- Volume 137, Issue 10, 1991
Volume 137, Issue 10, 1991
- Biochemistry
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Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2
A novel ethylene-forming enzyme that catalyses the formation of ethylene from 2-oxoglutarate was purified from a cell-free extract of Pseudomonas syringae pv. phaseolicola PK2. It was purified about 2800-fold with an overall yield of 53% to a single band of protein after SDS-PAGE. The purified enzyme had a specific activity of 660 nmol ethylene min–1 (mg protein)–1. The molecular mass of the enzyme was approximately 36 kDa by gel filtration and 42 kDa by SDS-PAGE. The isoelectric point and optimum pH were 5·9 and ca. 7·0–7·5 respectively. There was no homology between the N-terminal amino acid sequence of the ethylene-forming enzyme of Ps. syringae pv. phaseolicola PK2 and the sequence of the ethylene-forming enzyme of the fungus Penicillium digitatum IFO 9372. However, the two enzymes have the following properties in common. The presence of 2-oxoglutarate, l-arginine, Fe2+ and oxygen is essential for the enzymic reaction. The enzymes are highly specific for 2-oxoglutarate as substrate and l-arginine as cofactor. EDTA, Tiron, DTNB [5,5′-dithio-bis(2-nitrobenzoate)] and hydrogen peroxide are all effective inhibitors.
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The purification and characterization of 4-hydroxy-3-methoxycinnamic (ferulic) acid esterase from Streptomyces olivochromogenes
More LessA 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.
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The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans
More LessA 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.
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Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha
More LessAlcohol 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.
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Regulation of levels of purine biosynthetic enzymes in Bacillus subtilis: effects of changing purine nucleotide pools
More LessThe 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.
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Induction of secretory acid proteinase in Candida albicans
More LessCandida 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.
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Glucoamylase (exo-1,4-a-d-glucan glucanohydrolase, EC 3.2.1.3) is the major starch-degrading enzyme secreted by the phytopathogenic fungus Colletotrichum gloeosporioides
More LessActivity 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.
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A metabolic grid among versiconal hemiacetal acetate, versiconol acetate, versiconol and versiconal during aflatoxin biosynthesis
More LessDichlorvos 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.
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- Biotechnology
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Positive selection of antibiotic-producing soil isolates
More LessStepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produc antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0·1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7·7–8·0. Growth on proline (1 %) and humic acid (0·1%) as sole carbon/nitrogen sources, growth on asparagine as: nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.
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- Development And Structure
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Antibodies to the N-terminal sequence of GVPa bind to the ends of gas vesicles
More LessAntibodies were raised against intact gas vesicles of Anabaena flos-aquae, and against a synthetic peptide (GVPaNT) whose sequence is identical to the N-terminal region of the main gas vesicle protein, GVPa. A two-stage centrifugation procedure is described for separating gold-labelled antibodies bound to gas vesicles from unbound antibodies. The GVPaNT antibody bound to gas vesicles that had been previously rinsed with SDS to remove the outer gas vesicle protein, GVPc. Treatment with this antibody caused the gas vesicles to aggregate together end-to-end rather than side-by-side. The binding of the anti-GVPaNT-immunogold particles to the gas vesicle was restricted to the conical ends of the structure. These observations indicate that the sequence to which the GVPaNT antibodies were raised, residues 1 to 13 of the GVPa molecule, is exposed only at the outer surface of the cones and that it is normally obscured by GVPc. As GVPa forms both the conical ends and the cylindrical midsection of the gas vesicle, exposure of the N-terminal sequence only in the cones must be due to differences in the contact between adjacent GVPa molecules in the central cylinders and end-cones.
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The mechanical properties of the Microcystis gas vesicle
More LessGas 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.
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Immunodominant carbohydrate determinants in the multicellular stages of Dictyostelium discoideum
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.
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Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle
More LessVacuoles 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.
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- Genetics And Molecular Biology
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Identification of the exo loci required for exopolysaccharide synthesis in Agrobacterium radiobacter NCIB 11883
More LessWe initiated a genetic analysis of Agrobacterium radiobacter NCIB 11883 with particular reference to the (exo) genes required for exopolysaccharide synthesis. Following mutagenesis with nitrosoguanidine, several exo mutant strains were isolated and several of the mutations were corrected by DNA cloned in a newly constructed cosmid library. Analysis of various complementing cosmids by genetic and physical criteria indicated that exo loci were quite widely dispersed in the bacterial genome. Certain exo mutations were corrected by different cosmids that shared no homologous DNA; possible explanations for this are presented. Using phoA fusions, it was shown that some exo genes were, or were closely linked to, genes that specified polypeptides associated with the bacterial cell surface. By introducing the cloned exo genes of Rhizobium meliloti it was found that only one out of thirty exo mutants of A. radiobacter was corrected by a defined exo locus of the former species; further analysis indicated that this particular exo gene corresponded to exoB of R. meliloti. Finally, it was found that several A. radiobacter exo mutants were non-mucoid on media with dicarboxylic acids as sole carbon source but appeared to be wild-type when sugars were the source of carbon.
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Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-β-glucanase from Bacillus sp. KSM-330
More LessThe 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.
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Characterization of lip expression in Salmonella typhimurium: analysis of lip : : lac operon fusions
More LessStrains 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.
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Transcription of a stem/loop region of a tumour-amplified sequence induces bacterial aggregation
More LessA 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.
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Plasmid effects on secondary metabolite production by a streptomycete synthesizing an anthelmintic macrolide
More LessTransformation 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.
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Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1
More LessPhages 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.
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Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis
More LessA 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.
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Plasmid-mediated NAD independence in Haemophilus parainfluenzae
More LessThe location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5·25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0·1 to 0·2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid ‘curing’ effect with a frequency of‘curing’ of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5·25 kb plasmid.
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Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide
More LessLactococcin, a bacteriocin produced by Lactococcus lactis subsp. lactis ADRIA 85LO30, was purified as a 2·3–2·4 kDa peptide. Six non-bacteriocin-producing (Bac–) and non-immune (Imm–) strains were isolated after curing experiments. These strains had in common the loss or modification of two plasmids: pOS4 (32 kb) and pOS5 (70 kb). By comparing pOS5 and several modified plasmids, a DNA region from pOS5 of about 10 kb, which was necessary for wild-type bacteriocin production and immunity, was identified.
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The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene
More LessThe spontaneous and recessive mutation thn in the basidiomycete Schizophyllum commune suppresses the formation of aerial hyphae in the monokaryon and, if present as a double dose, the formation of both aerial hyphae and fruit-bodies in the dikaryon. In the monokaryon, the mutation prevents accumulation of mRNA of the Sc3 gene, and in the dikaryon it also prevents the accumulation of fruiting-specific mRNAs, including mRNAs of the Sc1 and Sc4 genes, which are homologous to the Sc3 gene. These three genes code for hydrophobins, a family of small hydrophobic cysteine-rich proteins. In the thn monokaryon, the only detectable change in synthesized proteins is the disappearance of an abundant protein of apparent M r = 28 K from the culture medium and from the cell walls. Protein sequencing shows that this is the product of the Sc3 gene. The Sc3 hydrophobin is present in the walls of aerial hyphae as a hot-SDS-insoluble complex. Submerged hyphae excrete large amounts of the hydrophobin into the medium.
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- Pathogenicity And Medical Microbiology
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Recovery of viable but non-culturable Campylobacter jejuni
More LessSuspensions of Campylobacter jejuni became non-culturable after storage in sterilized pond water at 4 °C for periods between 18 and 28 d, depending on the strain. Suspensions of four strains of C. jejuni that had been in water for 6 weeks, and shown to be non-culturable, were fed to suckling mice. Colonization of mice was established with two of the strains and failed with the other two strains. Examination of these suspensions under the electron microscope showed some cocci having the appearance of being viable, but most cocci and all remaining spiral forms showed extensive degeneration. The results indicate that non-culturable coccal forms of C. jejuni are capable of infecting mice but that this property may differ between strains.
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The type 4 pilin of Moraxella nonliquefaciens exhibits unique similarities with the pilins of Neisseria gonorrhoeae and Dichelobacter (Bacteroides) nodosus
More LessMoraxella nonliquefaciens is a bacterium which is part of the normal flora of the human upper respiratory tract and is an occasional cause of disease. Using a previously cloned type 4 pilin gene (tfpQ) from Moraxella bovis as a hybridization probe, we have cloned an 826 bp Sau3AI fragment which contains an M. nonliquefaciens type 4 pilin gene (tfpA) from strain NCTC 7784. The pilin gene is expressed in Escherichia coli. We have examined NCTC 7784 and nine other M. nonliquefaciens strains by genomic Southern hybridization using tfpA as a probe, and they all appeared to have more than one pilin gene. While the predicted amino acid sequence of the M. nonliquefaciens tfpA pilin has conserved regions as compared to pilins of M. bovis and M. lacunata, it also shows similarities to both the type 4 pilin of Neisseria gonorrhoeae and the type 4 pilin of Dichelobacter nodosus (formerly Bacteroides nodosus).
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- Physiology And Growth
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Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry
More LessThermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m 1, m 2, m 3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 °C, respectively. After cooling to 5 °C and reheating, peaks denoted f r, m r and p r were observed at 23, 73 and 94 °C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 °C and extending to just above 40 °C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 °C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 °C and is characterized by peaks m 1, m 2 and n. Peak m 3 at 75–76 °C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 °C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 °C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 °C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 °C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 °C caused by the remelting of ribosomal RNA. Peak pr at 94 °C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.
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Molar growth yields and bioenergetic parameters of extremely alkaliphilic Bacillus species in batch cultures, and growth in a chemostat at pH 10·5
More LessAlkaliphilic Bacillus species that grow at pH 10·5 must cope with a low protonmotive force (– 50 mV) due to a reversed transmembrane pH gradient at least 2 pH units more acid inside. Here we demonstrate that strictly alkaliphilic B. firmus RAB and two strains of B. alcalophilus (ATCC 27467 and DSM 485) grow exponentially in batch cultures with a doubling time of less than 1 h in 100 mm buffered medium, while the actual medium pH remains above 10·2. The ATCC strain continued to grow rapidly for at least 7 h, but the growth rate of the DSM strain declined dramatically after 3 h. However, both the B. alcalophilus strains, B. firmus RAB and facultatively alkaliphilic B. firmus OF4 were readily maintained for at least 24 h between pH 10·4 and 10·6 in a chemostat where nutrients were constantly replenished. A critical nutrient may be limiting in batch cultures of the DSM strain of B. alcalophilus. The facultative alkaliphile grew equally well in batch cultures at an initial pH of 7·5 or 10·5. Its molar growth yield (23 mg dry wt mmol−1) on malate (Y mal) was the same at the two pH values and was comparable to Y mal for B. subtilis grown at neutral pH. B. firmus RAB and B. alcalophilus ATCC 27467 grown at pH 10·5 also showed Y mal values at least as high as the neutralphile, indicating efficient use of the energy source even at low protonmotive force. Moreover, the phosphorylation potential of B. firmus OF4 grown at pH 7·5 (45·2 kJ mol−1)or pH 10·5 (46 kJ mol−1) was in a conventional range for bacteria.
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- Plant-Microbe Interactions
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Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase
More LessPseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for ⩽1·5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6·8) supplemented with 10 mm-l-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6·0) containing 1 mm-l-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH ~7 or pH ~6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.
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- Corrigendum
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