- Volume 136, Issue 9, 1990

A novel enzyme which catalyses the decarboxylation of carboxynorspermidine [H2N(CH2)3NHCH2CH2-CH(NH2)COOH] to yield norspermidine [H2N(CH2)3NH2], one of the unusual polyamines, was purified to apparent homogeneity (3100-fold) from cells of Vibrio alginolyticus. The enzyme has an apparent M r of 86000, with a pI of 4·25, and is a dimer composed of identical subunits with an apparent M r of 43500. The K m for carboxynorspermidine was 175 μm and for pyridoxal 5′-phosphate, 4·8 μm. The purified preparation had a specific activity of 24·1 μmol norspermidine produced min−1 (mg protein)−1. Carboxyspermidine, a structural homologue, was able fully to replace carboxynorspermidine as a substrate, to produce spermidine, but neither 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine nor lysine showed detectable substrate activity. The optimum pH was 8·25. Dithiothreitol greatly stimulated the enzyme activity, maximum stimulation being observed at more than 5 mm-dithiothreitol.

The membrane-bound aldehyde dehydrogenase from ‘Acetobacter rancens’ CCM 1774, solubilized from the membrane fraction by treatment with surfactants and subsequently purified to homogeneity, was characterized with respect to the M r of the dimeric enzyme (145000), pH optimum (5·1), pI value (5·3), substrate specificity towards straight-chain aldehydes, substrate inhibition and the effects of various inhibitors and ions. Methanol extracts of the membrane fraction and of the purified enzyme showed properties identical with pyrroloquinoline quinone as revealed by spectrophotometric methods and reactivity with apoquinoprotein glucose dehydrogenase. Pyrroloquinoline quinone could not be liberated by EDTA treatment. The enzyme activity of an apoenzyme could be partly restored by addition of membrane extracts containing pyrroloquinoline quinone adducts. The solubilized, purified quinoprotein aldehyde dehydrogenase and the membrane-bound enzyme differed in substrate binding, substrate inhibition, pH optimum and linkage to a c-type cytochrome which acts as electron acceptor in membrane fractions. Since double-reciprocal plots of initial reaction rates with various concentrations of aldehyde or electron acceptor showed intersecting lines, and different inhibition patterns were obtained for the two forms of the enzyme, a ping-pong kinetic behaviour with the two reactants was excluded. The kinetic mechanism was suggested to be changed after solubilization from a random-order type to a compulsory-order type, and is thus atypical for quinoproteins reported so far.

We have purified a specific phenol oxidase which is produced during conidiophore development in the fungus Aspergillus nidulans. Two active forms (A and B) have molecular masses of 50 and 48 kDa respectively; they have identical N-termini (24 residues). We have analysed the metal ion content of the B form; it is unusual in consisting of one zinc and two copper atoms per molecule. A temperature-sensitive mutant (ivoB192) produces a thermolabile enzyme, implying that ivoB is the structural locus. The natural substrate of the enzyme is N-acetyl-6-hydroxytryptophan, but it can be assayed colorimetrically or polarographically using hydroquinone monomethyl ether (HME) as substrate. It will also oxidize p-cresol, but not tyrosine, 3,4-dihydroxyphenylalanine or o-methoxyphenol. Colour development with HME substrate is strongly enhanced by high ammonium ion concentrations. Activity against HME is inhibited by 2,3-dihydroxynaphthalene, phenylhydrazine, diethyl dithiocarbamate and 8-hydroxyquinolene.

Batch cultures of a brewer’s strain of Saccharomyces cerevisiae, NCYC 227, in a defined medium exhibited characteristic flocculation during the late exponential phase of growth. However, early exponential cells were non-flocculent and flocculation of such cells could not be induced even in the presence of Ca2+. A specific glycoprotein, absent from the cell walls of non-flocculating exponentially growing cells of this yeast and those of non-flocculating strains of S. cerevisiae, was identified on the cell wall surface of flocculating exponentially growing cells of S. cerevisiae NCYC 227. Flocculent cells of S. cerevisiae NCYC 227 dispersed with EDTA and coated with monovalent Fab portions of the antibody showed reduced flocculation. Removal of the monovalent antibody portions from the cell surfaces induced cell flocculation in the presence of Ca2+. These results suggest a role for this glycoprotein in yeast cell flocculation.

Streptomyces sp. strain C5, an organism that normally produces baumycins, daunomycin and ε-rhodomycinone, was treaed with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). Mutants blocked at arious points in daunomycin and baumycin production were isolated by screening for altered pigmentation and absnce of bioactivity against Staphylococcus aureus. Examination of the mutants by thin-layer chromatography of their accumulated anthracycline metabolites, by cosynthesis assays, and by extract feeding experiments allowed a classification into six groups. Theses were: dauA, strains that accumulated no anthracyclines but with other blocked mutants cosynthesized anthracyclines (polyketide-synthase-minus mutants); dauG, regulatory mutants that, either alone or mixed with other blocked mutants, accumulated no anthracyclines; dauC, mutants that accumulated aklanonic acid; dauE, mutants that accumulated maggiemycin; dauF, mutants that accumulated aklavinone; and dauH, mutants that accumulated only ε-rhodomycinone. Mutant SC5-24 (dauE), which accumulated that shunt product maggiemycin, was re-mutagenized with NTG to obtain blocked mutants in preceding biosynthetic steps; the three grops of double mutants obtained accumulated aklanonic acid (dauC,E), aklanonic acid methyl ester (dauD,E) and akalviketone (dauE,F).

Aklanonic acid, a substituted anthraquinone, is the earliest stable intermediate isolated so far in the biosynthesis of anthracycline antibiotics. Desalted, soluble cell extracts of Streptomyces sp. C5 and Streptomyces peucetius (ATCC 29050) converted aklanonic acid to ε-rhodomycinone in an S-adenosyl-l-methionine- and NADPH-dependent sequence of reactions. Aklanonic acid was methylated to form aklanonic acid methyl ester (AAME), which was cyclized to form aklaviketone. Aklaviketone was reduced to aklavinone by an NADPH-linked reductase. When extracts of Streptomyces sp. C5 and S. peucetius were used, aklavinone was hydroxylated at C-11 by an NADPH- and oxygen-dependent reaction to form ε-rhodomycinone. Cell extracts of Streptomyces galilaeus strains ATCC 31133 and 31671 converted aklanonic acid to aklavinone in the same manner, but neither of the S. galilaeus strains hydroxylated aklavinone to ε-rhodomycinone. Mutants of Streptomyces sp. C5 blocked at steps in the conversion of aklanonic acid to aklavinone, and which accumulated aklanonic acid (SC5-39; dauC), AAME (SC5-138; dauD, dauE), and aklaviketone (SC5–159; dauE, dauF), lacked the expected enzymic activities. In a mutant (SC5–24; dauE) lacking aklaviketone reductase, but retaining aklavinone 11-hydroxylase activity, maggiemycin was formed as an apparent shunt product. An enzymic pathway for ε-rhodomycinone and maggiemycin formation is consistent with the data presented.

In the presence of S-adenosyl-l-methionine, cell-free extracts of Streptomyces sp. C5, Streptomyces peucetius ATCC 29050, Streptomyces insignis ATCC 31913 and Streptomyces coeruleorubidus ATCC 31276 O-methylated carminomycin and 13-dihydrocarminomycin to daunomycin and 13-dihydrodaunomycin, respectively. With the corresponding aglycones, carminomycinone and 13-dihydrocarminomycinone, as substrates, no methylated products were detected. Other 4-hydroxyanthracyclines such as aklavin and aclacinomycin A, and 4-hydroxyanthracyclinones such as ɛ-rhodomycinone and aklavinone, were not substrates for the 4-O-methyltransferase. These reaction specificities indicate that glycosylation of the anthracyclinone molecule must occur before 4-O-methylation, which means that 4-O-methylation of carminomycin is probably the terminal step in the biosynthesis of daunomycin, and that daunomycinone is not an intermediate in the pathway.

The recombinant plasmid pTK6 is composed of a 13·6 kb fragment from pTR2030 encoding phage resistance determinants for restriction/modification (R+/M+) and abortive phage infection (Hsp+) cloned into shuttle vector pSA3 (erythromycin resistance, Emr). Conjugal matings were performed to mobilize pTK6-encoded markers from Lactococcus lactis subsp. lactis MMS362 and MG1363. Emr transconjugants were recovered at 10−6 per input donor and harboured pTK6 or recombinant plasmids not found in either parental strain. The recombinant plasmids (pTRK78 and pTRK79) encoded Emr, Hsp+ and R+/M+, and transferred at high frequency in second-round matings. Mobilization of pTK6 from the otherwise plasmid-free donor, L. lactis MG1363, confirmed the presence of a conjugal element in this strain. Phage resistance in transconjugants containing pTRK78 and pTRK79 was markedly enhanced over pTK6-directed Hsp+ and R+/M+. In L. lactis LM2345 transconjugants, a reduction in plaque size was accompanied by a significant decrease in the efficiency of plaquing for phages c2 (10−2 to 10−6) and p2 (< 10−9). L. lactis NCK203 transconjugants containing pTRK78 or pTRK79 exhibited an additional 100-1000-fold reduction in the plaquing efficiency of 𝜙48 (10−4 to 10−5) over pTK6 imposed restriction (10−2). Increased resistance to phage was a consequence of the physical interaction of pTR2030-derived sequences on pTK6 with a conjugal element resident in the donor strains.

The grey-brown pigmentation of Aspergillus nidulans conidiophores depends on the functions of two ‘ivory’ loci. ivoB codes for a developmental specific phenol oxidase, and mutants accumulate its substrate N-acetyl-6-hydroxytryptophan. ivoA mutants are unable to make this substrate. ygA mutants are also poorly pigmented, and extracts require copper salts to activate both the phenol oxidase and conidial laccase. ivoA and ivoB mutants partially suppress the spore colour phenotype of ygA mutants. Comparisons of morphology, phenol oxidase and substrate accumulation in morphological mutants at the brlA locus suggest that the brlA protein regulates ivoA, ivoB and morphogenetic loci independently. The medA locus, which also affects morphology and pigmentation, may code for a modifier of brlA function.abaA mutants which are blocked at a later stage of development than brlA or medA mutants have low phenol oxidase levels, implying that by this stage of development the activity of the ivoB locus is declining.

Thirty-three neomycin- and temperature-sensitive mutants of Saccharomyces cerevisiae were isolated and characterized. The mutations fell into 29 complementation groups of neomycin sensitive mutations (nes). At the restrictive temperature (37 °C), ten mutants arrested at specific stages in the cell division cycle: four at early G1 stage, two at late G1 stage, one at S phase, and three at G2 stage. None of the G1-arresting mutants isolated in this experiment appeared to be allelic with cdc mutants examined, suggesting that these are new cdc mutants. The nes mutants were further characterized by determining their cross-sensitivity to G418, cycloheximide, LiCl and CaCl2.

The pma1.1 mutations of Saccharomyces cerevisiae and Schizosaccharomyces pombe decrease plasma-membrane ATPase activity. This study investigated how they affect different stress tolerances, and the extent and duration of the heat-shock response. pma1.1 mutants exhibited higher resistance to ethanol and osmotic stress, but lower tolerance to ultraviolet damage, as compared to wild-type cells. pma1.1 mutations also increased tolerance of the lethal temperature of 48 °C in cells in which no heat-shock response had been induced. However, after induction of a heat-shock response and elevated thermotolerance by a 25–38 °C upshift, then maintaining cells at 38 °C for 40 min, pma1.1 lowered subsequent tolerances of much higher lethal temperatures. Analysis of pulse-labelled S. cerevisiae proteins revealed reduced heat-shock protein synthesis in the pma1.1 mutant after a 25–38 °C heat shock. This may explain the greater increases in thermotolerance in wild-type as compared to pma1.1 cells after both were given identical 25–38 °C shocks. With more severe treatment (25–42 °C), heat-shock protein synthesis in wild-type cells, although initially high, was switched off more rapidly than in the pma1.1 mutant. These results indicate that plasma-membrane ATPase action exerts a major influence over several stress tolerances, as well as the extent and duration of heat-shock protein synthesis following induction of the heat-shock response.

Plasmid-like DNA (plDNA) was found in 48 out of 114 field isolates of Rhizoctonia solani. These 48 isolates were distributed as follows among the nine anastomosis groups (AG) and intraspecific groups (ISG) recognized among Japanese isolates of R. solani: 0 in AG-1 (sasakii type), 1 in AG-1 (web-blight type), 0 in AG-2-1, 11 in AG-2-2 (rush type), 10 in AG-2-2 (root rot type), 10 in AG-3,8 in AG-4,4 in AG-5 and 4 in AG-6. Each isolate carried one, two or three plDNAs identified by gel electrophoresis. Electron microscopic analysis revealed that all these plDNAs were linear molecules. The sequence homology among plDNAs found in representative isolates was examined by Southern blot analysis, using nick-translated plDNAs as probes. Considerable sequence homology was observed among plDNAs obtained from isolates within the same AG and ISG. The plDNAs occurring in the isolates of AG-2-2 were classified into two groups on the basis of the sequence homology.

Summary: A gene encoding a positive activator of the expression of extracellular polysaccharide (EPS) synthesis in the phytopathogen Erwinia amylovora has been isolated from a genomic library in Escherichia coli. The presence of the cloned gene in E. coli stimulated transcription of the genes encoding colanic acid biosynthesis and could complement res A mutations. Introduction of the gene on a multicopy plasmid into Er. amylovora caused a threefold increase in EPS expression. The nucleotide sequence of the gene (designated rcsA) was determined. This revealed a single open reading frame encoding an RcsA protein of 23·7 kDa. This was confirmed by minicell analysis in E. coli. The predicted amino acid sequence of this RcsA protein showed a high degree of homology to the RcsA protein of Klebsiella aerogenes, demonstrating the existence of a family of related RcsA activator proteins capable of stimulating EPS expression. The protein had no significant homology to known DNA-binding activator proteins, indicating, for the first time, that the RcsA family of activator proteins may stimulate expression of EPS synthesis indirectly by acting on other regulatory proteins.

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymelll-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.

Using transposon insertion mutagenesis with Tn1 or Tn5, we obtained Salmonella dublin mutant strains that showed either diminished serum resistance (five mutants) or diminished mouse lethality (two mutants). Detailed restriction cleavage analysis to determine the single sites of transposon insertion in an 80 kb plasmid (pTE800) indicated that a region for serum resistance was located within a 3·0 kb region of the SalI cleavage fragment 5 and the HindIII fragment 1. while the region for mouse lethality was within a 6·0 kb region of the SalI fragment 2 and the HindIII fragment 1. When the Tn1-containing SalI fragment 5 was reconverted, by homologous recombination, to the original SalI fragment 5 (9·6 kb), serum resistance was recovered to the same level as that of a parent strain 52401. Moreover, the change in the serum resistance correlated with changes in the neutral sugar composition of the LPS. The mutation in the plasmid in strain TE4-55 that gave diminished mouse lethality was also reversed by recombination with the cloned SalI fragment 2 (15·0 kb), with concomitant recovery of mouse lethality. These results indicate that the genetic region for serum resistance is different from that for mouse lethality, and that the gene for serum resistance is closely involved with the expression of the neutral sugar composition of the LPS of S. dublin.