- Volume 133, Issue 7, 1987
Volume 133, Issue 7, 1987
- Sgm Special Lecture
-
- Biochemistry
-
-
-
The Cycas revoluta-Nostoc Symbiosis: Enzyme Activities of Nitrogen and Carbon Metabolism in the Cyanobiont
More LessSUMMARY: A comparative study was made of enzymes involved in nitrogen and carbon metabolism in the cyanobiont directly isolated from Cycas revoluta coralloid roots, and in the cultured isolate Nostoc 7422. The symbiotic Nostoc showed high activity of glutamine synthetase and glutamate synthase, the primary ammonia-assimilating enzyme system in cyanobacteria. Ammoniaassimilating glutamate dehydrogenase (GDH) activity was undetectable, although the catabolic GDH activity was high. Both glutamate-oxaloacetic acid transaminase and malate dehydrogenase showed higher activities in the symbiotic Nostoc than in the cultured Nostoc strain. The symbiotic Nostoc did not fix CO2 in vivo although in cell-free extracts both ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase activities, similar to those in the cultured strain, were present.
-
-
-
-
Microalgae and Cyanobacteria as a Source of Glycosidase Inhibitors
More LessSUMMARY: Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of α-glucosidase, α-amylase and β-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.
-
- Biotechnology
-
-
-
Effect of Temperature on the Stability of Plasmid pTG201 and Productivity of xylE Gene Product in Recombinant Escherichia coli: Development of a Two-stage Chemostat with Free and Immobilized Cells
More LessSUMMARY: The effect of temperature on the stability of pTG201, a plasmid carrying the xylE gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida), and the production of catechol 2,3-dioxygenase in free and immobilized Escherichia coli during continuous culture have been studied at various temperatures. Immobilization of cells increased the stability of pTG201 considerably, even under conditions when expression of the xylE product was enhanced. Since xylE transcription was controlled by the γPR promoter and cI857 repressor, increasing derepression temperatures increased catechol 2,3-dioxygenase productivity and decreased pTG201 stability. A two-stage continuous culture system to overcome the impact of the high-level expression of the xylE gene on the stability of pTG201 is described. In the first stage, immobilized cells were grown in the repressed state in order to prevent loss of pTG201, whereas in the second stage, cultures were maintained in the derepressed state.
-
-
- Development And Structure
-
-
-
Isolation and Characterization of the Outer Membrane Proteins of Azospirillum brasilense
More LessSUMMARY: The outer membrane of Azospirillum brasilense was isolated from the total membrane fraction by sucrose density gradient centrifugation and by Sarkosyl extraction; both preparations showed an identical outer membrane protein profile in slab gels after electrophoresis under denaturing conditions. The profile showed a major 42 kDa protein constituting about 60% of the total outer membrane proteins. This major protein and a minor 40 kDa protein were tightly but non-covalently associated with peptidoglycan. In addition, a 23 kDa heat-modifiable protein, resistant to trypsin digestion, was detected in the outer membrane. Growth of A. brasilense under iron deficiency induced four additional high molecular mass proteins (87, 83, 78 and 72 kDa) in the outer membrane. A comparison of the outer membrane protein profiles of the three different Azospirillum species suggests that such profiles might be useful in taxonomic classification.
-
-
-
-
The Life Cycle and Growth Kinetics of an Anaerobic Rumen Fungus
More LessSUMMARY: The life cycle of an anaerobic fungus isolated from the rumen of sheep was studied and was found to be similar to that of the chytrids. The organism was monocentric. At 39 °C the duration of the life cycle varied from about 26 to 32 h. The zoospores were spherical or oval in shape and were polyflagellate (8 to 17 flagella per zoospore). During the first 6·5 h of growth there was a rapid development of an extensive, non-septate, highly branched rhizoidal system; during this period the ‘main’ rhizoid increased in length exponentially with a doubling time of 2·49 h. Between 6·5 to 9·5 h after inoculation, the rate of extension of the main rhizoid declined, and no further extension occurred after 9·5 h. The main rhizoid increased in width at its base from 2·2 to 15·0 μm during the first 13 h after inoculation, indicating that intercalary wall growth occurred. Nuclei were occasionally observed in the rhizomycelium using DAPI (4′,6-diamidino-2-phenylindole) staining. Zoosporangia varied in shape from spherical to columnar, and some columnar zoosporangia were observed to become spherical. The zoosporangium initially increased in volume at an exponential rate with a doubling time of 1·56 h. Between 14 to 20 h after inoculation, growth of the zoosporangium decelerated and little growth occurred after 20 h: the zoosporangium had a final volume of 2·5 × 105μm3. At about 21 h after inoculation, a septum was formed at the base of the zoosporangium, delimiting it from the rhizoidal system. The formation of this septum was correlated with the cessation of zoosporangial growth and the onset of zoosporogenesis. After zoosporogenesis, zoospores (about 88 zoospores per zoosporangium) were liberated through a pore formed in the zoosporangial wall opposite the main rhizoid. About 3 h after zoospore release the rhizoidal system became less refractile, suggesting that autolysis had occurred. Growth of the isolate was inhibited by nikkomycin (an inhibitor of chitin synthase), but not by amphotericin B or nystatin (antibiotics which bind to sterols).
-
- Ecology
-
-
-
Isolation of Anaerobic Fungi from Saliva and Faeces of Sheep
More LessSUMMARY: Anaerobic fungi were isolated from the faeces (isolates F1 and F2) and saliva (isolates S1 and S2) of a sheep. Isolate S1 and F1 were morphologically similar and both resembled Neocallimastix spp. They produced polyflagellate zoospores and were monocentric; the zoosporangium was supported by an extensive, highly branched, non-septate rhizomycelium. Isolate F1 utilized a variety of polysaccharides (including cellulose) and disaccharides, but of the eight monosaccharides tested, only fructose, glucose and xylose supported growth. Isolates S2 and F2 were morphologically similar to each other and resembled Sphaeromonas communis. These organisms were isolated on glucose but not on cellulose and they formed large spherical bodies which were closely associated with small ‘zoosporangia’; no rhizoids were observed. This is the first time that anaerobic fungi have been isolated from saliva and faeces of sheep. The ability of these organisms to survive in saliva and faeces, at reduced temperatures, suggests two possible routes of transfer of anaerobic fungi between animals.
-
-
-
-
Establishment of the Microflora and Anaerobic Fungi in the Rumen of Lambs
More LessSUMMARY: The establishment of different bacterial populations and fungi in the rumen was investigated in lambs reared under different conditions of diet and management. The rumen was rapidly colonized by an abundant microflora after birth. By day 2 strictly anaerobic bacteria predominated (109c.f.u. ml−1); their population increased slightly during the first week of life and again when the animals began to ingest solid feed (3 weeks). The composition of the microflora in the 2-10-d-old lambs was quite different from that of adult sheep. The aerobic and facultatively anaerobic bacterial count was 10-100-fold lower than the strictly anaerobic count during the first week, and decreased steadily afterwards. In flock-reared lambs, cellulolytic and methanogenic bacteria appeared very early after birth (3-4 d). At the end of the first week the population of these bacteria reached a level close to that generally observed in a mature rumen. The cellulolytic bacteria were also able to survive in the rumen of lambs fed cow's milk exclusively. Anaerobic fungi appeared later (8-10 d). They were present in all lambs studied until 3 weeks of age, and then disappeared in most of them when a solid diet was given.
-
- Genetics And Molecular Biology
-
-
-
Isolation, Complementation and Partial Characterization of Mutants of the Methanol Autotroph Xanthobacter H4-14 Defective in Methanol Dissimilation
More LessSUMMARY: Seven mutants of Xanthobacter H4-14, unable to grow on methanol but capable of growth on formate, were isolated and complemented with a chromosomal clone bank constructed in the broad-host-range cosmid pVK100. One mutant could not be complemented but the others fell into four distinct complementation groups that involved three different recombinant clones. All of the complementing regions were separated by at least 10 kbp. The five complementation classes had different phenotypic characteristics and were defective in different aspects of methanol and formaldehyde oxidation. Class I mutants were defective in methanol oxidation, class II mutants were impaired in formaldehyde oxidation, class III mutants appeared to be defective in a regulatory element involving the methanol oxidation system, and class IV mutants appeared to be defective in a regulatory element involving formaldehyde oxidation. Class V mutants exhibited a methanol-sensitive phenotype, which was correlated with an imbalance between methanol and formaldehyde dehydrogenase activities. Analysis of this class suggested it was defective in a repressor that regulated methanol dissimilation functions.
-
-
-
-
Secretion of Bacillus subtilis α-Amylase in the Periplasmic Space of Escherichia coli
SUMMARY: The Bacillus subtilis α-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis α-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.
-
-
-
The Effect of Lipophilic Weak Acids on the Segregational Stability of TOL Plasmids in Pseudomonas putida
More LessSUMMARY: The effect of various lipophilic weak acids on the stability of certain TOL plasmids was investigated. Benzoate induced deletion of TOL plasmid DNA in Pseudomonas putida MT15, followed by loss of the plasmid; this effect was pH- and concentration-dependent, suggesting that undissociated benzoic acid was a more effective curing agent than the benzoate anion. Plasmid loss always approached a frequency of 100% after a lag and apparently depended on the prior occurrence of deletions, although deleted plasmid was stably maintained in the absence of the acid. m-Toluate, acetate and butyrate also induced deletions and plasmid loss at high frequencies, although these acids were less effective than benzoate. Benzoate inhibited the growth of plasmid-containing cells rather than permitting faster growth of cured cells on benzoate. Similar results were obtained with P. putida strains MT20 and MT84, which contain different TOL plasmids. We suggest that lipophilic weak acids induced deletions, possibly by excision of a transposon-like region, and disrupted the segregation of deleted plasmid.
-
-
-
The Cloning of Chromosomal DNA Associated with Methicillin and Other Resistances in Staphylococcus aureus
More LessSUMMARY: Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for the tetracycline-resistance plasmid pT 181.
-
-
-
Resistance to Oleandomycin in Streptomyces antibioticus, the Producer Organism
More LessSUMMARY: Resistance to oleandomycin in Streptomyces antibioticus, the producer organism, was studied. The organism was highly resistant in vivo to the antibiotic but sensitive to other macrolides and lincosamides. Protein synthesis in vivo by mycelium of S. antibioticus was more resistant to oleandomycin than that by mycelium of Streptomyces albus G, an oleandomycin-sensitive strain, and this resistance was dependent on the age of the culture, older mycelium of S. antibioticus being more resistant to oleandomycin than young mycelium. [3H]Oleandomycin was capable of binding to the same extent to the 50S subunits of the ribosomes of both organisms. Oleandomycin also inhibited in vitro protein synthesis by ribosomes obtained from an oleandomycin-production medium at the time when maximum levels of oleandomycin were being produced. A clear difference between the ability of the two organisms to incorporate exogenous oleandomycin was observed. Thus, while S. albus G took up oleandomycin, S. antibioticus showed a decreased permeability to the antibiotic, suggesting a role for cell permeability in self-resistance.
-
-
-
Characterization of a Natural Cointegrate of the Pock-forming Plasmids pSK1*and pSK2*of Streptomyces kasugaensis MB273
More LessSUMMARY: Strains carrying only one species of pock-forming plasmid, designated as pSK3*, were isolated from two different derivative strains of Streptomyces kasugaensis MB273 which contained three species of plasmids, pSK1, pSK2 and pSK3. Single and double digestion of pSK3*with seven restriction endonucleases yielded fragments identical with those of pSK3 and assignable to those obtained from pSK1*and pSK2*. In particular, digestion with Bg/II alone or in combination with other restriction endonucleases afforded the same size fragments as those of pSK1*and pSK2*. Strains containing pSK3*induced pocks on lawns of strains carrying pSK1*or pSK2*and resisted pock formation by the latter strains. Therefore, it was concluded that pSK3*was a pSK3 derivative with elevated pock-forming ability and represented a composite plasmid consisting of two elements, pSK1*and pSK2*, without any loss of their plasmid functions. Deletion derivative plasmids constructed from the Bg/II fragments of pSK3*provided evidence supporting the above conclusion. Pock formation by a pSK3*-containing strain against strains carrying pSK1*, or pSK2*or no plasmid accompanied the transfer of pSK3*from the former to the latter. Segregation of pSK1*and pSK2*from pSK3*was observed in mycelium from pocks caused by pSK3*-containing strains and on subculture of pSK3*-containing strains.
-
-
-
Characterization and Localization of Plasmid Functions Involved in Pock Formation and Pock Resistance of Plasmid pSK3*of Streptomyces kasugaensis MB273
More LessSUMMARY: Deletion derivatives and recombinants of the plasmid pSK3*, which is a cointegrate of pSK1*and pSK2*in Streptomyces kasugaensis, were constructed and analysed for their ability to transfer and ‘pock’ on strains carrying pSK1*or pSK2*. Various deletions in the pSK1*and/or pSK2*regions of pSK3*were grouped into nine classes on the basis of their pock-forming ability and pock resistance. Analysis of these deletions and insertions provided tentative locations of DNA regions for two pock-resistance determinants (Porl and Por2), two pock-forming determinants (Poc1 and Poc2) consisting of plasmid transfer and spread determinants (Tra/Spr), and two replication determinants (Rep1 and Rep2) corresponding to the pSK1*and pSK2*regions of pSK3*. In particular, the Por2 function in the pSK2*region was determined to be located in a 1·35 kb segment.
-
-
-
Modulation of Competence for Genetic Transformation in Streptococcus pneumoniae
More LessSUMMARY: The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6·8 to 8·0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.
-
- Pathogenicity And Medical Microbiology
-
-
-
Purification and Characterization of an Elastolytic Protease of Vibrio vulnificus
More LessSUMMARY: Large amounts of a highly purified, extracellular elastolytic protease of Vibrio vulnificus were obtained by sequential ammonium sulphate precipitation and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The protease had an M r of about 50500 (estimated by SDS-PAGE), a pI of 5·7, and a temperature optimum range of 55 to 60 °C. The pH optimum and the results of inactivation studies suggested that the enzyme was a neutral metalloprotease. The protease had about 429 amino acid residues, and the first 20 amino-terminal amino acid residues were Ala-Gln-Ala-Asn-Gly-Thr-Gly-Pro-Gly-Gly-Asn-Ser-Lys-Thr-Gly-Arg-Tyr-Glu-Phe-Gly. The purified protease was toxic for mice (about 1·5 mg kg−1and 4·5 mg kg−1, intraperitoneal and intravenous LD50 values, respectively), and subcutaneous injection of the enzyme elicited rapid and extensive dermonecrosis.
-
-
-
-
Monoclonal Antibodies Directed against Surface-associated Polypeptides of Treponema pallidum Define a Biologically Active Antigen
More LessSUMMARY: Murine monoclonal antibodies (Mabs) were raised against two outer-membrane-associated polypeptides of Treponema pallidum (47 and 44 kDa). Three Mabs against each polypeptide were investigated further and only those directed against the 44 kDa polypeptide were demonstrated to have immobilizing activity. The specificity of the Mabs for T. pallidum was determined by Western blotting procedures and the surface association of the antigens was inferred by immunogold electron microscopy. The clear distinction between these two polypeptides in their biological activity could help to explain the pathobiology of syphilis infections as the 47 kDa antigen has been shown to be associated with the outer membrane of this organism. Inactivity of such a surface-located protein in antibody-mediated anti-treponemal mechanisms could account for the observed ability of this organism to survive in the face of strong antibody responses in infection.
-
-
-
Production of Murine Monoclonal Antibodies to the Major Axial Filament Polypeptide of Treponema pallidum
More LessSUMMARY: A suitable immunization protocol for the stimulation of a murine antibody response to the axial filament polypeptides of Treponema pallidum was established. A range of monoclonal antibodies (Mabs) specific for different epitopes of the major axial filament polypeptide (37 kDa) was generated which demonstrated diversity in their ability to react with other treponemal species. Immunogold electron microscopy located the 37 kDa antigen on the surface of the axial filament structure. The early appearance of specific antibody to this polypeptide in infected man and rabbit indicates that such Mabs are potentially useful for the diagnosis of early syphilis.
-
-
-
Nitrite Accumulatin during Anaerobic Nitrate Reduction by Binary Suspensions of Bacteria Isolated from the Achlorhydric Stomach
More LessSUMMARY: Binary suspensions of bacteria isolated from the gastric juice of achlorhydric patients were used to determine conditions which favour nitrite accumulation during nitrate reduction. Suspensions of Veillonella parvula and Haemophilus parainfluenzae accumulated nitrite during nitrate reduction in the absence of nitrite-reducing Neisseria subflava or Streptococcus sanguis. The maximum concentration of nitrite that transiently accumulated decreased predictably as the ratio of nitrite-removing bacteria to nitrite-accumulating bacteria increased. This ratio, but more importantly the bacterial density, determined the duration of nitrite accumulation. These results are correlated with the previously reported tendency of nitrite to accumulate in the gastric juice of hypogammaglobulinaemic and pernicious anaemic patients, and with the extremely high incidence of gastric cancer in the two groups.
-
-
-
Novobiocin-resistant Mutants of Streptococcus sanguis with Reduced Cell Hydrophobicity and Defective in Coaggregation
More LessSUMMARY: Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.
-
-
-
Oligonucleotide Probes Complementary to Variable Regions of Ribosomal RNA Discriminate between Mycoplasma Species
More LessSUMMARY: On the basis of information from computer-assisted sequence comparison of the Mycoplasma pneumoniae 16S ribosomal RNA (rRNA) sequences with sequences from various other mycoplasmal and bacterial species, we constructed M. pneumoniae-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Using a DNA/RNA dot blot hybridization procedure, it was possible to detect less than 1 × 103mycoplasmas. This test is a most sensitive assay for species-specific detection of bacteria. It can easily be adapted for detection and identification of other bacterial species and may have wide medical and industrial application.
-
-
-
Characterization of Mycobacterial Immunoprecipitates by Selective Staining of Enzymes
More LessSUMMARY: Immune precipitation patterns of Mycobacterium intracellulare, M. phlei and M. smegmatis were analysed by selective enzyme staining procedures in order to characterize individual mycobacterial antigens. Enzyme activity was shown in eight precipitinogens of M. intracellulare, seven of M. phlei, and six of M. smegmatis. The identification of mycobacterial precipitinogens as enzymes is important since only a few mycobacterial antigens have been functionally characterized.
-
-
-
Aggressiveness of Conidiobolus obscurus against the Pea Aphid: Influence of Cuticular Extracts on Ballistospore Germination of Aggressive and Non-aggressive Strains
More LessSUMMARY: Cuticular extracts of Acyrthosiphon pisum, the pea aphid, stimulated the formation of germ-tubes of ballistospores of aggressive strains of Conidiobolus obscurus, but showed no influence on those of non-aggressive strains. Aggressive strains were separated into two groups: (a) those stimulated by both (i) water- or alcohol-soluble cuticular extracts originating from honeydew and (ii) lipidic extracts of the cuticle, and (b) those stimulated by (ii) only. The hydrocarbon and polar fractions of the cuticular lipids were the most active. These results obtained with C. obscurus are compared to the effect of cuticular compounds on spore germination in other entomopathogenic fungi.
-
- Physiology And Growth
-
-
-
Regulation of Carbon and Nitrogen Flow by Glutamate Synthase in Neurospora crassa
More LessSUMMARY: A glycine-resistant Neurospora crassa mutant (am-132;glyr ), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic-acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.
-
-
-
-
Growth and Metabolism of Mannitol by Strains of Saccharomyces cerevisiae
More LessSUMMARY: Of 40 polyploid strains of Saccharomyces cerevisiae screened for growth on D-mannitol (5%, w/v), half grew well (5-20 mg dry biomass ml−1). Certain of these strains were unable to grow on low concentrations of mannitol (1-2%, w/v) and others, initially unable to grow on mannitol, exhibited long-term adaptation to growth. An NAD+-dependent D-mannitol dehydrogenase (EC 1.1.1.67) was detected in mannitol-grown yeast. Growth was dependent on mitochondrial function and was obligately aerobic. Measurement of products of metabolism and respiratory activity indicated that growth on mannitol allows catabolite derepression.
-
-
-
Metabolic Control of Phycocyanin Degradation in the Cyanobacterium Synechocystis PCC 6803: a Glucose Effect
More LessSUMMARY: Following transfer to medium lacking a nitrogen source, cells of Synechocystis PCC 6803 continued to divide, giving a doubling of cell number after 40 h. Phycocyanin degradation commenced immediately after the transfer, with a rapid phase lasting 5 h in which 50% of the phycocyanin disappeared and a slow second phase in which the phycocyanin content decreased to 10% of its initial value by 24 h. In the presence of glucose, a utilizable carbon source for this facultatively heterotrophic cyanobacterium, phycocyanin was degraded initially at a rate 60% of that observed in the absence of the sugar and proteolysis was almost completely inhibited after 6 h when only 27% of the phycocyanin had been lost; cell division ceased at this time. Photosynthetic O2 evolution decreased rapidly in the presence of glucose and the cells consumed O2 in the light after 7 h, indicative of a switch to oxidative metabolism; net O2 uptake in the absence of glucose occurred only after approximately 25 h. Inhibition of phycocyanin degradation by glucose required metabolism of the sugar, probably via the oxidative pentose phosphate cycle, and appeared to result from irreversible inactivation of the protease.
-
-
-
Ammonia is the Preferred Nitrogen Source in Several Rhizobia
More LessSUMMARY: When presented with an equimolar mixture of ammonia and L-glutamate, batch cultures of Rhizobium leguminosarum MNF3841 and ‘R. trifolii’ (R. leguminosarum biovar trifolii) TA1 used ammonia at a significantly faster rate than L-glutamate. The cowpea Rhizobium strain NGR234, however, used L-glutamate at a marginally faster rate than ammonia. R. leguminosarum MNF3841 also grew faster on ammonia than on L-glutamate as the nitrogen source.
Chemostat cultures of R. leguminosarum MNF3841 limited for phosphate did not release excess ammonia when grown on mannitol/L-glutamate, showing that L-glutamate catabolism was tightly regulated to meet the cells' nitrogen requirement. Further, the rate of consumption of ammonia was similar to that for L-glutamate when either was supplied as the sole nitrogen source. However, with L-histidine or L-alanine as a nitrogen source, large quantities of excess ammonia were released. When chemostat cultures of R. leguminosarum MNF3841 were supplied with an equimolar mixture of ammonia and L-glutamate, 89-100% of the nitrogen consumed was ammonia. Similarly, with mixtures of L-glutamate/L-histidine or L-glutamate/L-alanine, almost no L-glutamate was consumed, a result attributable to the release of excess ammonia from either L-histidine or L-alanine. The use of 14C-labelled fructose or L-glutamate showed that the intra- and extracellular L-glutamate pools were isolated, indicating that the ammonia preference must be exerted by a restriction in L-glutamate transport. L-Glutamate transport rates were low in chemostats containing L-glutamate/NH4Cl, an effect attributable in part to a significant repression of synthesis of the L-glutamate transport system by ammonia.
-
-
-
Metabolism of trans-2-Butene and Butane in Nocardia TB1
More LessSUMMARY: A bacterium capable of growth on trans-2-butene was isolated from soil and identified as a Nocardia sp. The isolate also grew on propane, butane, pentane and hexane; gaseous and volatile 1-alkenes did not support growth. Intact cells grown on trans-2-butene or butane oxidized all n-alkanes, 1-alkenes, 2-alkenes and alcohols tested. Simultaneous adaptation studies, inhibitor experiments and measurements of enzyme activities in crude extracts indicated a degradative route of trans-2-butene via crotonic acid and of butane via butyric acid. The key enzymes in the proposed pathways were induced by growth on either trans-2-butene or butane. The induction of these enzymes and the substrate specificities of the enzymes suggest a relation between trans-2-butene and butane degradation.
-
-
-
Mutations Affecting Penicillin-binding Proteins 2a, 2b and 3 in Bacillus subtilis Alter Cell Shape and Peptidoglycan Metabolism
More LessSUMMARY: Bacillus subtilis mutants with altered penicillin-binding proteins (PBPs), or altered expression of PBPs, were isolated by screening for changes in susceptibility to β-lactam antibiotics. Mutations affecting only PBPs 2a, 2b and 3 were isolated. Cell shape and peptidoglycan metabolism were examined in representative mutants. Cells of a PBP 2a mutant (UB8521) were usually twisted whereas PBP 2b (UB8524) and 3 (UB8525) mutants produced helices, particularly after growth at 41 °C. The PBP 2a mutant (UB8521) had a higher peptidoglycan synthetic activity than its parent strain whereas the opposite applied to the PBP 2b mutant UB8524. The PBP 3 mutant (UB8525) had a similar peptidoglycan synthetic activity to that of the parent strain when grown at 37 °C, but 40% higher activity after growth at 41°C. The PBP 2a mutant (UB8521) exhibited the same wall thickening activity as the parent, but the PBP 2b and 3 mutants (UB8524 and UB8525) were partially defective in this respect. The changes in the susceptibility of PBP 2a, 2b and 3 mutants to β-lactam antibiotics imply that these PBPs are killing targets, consistent with the fact that these PBPs are also important for shape determination and peptidoglycan synthesis.
-
-
-
Progress of O-Acetylation and Cross-linking of Peptidoglycan in Neisseria gonorrhoeae Grown in the Presence of Penicillin
More LessSUMMARY: The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The β-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by β-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2·5 × MIC).
-
-
-
Iron Transport in Azospirillum brasilense: Role of the Siderophore Spirilobactin
More LessSUMMARY: A catechol-type siderophore was secreted by Azospirillum brasilense in the growth medium when the cells became iron-deficient. The siderophore, which we call spirilobactin, was purified and a partial characterization of its structure by hydrolysis revealed that it contained 2,3-dihydroxybenzoic acid, ornithine and serine in equimolar amounts. A high-affinity iron transport system capable of 59Fe uptake from a 59Fe(III)-spirilobactin complex was also induced in A. brasilense grown under iron deficiency. Kinetic studies on the iron uptake system revealed that it was carrier-mediated, with K m and V max values of 0·23 μm and 0·27 nmol Fe min−1(mg cell protein)−1, respectively. Dependence of iron uptake on an energized membrane, its insensitivity to arsenate and inhibition by protonophores suggest that transport is driven by the proton gradient across the membrane. Iron-deficient Azospirillum lipoferum transported 59Fe from the 59Fe(III)-spirilobactin complex (at a rate 3-fold lower than that of A. brasilense) but A. amazonense failed to show uptake of iron under the same conditions.
-
-
-
Fractionation of the Providencia stuartii Cell Envelope
More LessSUMMARY: Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.
-
-
-
Factors Reducing and Promoting the Effectiveness of Proline as an Osmoprotectant in Escherichia coli K12
More LessSUMMARY: Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/Δ1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.
-
-
-
The Role of Osmotic Effects in Haloadaptation of Vibrio costicola
More LessSUMMARY: Growth rates of Vibrio costicola showed a broad optimum between 0·8 and 1·5 M-NaCl, and there was no growth above 3·3 M-NaCl in a peptone-based medium. The minimum requirement of 0·5 M-NaCl for growth in NaCl alone was reduced to 0·3 M-NaCl when the total solute concentration was raised to 0·5 to 1·0 M equivalent with sucrose or glycerol. Compared with equivalent NaCl concentrations, higher concentrations of sucrose were more inhibitory to growth, whereas glycerol had less effect. Increasing the medium NaCl concentration suddenly by 2- or 3-fold with either a constant starting, or final, salt concentration showed that, after the shift-up, the lag in growth, the rate of growth, and the inhibition of phospholipid synthesis depended both on the final NaCl concentration and the magnitude of the shift in salinity. The time-courses of phospholipid synthesis following a 2-or 3-fold shift-up in NaCl or sucrose media were very similar and exhibited a relative increase in phosphatidylglycerol synthesis over that of phosphatidylethanolamine. This ‘switch-over’ was not seen following shift-up in glycerol media when there was also a stimulation, rather than inhibition, of phospholipid synthesis. It is concluded that during phenotypic haloadaptation of V. costicola, osmotic effects play a significant part in the sensing of and response to raised external salinity.
-
-
-
A Mathematical Method for Analysing Plasmid Stability in Micro-organisms
More LessSUMMARY: A mathematical model describing the instability of plasmids in micro-organisms has been developed. The model is based on the assumption that the overall causes of plasmid instability are described by the segregational instability of the plasmid, R (i.e. the rate at which plasmid-free cells are generated from plasmid-bearing cells), and the growth rate difference, dμ (i.e. the difference in growth rate between plasmid-free and plasmid-bearing cells). A method for determining the values of R and dμ (accompanied by 95% confidence limits) for any plasmid-bearing micro-organism is described. This method is based on the observation that, depending on the plasmid, various exponential patterns of plasmid instability are observed. The stability of Escherichia coli 1B373(pMG169), where dμ >> R, and E. coli RV308(pHSG415), where R >> dμ, are analysed in order to demonstrate the method.
-
-
-
Investigation of the Effect of Growth Environment on the Stability of Low-copy-number Plasmids in Escherichia coli.
SUMMARY: The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dμ) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.
-
- Systematics
-
-
-
Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase Enzyme Electrophoretic Patterns Amongst Strains of Bacteroides fragilis
More LessSUMMARY: Fifty-two strains of Baeteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.
-
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)