- Volume 137, Issue 10, 1991

A 4-hydroxy-3-methoxycinnamic acid (ferulic acid) esterase has been purified from the extracellular broth of cultures of Streptomyces olivochromogenes after growth on oat spelt xylan. The purification procedure utilizes ion exchange on DEAE-BioGel A, anion exchange on Mono Q, gel filtration and hydrophobic interaction chromatography. The purified enzyme appeared as a single band on SDS-PAGE, with an apparent M r of 29000. Two bands, at pI 7·9 and 8·5, were observed on isoelectric focusing. With methyl ferulate as substrate, the pH and temperature optima were 5·5 and 30 °C respectively, with a K m of 1·86 mm and V max of 0·3 μmol min–1 mg–1. The purified enzyme released ferulic acid from de-starched wheat bran only in the presence of xylanase.

A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans. Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions - that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system. The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein. The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa. The M-protein is active in the ‘physiological’ assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde. It has its maximum effect when the ratio of M-protein: MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95 %, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in V max and a significant (about one order) decrease in the K m of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k=0·78 h−1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (k app = 0·27 h−1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as d-amino acid oxidase from Candida boidinii.

The genes encoding the enzymes of IMP biosynthesis in Bacillus subtilis constitute the pur operon, whereas the genes encoding GMP biosynthetic enzymes, guaA (GMP synthetase) and guaB (IMP dehydrogenase), and the purA gene encoding adenylosuccinate (sAMP) synthetase all occur as single units. The purB gene encodes an enzyme involved in both IMP and AMP biosynthesis and is located in the pur operon. The levels of purine biosynthetic enzymes (except for GMP synthetase) were repressed in cells grown in the presence of purine compounds. Transcription of the pur operon is regulated negatively by adenine and guanine compounds. Our results suggest that ATP and guanine (or hypoxanthine) act as low molecular mass repressors. The level of IMP dehydrogenase was repressed by guanosine, but not in the presence of adenine, and was negatively correlated with the GTP/ATP pools ratio. The level of sAMP synthetase was repressed by adenine and increased by guanosine, and was positively correlated with the GTP/ATP pools ratio. It appears that the mode of regulating purine biosynthetic enzyme levels coincides with the cellular need for the individual enzymes.

Candida albicans and some other pathogenic Candida species, when grown in a medium containing a protein as a sole source of nitrogen, secrete an acid proteinase. Culture supernatants were assayed for proteinase activity, and were also analysed by Western blotting with antibodies raised and affinity-purified against proteinase of C. albicans. Proteinases secreted by C. tropicalis and C. parapsilosis were antigenically related to that of C. albicans, but had different molecular masses. The proteinases secreted by C. lipolytica, C. rugosa and C. lusitaniae were not antigenically related. The kinetics of proteinase secretion by C. albicans were monitored by activity and by Western blotting. With BSA as the nitrogen source, proteinase secretion increased exponentially until about 16 h. Culture supernatants of BSA-grown cultures accumulated proteinase to about a 1000-fold higher level than those of ammonium-sulphate-grown cultures. In vivo labelling experiments showed that proteinase was not detectably accumulated in the cells, but was secreted immediately after synthesis. Immunoprecipitation of in vitro translated poly(A)-containing RNA identified a putative pre-protein of about 54 kDa. As well as BSA, other proteins (haemoglobin, ovalbumin, histone), peptone and tryptone, when used as nitrogen sources, could induce proteinase, but to different levels. When Casamino acids or an amino acid mixture (equivalent to the composition of BSA) was used as nitrogen source, no induction was observed. Ammonium sulphate, or any other ammonium salt, repressed secretion of proteinase.

Activity assays based on selective product detection were used to identify the classes of amylase present in culture filtrates of the phytopathogen Colletotrichum gloeosporioides. When transferred from a glucose medium to a medium containing starch as sole carbohydrate, C. gloeosporioides secreted amylolytic activity into liquid shake cultures starting 2-3 d after inoculation, and produced levels of activity up to 0.45 IU ml−1 (assayed by the p-hydroxybenzoic acid hydrazide — PAHBAH — reducing sugar method), about 20 times higher than the activity in comparable glucose-grown controls, suggesting that starch (or a starch degradation product) was required to induce amylase synthesis. In four separate experiments, the ratio of total reducing sugar production (assayed by the PAHBAH method) to glucose production (assayed by the glucose-specific glucose oxidase — GO — method) was about 1.0 in most samples of culture filtrate, clearly demonstrating that the major amylolytic enzyme secreted by C. gloeosporioides is a glucoamylase (exo-1,4-a-d-glucan glucanohydrolase, EC 3.2.1.3, synonym amylogluco-sidase). On one occasion only (a 7 d sample in one experiment) was evidence obtained for a-amylase (endo-1,4-a-d-glucan glucanohydrolase, EC 3.2.1.1) also being present in the culture filtrate (PAHBAH/GO ratio 1.37); the presence of a-amylase in this sample was confirmed by native polyacrylamide gel electrophoresis using a starch-iodine activity stain.

Dichlorvos treatment of aflatoxigenic Aspergillus parasiticus SYS-4 (NRRL 2999) or a versicolorin A-accumulating mutant, NIAH-9, resulted in accumulation of versiconol acetate (VOAc) and versiconal hemiacetal acetate (VHA), whereas the production of aflatoxins, versicolorin A (VA), and versiconol (VOH) decreased. In feeding experiments using another non-aflatoxigenic mutant, NIAH-26, aflatoxins were newly produced from each of VHA, VOAc, VOH, versicolorin B (VB) and versicolorin C (VC). In these experiments, aflatoxin production from VHA or VOAc was inhibited by dichlorvos, whereas that from each of VOH, VB and VC was insensitive to dichlorvos. In cell-free experiments using the cytosol fraction of NIAH-26, VHA was converted to VC (or VB) and a substance tentatively identified as versiconal (VHOH). By further addition of NADH or NADPH to the same reaction mixture, VOAc and VOH were also formed together with VC (VB) and VHOH. VOH was produced from VOAc irrespective of nicotinamide adenine nucleotide. Also, the incubation of VOH in the presence of NAD or NADP led to the formation of VC (VB). The production of VC (VB) and VHOH from VHA, and that of VOH from VOAc was inhibited by dichlorvos, whereas the production of VOAc from VHA, and that of VC (VB) from VOH, was insensitive to dichlorvos. These results indicate that a metabolic grid catalysed by dehydrogenase and esterase among VHA, VOAc, VOH and VHOH, and a reaction from VHOH to VC (VB) are involved in aflatoxin biosynthesis. These enzyme activities were also detected when yeast extract peptone medium was used, or when A. oryzae SYS-2 was examined.

Gas vesicles isolated from the cyanobacterium Microcystis sp. retained their high stability and critical pressure on storage and could be held under a sustained pressure of 0·5 MPa without collapse. Application and release of pressures up to 0·5 MPa gave reversible changes in volume that indicated an elastic compressibility of 0·0087 MPa−1, which was linear with pressure. The elastic bulk modulus of the Microcystis gas vesicle was 115 MPa. The average yield pressure of the gas vesicles, 4·3 MPa, was determined by infiltrating them with gas at high pressure and then releasing the pressure so that they exploded. From these two measurements the proteinaceous wall of the gas vesicle was calculated to have a Young’s modulus of 3·8 GPa and a yield stress of 78 MPa. These values correspond closely to those of nylon, which has a similar secondary structure to that of the gas vesicle protein. The critical collapse pressure of the Microcystis gas vesicle, 0·8 MPa, is substantially higher than the theoretical buckling pressure, 0·2 MPa, for an unstiffened homogeneous cylinder of similar relative dimensions. The stiffening of the gas vesicle may be provided by its outer layer of GVPc, since the removal of this protein decreases the critical pressure to 0·23 MPa. The volumetric compressibility of the gas vesicle generates a small reversible change in turbidity (0·008 MPa−1). Spectrophotometric measurements showed that a gas vesicle suspension containing 1 μl ml−1 of gas gives a pressure-sensitive optical density of 2·03 cm−1 at a wavelength of 500 nm.

Two families of glycoprotein are defined in Dictyostelium discoideum by the presence of different glycoconjugates, both of which are highly immunogenic in mice. The previously described monoclonal antibodies MUD50 and MUD62 recognize the glycoconjugates and identify the respective glycoprotein families. Both types of glycosylation occur on vegetative and developmentally regulated glycoproteins. The immunodominant components of both families are reportedly O-linked sugars, but Western blots do not identify any glycoprotein that has both O-glycans, suggesting that there are two independently processed types of O-linked glycosylation in D. discoideum. The synthesis of the two O-glycan families is affected by glycosylation-defective mutations. Strains with a mutation at the modB locus lack one of these glycosylation types (that recognized by MUD50) and this mutation alters the size of two minor glycoproteins in the second family. Two new mutants, HU2470 (mod-352) and HU2471 (mod-353), lack the epitope recognized by MUD62. The two mutations map to different chromosomes. The mod-353 mutation also affects the size of PsA, a cell surface glycoprotein carrying the modB-dependent O-glycan.

Vacuoles of Saccharomyces cerevisiae were visualized by phase-contrast microscopy. Visualization was enhanced by adding polyvinylpyrrolidone. Vacuolar segregation during the cell cycle was analysed in 42 individual cells of strain X2180 by time-lapse photomicrography. Within 15 min of bud emergence, more than 80% of the cells contained a vacuolar segregation structure in the form of either a tubule or an alignment of vesicles. The structure emerged from one point of the mother vacuole, then elongated and moved into the bud in a few minutes. The vacuolar segregation structure disappeared, usually within 20 min, before nuclear migration, leaving a separate vacuole in the bud. To test the generality of this observation several strains were grown in the presence of the vacuolar vital dye fluorescein isothiocyanate. The bud size was used to measure progress in the cell cycle. All strains formed vacuolar segregation structures in cells with small buds, although with variations in duration and timing in the cell cycle. In the presence of nocodazole vacuolar segregation occurred normally, thus, microtubules seem not to be essential in this process.

The gene encoding an acid endo-1,4-β-glucanase from Bacillus sp. KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The recombinant plasmid contained a 3.1 kb HindIII insert, 1·8 kb of which was sufficient for the expression of endoglucanase activity in E. coli HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an M r of 51882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4. However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.

Strains of Salmonella typhimurium which have an auxotrophic requirement for lipoic acid were isolated by mutagenesis with the transposable element Mu dJ. The chromosomal location of these insertion mutations was determined to be at 14 map units by bacteriophage P22-mediated cotransduction. The lip gene is transcribed in the clockwise direction relative to the S. typhmurium genetic map. Strains with lip : : lac operon fusions were used to characterize the transcriptional activity of the lip promoter. Transcription of the lip gene is not regulated by catabolite repression or lipoic acid concentration. The data indicate that the lip gene product is expressed constitutively at a low level.

A sequence of human DNA, amplified in a lung tumour, was shown to induce expression-dependent effects on Escherichia coli phenotype. Previous studies have demonstrated the induction of abnormal plasmid supercoiling and bacterial aggregation in host strains harbouring various constructs encoding this region. Subcloning identified a short stretch of 50 bp crucial for these effects. This study details further characterization of the active sequence. Computer analysis of the region predicted the formation of a stem/loop structure in transcribed RNA. Transcription, in the absence of translation, of a 22 bp sequence comprising this structure was sufficient to induce bacterial aggregation. Site-directed and random mutagenesis of the sub-region were carried out in order to identify critical factors in the induction of this phenotypic shift. It was found that changes in the loop sequence modulated activity, and mutations increasing predicted stem stability produced more active constructs. The wild-type sequence induced high level aggregation in only a limited number of strains but using the mutagenesis data, a sequence was synthesized that induced high level aggregation in most E. coli strains tested.

Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity). However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains. A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before. All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium. Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used. In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton. The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production. No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC 8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC 8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

A 3·8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance. The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light. E. coli containing the 3·8 kb PstI fragment produced a 38–40 kDa protein which cross-reacted with antibodies raised against the E. coli RecA protein. The cloned DNA thus probably encodes a RecA homologue.