensitive ATPase at pH 8.5, and sterol content) as mitochondrial and plasma-membrane fractions, respectively. The ATPase activity associated with the plasma-membrane fraction exhibited a pH optimum at 6.5, was insensitive to oligomycin and was inhibited by vanadate. It can therefore be used as an intrinsic plasma-membrane marker.

Adherence of silica microbeads to the protoplasts effectively increased the density of the plasma membrane so that it could be obtained in a purified state by a single density gradient centrifugation. A third ATPase, presumably associated with the tonoplast membrane, was also demonstrated.

A mercuric ion-reducing flavoprotein was purified from Thiobacillus ferrooxidans TFI 29 by using dye-matrix affinity chromatography. The isolated enzyme had a molecular weight of 130000 and was composed of two subunits (54000 and 62000). The visible absorbance spectrum of the oxidized enzyme had a maximum at 455 nm. Upon addition of NADPH a new absorbance at 540 nm appeared, which was attributed to a charge transfer complex. The K m for mercuric chloride was determined to be 8·9μ m and the enzyme was shown to have a turnover number of 746 min−1 per FAD. A comparison of these physical properties, as well as metal ion inhibition and pH profiles, indicate that the enzyme from T. ferrooxidans is very similar in structure and function to mercuric reductases isolated from other bacterial sources.

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semi-rough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions. It is suggested that, in these PA mutants, changes in LPS composition modify their O-antigenicity and also expose other outer membrane components to varying extents. It is these protein and rough core components which are responsible for the cross-reactivity with heterologous O-antisera.

After an initial period of growth in medium made up in D2O, most strains of pneumococcus tested dramatically lost viability, the extent of the loss depending on the strain and on the amount of contaminating H2O in the D2O. This was followed by a recovery period. Once a strain was ‘adapted’, the ability to grow in D2O-medium without cell death was inherited, even after passage through H2O-medium, indicating the selection of mutants. Cultures that had not reached ‘full adaptation’ also exhibited cell death if transferred into either D2O-medium or H2O-medium, supporting the conclusion that the presence of hydrogen and deuterium together caused the toxicity.

‘Adapted’ cells exhibited an increased mutation frequency to a variety of antibiotic resistances, the propensity for this appearing in the death phase of ‘adaptation’. The specific transforming activity of DNA preparations from cultures undergoing ‘adaptation’ decreased before DNA synthesis ceased indicating damage to the DNA. The integration efficiency of a low-efficiency marker also dropped during ‘adaptation’ before returning to the initial value when measured in a Hex- recipient, but remained constant in a Hex+ recipient, suggesting that the Hex system may be involved in repair of the DNA damage. ‘Adapted’ organisms showed evidence of possessing higher Hex activity and were also able to repair lesions caused by UV-irradiation better than the wild-type.

The first three steps in quinic acid degradation in Aspergillus nidulans are catalysed by highly inducible enzymes encoded by a gene cluster regulated by an adjacent control region. Analysis of two non-inducible mutants has been done in diploid strains, where qutA8 is recessive and all three enzyme activities are fully induced in heterozygous qutA8/qutA+ diploids. In contrast, qutA4/qutA+ heterozygous diploids show semi-dominance of the mutant allele, giving markedly diminished growth on quinic acid and 30–40% decrease of enzyme induction. Strikingly, the qutA4/qutA8 heterozygous diploid grows to the same degree on quinic acid as the qutA4/qutA+ heterozygote and shows the same level of enzyme induction, whereas both the homozygous mutant diploids do not grow on quinic acid and show no enzyme induction. Therefore the two mutant genomes complement, identifying two distinct regulatory gene functions. A genetic model is proposed of a negatively acting gene (qutA) repressing expression of a positively acting gene (qutD, previously designated qutA8+ ) whose product is in turn required for expression of the three structural genes. The qutA4 mutation is interpreted to produce an altered repressor insensitive to quinic acid, and the qutD8 mutation the loss of activator protein. Close similarity in the regulation of the quinic acid gene cluster in Neurospora crassa suggests that the two types of control mutation, qals and qalF, described for N. crassa may also reflect two regulatory genes.

Strains of Aspergillus nidulans containing informational suppressors were grown on medium containing antibiotics known to affect protein synthesis at the ribosomal level. These strains reacted in the anticipated manner: presumed ribosomal suppressors suaA101, suaA105, suaC109 and sua-115 were sensitive or even hypersensitive to aminoglycoside antibiotics, whereas presumed tRNA-like suppressors suaB111, suaD103 and D108 were only slightly sensitive or wild-type in response. Hygromycin and paromomycin were the most useful antibiotics. All the antibiotics reduced the colony radial growth rate, K r, increased the lag phase and produced wrinkled morphology. Hygromycin was the most toxic. Resistant sectors were produced on paromomycin and hygromycin. The selective action of ‘misreading’ antibiotics on suaA and suaC strains is further evidence that these are ribosomal suppressors, whereas suaB and suaD may code for altered tRNA molecules. The results imply that hygromycin or paromomycin could be used for isolating ribosomal suppressors.

Spontaneous revertants of the leaky adE20 mutant of Aspergillus nidulans were obtained as vigorous sectors emerging from stunted colonies on adenine-free medium. Among the genetically heterogeneous sectors up to about 20% were recognized unequivocally as having an additional chromosome segment bearing adE20; two doses of this leaky allele permitted growth without added adenine. Eleven spontaneous duplication strains of independent origin were analysed genetically. Eight carried the duplicate segment on chromosome IIR; three of these, phenotypically similar to all eight, were analysed in detail and were shown - within the limits of such genetic analysis - to have a large, terminal segment of IR duplicated and attached terminally and uninverted to IIR. One strain had a duplication, possibly tandem, on IR and two had duplications attached elsewhere in the genome. The results suggested a preferential site for the initiation of duplicate segments in this system, as well as a preferential site for their attachment. Agents known to modify instability of a previously studied Dp(IR → IIR) strain affected the frequency of duplications among selected adE20 revertant sectors and/or the genomic locations of duplicated segments. Trypan blue and coumarin, which enhance Dp(IR → IIR) instability in a specific way, and Co2+, which stabilizes Dp(IR → IIR), gave 14, 50 and 62% duplication sectors respectively, among revertants. Duplications selected in the presence of Co2+ had mainly IIR attachments; of those from trypan blue and coumarin, about one-quarter were attached to IIL and none to IIR.

Mutants of Saccharomyces cerevisiae that are derepressed for meiosis and spore formation have been isolated and characterized genetically. All are the result of single, recessive nuclear mutations that fall into four linkage groups. Three of these groups are represented by spd1, spd3 and spd4 mutations, which in homozygous diploids confer poor growth and extensive sporulation on a range of non-fermentable media. Haploids carrying any of these mutations are arrested under these conditions in the G1 phase of the cell division cycle as large unbudded cells. The alleles of the spd2 mutation complemented all other mutations but were very closely linked to the spd1 locus. The fourth linkage group was represented by a mutation conferring temperature-sensitive growth and derepressed sporulation on homozygous diploids grown between 25 °C and 30 °C on media containing galactose or glycerol, but not glucose, as energy source. Above 30 °C this mutant lysed on all media. The mutation it carried failed to complement available cdc25 mutations. These data bring to five the number of loci at which mutation can lead to derepressed sporulation (spd1, spd3, spd4, cdc25 and cdc35). The spd1 locus has been mapped 13·9 cM to the left of the centromere on chromosome XV, adjacent to the SUP3 gene. Diploid strains homozygous for spd mutations are genetically unstable, giving rise to asporogenous mutants at high frequency, usually as the result of a second mutation unlinked to the spd mutation. Diploids homozygous for these mutations, and for spd mutations, show an altered regulation of the formulation of at least three polypeptides normally subject to carbon source repression.

Mutants of Saccharomyces cerevisiae that are unable to initiate sporulation, but can continue vegetative growth under conditions in which the wild-type strain sporulates, have been isolated and characterized. The mutations arose spontaneously as suppressors of the spd1 mutations, restoring the ability of spd1 mutants to grow on glycerol, and also spontaneously in cultures of a wild-type diploid strain undergoing sporulation in continuous culture. The mutations all conferred asporogeny, and were recessive in this respect to the wild-type, but dominant in acting as suppressors of the spd1 mutation. They fell into three complementation groups which corresponded to three unlinked loci, designated spo50, spo51 and spo53. None of these mutations was closely linked to the other initiation mutations defined by the spd1, spd3, spd4, cdc25, cdc28 loci, nor to the cell size control mutations whi1 and whi2. Loose linkage was detected between spd1 and spo53, and spo50, spd3 and spo53 were linked to their respective centromeres. The spo50, spo51 and spo53 mutations are not nonsense suppressors. Mutations in all three genes conferred similar highly pleiotropic phenotypes including: asporogeny; dominant suppression of both spd1 and spd3 mutations; aberrant cell morphology and viability loss on starvation; constitutive ability to reduce tetrazolium (which is subject to carbon source repression in the wild-type); and complete repression of the synthesis of several polypeptides that are subject to carbon source repression in the wild-type strain and derepressed in spd1 mutants derepressed for sporulation. A diploid strain homozygous for the spo50 mutation did not undergo either premeiotic DNA replication or meiotic recombination when transferred to sporulation media.

The plasmid pAB65, derived from a specialized transducing phage carrying DNA from about 52 min on the Escherichia coli genome, coded for two polypeptides of M r approx. 34000. The expression of one was regulated by cyst(e)ine and the cysB gene product and the other by the cysB gene product only. One of these polypeptides was a subunit of O-acetylserine (thiol)-lyase (EC 4.2.99.8); the other, associated with the E. coli membrane, was the N-terminus of the product of the ? ben gene. The pattern of peptide synthesis directed by plasmids carrying smaller DNA fragments indicated that the gene for O-acetylserine (thiol)-lyase was transcribed clockwise. The spectrum, amino acid composition and subunit number of the enzyme were determined. The enzyme appears homologous with the Salmonella typhimurium cysK gene product. This provides further evidence for the inversion of this region of the genome.

Plasmid D, a hybrid of plasmids P-lac and R1drd19, mediates polarized chromosome mobilization from one origin in Proteus mirabilis strain PM5006, while the parental plasmids neither individually nor combined mobilize this chromosome. To elucidate its acquired mobilizing ability plasmid D was characterized physically in relation to P-lac and R1drd19. Restriction patterns of these plasmids were compared and it was shown that D consists of P-lac and only the r-determinant (r-det) of R1drd19. A mechanism for the formation of plasmid D, via transduction of the r-det and subsequent transposon-like integration into P-lac, involving insertion sequence IS1, was suggested. Evidence for aberration in plasmid D DNA as a result of r-det integration into P-lac was attributed to IS1 elements which flank the r-det. Recombination regions of parental plasmid DNA were located on HindIII fragments a and of plasmid D and were subsequently inserted in vitro into IncP-1 plasmid RP4 that fails to mobilize the P. mirabilis chromosome. RP4:: HindIII a plasmids did not mobilize the latter chromosome, but rendered the Proteus host lac+. RP4:: HindIII plasmids pMC1 and pMC17, containing the fragment in opposite orientations, mobilized the P. mirabilis chromosome. For pMC17, mobilization was indistinguishable from that of plasmid D, i.e. having the same orientation and the same single origin. However, mobilization promoted by pMC1 was from two distinctly different origins, different from that of pMC17. This apparently deviates from known examples where inversion of homologous DNA inserted into plasmids leads to mobilization from the same origin but in reverse direction.

Escherichia coli W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins. By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the EcoRI site (4·0 kb on the restriction map) and the PvuII site (3·68 kb) of the ColE3-CA38 plasmid. This placed the colicin E8 immunity gene between the colicin E3 immunity gene and lys, the region which determined mitomycin C sensitivity. Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes. In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity. The phenotype conferred by plasmids with a transposon inserted into the lys region of ColE3-CA38 was dependent upon the site of insertion.